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Inhibition of cytokine secretion by scrophuloside A3 and gmelinoside L isolated from Verbascum blattaria L. by high-performance countercurrent chromatography
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Inhibition of cytokine secretion by scrophuloside A3 and gmelinoside L isolated from Verbascum blattaria L. by high-performance countercurrent chromatography Simon-Vlad Lucaa,b, , Monika Ewa Czerwińskac, Laurent Marcourte, Anca Mirona, Ana Clara Aprotosoaiea, Nina Ciocarland, Jean-Luc Wolfendere, Sebastian Granicac, Krystyna Skalicka-Woźniakb ⁎
a
Department of Pharmacognosy, Grigore T. Popa University of Medicine and Pharmacy Iasi, Universitatii 16, 700115, Iasi, Romania Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, Chodźki 1, 20-093, Lublin, Poland Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw, Banacha 1, 02-097, Warsaw, Poland d Botanical Garden, Academy of Sciences of Moldova, Padurii 18, MD-2002, Chisinau, Republic of Moldova e School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU, Michel Servet 1, 1211, Geneva 4, Switzerland b c
ARTICLE INFO
ABSTRACT
Keywords: Verbascum blattaria L. Catalpol derivatives HPCCC Neutrophils Inflammation
The purpose of this study was to analyze the HPLC-DAD-ESI-Q-TOF-MS/MS profile of the methanolic extract obtained from the aerial parts of Verbascum blattaria L. and develop efficient high-performance countercurrent chromatography (HPCCC) methods for isolation of its major iridoid glycosides. Additionally, the potential antiinflammatory activities of the extract and two of the isolated compounds were evaluated using a human neutrophil model. LC–MS analysis led to the identification of 35 constituents, of which 18 iridoids, eight saponins, three phenylethanoids, four flavonoids and two phenolic acids. Scropolioside F (A, 2 mg), scrophuloside A3 (B, 16 mg) and gmelinoside L (C, 13 mg) were obtained from 300 mg of the crude methanolic extract by HPCCC with the help of two different biphasic solvent systems: n-hexane/ethyl acetate/n-butanol/water (1/2/1/2; v/v/v/v) and n-hexane/ethyl acetate/methanol/water (1/19/1/19; v/v/v/v). The crude methanolic extract (100 μg/mL) down-regulated lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) release in a comparable extent to dexamethasone (25 μM). Gmelinoside L (50 μM) was more active than scrophuloside A3, producing inhibition percentages of 43.52% and 41.28% on interleukin 8 (IL-8) and TNF-α level, respectively. The HPCCC methods developed in the current work may serve to large scale isolation of iridoid glycosides from Verbascum species or other iridoid abundant-genera.
1. Introduction Verbascum genus (mullein, Scrophulariaceae) comprises annual, biennial and perennial herbs with deep tap roots or even small shrubs. The genus is represented by about 360 species, distributed in Asia, Europe and North America. West and Central Asia (especially Anatolia) are the main sites of biodiversity, with around 230 species documented and a high rate of endemism (84%). Flora Europaea includes 95 species, with 23 of them also present in Romanian flora (Alipieva et al., 2014; Ferguson, 1972). Mullein roots, leaves and flowers have been used for centuries in the traditional folk medicine of many countries as expectorant, mucolytic, demulcent and diuretic to treat respiratory disorders, hemorrhoids, diarrhea, rheumatic pain, wounds, fungal
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infections and other inflammatory skin conditions (Tatli and Akdemir, 2006). Nowadays, a wide variety of mullein-based pharmaceutical products (dried plant material for herbal teas, aqueous, alcoholic and oily extracts, capsules and tablets) are available in drug stores all over Europe and USA (Alipieva et al., 2014). Iridoids and, in particular, acylated iridoid diglycosides are considered to be the most abundant constituents of Verbascum genus. Nevertheless, more than 200 plant metabolites (iridoids, phenylethanoids, neolignan glycosides, flavonoids, saponins, phenolic acids and polysaccharides) have been previously reported in Verbascum species (Alipieva et al., 2014). In one of our previous reports (Luca et al., 2018), it was shown that, out of 53 metabolites identified in the extracts of V. ovalifolium Donn ex Sims, 21 were mono-, di- or tri-acyl catalpol-type
Corresponding author at: Grigore T. Popa University of Medicine and Pharmacy Iasi, Department of Pharmacognosy, Universitatii 16, 700115, Iasi, Romania. E-mail address: [email protected] (S.-V. Luca).
https://doi.org/10.1016/j.phytol.2019.02.032 Received 29 November 2018; Received in revised form 16 February 2019; Accepted 28 February 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Simon-Vlad Luca, et al., Phytochemistry Letters, https://doi.org/10.1016/j.phytol.2019.02.032
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iridoid diglycosides. Moreover, 18 of the 23 compounds isolated from V. thapsus L. were 6-O-α-L-rhamnopyranosylcatalpol esters (Warashina et al., 1991). Although numerous such derivatives have been already isolated from various Verbascum species (Alipieva et al., 2014), their preparative purification is currently still managed by the route of conventional methods, which are time consuming, require large amounts of organic solvents and repeated chromatographic steps on silica gel and Sephadex LH-20 columns for completion. The overall yields are poor because the hydroxyl groups in the iridoid glycosides make these compounds strongly adsorbed into the solid matrix during separation (Chen et al., 2017). Countercurrent chromatography (CCC) is a support-free liquid-liquid partitioning chromatographic technique in which both the stationary and mobile phases are composed of two immiscible liquids that create a biphasic solvent system. Due to column configuration and centrifugal force, one phase (the stationary one) is retained in the column without the use of an adsorptive matrix, while the second phase (the mobile one) is passed through, making thus contact with the stationary phase (Conway, 2000). The components of a mixture can be therefore separated on the basis of their affinities for one of the two layers (Koziol et al., 2018). As compared to conventional chromatography, CCC is endowed with a series of advantages, such as: the lack of irreversible adsorption, higher loading capacity, low solvent consumption, maximum sample recovery, minimum denaturation of constituents, possibility to work in both normal and reversed-phase modes with the same solvent system and easy scale-up from analytical to preparative scale (Friesen et al., 2015). Despite the fact that there are numerous reports assessing the biological activities of various Verbascum species (Alipieva et al., 2014; Tatli and Akdemir, 2006), there is a very limited number of data related to the activity of acylated iridoid diglycosides. Nigroside VI [6-O-(2′'-Otrans-p-coumaroyl)-α-L-rhamnopyranosylaucubin] and/or nigroside III [6-O-(2′'-O-trans-p-coumaroyl)-α-L-rhamnopyranosylaucubin] from V. xanthophoeniceum Griseb. have been shown to suppress the release of pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), nitric oxide, interferon gamma (IFN-γ)-induced protein-10 in stimulated peritoneal macrophages and keratinocytes (Dimitrova et al., 2012; Georgiev et al., 2012). In a recent study, the Z and E isomers of 6-O-(2′'-O-p-coumaroyl-3′'-O-menthiafoloyl)-α-Lrhamnopyranosylcatalpol from V. nobile Velen. have been proven to inhibit the growth of concanavalin A (Con A)-stimulated Jurkat T cells and Con A-induced extracellular signal-regulated kinase (ERK) phosphorylation, alter the dynamic of cell proliferation of murine CD3 T cells and inhibit the expression of early activation marker CD69 and intracellular level of IFN-γ in murine CD3+ T cells (Dimitrova et al., 2018). Verbascum blattaria L. (moth mullein) is a biennial plant of Scrophulariaceae family, native to Europe, Western and Central Asia, North Africa and naturalized to North America (USA and Canada) (Gross and Werner, 1978). Previous phytochemical investigations on V. blattaria L. have pointed out that moth mullein might be a promising source mostly of iridoid glycosides. Catalpol together with four aucubin (aucubin, 6-O-β-D-glucopyranosylaucubin, 6-O-β-D-xylopyranosylaucubin, sinuatol), three ajugol (ajugol, 8-O-acetylajugol, laterioside) and three harpagide derivatives (harpagide, 8-O-acetylharpagide harpagide acetate, harpagoside) were identified by thin layer chromatography in the ethanolic (70%) extract (Grabias and Swiatek, 1987). Moreover, from the ethyl acetate soluble fraction, two iridoid monoglycosides (harpagoside, laterioside) and two flavonoids (kaempferol-3-O-β-D-glucoside and liquiritigenin) were isolated for the first time by Youn et al. (2015). The aim of the current study was to provide a comprehensive survey of the secondary metabolite composition of V. blattaria L. by HPLCDAD-ESI-Q-TOF-MS/MS. High-performance countercurrent chromatography (HPCCC) was applied to obtain the main iridoid glycosides in a pure form, in sufficient amounts to perform biological activity studies. The effect of the extract and two of the isolated compounds on IL-8 and
TNF-α production was evaluated using a human neutrophil model. 2. Experimental 2.1. Chemicals Analytical grade methanol, ethyl acetate, n-butanol and n-hexane were purchased from POCh (Gliwice, Poland), whereas HPLC grade acetonitrile and formic acid were provided by J.T. Baker (Deventer, the Netherlands). L-glutamine, dexamethasone, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), Hanks’ balanced salt solution (HBSS), RPMI 1640 medium and fetal bovine serum (FBS) were provided by Sigma-Aldrich (St. Louis, MO, USA). Phosphate buffered saline (PBS) was purchased from Gibco (Gibco, HK, China). LPS from Escherichia coli 0111:B4 was purchased from Merck (Kenilworth, NJ, USA). Human ELISA sets (IL-8 and TNF-α) and propidium iodide (PI) were obtained from BD Biosciences (Franklin Lake, NJ, USA). 2.2. Plant material and extraction The aerial parts of V. blattaria L. were collected in Răscăieţi (Ștefan Vodă County, Republic of Moldova) in June 2014 and identified by one of the authors (N.C.). Voucher specimen VB2806/2014 was deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Grigore T. Popa University of Medicine and Pharmacy, Iasi (Romania). The air-dried, ground and pulverized plant material (5 g) was subjected to ultrasound extraction for 30 min at room temperature with methanol (3 × 50 mL). Extracts were combined, dried under the vacuum and 0.825 g crude methanolic extract (yield: 16.5%) resulted. 2.3. Metabolite profiling by HPLC-DAD-ESI-Q-TOF-MS/MS Metabolite profiling by HPLC-DAD-ESI-Q-TOF-MS/MS was performed on an Agilent 1200 HPLC (Agilent Technologies, Santa Clara, USA) equipped with a G1329B auto-sampler, G1312C binary pump, G1316 A thermostat, G1315D diode array detector (DAD), nitrogen generator (Parker Hannifin Corp., Cleveland, USA), compressed air generator (Jun-Air Oxymed, Łódź, Poland) and Agilent G6530B ESI-QTOF-MS. The separation was carried out on a Phenomenex Gemini C18 (100 × 2 mm, 3 μm) column, with (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile as mobile phase, under the following elution gradient: 0–45 min 0–60% B, 45–50 min 60–90% B min; the injection volume, flow-rate and column temperature were 10 μL, 0.2 mL/min and 25 °C, respectively, with UV spectra recorded from 200 to 400 nm. ESI-Q-TOF-MS data were acquired in negative ionization modes with the following parameters: 100-1500 m/z; gas temperature 300 °C; gas flow 12 L/min; nebulizer pressure 40 psig; capillary voltage 4000 V; skimmer 65 V; fragmentor 140 V; fixed collision energies 10 and 40 V. Data were processed with a MassHunter Qualitative Analysis Navigator B.08.00 software (Agilent). 2.4. HPCCC separation 2.4.1. HPCCC solvent system selection and preparation The suitable solvent systems were selected according to the partition coefficients (K values) in a series of shake flask experiments, as follows: about 1 mg of sample was added to 4 mL of pre-equilibrated two-phase solvent systems. Each flask was rigorously shaken and left to stand at room temperature. After the equilibrium was reached, 0.5 mL of upper and lower phases were taken, evaporated to dryness, re-dissolved in 1 mL of methanol and analyzed by HPLC-DAD. The partition coefficients were calculated as the ratios between the HPLC peak areas of target compounds in the stationary and mobile phases, respectively. The previously selected two phase solvent systems were prepared in a separation funnel (2 L) according to the volume ratios and thoroughly equilibrated after shaking at room temperature. The upper and lower 2
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Fig. 1. Chromatographic profile of V. blattaria extracts (a) HPLC-DAD-ESI-Q-TOF-MS base peak chromatogram; identity of compounds as in Table S1 and (b) HPLCDAD chromatogram (280 nm).
2.6. Structure elucidation
phases were labeled in two different recipients and both degassed for 10 min prior to use.
Compounds were identified by HPLC-DAD-ESI-Q-TOF-MS/MS (performed as described in 2.3) and NMR analyses. 1H-NMR, 13C-NMR, COSY, HSQC, HMBC and ROESY analyses were performed on a Bruker Avance Neo 600 MHz NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a QCI 5 mm Cryoprobe and a SampleJet automated sample changer. CD3OD (δH 3.31 ppm; δC 49.0 ppm) was used as internal standard for 1H and 13C NMR.
2.4.2. HPCCC separation procedure HPCCC separations of the crude methanolic extract of V. blattaria L. were carried out using a Spectrum HPCCC (Dynamic Extractions Co. Ltd., Slough, Berkshire, UK) equipped with two bobbins that fitted both the analytical (22 mL, 0.8 mm i.d.) and semi-preparative (136 mL, 1.6 mm i.d.) coils and connected to an Alpha 10 pump and a Sapphire UV detector (ECOM, Prague, Czech Republic). The semi-preparative column was first entirely filled with the upper organic phase as the stationary phase; subsequently, the lower aqueous phase was pumped into the column at a flow rate of 4.2 or 6 mL/min, while the apparatus was run at 1600 rpm (reversed-phase or head-to-tail mode). After the hydrodynamic equilibrium was attained, samples (300 mg of crude methanolic extract dissolved in a mixture of 3 mL lower phase and 3 mL upper phase) were injected through a 6 mL injection valve. The effluent from the outlet of the column was continuously monitored at 280 or 310 nm and one minute fractions (4.2 mL or 6 mL each, depending on the flow-rate) were collected from the moment of injection until the end of the chromatographic separation. The purity of all fractions was checked by analytical HPLC-DAD; similar fractions containing target compound were pooled together.
2.7. Evaluation of IL-8 and TNF-α secretion Buffy coats were obtained and prepared as previously reported (Czerwińska et al., 2018). Neutrophil viability was determined by staining with PI using the protocol described by Pawłowska et al. (2018). Neutrophils (2 × 106) were cultured in a 24-well plate in RPMI 1640 medium with 10% FBS, 10 mM HEPES and 2 mM ʟ-glutamine for 24 h at 37 °C with 5% CO2 in the absence or presence of extract (25–100 μg/mL) or compounds (12.5–50 μM), added 30 min before the stimulation with LPS (100 ng/mL). The released IL-8 and TNF-α into cell supernatants were measured by ELISA following the indications of the manufacturer. The effect on IL-8 and TNF-α secretion was calculated as the percentage of released agent in comparison with stimulated control without tested sample. Dexamethasone (1–25 μM) was used as a positive control in the cytokine secretion assay. Each extract/compound was tested in triplicate in three independent experimental sets.
2.5. Analytical HPLC-DAD analysis
2.8. Statistical analysis
The partition coefficients of target compounds and purity of all HPCCC and semi-preparative collected fractions were established by analytical HPLC-DAD experiments performed on a Shimadzu 20 A series HPLC (Shimadzu Corp., Kyoto, Japan) equipped with a DGC-20 A 3R automatic degasser, LC-20 AD quaternary pump, SIL-20 A HT autosampler and SPD-M20 A DAD. The separation was performed on an Agilent Zorbax Eclipse XDB-C18 (250 × 4.6 mm, 5 μm) column with (A) 0.1% formic acid in water and (B) acetonitrile as mobile phase, under the following elution gradient: 0–5 min 10–25% B, 5–10 min 25–40% B, 10–20 min 40–70% B, 25–27 min 70–100% B, 27–35 min 100–10% B; the injection volume, flow-rate and column temperature were 10 μL, 1 mL/min and 25 °C, respectively; UV spectra were recorded at 280 and 310 nm.
The results were expressed as a means ± S.E.M. Statistical significance of differences between means was established by ANOVA with Turkey’s post-hoc test. P values below 0.05 were considered statistically significant. All analyses were performed using Statistica 10 (StatSoft). 3. Results and discussion 3.1. Metabolite profiling by HPLC-DAD-ESI-Q-TOF-MS/MS Metabolite profiling was initially performed by HPLC-DAD-ESI-QTOF-MS/MS on the crude methanolic extract of V. blattaria L. It showed 3
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a high occurrence of iridoid glycosides, together with other minor metabolites, such as: phenylethanoid glycosides, flavonoids, saponins and phenolic acids. The representative base peak chromatogram of the HPLC-DAD-ESI-Q-TOF-MS analysis is depicted in Fig. 1A, whereas peak assignments are given in Table S1.
tetradesmosidic, were identified in V. blattaria L. Their assignments were performed based on the number of hexose units lost from their pseudomolecular ions and by searching the corresponding aglycon in literature data on Verbascum saponins, since the saponin aglycons do not generally suffer further relevant fragmentations. Moreover, it was observed the following order of neutral hexose losses: rhamnosyl/glucosyl → glucosyl/rhamnosyl → glucosyl → fucosyl, suggesting that fucose is bound directly to the aglycon. Peak 27 presented a pseudomolecular ion [M−H]– at m/z 1089.5839 that suggested the molecular formula of C54H90O22. The fragment ions resulted from the successive losses of glucose (m/z 927), rhamnose (m/z 781), glucose (m/z 619) and fucose (m/z 473) allowed the tentative annotation as songarosaponin B (Seifert et al., 1991), whereas compound 35 ([M−H]– at m/ z 925.5162, C48H78O17) was assigned to desrhamnosylverbascosaponin based on the report of Klimek (1996). After the loss of all hexose residues, peaks 28 ([M−H]– at 1087.5702, C54H88O22) and 29 ([M−H]– at 941.5121, C48H78O18) yielded the same aglycon with m/z 471, the difference between them appearing due to an extra rhamnose within the structure of compound 28. These constituents were tentatively identified as buddlejasaponins I and IV, respectively (Hartleb and Seifert, 1995). HRMS spectra of peaks 31 ([M−H]– at 1103.5655) and 32 ([M−H]– at 1103.5980) suggested two different molecular formulas, C54H88O23 and C55H92O22, respectively, being ascribed to mulleinsaponin IV (Miyase et al., 1997) and ilwensisaponin C, respectively (Calis et al., 1993; Tatli et al., 2004). Finally, compounds 33 and 34 ([M−H]– at m/z 1071.5719, C54H88O21) were identified as verbascosaponin (Schroder and Haslinger, 1993) and songarosaponin A (Seifert et al., 1991). Songarosaponins A–B were reported in V. songaricum Schrenk, buddlejasponins I-IV in V. thapsiforme Schrad., verbascosaponin and derhamnosylverbascosaponin in V. phlomoides L., mulleinsaponin IV in V. fruticulosum Post., whereas ilwensisaponin C was found in V. pterocalycinum var. mutense Hub.-Mor. (Alipieva et al., 2014).
3.1.1. Iridoid glycosides Eighteen iridoid glycosides were identified in V. blattaria extract; of these, 3 monoglycosides and 15 diglycosides. In our previous work (Luca et al., 2018) the approach used for the efficient dereplication of acylated catalpol-type iridoid diglycosides has been described. In brief, the deprotonated molecular ion [M−H]– recorded in the negative ion mode exhibited the loss of the glucose moiety (162 Da), invariably followed by a characteristic loss of a C5H6O3 fragment (114 Da) from the catalpolgenin moiety. The complete breakage of the aglycon part generates a negatively charged ion that contains the acyl residue(s) linked to the second sugar unit (rhamnose). Depending on their masses, all hexose losses were attributed to glucose or rhamnose, because of the consistencies with known compounds previously isolated from this genus. Further fragmentations yielded smaller mass, but highly abundant, ions assigned to the deprotonated, dehydrated or decarboxylated forms of the cinnamic acid derivatives, such as p-coumaric, caffeic or ferulic acids, within the structure of these compounds. By applying these general rules, three monacyl (6, 12, 13), ten diacyl (16-19, 21-24, 26, 30) and two triacyl (14, 25) catalpol-type iridoid diglycosides have been tentatively identified (Table S1). This subcategory of specialized metabolites is known to be highly specific to Verbascum L., with more than 30 representatives, such as saccatoside (Tatli et al., 2006), verbaspinoside (Kalpoutzakis et al., 1999), verbascoside A (Warashina et al., 1991), buddlejoside A5 (Akdemir et al., 2004), pulverulentoside I (Seifert et al., 1989), reported in numerous mullein species. The three iridoid monoglycosides were assigned as follows: peak 2 ([M−H]– at m/z 361.1152.; fragment ions at m/z 343, 199, 181 and 169) to catalpol (also confirmed by standard); peak 3 ([M−H]– at m/z 347.1365; ion fragments at m/z 185, 167, 149, 131, 119 and 89) to ajugol; and peak 20 ([M−H]– at m/z 477.1744; fragments ions at m/z 329, 147, 103) to laterioside (Luca et al., 2018). Catalpol and ajugol are frequent in Verbascum species (Alipieva et al., 2014), whereas laterioside has been previously reported only in V. blattaria L. (Youn et al., 2015).
3.1.4. Flavonoids Four flavonoid glycosides were identified in V. blattaria L. Peak 4 was tentatively labeled as apigenin pentoside, previously reported in V. ovalifolium Donn ex Sims (Luca et al., 2018). After successive losses of glucuronide (176 Da) and glucose (162 Da), compound 11 ([M−H]– at m/z 607.1319) generated the aglycon apigenin (at m/z 269) that further gave characteristic ion fragments (Fabre et al., 2001); thence, for this compound, the structure of apigenin glucoside glucuronide was proposed. Since peak 10 ([M−H]– at m/z 447.0946) produced, after the loss of 162 Da, the aglycon fragment ion at m/z 285 characteristic to luteolin (Fabre et al., 2001), it was unequivocally assigned to luteolin7-O-glucoside, by also comparing its retention time, UV and MS spectra with that of an authentic standard. In addition to compound 10, peak 7 ([M−H]– at m/z 623.1285) contained one glucuronide residue, being thus tentatively identified as luteolin glucoside glucuronide. Compounds 7 and 11 have not been previously reported in Verbascum species, whereas flavonoid glucuronides, such as apigenin-7-O-glucuronide and luteolin-7-O-glucuronide, have been identified in V. lychnitis L. and V. nigrum L. (Alipieva et al., 2014).
3.1.2. Phenylethanoid glycosides Three phenylethanoid glycosides were identified in V. blattaria L. Compound 9 exhibited [M−H]– at m/z 623.1990 and the following fragment ions (m/z): 461 [M–caffeoyl–H]–, 315 [M–caffeoyl–rhamnosyl–H]–, 179 [caffeate]–, 161 [anydro-caffeate]– and 153 [hydroxytyrosol-H]–. The comparison of retention time, UV and MS data with that of the commercial standard led to its unequivocal identification as verbascoside. Compound 8 ([M−H]– at m/z 755.2415), generated some additional fragment ions (at m/z 593 [M–caffeoyl–H]–, 461 [M–caffeoyl–pentosyl–H]–, 447 [M–caffeoyl–rhamnosyl–H]–, 315 [M–caffeoyl–pentosyl–rhamnosyl–H]–, 179 [cafeate]–, 161 [caffeoyl–H]–, 135 [caffeate−CO2]–) when compared to verbascoside, due to the presence of a pentosyl unit in its structure. It was tentatively identified as angoroside A (Gross et al., 1987; Zhou et al., 2014). Peak 15 ([M−H]– at m/z 783.2526) showed several main fragment ions at m/z 637 [M–rhamnosyl–H]–, 607 [M–feruloyl–H]–, 475 [M–feruloyl–pentosyl–H]–, 461 [M–feruloyl–rhamnosyl–H]–, 329 [M–feruloyl–rhamnosyl–pentosyl–H]– and 193 [ferulate]–. These MS/MS data were in agreement with the structure of angoroside C (Calis et al., 1988; Tong et al., 2009). Verbascoside is one them most frequently encountered constituent in Verbascum L. (Alipieva et al., 2014), whereas angorosides A/ C have been isolated from some species, such as V. spinosum L. (Kalpoutzakis et al., 1999).
3.1.5. Phenolic acids Additionally, two phenolic acids have been identified in V. blattaria L. Compound 1 showed [M−H]– at m/z 341.0890 and three fragment ions at m/z 179 [caffeate]–, 161 [anhydro-caffeate]– and 135 [decarboxy-caffeate]–, being tentatively characterized as caffeic acid glucoside. Compound 5, with a parent ion at m/z 337.0944 and fragment ions at m/z 191 [quinate]–, 163 [p-coumarate]– and 145 [anhydro-pcoumarate]–, was tentatively ascribed to p-coumaroyl quinic acid. Both compounds were previously reported in V. ovalifolium Donn ex Sims (Luca et al., 2018).
3.1.3. Saponins Eight oleanan-type triterpene saponins, two tridemosidic and six 4
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3.2. HPCCC separation Selection of the two-phase solvent system is the most important feature in achieving a successful HPCCC separation. A good solvent system requires several considerations, such as: (i) a short settling time (< 30 s); (ii) a partition coefficient of the target constituent within a suitable range (usually between 0.2 and 5); and (iii) a separation factor (α = K2/K1, K2 > K1) between any two compounds higher than 1.5 (Yue et al., 2013). HPLC-DAD profile of the crude methanolic extract of V. blattaria L. (Fig. 1B) suggested the presence of three major acylated iridoid diglycosides, as also confirmed by dereplication based on HRMS and MS/MS. Various mixtures of ethyl acetate/n-butanol/water (EBWat), n-hexane/ ethyl acetate/n-butanol/water (HEBWat) and n-hexane/ethyl acetate/ methanol/water (HEMWat) were screened for the partition coefficients of the three target compounds. Previously, EBWat family has proven successful in CCC isolation of iridoids from numerous plant sources, such as catalpol from Rehmannia glutinosa Libosch. (Tong et al., 2015), geniposide from Gardenia jasminoides Ellis (Wang et al., 2015), geniposidic acid from Eucommia ulmoides Oliv. (Dai et al., 2013), harpagoside from Scrophularia ningpoensis Hemsl. (Tong et al., 2006) or loganic acid from Gentiana scabra Bunge (Chen et al., 2017). However, in the case of V. blattaria L., EBWat 5/1/5 v/v/v (I) and EBWat 2/1/3 v/v/ v (II) gave suitable partition coefficients only for compound A, whereas the other two constituents had K values higher than 5 (Table 1). When n-hexane was added to the system, as in system III, the partition coefficient of compound B significantly decreased to 1.91, but it remained unsolved for the third analyte. Therefore, less polar solvent systems were tested, such as those belonging to HEMWat family (IV and V); consequently, the K values of compound C were situated within the range of 0.2–5 in both cases, but those of the first two constituents were lower than 0.2. Since one solvent system to simultaneously and efficiently target all three compounds was not found, mainly due to constituents’ different polarity, solvent systems III and IV were subsequently selected for two parallel sets of semi-preparative HPCCC experiments. Compounds A (HPCCC elution time 24–25, 2 mg) and B (HPCCC elution time 42–44 min, 16 mg) were obtained after separation with HEBWat 1/2/1/2 v/v/v/v (300 mg crude extract, 1600 rpm, 4.2 mL/ min, 310 nm, stationary phase retention 67%; upper phase as stationary phase), whereas compound C (HPCCC elution time 23–25 min, 13 mg) was afforded after separation with HEMWat 1/19/1/19 v/v/v/v (300 mg, 1600 rpm, 6 mL/min, 280 nm, stationary phase retention 65%; upper phase as stationary phase) (Fig. S1). The identity of the three compounds (Fig. 2) was established by HPLC-DAD-ESI-Q-TOF-MS/MS, 1D NMR, 2D NMR analyses and comparison of these spectral data with those reported in the available literature, as follows: (A) scropolioside F (Tatli et al., 2006); (B) scrophuloside A3 (Miyase and Mimatsu, 1999); and (C) gmelinoside L (Hosny and Rosazza, 1998). Of these, only compound A has been previously reported in other Verbascum species (Tatli et al., 2006), while the other two are new for this genus, with scrophuloside A3 isolated
Fig. 2. Chemical structures of isolated compounds from V. blattaria (A) scropolioside F (B) scrophuloside A3 and (C) gmelinoside L.
only from Scrophularia nodosa L. (Miyase and Mimatsu, 1999) and gmelinoside L from Gmelina arborea Roxb. and G. hainanensis Oliv. (Hosny and Rosazza, 1998; Xiong et al., 2018). Thus, this is the first report where countercurrent separation techniques are used as chromatographic approach for the efficient purification of these constituents. The methods developed in the current study are extremely fast (less than 45 min), which might make them suitable for the highscale HPCCC purification of identical or similar acylated iridoid diglycosides from other Verbascum species or other plant sources containing this category of metabolites. 3.3. Evaluation of IL-8 and TNF-α secretion In the last part of the current study, the crude methanolic extract of V. blattaria L., scrophuloside A3 and gmelinoside L were investigated as potential IL-8 and TNF-α inhibitors in LPS-stimulated neutrophils. None of the samples were cytotoxic to neutrophils at all tested concentrations based on cytometric experiments (data not shown). Neutrophils (polymorphonuclear cells, PMNs) are the most abundant leukocytes in the blood and constitute the first line of host defense against numerous infectious pathogens (Rosales et al., 2016). After migration to the infection and inflammation sites, they recognize and phagocyte invading microorganisms via processes that involve molecular mediators, such as reactive oxygen species, proteases (elastase, metalloproteinases), chemokines sand cytokines (IL-8, TNF-α). Furthermore, it is claimed that overstimulation of PMNs initiates advanced non-infectious inflammatory responses. IL-8 and TNF-α are two of the most important chemokines produced by PMNs, being involved in T lymphocyte and basophile chemotaxis, degranulation and PMNs endothelial adhesion. The decrease of IL-8 and TNF-α production results in prevention of inflammatory response progression (Czerwińska et al., 2018; Rosales et al., 2016). The crude methanolic extract of V. blattaria L. was able to significantly inhibit both IL-8 (65.31–71.12% of LPS + control) and TNF-α secretion (16.07–47.25%) at 50 and 100 μg/mL (Fig. 3). It is worth mentioning that the effect produced by the extract (100 μg/mL) on TNF-α release is comparable to that obtained for the positive control (dexamethasone) at 25 μM (19.46%). Scrophuloside A3 (B) decreased the production of IL-8 at all tested concentrations (between 62.62–66.16% of LPS + control), whereas TNF-α release was reduced
Table 1 Partition coefficient values of isolated compounds from V. blattaria. No.
Solvent system
KA
KB
KC
I II III IV V
EBWat (2/1/3; v/v/v) EBWat (5/1/5; v/v/v) HEBWat (1/2/1/2; v/v/v/v) HEMWat (1/19/1/19; v/v/v/v) HEMWat (1/9/1/9; v/v/v/v)
2.76 0.99 0.31 0.05 0.01
11.76 6.00 1.91 0.15 0.19
18.75 8.92 6.17 1.05 0.64
EBWat ethyl acetate/n-butanol/water; HEBWat n-hexane/ethyl acetate/n-butanol/water; HEMWat n-hexane/ethyl acetate/methanol/water; values in bold indicate that these solvent systems were further used for the high-performance counter-current chromatography separation of those target compounds. 5
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Fig. 3. Effect of the crude methanolic extract (25–100 μg/mL), scrophuloside A3 (B, 12.5–50 μM), gmelinoside L (C, 12.5–50 μM) and dexamethasone (Dex, 1–25 μM) on (a) IL-8 and (b) TNF-α secretion in LPS-stimulated (100 ng/mL) neutrophils. Data were expressed as mean ± SEM of three separate experiments performed with cells isolated from independent donors assayed in triplicate. Statistical significance: *p < 0.05 and **p < 0.001 vs. stimulated control; #p < 0.001 vs. nonstimulated control.
only at 25 μM (63.48%) and 50 μM (47.70%). Gmelinoside L (C) produced significant inhibitory effects on IL-8 (43.52–48.25%) and TNF-α (41.28–64.64%) production at 25–50 μM. However, the effects of both tested compounds were weaker than those of dexamethasone at all tested concentrations. Despite the fact that there are numerous reports assessing the antiinflammatory potential of extracts of various Verbascum species (Alipieva et al., 2014; Tatli and Akdemir, 2006), there is a very limited number of data related to the activity of acylated iridoid diglycosides, which are known as one of the most abundant constituents in mulleins. For instance, the crude methanolic extract of V. xanthophoeniceum Griseb. (500 μg/mL) together with nigroside VI (250 μg/mL) significantly down-regulated IL-6 and TNF-α production in LPS-stimulated peritoneal macrophages (Dimitrova et al., 2012). However, when tested on IFN-γ-stimulated NHK keratinocytes, nigroside VI (10–50 μM) did not significantly reduced IL-8 secretion. Nevertheless, nigroside III was inactive in all the above assays (Georgiev et al., 2012). Overall, this is the first study reporting that scrophuloside A3 and gmelinoside L significantly influence IL-8 and TNF-α secretion in LPS-stimulated neutrophils.
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Acknowledgments The School of Pharmaceutical Sciences of the University of Geneva (Prof. J-L. Wolfender) is thankful to the Swiss National Science Foundation for the support in the acquisition of the NMR 600 MHz (SNF R’Equip grant 316030_164095). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.02.032. References Akdemir, Z.S., Tatli, I.I., Bedir, E., Khan, I.A., 2004. Acetylated iridoid glycosides from Verbascum lasianthum Boiss. Turk. J. Chem. 28, 101–109. Alipieva, K.I., Orhan, I.E., Cankaya, I.I.T., Kostadinova, E.P., Georgiev, M.I., 2014. Treasure from garden: chemical profiling, pharmacology and biotechnology of mulleins. Phytochem. Rev. 13, 417–444. Calis, I., Gross, G.A., Sticher, O., 1988. Two phenylpropanoid glycosides from Scrophularia scopolii. Phytochemistry 27, 1465–1468. Calis, I., Zor, M., Basaran, A.A., Wright, A.D., Sticher, O., 1993. Ilwensisaponins a, B, C, and d: triterpene saponins from Scrophularia ilwensis. Helv. Chim. Acta 76, 1352–1360. Chen, B., Peng, Y., Wang, X., Li, Z., Sun, Y., 2017. Preparative separation and purification of four glycosides from Gentianae radix by high-speed counter-current chromatography and comparison of their anti-NO production effects. Molecules 22, 2002. Conway, W.D., 2000. Countercurrent chromatography and high speed countercurrent chromatography: instrumentation. In: Wilson, I.D. (Ed.), Encyclopedia of Separation Science. Academic Press, Oxford, pp. 365–374. Czerwińska, M.E., Dudek, M.K., Pawłowska, K.A., Pruś, A., Ziaja, M., Granica, S., 2018.
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Zhang, H., 2015. Systematic separation and purification of iridoid glycosides and crocetin derivatives from Gardenia jasminoides Ellis by high-speed counter-current chromatography. Phytochem. Anal. 26, 202–208. Warashina, T., Miyase, T., Ueno, A., 1991. Iridoid glycosides from Verbascum thapsus L. Chem. Pharm. Bull. 39, 3261–3264. Xiong, J., Wu, X.Y., Wang, P.P., Lau, C., Fan, H., Ma, G.L., Tang, Y., Li, J., Hu, J.F., 2018. Acylated iridoid diglycosides from the cultivated endangered ornamental tree Gmelina hainanensis. Phytochem. Lett. 25, 17–21. Youn, I.S., Han, A.R., Roh, M.S., Seo, E.K., 2015. Constituents of the leaves of Verbascum blattaria. Nat. Prod. Commun. 10, 445–446. Yue, H.L., Zhao, X.H., Wang, Q.L., Tao, Y.D., 2013. Separation and purification of watersoluble iridoid glucosides by high speed counter-current chromatography combined with macroporous resin column separation. J. Chromatogr B. 936, 57–62. Zhou, W., Shan, J., Wang, S., Ju, W., Meng, M., Cai, B., Di, L., 2014. Simultaneous determination of caffeic acid derivatives by UPLC-MS/MS in rat plasma and its application in pharmacokinetic study after oral administration of Flos Lonicerae-Fructus Forsythiae herb combination. J. Chromatogr. B 949-950, 7–15.
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Inhibition of cytokine secretion by scrophuloside A3 and gmelinoside L isolated from Verbascum blattaria L. by high-performance countercurrent chromatography Simon-Vlad Lucaa,b, , Monika Ewa Czerwińskac, Laurent Marcourte, Anca Mirona, Ana Clara Aprotosoaiea, Nina Ciocarland, Jean-Luc Wolfendere, Sebastian Granicac, Krystyna Skalicka-Woźniakb ⁎
a
Department of Pharmacognosy, Grigore T. Popa University of Medicine and Pharmacy Iasi, Universitatii 16, 700115, Iasi, Romania Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, Chodźki 1, 20-093, Lublin, Poland Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw, Banacha 1, 02-097, Warsaw, Poland d Botanical Garden, Academy of Sciences of Moldova, Padurii 18, MD-2002, Chisinau, Republic of Moldova e School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU, Michel Servet 1, 1211, Geneva 4, Switzerland b c
ARTICLE INFO
ABSTRACT
Keywords: Verbascum blattaria L. Catalpol derivatives HPCCC Neutrophils Inflammation
The purpose of this study was to analyze the HPLC-DAD-ESI-Q-TOF-MS/MS profile of the methanolic extract obtained from the aerial parts of Verbascum blattaria L. and develop efficient high-performance countercurrent chromatography (HPCCC) methods for isolation of its major iridoid glycosides. Additionally, the potential antiinflammatory activities of the extract and two of the isolated compounds were evaluated using a human neutrophil model. LC–MS analysis led to the identification of 35 constituents, of which 18 iridoids, eight saponins, three phenylethanoids, four flavonoids and two phenolic acids. Scropolioside F (A, 2 mg), scrophuloside A3 (B, 16 mg) and gmelinoside L (C, 13 mg) were obtained from 300 mg of the crude methanolic extract by HPCCC with the help of two different biphasic solvent systems: n-hexane/ethyl acetate/n-butanol/water (1/2/1/2; v/v/v/v) and n-hexane/ethyl acetate/methanol/water (1/19/1/19; v/v/v/v). The crude methanolic extract (100 μg/mL) down-regulated lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) release in a comparable extent to dexamethasone (25 μM). Gmelinoside L (50 μM) was more active than scrophuloside A3, producing inhibition percentages of 43.52% and 41.28% on interleukin 8 (IL-8) and TNF-α level, respectively. The HPCCC methods developed in the current work may serve to large scale isolation of iridoid glycosides from Verbascum species or other iridoid abundant-genera.
1. Introduction Verbascum genus (mullein, Scrophulariaceae) comprises annual, biennial and perennial herbs with deep tap roots or even small shrubs. The genus is represented by about 360 species, distributed in Asia, Europe and North America. West and Central Asia (especially Anatolia) are the main sites of biodiversity, with around 230 species documented and a high rate of endemism (84%). Flora Europaea includes 95 species, with 23 of them also present in Romanian flora (Alipieva et al., 2014; Ferguson, 1972). Mullein roots, leaves and flowers have been used for centuries in the traditional folk medicine of many countries as expectorant, mucolytic, demulcent and diuretic to treat respiratory disorders, hemorrhoids, diarrhea, rheumatic pain, wounds, fungal
⁎
infections and other inflammatory skin conditions (Tatli and Akdemir, 2006). Nowadays, a wide variety of mullein-based pharmaceutical products (dried plant material for herbal teas, aqueous, alcoholic and oily extracts, capsules and tablets) are available in drug stores all over Europe and USA (Alipieva et al., 2014). Iridoids and, in particular, acylated iridoid diglycosides are considered to be the most abundant constituents of Verbascum genus. Nevertheless, more than 200 plant metabolites (iridoids, phenylethanoids, neolignan glycosides, flavonoids, saponins, phenolic acids and polysaccharides) have been previously reported in Verbascum species (Alipieva et al., 2014). In one of our previous reports (Luca et al., 2018), it was shown that, out of 53 metabolites identified in the extracts of V. ovalifolium Donn ex Sims, 21 were mono-, di- or tri-acyl catalpol-type
Corresponding author at: Grigore T. Popa University of Medicine and Pharmacy Iasi, Department of Pharmacognosy, Universitatii 16, 700115, Iasi, Romania. E-mail address: [email protected] (S.-V. Luca).
https://doi.org/10.1016/j.phytol.2019.02.032 Received 29 November 2018; Received in revised form 16 February 2019; Accepted 28 February 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Simon-Vlad Luca, et al., Phytochemistry Letters, https://doi.org/10.1016/j.phytol.2019.02.032
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iridoid diglycosides. Moreover, 18 of the 23 compounds isolated from V. thapsus L. were 6-O-α-L-rhamnopyranosylcatalpol esters (Warashina et al., 1991). Although numerous such derivatives have been already isolated from various Verbascum species (Alipieva et al., 2014), their preparative purification is currently still managed by the route of conventional methods, which are time consuming, require large amounts of organic solvents and repeated chromatographic steps on silica gel and Sephadex LH-20 columns for completion. The overall yields are poor because the hydroxyl groups in the iridoid glycosides make these compounds strongly adsorbed into the solid matrix during separation (Chen et al., 2017). Countercurrent chromatography (CCC) is a support-free liquid-liquid partitioning chromatographic technique in which both the stationary and mobile phases are composed of two immiscible liquids that create a biphasic solvent system. Due to column configuration and centrifugal force, one phase (the stationary one) is retained in the column without the use of an adsorptive matrix, while the second phase (the mobile one) is passed through, making thus contact with the stationary phase (Conway, 2000). The components of a mixture can be therefore separated on the basis of their affinities for one of the two layers (Koziol et al., 2018). As compared to conventional chromatography, CCC is endowed with a series of advantages, such as: the lack of irreversible adsorption, higher loading capacity, low solvent consumption, maximum sample recovery, minimum denaturation of constituents, possibility to work in both normal and reversed-phase modes with the same solvent system and easy scale-up from analytical to preparative scale (Friesen et al., 2015). Despite the fact that there are numerous reports assessing the biological activities of various Verbascum species (Alipieva et al., 2014; Tatli and Akdemir, 2006), there is a very limited number of data related to the activity of acylated iridoid diglycosides. Nigroside VI [6-O-(2′'-Otrans-p-coumaroyl)-α-L-rhamnopyranosylaucubin] and/or nigroside III [6-O-(2′'-O-trans-p-coumaroyl)-α-L-rhamnopyranosylaucubin] from V. xanthophoeniceum Griseb. have been shown to suppress the release of pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), nitric oxide, interferon gamma (IFN-γ)-induced protein-10 in stimulated peritoneal macrophages and keratinocytes (Dimitrova et al., 2012; Georgiev et al., 2012). In a recent study, the Z and E isomers of 6-O-(2′'-O-p-coumaroyl-3′'-O-menthiafoloyl)-α-Lrhamnopyranosylcatalpol from V. nobile Velen. have been proven to inhibit the growth of concanavalin A (Con A)-stimulated Jurkat T cells and Con A-induced extracellular signal-regulated kinase (ERK) phosphorylation, alter the dynamic of cell proliferation of murine CD3 T cells and inhibit the expression of early activation marker CD69 and intracellular level of IFN-γ in murine CD3+ T cells (Dimitrova et al., 2018). Verbascum blattaria L. (moth mullein) is a biennial plant of Scrophulariaceae family, native to Europe, Western and Central Asia, North Africa and naturalized to North America (USA and Canada) (Gross and Werner, 1978). Previous phytochemical investigations on V. blattaria L. have pointed out that moth mullein might be a promising source mostly of iridoid glycosides. Catalpol together with four aucubin (aucubin, 6-O-β-D-glucopyranosylaucubin, 6-O-β-D-xylopyranosylaucubin, sinuatol), three ajugol (ajugol, 8-O-acetylajugol, laterioside) and three harpagide derivatives (harpagide, 8-O-acetylharpagide harpagide acetate, harpagoside) were identified by thin layer chromatography in the ethanolic (70%) extract (Grabias and Swiatek, 1987). Moreover, from the ethyl acetate soluble fraction, two iridoid monoglycosides (harpagoside, laterioside) and two flavonoids (kaempferol-3-O-β-D-glucoside and liquiritigenin) were isolated for the first time by Youn et al. (2015). The aim of the current study was to provide a comprehensive survey of the secondary metabolite composition of V. blattaria L. by HPLCDAD-ESI-Q-TOF-MS/MS. High-performance countercurrent chromatography (HPCCC) was applied to obtain the main iridoid glycosides in a pure form, in sufficient amounts to perform biological activity studies. The effect of the extract and two of the isolated compounds on IL-8 and
TNF-α production was evaluated using a human neutrophil model. 2. Experimental 2.1. Chemicals Analytical grade methanol, ethyl acetate, n-butanol and n-hexane were purchased from POCh (Gliwice, Poland), whereas HPLC grade acetonitrile and formic acid were provided by J.T. Baker (Deventer, the Netherlands). L-glutamine, dexamethasone, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), Hanks’ balanced salt solution (HBSS), RPMI 1640 medium and fetal bovine serum (FBS) were provided by Sigma-Aldrich (St. Louis, MO, USA). Phosphate buffered saline (PBS) was purchased from Gibco (Gibco, HK, China). LPS from Escherichia coli 0111:B4 was purchased from Merck (Kenilworth, NJ, USA). Human ELISA sets (IL-8 and TNF-α) and propidium iodide (PI) were obtained from BD Biosciences (Franklin Lake, NJ, USA). 2.2. Plant material and extraction The aerial parts of V. blattaria L. were collected in Răscăieţi (Ștefan Vodă County, Republic of Moldova) in June 2014 and identified by one of the authors (N.C.). Voucher specimen VB2806/2014 was deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Grigore T. Popa University of Medicine and Pharmacy, Iasi (Romania). The air-dried, ground and pulverized plant material (5 g) was subjected to ultrasound extraction for 30 min at room temperature with methanol (3 × 50 mL). Extracts were combined, dried under the vacuum and 0.825 g crude methanolic extract (yield: 16.5%) resulted. 2.3. Metabolite profiling by HPLC-DAD-ESI-Q-TOF-MS/MS Metabolite profiling by HPLC-DAD-ESI-Q-TOF-MS/MS was performed on an Agilent 1200 HPLC (Agilent Technologies, Santa Clara, USA) equipped with a G1329B auto-sampler, G1312C binary pump, G1316 A thermostat, G1315D diode array detector (DAD), nitrogen generator (Parker Hannifin Corp., Cleveland, USA), compressed air generator (Jun-Air Oxymed, Łódź, Poland) and Agilent G6530B ESI-QTOF-MS. The separation was carried out on a Phenomenex Gemini C18 (100 × 2 mm, 3 μm) column, with (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile as mobile phase, under the following elution gradient: 0–45 min 0–60% B, 45–50 min 60–90% B min; the injection volume, flow-rate and column temperature were 10 μL, 0.2 mL/min and 25 °C, respectively, with UV spectra recorded from 200 to 400 nm. ESI-Q-TOF-MS data were acquired in negative ionization modes with the following parameters: 100-1500 m/z; gas temperature 300 °C; gas flow 12 L/min; nebulizer pressure 40 psig; capillary voltage 4000 V; skimmer 65 V; fragmentor 140 V; fixed collision energies 10 and 40 V. Data were processed with a MassHunter Qualitative Analysis Navigator B.08.00 software (Agilent). 2.4. HPCCC separation 2.4.1. HPCCC solvent system selection and preparation The suitable solvent systems were selected according to the partition coefficients (K values) in a series of shake flask experiments, as follows: about 1 mg of sample was added to 4 mL of pre-equilibrated two-phase solvent systems. Each flask was rigorously shaken and left to stand at room temperature. After the equilibrium was reached, 0.5 mL of upper and lower phases were taken, evaporated to dryness, re-dissolved in 1 mL of methanol and analyzed by HPLC-DAD. The partition coefficients were calculated as the ratios between the HPLC peak areas of target compounds in the stationary and mobile phases, respectively. The previously selected two phase solvent systems were prepared in a separation funnel (2 L) according to the volume ratios and thoroughly equilibrated after shaking at room temperature. The upper and lower 2
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Fig. 1. Chromatographic profile of V. blattaria extracts (a) HPLC-DAD-ESI-Q-TOF-MS base peak chromatogram; identity of compounds as in Table S1 and (b) HPLCDAD chromatogram (280 nm).
2.6. Structure elucidation
phases were labeled in two different recipients and both degassed for 10 min prior to use.
Compounds were identified by HPLC-DAD-ESI-Q-TOF-MS/MS (performed as described in 2.3) and NMR analyses. 1H-NMR, 13C-NMR, COSY, HSQC, HMBC and ROESY analyses were performed on a Bruker Avance Neo 600 MHz NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a QCI 5 mm Cryoprobe and a SampleJet automated sample changer. CD3OD (δH 3.31 ppm; δC 49.0 ppm) was used as internal standard for 1H and 13C NMR.
2.4.2. HPCCC separation procedure HPCCC separations of the crude methanolic extract of V. blattaria L. were carried out using a Spectrum HPCCC (Dynamic Extractions Co. Ltd., Slough, Berkshire, UK) equipped with two bobbins that fitted both the analytical (22 mL, 0.8 mm i.d.) and semi-preparative (136 mL, 1.6 mm i.d.) coils and connected to an Alpha 10 pump and a Sapphire UV detector (ECOM, Prague, Czech Republic). The semi-preparative column was first entirely filled with the upper organic phase as the stationary phase; subsequently, the lower aqueous phase was pumped into the column at a flow rate of 4.2 or 6 mL/min, while the apparatus was run at 1600 rpm (reversed-phase or head-to-tail mode). After the hydrodynamic equilibrium was attained, samples (300 mg of crude methanolic extract dissolved in a mixture of 3 mL lower phase and 3 mL upper phase) were injected through a 6 mL injection valve. The effluent from the outlet of the column was continuously monitored at 280 or 310 nm and one minute fractions (4.2 mL or 6 mL each, depending on the flow-rate) were collected from the moment of injection until the end of the chromatographic separation. The purity of all fractions was checked by analytical HPLC-DAD; similar fractions containing target compound were pooled together.
2.7. Evaluation of IL-8 and TNF-α secretion Buffy coats were obtained and prepared as previously reported (Czerwińska et al., 2018). Neutrophil viability was determined by staining with PI using the protocol described by Pawłowska et al. (2018). Neutrophils (2 × 106) were cultured in a 24-well plate in RPMI 1640 medium with 10% FBS, 10 mM HEPES and 2 mM ʟ-glutamine for 24 h at 37 °C with 5% CO2 in the absence or presence of extract (25–100 μg/mL) or compounds (12.5–50 μM), added 30 min before the stimulation with LPS (100 ng/mL). The released IL-8 and TNF-α into cell supernatants were measured by ELISA following the indications of the manufacturer. The effect on IL-8 and TNF-α secretion was calculated as the percentage of released agent in comparison with stimulated control without tested sample. Dexamethasone (1–25 μM) was used as a positive control in the cytokine secretion assay. Each extract/compound was tested in triplicate in three independent experimental sets.
2.5. Analytical HPLC-DAD analysis
2.8. Statistical analysis
The partition coefficients of target compounds and purity of all HPCCC and semi-preparative collected fractions were established by analytical HPLC-DAD experiments performed on a Shimadzu 20 A series HPLC (Shimadzu Corp., Kyoto, Japan) equipped with a DGC-20 A 3R automatic degasser, LC-20 AD quaternary pump, SIL-20 A HT autosampler and SPD-M20 A DAD. The separation was performed on an Agilent Zorbax Eclipse XDB-C18 (250 × 4.6 mm, 5 μm) column with (A) 0.1% formic acid in water and (B) acetonitrile as mobile phase, under the following elution gradient: 0–5 min 10–25% B, 5–10 min 25–40% B, 10–20 min 40–70% B, 25–27 min 70–100% B, 27–35 min 100–10% B; the injection volume, flow-rate and column temperature were 10 μL, 1 mL/min and 25 °C, respectively; UV spectra were recorded at 280 and 310 nm.
The results were expressed as a means ± S.E.M. Statistical significance of differences between means was established by ANOVA with Turkey’s post-hoc test. P values below 0.05 were considered statistically significant. All analyses were performed using Statistica 10 (StatSoft). 3. Results and discussion 3.1. Metabolite profiling by HPLC-DAD-ESI-Q-TOF-MS/MS Metabolite profiling was initially performed by HPLC-DAD-ESI-QTOF-MS/MS on the crude methanolic extract of V. blattaria L. It showed 3
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a high occurrence of iridoid glycosides, together with other minor metabolites, such as: phenylethanoid glycosides, flavonoids, saponins and phenolic acids. The representative base peak chromatogram of the HPLC-DAD-ESI-Q-TOF-MS analysis is depicted in Fig. 1A, whereas peak assignments are given in Table S1.
tetradesmosidic, were identified in V. blattaria L. Their assignments were performed based on the number of hexose units lost from their pseudomolecular ions and by searching the corresponding aglycon in literature data on Verbascum saponins, since the saponin aglycons do not generally suffer further relevant fragmentations. Moreover, it was observed the following order of neutral hexose losses: rhamnosyl/glucosyl → glucosyl/rhamnosyl → glucosyl → fucosyl, suggesting that fucose is bound directly to the aglycon. Peak 27 presented a pseudomolecular ion [M−H]– at m/z 1089.5839 that suggested the molecular formula of C54H90O22. The fragment ions resulted from the successive losses of glucose (m/z 927), rhamnose (m/z 781), glucose (m/z 619) and fucose (m/z 473) allowed the tentative annotation as songarosaponin B (Seifert et al., 1991), whereas compound 35 ([M−H]– at m/ z 925.5162, C48H78O17) was assigned to desrhamnosylverbascosaponin based on the report of Klimek (1996). After the loss of all hexose residues, peaks 28 ([M−H]– at 1087.5702, C54H88O22) and 29 ([M−H]– at 941.5121, C48H78O18) yielded the same aglycon with m/z 471, the difference between them appearing due to an extra rhamnose within the structure of compound 28. These constituents were tentatively identified as buddlejasaponins I and IV, respectively (Hartleb and Seifert, 1995). HRMS spectra of peaks 31 ([M−H]– at 1103.5655) and 32 ([M−H]– at 1103.5980) suggested two different molecular formulas, C54H88O23 and C55H92O22, respectively, being ascribed to mulleinsaponin IV (Miyase et al., 1997) and ilwensisaponin C, respectively (Calis et al., 1993; Tatli et al., 2004). Finally, compounds 33 and 34 ([M−H]– at m/z 1071.5719, C54H88O21) were identified as verbascosaponin (Schroder and Haslinger, 1993) and songarosaponin A (Seifert et al., 1991). Songarosaponins A–B were reported in V. songaricum Schrenk, buddlejasponins I-IV in V. thapsiforme Schrad., verbascosaponin and derhamnosylverbascosaponin in V. phlomoides L., mulleinsaponin IV in V. fruticulosum Post., whereas ilwensisaponin C was found in V. pterocalycinum var. mutense Hub.-Mor. (Alipieva et al., 2014).
3.1.1. Iridoid glycosides Eighteen iridoid glycosides were identified in V. blattaria extract; of these, 3 monoglycosides and 15 diglycosides. In our previous work (Luca et al., 2018) the approach used for the efficient dereplication of acylated catalpol-type iridoid diglycosides has been described. In brief, the deprotonated molecular ion [M−H]– recorded in the negative ion mode exhibited the loss of the glucose moiety (162 Da), invariably followed by a characteristic loss of a C5H6O3 fragment (114 Da) from the catalpolgenin moiety. The complete breakage of the aglycon part generates a negatively charged ion that contains the acyl residue(s) linked to the second sugar unit (rhamnose). Depending on their masses, all hexose losses were attributed to glucose or rhamnose, because of the consistencies with known compounds previously isolated from this genus. Further fragmentations yielded smaller mass, but highly abundant, ions assigned to the deprotonated, dehydrated or decarboxylated forms of the cinnamic acid derivatives, such as p-coumaric, caffeic or ferulic acids, within the structure of these compounds. By applying these general rules, three monacyl (6, 12, 13), ten diacyl (16-19, 21-24, 26, 30) and two triacyl (14, 25) catalpol-type iridoid diglycosides have been tentatively identified (Table S1). This subcategory of specialized metabolites is known to be highly specific to Verbascum L., with more than 30 representatives, such as saccatoside (Tatli et al., 2006), verbaspinoside (Kalpoutzakis et al., 1999), verbascoside A (Warashina et al., 1991), buddlejoside A5 (Akdemir et al., 2004), pulverulentoside I (Seifert et al., 1989), reported in numerous mullein species. The three iridoid monoglycosides were assigned as follows: peak 2 ([M−H]– at m/z 361.1152.; fragment ions at m/z 343, 199, 181 and 169) to catalpol (also confirmed by standard); peak 3 ([M−H]– at m/z 347.1365; ion fragments at m/z 185, 167, 149, 131, 119 and 89) to ajugol; and peak 20 ([M−H]– at m/z 477.1744; fragments ions at m/z 329, 147, 103) to laterioside (Luca et al., 2018). Catalpol and ajugol are frequent in Verbascum species (Alipieva et al., 2014), whereas laterioside has been previously reported only in V. blattaria L. (Youn et al., 2015).
3.1.4. Flavonoids Four flavonoid glycosides were identified in V. blattaria L. Peak 4 was tentatively labeled as apigenin pentoside, previously reported in V. ovalifolium Donn ex Sims (Luca et al., 2018). After successive losses of glucuronide (176 Da) and glucose (162 Da), compound 11 ([M−H]– at m/z 607.1319) generated the aglycon apigenin (at m/z 269) that further gave characteristic ion fragments (Fabre et al., 2001); thence, for this compound, the structure of apigenin glucoside glucuronide was proposed. Since peak 10 ([M−H]– at m/z 447.0946) produced, after the loss of 162 Da, the aglycon fragment ion at m/z 285 characteristic to luteolin (Fabre et al., 2001), it was unequivocally assigned to luteolin7-O-glucoside, by also comparing its retention time, UV and MS spectra with that of an authentic standard. In addition to compound 10, peak 7 ([M−H]– at m/z 623.1285) contained one glucuronide residue, being thus tentatively identified as luteolin glucoside glucuronide. Compounds 7 and 11 have not been previously reported in Verbascum species, whereas flavonoid glucuronides, such as apigenin-7-O-glucuronide and luteolin-7-O-glucuronide, have been identified in V. lychnitis L. and V. nigrum L. (Alipieva et al., 2014).
3.1.2. Phenylethanoid glycosides Three phenylethanoid glycosides were identified in V. blattaria L. Compound 9 exhibited [M−H]– at m/z 623.1990 and the following fragment ions (m/z): 461 [M–caffeoyl–H]–, 315 [M–caffeoyl–rhamnosyl–H]–, 179 [caffeate]–, 161 [anydro-caffeate]– and 153 [hydroxytyrosol-H]–. The comparison of retention time, UV and MS data with that of the commercial standard led to its unequivocal identification as verbascoside. Compound 8 ([M−H]– at m/z 755.2415), generated some additional fragment ions (at m/z 593 [M–caffeoyl–H]–, 461 [M–caffeoyl–pentosyl–H]–, 447 [M–caffeoyl–rhamnosyl–H]–, 315 [M–caffeoyl–pentosyl–rhamnosyl–H]–, 179 [cafeate]–, 161 [caffeoyl–H]–, 135 [caffeate−CO2]–) when compared to verbascoside, due to the presence of a pentosyl unit in its structure. It was tentatively identified as angoroside A (Gross et al., 1987; Zhou et al., 2014). Peak 15 ([M−H]– at m/z 783.2526) showed several main fragment ions at m/z 637 [M–rhamnosyl–H]–, 607 [M–feruloyl–H]–, 475 [M–feruloyl–pentosyl–H]–, 461 [M–feruloyl–rhamnosyl–H]–, 329 [M–feruloyl–rhamnosyl–pentosyl–H]– and 193 [ferulate]–. These MS/MS data were in agreement with the structure of angoroside C (Calis et al., 1988; Tong et al., 2009). Verbascoside is one them most frequently encountered constituent in Verbascum L. (Alipieva et al., 2014), whereas angorosides A/ C have been isolated from some species, such as V. spinosum L. (Kalpoutzakis et al., 1999).
3.1.5. Phenolic acids Additionally, two phenolic acids have been identified in V. blattaria L. Compound 1 showed [M−H]– at m/z 341.0890 and three fragment ions at m/z 179 [caffeate]–, 161 [anhydro-caffeate]– and 135 [decarboxy-caffeate]–, being tentatively characterized as caffeic acid glucoside. Compound 5, with a parent ion at m/z 337.0944 and fragment ions at m/z 191 [quinate]–, 163 [p-coumarate]– and 145 [anhydro-pcoumarate]–, was tentatively ascribed to p-coumaroyl quinic acid. Both compounds were previously reported in V. ovalifolium Donn ex Sims (Luca et al., 2018).
3.1.3. Saponins Eight oleanan-type triterpene saponins, two tridemosidic and six 4
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3.2. HPCCC separation Selection of the two-phase solvent system is the most important feature in achieving a successful HPCCC separation. A good solvent system requires several considerations, such as: (i) a short settling time (< 30 s); (ii) a partition coefficient of the target constituent within a suitable range (usually between 0.2 and 5); and (iii) a separation factor (α = K2/K1, K2 > K1) between any two compounds higher than 1.5 (Yue et al., 2013). HPLC-DAD profile of the crude methanolic extract of V. blattaria L. (Fig. 1B) suggested the presence of three major acylated iridoid diglycosides, as also confirmed by dereplication based on HRMS and MS/MS. Various mixtures of ethyl acetate/n-butanol/water (EBWat), n-hexane/ ethyl acetate/n-butanol/water (HEBWat) and n-hexane/ethyl acetate/ methanol/water (HEMWat) were screened for the partition coefficients of the three target compounds. Previously, EBWat family has proven successful in CCC isolation of iridoids from numerous plant sources, such as catalpol from Rehmannia glutinosa Libosch. (Tong et al., 2015), geniposide from Gardenia jasminoides Ellis (Wang et al., 2015), geniposidic acid from Eucommia ulmoides Oliv. (Dai et al., 2013), harpagoside from Scrophularia ningpoensis Hemsl. (Tong et al., 2006) or loganic acid from Gentiana scabra Bunge (Chen et al., 2017). However, in the case of V. blattaria L., EBWat 5/1/5 v/v/v (I) and EBWat 2/1/3 v/v/ v (II) gave suitable partition coefficients only for compound A, whereas the other two constituents had K values higher than 5 (Table 1). When n-hexane was added to the system, as in system III, the partition coefficient of compound B significantly decreased to 1.91, but it remained unsolved for the third analyte. Therefore, less polar solvent systems were tested, such as those belonging to HEMWat family (IV and V); consequently, the K values of compound C were situated within the range of 0.2–5 in both cases, but those of the first two constituents were lower than 0.2. Since one solvent system to simultaneously and efficiently target all three compounds was not found, mainly due to constituents’ different polarity, solvent systems III and IV were subsequently selected for two parallel sets of semi-preparative HPCCC experiments. Compounds A (HPCCC elution time 24–25, 2 mg) and B (HPCCC elution time 42–44 min, 16 mg) were obtained after separation with HEBWat 1/2/1/2 v/v/v/v (300 mg crude extract, 1600 rpm, 4.2 mL/ min, 310 nm, stationary phase retention 67%; upper phase as stationary phase), whereas compound C (HPCCC elution time 23–25 min, 13 mg) was afforded after separation with HEMWat 1/19/1/19 v/v/v/v (300 mg, 1600 rpm, 6 mL/min, 280 nm, stationary phase retention 65%; upper phase as stationary phase) (Fig. S1). The identity of the three compounds (Fig. 2) was established by HPLC-DAD-ESI-Q-TOF-MS/MS, 1D NMR, 2D NMR analyses and comparison of these spectral data with those reported in the available literature, as follows: (A) scropolioside F (Tatli et al., 2006); (B) scrophuloside A3 (Miyase and Mimatsu, 1999); and (C) gmelinoside L (Hosny and Rosazza, 1998). Of these, only compound A has been previously reported in other Verbascum species (Tatli et al., 2006), while the other two are new for this genus, with scrophuloside A3 isolated
Fig. 2. Chemical structures of isolated compounds from V. blattaria (A) scropolioside F (B) scrophuloside A3 and (C) gmelinoside L.
only from Scrophularia nodosa L. (Miyase and Mimatsu, 1999) and gmelinoside L from Gmelina arborea Roxb. and G. hainanensis Oliv. (Hosny and Rosazza, 1998; Xiong et al., 2018). Thus, this is the first report where countercurrent separation techniques are used as chromatographic approach for the efficient purification of these constituents. The methods developed in the current study are extremely fast (less than 45 min), which might make them suitable for the highscale HPCCC purification of identical or similar acylated iridoid diglycosides from other Verbascum species or other plant sources containing this category of metabolites. 3.3. Evaluation of IL-8 and TNF-α secretion In the last part of the current study, the crude methanolic extract of V. blattaria L., scrophuloside A3 and gmelinoside L were investigated as potential IL-8 and TNF-α inhibitors in LPS-stimulated neutrophils. None of the samples were cytotoxic to neutrophils at all tested concentrations based on cytometric experiments (data not shown). Neutrophils (polymorphonuclear cells, PMNs) are the most abundant leukocytes in the blood and constitute the first line of host defense against numerous infectious pathogens (Rosales et al., 2016). After migration to the infection and inflammation sites, they recognize and phagocyte invading microorganisms via processes that involve molecular mediators, such as reactive oxygen species, proteases (elastase, metalloproteinases), chemokines sand cytokines (IL-8, TNF-α). Furthermore, it is claimed that overstimulation of PMNs initiates advanced non-infectious inflammatory responses. IL-8 and TNF-α are two of the most important chemokines produced by PMNs, being involved in T lymphocyte and basophile chemotaxis, degranulation and PMNs endothelial adhesion. The decrease of IL-8 and TNF-α production results in prevention of inflammatory response progression (Czerwińska et al., 2018; Rosales et al., 2016). The crude methanolic extract of V. blattaria L. was able to significantly inhibit both IL-8 (65.31–71.12% of LPS + control) and TNF-α secretion (16.07–47.25%) at 50 and 100 μg/mL (Fig. 3). It is worth mentioning that the effect produced by the extract (100 μg/mL) on TNF-α release is comparable to that obtained for the positive control (dexamethasone) at 25 μM (19.46%). Scrophuloside A3 (B) decreased the production of IL-8 at all tested concentrations (between 62.62–66.16% of LPS + control), whereas TNF-α release was reduced
Table 1 Partition coefficient values of isolated compounds from V. blattaria. No.
Solvent system
KA
KB
KC
I II III IV V
EBWat (2/1/3; v/v/v) EBWat (5/1/5; v/v/v) HEBWat (1/2/1/2; v/v/v/v) HEMWat (1/19/1/19; v/v/v/v) HEMWat (1/9/1/9; v/v/v/v)
2.76 0.99 0.31 0.05 0.01
11.76 6.00 1.91 0.15 0.19
18.75 8.92 6.17 1.05 0.64
EBWat ethyl acetate/n-butanol/water; HEBWat n-hexane/ethyl acetate/n-butanol/water; HEMWat n-hexane/ethyl acetate/methanol/water; values in bold indicate that these solvent systems were further used for the high-performance counter-current chromatography separation of those target compounds. 5
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Fig. 3. Effect of the crude methanolic extract (25–100 μg/mL), scrophuloside A3 (B, 12.5–50 μM), gmelinoside L (C, 12.5–50 μM) and dexamethasone (Dex, 1–25 μM) on (a) IL-8 and (b) TNF-α secretion in LPS-stimulated (100 ng/mL) neutrophils. Data were expressed as mean ± SEM of three separate experiments performed with cells isolated from independent donors assayed in triplicate. Statistical significance: *p < 0.05 and **p < 0.001 vs. stimulated control; #p < 0.001 vs. nonstimulated control.
only at 25 μM (63.48%) and 50 μM (47.70%). Gmelinoside L (C) produced significant inhibitory effects on IL-8 (43.52–48.25%) and TNF-α (41.28–64.64%) production at 25–50 μM. However, the effects of both tested compounds were weaker than those of dexamethasone at all tested concentrations. Despite the fact that there are numerous reports assessing the antiinflammatory potential of extracts of various Verbascum species (Alipieva et al., 2014; Tatli and Akdemir, 2006), there is a very limited number of data related to the activity of acylated iridoid diglycosides, which are known as one of the most abundant constituents in mulleins. For instance, the crude methanolic extract of V. xanthophoeniceum Griseb. (500 μg/mL) together with nigroside VI (250 μg/mL) significantly down-regulated IL-6 and TNF-α production in LPS-stimulated peritoneal macrophages (Dimitrova et al., 2012). However, when tested on IFN-γ-stimulated NHK keratinocytes, nigroside VI (10–50 μM) did not significantly reduced IL-8 secretion. Nevertheless, nigroside III was inactive in all the above assays (Georgiev et al., 2012). Overall, this is the first study reporting that scrophuloside A3 and gmelinoside L significantly influence IL-8 and TNF-α secretion in LPS-stimulated neutrophils.
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