Metabolite Profiling and Fingerprinting of Hypericum Species: a Comparison of MS and NMR Metabolomics

Home , Hypericaceae, Hypericum gentianoides, Hypericum polyphyllum

Metabolomics (2014) 10:574–588 DOI 10.1007/s11306-013-0609-7

ORIGINAL ARTICLE

Metabolite proﬁling and ﬁngerprinting of Hypericum species: a comparison of MS and NMR metabolomics

Andrea Porzel • Mohamed A. Farag • Julia Mu¨lbradt • Ludger A. Wessjohann

Received: 16 August 2013 / Accepted: 15 November 2013 / Published online: 7 December 2013 Ó Springer Science+Business Media New York 2013

Abstract Hypericum perforatum, commonly known as comparable score plots in principal component analysis St. John’s wort, is a popular herbal supplement used for the were derived from both MS and NMR datasets, loading treatment of mild to moderate depression. The major sec- plots reveal, that different set of metabolites contribute for ondary metabolites of St. John’s wort extracts include species segregation in each dataset. Major peaks in 1H phenylpropanoids, flavonoids, xanthones, phloroglucinols, NMR and MS spectra contributing to species discrimina- and naphthodianthrones. There are over 400 species in the tion were assigned as those of hyperforins, lipids, chloro- genus Hypericum world-wide, most of which are little or genic and shikimic acid. Shikimic acid and its downstream not characterized in terms of phytochemical or pharma- phenylpropanoids were more enriched in H. perforatum, H. cological properties. Metabolomics techniques were used androsaemum, H. kouytchense and H. inodorum extracts; to investigate the natural product diversity within the genus whereas a novel hyperforin was found exclusively in H. Hypericum (Hypericaceae) and its correlation to bioactiv- polyphyllum. Next to H. perforatum, H. polyphyllum and ity, exemplified by cytotoxic properties. Utilizing nuclear H. tetrapterum show the highest levels of hypericins, and magnetic resonance (NMR) fingerprinting and mass spec- H. perforatum and H. polyphyllum are highest in trometry (MS) metabolic profiling techniques, MS and phloroglucinols, suggesting that the latter species might be NMR spectra of extracts from H. perforatum, H. poly- used as an alternative to St. John’s wort. However, the phyllum, H. tetrapterum, H. androsaemum, H. inodorum, major hyperforin-type compound in H. polyphyllum pos- H. undulatum and H. kouytchense were evaluated and sesses a novel constitution of yet unknown bioactivity. submitted to statistical multivariate analyses. Although Anti-cancer in vitro assays to evaluate the ability of extracts from Hypericum species in inhibiting prostate and colon cancer growth suggest that such bioactivity might be predicted by gross metabolic profiling. Electronic supplementary material The online version of this article (doi:10.1007/s11306-013-0609-7) contains supplementary Keywords H. perforatum H. polyphyllum material, which is available to authorized users. 1H NMR-based metabolomics LC–MS Hyperforin Anticancer activity prediction A. Porzel M. A. Farag J. Mu¨lbradt L. A. Wessjohann (&) Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany Abbreviations e-mail: [email protected] ESI Electrospray ionisation LC Liquid chromatography M. A. Farag Pharmacognosy Department, College of Pharmacy, Cairo MS Mass spectrometry University, Kasr El Aini St., P.B. 11562, Cairo, Egypt MSn Tandem mass spectrometry NMR Nuclear magnetic resonance Present Address: PDA Photodiode array detection J. Mu¨lbradt FB Biologie/Chemie, Universita¨t Bremen, NW2/Leobener Str., PCA Principal component analysis 28359 Bremen, Germany HCA Hierarchical cluster analysis 123 A comparison of MS and NMR metabolomics 575

1 Introduction annulatophenone, hypericophenonoside, otogirin, itali- dipyrone, chinesins, and sampsonines (Hillwig et al. 2008). In recent years, the use of herbs as dietary supplements has With regard to the concentrations of active components, increased dramatically in many countries. The World significant differences were found between different spe- Health Organization estimates (WHO Fact sheet No. 134, cies in Hypericum (Umek et al. 1999; Kitanov 2001). December 2008) that up to 80 % of the world’s population The increasing attention on St. John’s wort relies on its relies on traditional medicinal systems, and in many of efficacy as an antidepressant medicine, which has been these, herbal medicines play a key role in human health. demonstrated in numerous clinical trials challenging the Among these products, Hypericum perforatum (St. John’s conventional tricyclic antidepressants (Fornal et al. 2001; wort) is a very popular phytomedicine, recognized as one Gobbi and Mennini 2001). The antidepressant activity of of the top five herb remedies (Giese 1999). H. perforatum H. perforatum was first attributed to naphthodianthrones has been reported as an antidepressant, antiviral, antimi- hypericin, pseudohypericin, protohypericin and proto- crobial, anti-inflammatory, and a healing agent (Brolis pseudohypericin (Meruelo et al. 1988). Recent studies et al. 1998). It encompasses a myriad of natural product revealed that the phloroglucinol hyperforin and its deriva- groups (Fig. 1) including naphthodianthrones primarily tive adhyperforin, can also inhibit various neurotransmitter represented by hypericin and pseudohypericin, flavonoids receptors (Laakmann et al. 1998a, b). Additionally, flavo- such as hyperoside, rutin, quercetin and astilbin, and a third noids present in St. John’s wort extracts have been shown group of phloroglucinol derivatives such as hyperforin and to have anti-depressant activities (Butterweck et al. 1997). adhyperforin (Nahrstedt and Butterweck 1997). Other Moreover, hyperforin has been shown to display antibac- phloroglucinols already identified within Hypericum spe- terial activity (Schempp et al. 2002b) and was also pro- cies include, perforatumone, uliginosin, bromouliginosin, posed to act as a novel anticancer drug by induction of drummondins, saroaspidins, hyperbrasilols, paglucinol, apoptosis (Schempp et al. 2002a).

a b

Fig. 1 Major groups of natural products; phloroglucinols (a), naph- carbon numbering system for each compound is used throughout the thodianthrones (b) phenolic/organic acids (c) and ﬂavonoids manuscript for NMR assignment, and thus is based on analogy rather (d) detected in Hypericum species discussed in the manuscript. The than IUPAC rules

123 576 A. Porzel et al.

Up to now, H. perforatum has been the primary source For this study, a one pot extraction method compatible for the naphthodianthrones/phloroglucinols or dietary with both NMR and LC–MS metabolomics was developed. supplements containing Hypericum plant material. Other Extracts derived from seven Hypericum species were species such as H. hirsutum, H. maculatum, H. tetrapterum, evaluated using the aforementioned technology platforms, H. grandifolium, H. montanum and H. humifusum were and processed data were further subjected to multivariate also found to contain a certain amount of theses metabo- analysis using PCA and hierarchical cluster analysis (HCA) lites (Butterweck et al. 1997; Umek et al. 1999; Smelce- to help reveal for compositional differences among species. rovic and Spiteller 2006a, c; Bonkanka et al. 2008). Our focus on flowers was based on results revealing that Although there are many species present in nature, classi- among H. perforatum aerial organs, flower and floral bud fication of which might be intricate in terms of taxonomy, are distinct in having the highest levels of hyperforins and there are only few reports on the systematic untargeted hypericins (Bonkanka et al. 2008). Except for H. tet- metabolic analysis of other Hypericum species. For reliable rapterum, the additional species studied have not been differentiation and chemical analysis, a broad systematic chemically profiled previously. As well, previous phyto- method monitoring the whole set or at least the medicinally chemical analyses mostly aimed at targeted quantification relevant metabolites (metabolite profiling) is deemed of and analysis of metabolites already well characterized in H. interest. Also, chemical analyses that are based on the perforatum (Kusari et al. 2009; Sagratini et al. 2008; natural composition of metabolites, rather than detection of Smelcerovic and Spiteller 2006; Smelcerovic et al. 2006c). a single constituent, are favored as they cover additionally In this study, data collected using MS and NMR techniques or synergistically relevant components and can confirm the were found to be complementary, and only by combining efficacy of H. perforatum medical preparations (Politi et al. them, a clearer and quantitative interpretation of the Hy- 2009; Rasmussen et al. 2006; Poutaraud et al. 2001). pericum species metabolome could be achieved. This With the recent developments in plant metabolomics comparative metabolomic approach can also provide a new techniques, it is now possible to detect several hundred platform for global analyses of Hypericum pharmaceuticals metabolites simultaneously and to compare samples reli- and or other phytomedicines. ably to identify differences and similarities in an untargeted manner. To obtain the most complete metabolite profile, it is necessary to use a wide spectrum of extraction and 2 Experimental analytical techniques which are rapid, reproducible, stable in time and require only a very simple sample preparation. 2.1 Plant material Most commonly, metabolomics is heavily supported by mass spectrometry (MS). Nuclear magnetic resonance Hypericum perforatum and H. polyphyllum ‘Grandiflorum’ (NMR) has emerged as parallel technology that can pro- seedlings were obtained from Staudenga¨rtnerei Gaißmayer vide an overview of the metabolome and provides high- GmbH and Co. KG (Illertissen, Germany). Seedlings of H. power structural elucidation and absolute quantification. tetrapterum were provided by prime factory GmbH and The combination of liquid chromatography (LC)–MS and Co. KG (Hennstedt, Germany). Seeds of H. inodorum, H. NMR is a powerful method for identifying metabolites. kouytchense, and H. undulatum were obtained from N. Metabolomic fingerprinting through 1H NMR spectroscopy L. Chrestensen Erfurter Samen- und Pflanzenzucht GmbH has been profusely used for H. perforatum and or its (Erfurt, Germany) and seeds of H. androsaemum from commercial preparations analysis coupled to principal Jelitto Staudensamen GmbH (Schwarmstedt, Germany). component analysis (PCA) and PARFAC analysis (Tatsis All seedlings of Hypericum species were grown side by et al. 2007; Schmidt et al. 2008). However, the application side in the same field at the Leibniz Institute of Plant of NMR poses the problem of overlapping 1H NMR signals Biochemistry, Halle, Germany. Flowers were harvested on that hinder robust metabolite identification. While NMR the same day in the middle of August 2009 and 2010. The provides the valuable metabolite signature of a complex collected plant material was immediately frozen in liquid plant extract, LC–MS resolves individual chemical com- nitrogen, and kept at -80 °C until further analyzed. ponents into separate peaks, enhancing the opportunity to mine and uncover novel metabolites. Successful applica- 2.2 Chemicals and reagents tions of LC–MS in phytomedicinal quality control analysis include authentication of medicinal ginger (Jiang et al. Methanol-d4 (99.80 % D), acetone-d6 (99.80 % D) and 2006), analysis of green tea (Pongsuwan et al. 2007), hexamethyldisiloxane (HMDS) were purchased from Gingko biloba (Agnolet et al. 2010), licorice (Farag et al. Deutero GmbH (Kastellaun, Germany). For NMR quanti- 2012b), hop (Farag et al. 2012a), and H. perforatum (Farag fication and calibration of chemical shifts, HMDS was and Wessjohann 2012). added to a final concentration of 0.94 mM. Acetonitrile and 123 A comparison of MS and NMR metabolomics 577 acetic acid (LC–MS grade) were obtained from J. T. Baker LC (HPLC) and equipped with a HYPERSIL GOLD RP18- (The Netherlands), milliQ water was used for LC analysis. column (5 mm, 150 9 1 mm, ThermoScientific). For Chromoband C18 (500 mg, 3 mL) cartridge was purchased HPLC a gradient system was used starting from ammonium from Macherey and Nagel (Du¨ren, Germany). Hyperforin, acetate in water 50 mM:CH3CN = 95:5 to 100 % CH3CN hypericin, pseudohypericin, rutin, hyperoside, isoquerce- within 35 min, then isocratically for further 10 min; flow trin, and kaemferol-3-O-glucoside were all purchased from rate 70 lL/min. All mass spectra were averaged and Chromadex (Wesel, Germany). All other chemicals and background subtracted. The Xcalibur 2.0 software was standards were from Sigma Aldrich (St. Louis, MO, USA). used for the data evaluation. The MSn spectra were recorded during the HPLC run by 2.3 Extraction procedure and sample preparation using the following conditions: MS/MS analysis with a for NMR and MS analyses starting collision-induced dissociation energy of 20 eV and an isolation width of ±2 amu in a data dependant mode. Freeze dried Hypericum flowers were ground with a pestle in a mortar using liquid nitrogen. For each specimen, three 2.5 High resolution UPLC–MS analysis biological replicates were provided and extracted in parallel under identical conditions. The powder (60 mg) was Chromatographic separations were performed on an Ac- homogenized with 5 mL 100 % MeOH containing 8 lg/ quity UPLC system (Waters) equipped with a HSS T3 mL umbelliferone (an internal standard for relative quan- column (100 9 1.0 mm, particle size 1.8 lm; Waters) tification using LC–MS) using a Turrax mixer (11,000 applying the following binary gradient at a flow rate of 150 RPM) for five 20 s periods. To prevent heating, a period of lL/min: 0–1 min, isocratic 95 % A (50 mM ammonium 1 min separated each mixing period. Extracts were then acetate in water), 5 % B (acetonitrile); 1–16 min, linear vortexed vigorously and centrifuged at 3,0009g for 30 min from 5 to 95 % B; 16–18 min, isocratic 95 % B; to remove plant debris. 18–20 min, isocratic 5 % B. The injection volume was 3.1 For NMR analysis, 4 mL of the supernatant were ali- lL (full loop injection). Eluted compounds were detected quoted using a syringe and the solvent was evaporated from m/z 100–1,000 using a MicroTOF-Q hybrid quadru- under a stream of nitrogen till dryness. Dried extracts were pole time-of-flight mass spectrometer (Bruker Daltonics) resuspended with 800 lL 100 % methanol-d4 containing equipped with an Apollo II electrospray ion source in HMDS (0.94 mM), which served as an internal chemical positive and negative ion modes using the following shift NMR standard. After centrifugation (13,0009g for instrument settings: nebulizer gas, nitrogen, 1.6 bar; dry 1 min), the supernatant was transferred to a 5 mm NMR gas, nitrogen, 6 L/min, 190 °C; capillary, -5,500 V tube. (?4,000 V); end plate offset, -500 V; funnel 1 RF,

For LC–MS analyses, 500 lL were aliquoted and placed 200 Vpp; funnel 2 RF, 200 Vpp; in-source CID energy, 0 V; on a (500 mg) C18 cartridge preconditioned with methanol hexapole RF, 100 Vpp; quadrupole ion energy, 5 eV; col- and water. Samples were then eluted using 2 9 3mL lision gas, argon; collision energy, 10 eV; collision RF methanol, the eluent was evaporated under a nitrogen 200/400 Vpp (timing 50/50); transfer time, 70 ls; prepulse stream and the obtained dry residue was resuspended in storage, 5 ls; pulser frequency, 10 kHz; spectra rate, 3 Hz. 1 mL methanol. Two lL were used for LC–MS analysis. Internal mass calibration of each analysis was performed All 1H NMR spectra for multivariate data analysis were by infusion of 20 lL 10 mM lithium formate in isopro- acquired consecutively within a 48 h time-interval with panol/water, 1/1 (v/v), at a gradient time of 18 min using a samples prepared immediately before data acquisition. It diverter valve. should be noted that extraction was conducted under minimal light condition to prevent decomposition of hy- 2.6 NMR analysis perforins. All wet samples were processed immediately to avoid oxidation or light promoted artefact formation. All spectra were recorded on an Agilent (Varian) VNMRS 600 NMR spectrometer operating at a proton 2.4 LC–MS analysis NMR frequency of 599.83 MHz using a 5 mm inverse detection cryoprobe. The spectra were referenced to n 1 The LC–ESI (electrospray ionisation) and ESI–MS (tan- internal HMDS at 0.062 ppm for H NMR and to CD3OD dem mass spectra) were obtained from a LCQ Deca XP signals at 49.0 ppm for 13C NMR. 1H NMR spectra were MAX system (ThermoElectron, San Jose, USA) equipped recorded with the following parameters: digital resolution with a ESI source (electrospray voltage 4.0 kV, sheath gas 0.37 Hz/point (32 K complex data points), pulse nitrogen, capillary temperature 275 °C). The Ion Trap MS width = 3 ls (45°), relaxation delay = 23.7 s, acquisition system is coupled with a Surveyor micro-high-performance time = 2.7 s, number of transients = 160. Zero filling up 123 578 A. Porzel et al. to 128 K and an exponential window function with 2.9 NMR data processing and PCA analysis lb = 0.4 was used prior to Fourier transformation. 2D NMR spectra were recorded using standard CHEMPACK The 1H NMR spectra were automatically Fourier trans- 4.1 pulse sequences (gDQCOSY, gHSQCAD, gHMB- formed to ESP files using ACD/NMR Manager lab version CAD) implemented in Varian VNMRJ 2.2C spectrometer 10.0 software (Toronto, Canada). Spectral intensities were software. The heteronuclear single-quantum correlation reduced to integrated regions, referred to as buckets, of 1 (HSQC) experiment was optimized for JCH = 146 Hz equal width (0.04 ppm) within the region of d 11.4 to with DEPT-like editing and 13C-decoupling during -0.4 ppm. The regions between d 5.0–4.7 and 3.4–3.25 acquisition time. The heteronuclear multiple bond corre- corresponding to residual water and methanol signals, lation (HMBC) experiment was optimized for a long- respectively, were removed prior to multivariate analyses. 1 range coupling of 8 Hz, a two-step JCH filter was used PCA was performed with R package (2.9.2) using custom- (130–165 Hz). written procedures after normalizing to HMDS signal and exclusion of solvent regions. 2.7 MS data processing and PCA analysis 2.10 NMR quantification Relative quantification of Hypericum metabolite profiles For the quantification of metabolites using NMR spec- was performed using XCMS data analysis software, troscopy, the peak area of selected proton signals belonging which can be downloaded for free as an R package from to the target compounds, and the peak area of the IS the Metlin Metabolite Database (http://137.131.20.83/ (HMDS) were integrated manually for all the samples. The download/; Smith et al. 2006). This software allows peak following equation was applied for the calculations. alignments, matching and comparison (Smith et al. 2006). Native LC–MS files from Xcalibur 1.4 (Thermo Fisher IT xSt mT ¼ MT cSt vSt; Scientific, Inc., Waltham, MA) were first converted into ISt xT netCDF files using the File Converter tool. Files were mT is the mass of the target compound in the solution used arranged in one folder that was set as the file source. 1 for H NMR measurement (lg), MT is the molecular Peaks were subsequently extracted using XCMS under R weight of target compound (g/mol), IT is the relative 2.9.2 environment with the signal-to-noise ratio set at 4. integral value of 1H NMR signal of the target compound, After peak extraction and grouping, nonlinear retention 1 ISt is the relative integral value of H NMR signal of the time (rt) correction of peaks was accomplished in two standard compound, xSt is the number of protons belonging iterative cycles with descending bandwidth (bw). This 1 to the H NMR signal of the standard compound, xT is the was accomplished by manually decreasing the bw number of protons belonging to the 1H NMR signal of the parameter (from 30 to 10 s). The resulting peak list was target compound, cSt is the concentration of standard further processed using the Microsoft Excel software compound in the solution used for 1H NMR measurement (Microsoft, Redmond, WA), where the ion features were 1 (mmol/L) and vSt volume of solution used for HNMR normalized to the total integrated area (1,000) per sample measurement (mL). and imported into the R 2.9.2 software package for PCA. Absolute peak area values were autoscaled (the mean area 2.11 Cytotoxicity assay value of each feature throughout all samples was subtracted from each individual feature area and the result Human prostate PC3 cancer cell line was obtained from divided by the standard deviation) prior to PCA. This the Deutsche Sammlung von Mikroorganismen und provides similar weights for all the variables, as described Zellkulturen, Braunschweig (DMSZ ACC code 465) and elsewhere (van den Berg et al. 2006). PCA was then the HT29 colon cancer cell line was obtained from the performed on the MS-scaled data to visualize general Medical Immunology Department at Martin Luther-Uni- clustering, trends, and outliers among the samples on the versita¨t Halle-Wittenberg (Professor Seliger). The cells scores plot. were grown as monolayers in adherent cell lines and were routinely cultured in RPMI (Roswell Park Memorial 2.8 Hierarchical cluster analysis (HCA) Institute) 1640 supplemented with 10 % heat-inactivated 3 fetal bovine serum and 1 % L-glutamine in 75 cm poly- HCA and the creation of heat maps of data from non- styrene flasks (Corning Life Sciences, UK) and maintained targeted analysis (LC–MS) were performed using two R at 37 °C in a humidified atmosphere with 5 % CO2. Cells packages, Heatplus, and gplots according to (Bilia et al. were plated at a density of 1 9 104/well in 96-well plates. 2001; Xie et al. 2009). They were allowed to attach to the plate for 24 h. After 123 A comparison of MS and NMR metabolomics 579

24 h, the media were replaced with RPMI media con- perforatum. To accomplish that goal, samples of freshly taining resin extracts. Dried extracts prepared as in Sect. frozen Hypericum, field-grown at the same time under 3.3 were initially dissolved in DMSO at a concentration of identical conditions, were used to ensure that environ- 2 mg/mL and further diluted with RPMI medium. Three mental effects were eliminated in this study. The adopted concentrations were used (5, 10, 50 and 100 lg/mL) from approach is focused on developing fast and effective ana- each extract. The maximum DMSO concentration in the lytical methods for metabolomic fingerprinting of Hyperi- assay was 0.1 % and is not cytotoxic to the tumor cells. cum extracts by direct analyses of extract solutions using After 72 h, the medium was taken out and 100 lL of XTT- 1H NMR, ideally without any preliminary chromatographic solution (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phe- separation step, in parallel with chromatographic nylamino) carbonyl]-2H-tetrazolium hydroxide; Roche (hyphenated) MS techniques. Owing to the complexity of Applied Science, Mannheim, Germany) was added to each the acquired data, as reflected in the complexity of spectral well at a final concentration of XTT of 0.3 mg/mL. The data, statistical multivariate analyses, e.g. HCA and PCA plates were incubated at 37 °C for another 4 h. Absorbance were performed to ensure good analytical rigorousness and was measured at 490 nm against a reference wavelength at define both similarities and differences among samples. 650 nm using a microplate reader (Beckman Coulter, DTX 880 Multimode Reader). The mean of triplicate experi- 3.1 Development of a single pot extraction method ments for each dose was used to calculate the growth IC50 for MS and NMR analyses and repeated in two passages for each cancer cell line. Digitonin was used as a positive drug control in DMSO. To allow for a comparative analysis of the metabolite data derived from these different technology platforms, a one 2.12 Total phenolics (TP) quantification pot extraction method was developed for extraction that is found compatible for both NMR and MS metabolomics. A spectrophotometric method using Folin–Ciocalteau Several solvents were initially tested including acetone, reagent was used to determine the TP content in Hypericum methanol, and methanol/water 80/20 (v/v). Solvent selec- flowers. Extracts were prepared by cold extraction with tion for sample preparation was evaluated with respect to shaking over 3 h using 100 % MeOH. Plant debris was reproducibility, quality and recovery of Hypericum sec- removed by centrifugation and the solvent was removed by ondary metabolites as revealed by 1H NMR. Compared evaporation under nitrogen followed by lyophilisation for with MS, 1H NMR can detect metabolites universally, thus 6 h. The dry residue was dissolved in a calculated volume providing an unbiased picture of differences among of methanol to obtain a solution of a concentration of extraction methods. Compared with acetone, methanol 10 mg/mL. Folin–Ciocalteau reagent (100 lL) was added shows a better recovery rate of phenolics based on the to a test tube containing 20 lL of the extract solution. NMR signals as is evident from its respective resonances at Contents were mixed, and a saturated aqueous sodium d 5.5–8 ppm, i.e. of the olefinic/aromatic region and by carbonate solution (200 lL) was added. The volume was overall signals splitting (data not shown). Previously, adjusted to 2 mL by the addition of 0.68 mL of milliQ methanol was shown to possess high efficiency for the water and the contents were mixed vigorously. The tubes extraction of hypericins, hyperforin and flavonoids from H. were allowed to stand at 28 °C for 25 min and then cen- perforatum with sonication (Gioti et al. 2009; Smelcerovic trifuged for 5 min at 2,4359g. Absorbance of the super- et al. 2006b). It should be noted that modifying solvent natant was read at 760 nm. Blank samples of each extract polarity by addition of water at a concentration of 20 % (without addition of Folin–Ciocalteau reagent) were used enhanced the recovery of sugars (3.5–5 ppm), but nega- for background subtraction. Gallic acid was used as stan- tively affected the signal intensity of hyperforins and more dard for the calibration curve at concentrations of 0.01, 0.1, dramatically that of lipids. Owing to the solubility of 1 and 5 mg/mL. The assays were carried out in triplicate. hypericin and hyperforin in water diluted organic solvent (Gioti et al. 2009), 100 % methanol was chosen for pre- paring the bulk extract from freeze dried samples, further 3 Results and discussion aliquoted for both MS and NMR sample preparation.

The major goal of this study was to investigate Hypericum 3.2 LC–MS peak identification secondary metabolites in an untargeted, holistic manner in the context of its genetic diversity and its effect on bio- Except for H. perforatum, the use of LC–MS for metabolite logical activity so as to set a framework for metabolite profiling has not been previously reported from the other pattern based taxonomy, quality control analysis, and to investigated species (Tatsis et al. 2007; Farag and Wes- help identify alternatives to the important botanical H. sjohann 2012). Chemical constituents of H. perforatum 123 580 A. Porzel et al. were analyzed by reversed-phase HPLC/(-)ESI–MS, and adhyperforin. Note the mass difference of 14 amu eluting with gradient mobile phase consisting of methanol between peaks (C26 and C27), similar to that observed for and ammonium acetate. The reverse-phase HPLC–MS hyperforin and adhyperforin. method reported here was similar to that previously Hyperfirin (C29), regarded as biosynthetic intermediate reported (Liu et al. 2005); however the present method of hyperforin (Tatsis et al. 2007; Adam et al. 2002), was used slightly different mobile phases and a steeper gradi- present in H. perforatum, though at much lower levels. H. ent. This steeper gradient of 50 mM ammonium acetate tetrapterum gave two peaks for phloroglucinols (C21 and (pH 5) and acetonitrile allowed for the elution of all ana- C24) with similar tandem MSn spectra to that of hyperfo- lytes i.e. hyperforins, flavonoids and naphthodianthrones rins, albeit both show a UV/Vis maximum at 352 nm (hypericins) within 60 min compared to the 100 min ana- suggestive of highly conjugated phloroglucinol derivatives. lysis time previously reported. It should be noted that ini- The identification of these novel phloroglucinols is still tial attempts to optimize chromatographic separation of under investigation. Other classes of identified phloroglu- different classes of compounds using acetic acid (pH 1.9) cinols include hypersampsones E/D isomers (peaks C30 as elution gradient prevented hypericin peaks from being and C32), and were found exclusively in H. inodorum, detected (Farag and Wessjohann 2012). One possible H. undulatum and H. androsaemum. Furohyperforin, an explanation is the acidic nature of the chosen mobile phase oxidation product of hyperforin (peak C23) was detected (pH 1.9) compared to ammonium acetate (pH 5) as an only in H. perforatum, albeit at trace levels, suggestive that eluent, which might not be appropriate for the elution and hyperforins were not subject to major chemical degradation further detection of these special constituents. Indeed, during extraction (Lee et al. 2006). In comparison, naph- separation methods reported in the literature using organic thodianthrones are more stable due to their aromatic con- acid in aqueous phase failed to detect hypericins in H. jugation (Liu et al. 2005), except that the open-ring proto- perforatum extracts (Tatsis et al. 2007; Schmidt et al. forms, i.e. protohypericin and protopseudohypericin, are 2008). less stable than the cyclized counterparts hypericin and LC–MS base peak chromatograms of the seven Hyper- pseudohypericin (close-ring structures). In this analysis, icum species is presented in (Supplementary Fig. 1). The protopseudohypericin was detected (peak C18) as a major identities, retention times (rt), UV/Vis characteristics, and naphthodianthrone, especially in the H. polyphyllum observed molecular and fragment ions for individual extract (Supplementary Fig. 2). Previous reports revealed components are presented in Table 1. Metabolite assign- that H. polyphyllum accumulates hypericins in its leaves ments were made by comparing rt, UV/Vis spectra and MS and flowers (Osinska 2003; Brunarska 1962). data (accurate mass, isotopic distribution and fragmenta- Several sugar conjugates of quercetin and myricetin, and tion pattern in negative ion mode) of the compounds apigenin aglycones (i.e. peaks C3–C8 in Table 1) were detected with Hypericum compounds reported in the liter- identified in most extracts. These conjugates exhibit the ature and the dictionary of natural products database pre-dominant losses of 146 amu (deoxyhexose), 162 amu (Wiley, CRC). Identifications were confirmed with stan- (hexose) or 176 amu (glucuronic acid), in the MSn spec- dard compounds whenever available in-house. About 38 trum diagnostic for flavonoid-O-glycosides, and were metabolites, including phloroglucinols, flavonoids, and assigned as non-prenylated flavonoids. naphthodianthrones were resolved of which 29 were While phloroglucinols and flavonoids were found in all identified. Phloroglucinols accounted for the highest examined species, naphthodianthrones were only identified abundance among all species (Supplementary Fig. 1). in H. perforatum, H. polyphyllum and H. tetrapterum as is Except for H. tetrapterum and H. polyphyllum, hyperforin evident from reconstructed UV chromatograms for peaks and adhyperforin (peaks C34 and C36) with [M - H]- at displaying UV/Vis spectra in the range of 500–600 nm, i.e. 535 and 549 amu, were identified as the major PC of Hy- the typical absorption region for hypericins (Supplemen- pericum extracts and in agreement with literature. Moni- tary Fig. 2). A quantitative analysis for the different classes toring MSn data, the predominant loss of 69, 138 and 152 of compounds identified in Hypericum flowers is presented - - amu corresponding to [M-H–C5H9] , [M-H–C5H9–C5H9] in (Supplementary Table 1), which includes the relative n and [M-H–C5H9–C5H9–C6H11], respectively, in the MS quantification of each metabolite to the recovered amount spectrum was diagnostic for the presence of isoprenyl of spiked umbelliferone (IS) to allow for comparison across groups in hyperforins (Heinke et al. 2013; Keller et al. species. 2003). A total of 13 peaks show this pattern and the number of isoprenyl groups were predicted. The H. polyphyllum 3.3 Multivariate PCA analysis of LC–MS data extract showed two major peaks (C26 and C27) with a measured m/z value [M - H]- at 481 and 495, suggesting Although different metabolite patterns were observed by for one less prenyl chain compared to those of hyperforin visual inspection of the LC–MS traces of the different 123 A comparison of MS and NMR metabolomics 581

Table 1 Compounds tentatively assigned in Hypericum species ﬂower methanol extract using LC–MS Peaks Rt (min) UV Names [M - H]- Error El. Comp. [M - H]- MSn ions (m/z) (ppm)

C1 2.27 292 shd, 325 5-O-caffeoylquinic acid (chlorogenic acid) 353.0869 0.01 C16H17O9 353 239

C2 2.58 292 shd, 325 O-caffeoylquinic acid 353.0856 -3.6 C16H17O9 353 239, 119

C3 6.94 270, 365 Myricetin 3-O-glucoside 479.0802 -4.1 C21H19O13 479 239

C4 7.95 270, 355 Miquelianin 477.0691 5.2 C21H17O13 477 301

C5 9.02 255, 345 Rutin 609.1446 -0.5 C27H29O16 609 301

C6 8.55 265, 355 Quercetin-3-O-galactoside (hyperoside) 463.0885 0.5 C21H19O12 463 301

C7 9.5 290, 330 Dihydroquercetrin (astilbin) 449.1108 6.3 C21H21O11 449 303

C8 10.64 255, 350 Quercetin-3-O-rhamnoside (isoquercetrin) 447.0924 2.0 C21H19O11 447 301

C9 10.88 330 Unknown 263.1293 5.3 C15H19O4 263 119

C10 13.63 260 shd, 360 Quercetin 301.0329 -5.8 C15H9O7 301 263

C11 14.11 285, 355 Unidentiﬁed 393.2267 -0.6 C22H33O6 393 323

C12 15.46 270, 335 Amentaﬂavone 537.0809 -1.7 C30H17O10 537 443, 385 0 C13 16.94 272 Enaimeone C/1 -hydroxyialibinone D 375.215 -3.7 C22H31O5 375 194

C14 18.54 300 shd, 470 Skyrin-2-O-glucopyranoside 699.1299 -6.4 C36H27O15 699 519, 537

C15 19.3 275, 365 Quercetin-7-O-arabinoside 433.2371 -0.1 C28H33O4 433 364, 321

C16 19.47 225, 345 Unidentiﬁed 359.2241 7.2 C22H31O4 359 315, 290

C17 20.66 nd Hyperatomanin/hypercalyxone A 399.2527 3.1 C25H35O4 399 330, 287

C18 20.94 370, 535, 575 Protopseudohypericin 521.0857 4.0 C30H17O9 521

C19 21.26 290, 305, 355 7-Epiclusianone 501.3027 6.3 C33H41O4 501 432

C20 21.42 325, 545, 585 Pseudohypericin 519.0702 3.9 C30H15O9 519 467

C21 21.77 351 Unknown phloroglucinol I 413.2781 -2.4 C19H41O9 413 275

C22 22.37 370 7-Epiclusianone isomer 501.3017 4.3 C33H41O4 501 363, 321

C23 22.57 nd Furohyperforin 551.3688 -6.5 C35H51O5 551 481

C24 22.77 353 Unidentiﬁed phloroglucinol II 427.2907 2.3 C20H43O9 427 289

C25 22.82 280 Unidentiﬁed phloroglucinol III 467.3181 6.4 C30H43O4 467 423, 32

C26 22.92 230, 285 Hyperpolyphyllirin 481.3312 1.0 C31H45O4 481 437, 411

C27 23.87 230, 285 Unidentiﬁed phloroglucinol IV 495.3472 1.8 C32H47O4 495 467, 426, 357

C28 23.9 nd Unidentiﬁed 507.3451 -2.4 C33H47O4 507 345

C29 24.64 nd Hyperﬁrin 467.3134 -3.6 C30H43O4 467 329, 287

C30 24.75 240, 278 Hypersampsone E/D 569.3662 7.5 C38H49O4 569 500, 431, 417

C31 25.34 355 Adhyperﬁrin 481.3356 -1.8 C24H49O9 481 276, 233

C32 25.51 240, 275 Hypersampsone E/D 569.3645 4.5 C38H49O4 569 500, 431, 417

C33 25.92 370 Unidentiﬁed phloroglucinol V 569.3593 -4.6 C38H49O4 569 431, 309

C34 26.31 235, 290 Hyperforin 535.3787 2.2 C35H51O4 535 466, 383

C35 26.77 230, 290, 310 Geranyl phlorisobutyrophenone 331.1926 6.9 C20H27O4 331 207

C36 27.4 230, 290 Adhyperforin 549.3967 6.5 C36H53O4 549 480, 397

C37 28.09 nd Unidentiﬁed 495.3517 7.6 C32H47O4 495 451, 290, 247

C38 28.05 330, 545, 585 Hypericin 503.0762 2.1 C30H15O8 503 species, we attempted to analyze LC–MS spectra in a more mass signals were extracted by XCMS from the LC–MS holistic way using PCA to explore the relative variability data sets acquired in negative and positive ionisation mode, within the different species (Goodacre et al. 2000). Extracts respectively. Similar PCA results were observed in positive were analysed in both positive and negative ion electro- and negative mode and thus only the results derived from spray ionization HPLC–(ESI)MS modes as changes in ESI the richer negative mode data are presented. The main PC polarity can often circumvent or signiﬁcantly alter com- to differentiate between species, i.e. PC1, accounts for petitive ionization and suppression effects revealing 54 % of Hypericum variance (Fig. 2a). On the right side of otherwise suppressed metabolite signals (de Rijke et al. the plot, samples for H. kouytchense, H. polyphyllum and 2003). From the seven species, altogether 1,243 and 1,015 H. tetrapterum are positioned (positive PC1 values),

123 582 A. Porzel et al.

Fig. 2 LC–MS (m/z 100–1,000) principal component analyses of metabolites identification using LC–MS (n = 3). H. perforatum (e, Hypericum species in negative ionization mode. a Score plot of PC1 diamonds), H. polyphyllum (9, times), H. tetrapterum (5, down and -2 scores. b Loading plot for PC1 contributing mass peaks and pointing triangles), H. andorsaemum ( , box with times symbol), H. their assignments: C21 unidentified phloroglucinol I, C26 hyperpoly- inodorum (s, circles), H. undulatum (?, plus), H. kouytchense (4, up phyllirin, C30 hypersamspone E/D, C34 hyperforin, C36 adhyperfo- pointing triangles) rin. Peak numbers corresponds to those listed in Table 1 for whereas on the far left side, H. perforatum, H. androsae- perforatum is positioned in between these clusters as it mum, H. inodorum, and H. undulatum are located (negative shows characteristics of both. It is clustering close to H. PC1 values). The scores plot shows that reproducibility of polyphyllum and H. tetrapterum due to MS signals for the triplicate Hypericum samples was good. Discrimination napthodianthrones (hypericins) found only in the afore- between H. polyphyllum and H. tetrapterum is also optimal mentioned group 1A species as revealed from clustering along PC2. Examination of the loadings plot suggested that heat map plot (dashed box A, Supplementary Fig. 3) and the variables referring to the MS signals of hyperforins and reconstructed LC–UV/Vis chromatograms (Supplementary phloroglucinol (peaks C21, C26, C30, C34 and C36, see Fig. 2). Also, next to H. perforatum, H. kouytchense shows Table 1) contributed the most to the discrimination of the highest levels of flavonoids as revealed from the den- species (Fig. 2b). sity of corresponding MS signals in the cluster heat map (dashed box C in Supplementary Fig. 3). There are other 3.4 Multivariate HCA analysis of Hypericum species clusters of peaks which are typical for a single species for H. tetrapterum, H. polyphyllum, H. perforatum and H. ko- Like PCA, HCA is an unsupervised data analysis method uytchense, i.e. which might be used for a determination of with no need for prior knowledge of the sample. Such these species in preparations, e.g. to exclude adulteration, methods allow the clustering of the samples according to while H. androsaemum, H. undulatum and H. inodorum do intrinsic variance. Cluster analysis of the different Hyper- not show equally distinct clusters. icum species based on metabolite data from (Table 1) was used as an additional exploratory tool to assess the heter- 3.5 Visual inspection of 1H NMR spectra ogeneity between different genotypes. HCA clearly shows and assignments of metabolites two major clusters, of two and four genotypes (Supple- mentary Fig. 3), referred to as groups 1A and 1B, respec- The use of NMR for metabolite fingerprinting of H. per- tively. Inspection of group 1A shows that H. tetrapterum foratum has been previously reported (Bilia et al. 2001) and and H. polyphyllum are more closely related. In both spe- more recently a LC/DAD/SPE/NMR technique has been cies hyperforin/adhyperforin type phloroglucinols (peaks applied for identification of the major constituents present C34 and C36) were absent and thus HCA results are in full in Greek H. perforatum (Tatsis et al. 2007). In this study in agreement with PCA (PC1) results (Fig. 3). Cluster 1B we focus on the yet uncharacterized species H. polyphyllum includes H. kouytchense, H. androsaemum, H. undulatum to prove the unambiguous occurrence of hyperforins and and H. inodorum, as all these species show MS signals of other metabolites indicated by the LC–MS survey in order benzoylphloroglucinol derivatives (peaks C30 and C32). H. to evaluate its potential as H. perforatum substitute. 123 A comparison of MS and NMR metabolomics 583

Fig. 3 Details of the 1H NMR spectrum of Hypericum a sugars polyphyllum ﬂowers phloroglucinol demonstrating both signal HMDS phenolics richness and dynamic range of extract NMR spectra (a). Expanded spectral region from 0.85 to 1.7 ppm for hyperforins 8N7 6 4 2 0 (b), sugars 3.5–6 ppm (c), N3 phenolics 5.5–8 ppm (d) with b N3 N7 N3 assigned peaks: N1 astilbin, N2 chlorogenic acid, N3 fatty acids, N4 fructose, N5 b-glucose, N6 a-glucose, N7 hyperpolyphyllirin, N8 quercetin conjugates, N9 1.7 1.5 1.3 1.1 0.9 shikimic acid. The assignments c N4 were established using NMR N5 N6 spectra of standards. Signal numbers correspond to those listed in Table 2 for metabolite 1 5N2 4.5 4 3.5 identiﬁcation using H NMR N8+N9 N8 d N8 N8 N2 N2 N2

7.5 7.0 6.5 6.0 ppm

1H- and 13C NMR signals (Table 2) were assigned in show strong HMBC correlations to two carbonyl signals at comparison with spectra of authentic samples as well as on 212.8 and 187.0 ppm, to a quaternary carbon at 58.0 ppm the basis of 2D-NMR analysis using 1H–1H COSY and a methylene carbon at 42.4 ppm (Supplementary (homonuclear correlation), HSQC, and HMBC. Figure 3 Fig. 5). The latter can be readily assigned to C8 because of shows a representative 1H NMR spectrum from H. poly- the H8a/b HMBC correlation to C6 (47.6 ppm) and C7 phyllum. By means of NMR analysis, major classes of (44.0 ppm). C6 and C7 in turn are assigned according to constituents present in the extracts of St. John’s wort, such their HMBC correlations with H3-14. Furthermore, H8a/b as prenylated phloroglucinols and flavonoids, could be show correlations to the same two carbonyl signals as the identified in this species in addition to sugars and lipids. CH3 singlet at 1.15 ppm does (212.8 and 187.0 ppm), The 1H NMR spectrum is characterized by three main whereas only one of them (187.0 ppm) shows a correlation regions: a low-field region between 8.0 and 5.5 ppm with with the methylene protons of a prenyl side chain signals mainly due to aromatic protons of quercetin con- (3.04 ppm, CH2-26). Accordingly, the carbon signals at jugates and olefinic protons of chlorogenic acid as well as 187.0 and 212.8 ppm were assigned to C2 and C9, prenyl side chains of phloroglucinols; a mid-low field respectively. As a consequence, the methyl group showing region between 5.5 and 4.0 ppm with intensive signals due a 1H and 13C signal at 1.15 and 17.7 ppm, respectively, has to anomeric protons of sugar units, and a high-field region to be attached to C1 (58.0 ppm). Thus, the main phloro- between 3.2 and 0.8 ppm with strong signals due to ali- glucinol compound of H. polyphyllum flowers differs from phatic protons of hyperforins and fatty acids. hyperforin by the replacement of one prenyl side by a A detailed inspection of the 2D NMR correlation signal methyl group (Fig. 4). The structure of this compound pattern and comparison with the 2D NMR spectra of designated as ‘‘hyperpolyphyllirin’’, which to the best of authentic hyperforin revealed that the major phloroglucinol our knowledge has not been described previously [Sci- compound of H. polyphyllum flowers contains only three Finder 2013-10-23], was unambiguously elucidated from prenyl side chains (Supplementary Fig. 4), but exhibits an the crude extract of H. polyphyllum flowers in the course of additional 1H NMR singlet at 1.15 ppm, sharing HMBC our metabolomics investigation. This impressively dem- correlations to phloroglucinol carbon signals with prenyl onstrates the potential of NMR as an important tool for side chain proton signals. Owing to its HSQC correlation to metabolomics, which provides comprehensive structural the carbon signal at 17.7 ppm, the proton singlet at information. The NMR data of hyperpolyphyllirin are 1.15 ppm belongs to a methyl group. The methyl protons summarized in Supplementary Table 2 and in comparison

123 584 A. Porzel et al.

30 29 Table 2 Resonance assignments with chemical shifts of constituents 28 identiﬁed from 600 MHz 1H and 1H–13C NMR spectra of Hypericum polyphyllum (methanol-d4) 27 Numbers 26 Compounds Assignments 1H (multiplicity) 13C 3 O 2 OH 12 N1 4 Astilbin CH, 6 5.88 d (1.9 Hz) 97.0 1 5 11 CH, 8 5.92 d (1.9 Hz) 95.8 9 10 31 13 N2 O O Chlorogenic acid CH2, 2 axial 2.59 dd (15. 41.0 6 8 7 Hz) 15 7 b CH2,2 2.78 41.0 21 14 16 equatorial 22 17 CH, 20 6.28 d (16.0 Hz) 115.5 0 b 23 18 CH, 9 6.77 d 116.4 24 25 20 19 CH, 30 7.56 d (16.0 Hz) 147.3 CH, 50 7.05 d (1.5 Hz) 115.1 Fig. 4 Proposed structure of the novel phloroglucinol ‘‘hyperpolyphyllirin’’ from Hypericum polyphyllum N3

Fatty acids CH3, terminal 0.90 t (7.3 Hz) 14.5 (CH2–)n 1.29 30.8 to those of hyperforin. It should be noted, that hyperpoly- (CH2–)n 1.33 30.3 phyllirin has the same molecular formula, C31H46O4,as N4 adhyperfirin (Supplementary Fig. 6) and can therefore not Fructose CH, 4 4.02 d (7.7 Hz) 77.0 be distinguished by standard MS. Adhyperfirin has not yet N5 been isolated, its constitution was proposed based on MS b-Glucose CH, 1 4.47 (d, 7.7 Hz) 98.02 data only in analogy to adhyperforin (Tatsis et al. 2007; M 6 Alali and Tawaha 2009; Alali et al. 2009; Hoelscher et al. a-Glucose CH, 1 5.11 (d, 3.7 Hz) 94.0 2009). Some 1H NMR data were shown for the compound N7 proposed to be adhyperfirin (Tatsis et al. 2007), however, Hyperpolyphyllirin CH , 13 1.05 (d, 6.7 Hz) 22.3 3 data of a methyl triplet, important for the verification of the CH , 12 1.08 (d, 6.3 Hz) 21.1 3 suggested 2-methyl-butanoyl side chain, are missing. It CH , 14 0.97 s 15.1 3 cannot be excluded that a metabolite assigned to be ad- CH , 19 1.56 s 18.0 3 hyperfirin based on MS data only, has in fact the herein CH , 35 1.59 s 17.8 3 proposed constitution of hyperpolyphyllirin. A similar CH , 34 1.61 s 26.4 3 analytical problem can be found in the distinction between CH , 20 1.64 s 26.0 3 secohyperforin (Supplementary Fig. 6), and hyperfirin, CH , 30 1.65 s 26.0 3 based on MS data only. Secohyperforin was characterized CH , 29 1.69 s 18.1 3 by online LC–2D-NMR experiments (Charchoglyan et al. CH, 22 5.01 t (7.0 Hz) 124.4 2007) and compared with hyperforin, it lacks a prenyl CH, 27 5.05 t (7.0 Hz) 126.8 group as hyperfirin does (Supplementary Fig. 6). Secohy- CH, 17 5.16 t (7.0 Hz) 125.3 perforin, was proposed to be a result from a branch point in N8 the biosynthesis of prenylated acylphloroglucinols Quercetin CH, 100 5.31 d (7.7 Hz) 103.1 conjugates (glucose) (Charchoglyan et al. 2007). Whether hyperpolyphyllirin CH, 6 6.20 d (2.2 Hz) 99.7 follows a similar biosynthetic route has yet to be investi- CH, 8 6.36 d (2.2 Hz) 94.6 gated, although it should be noted that the presence of a CH, 20 7.81 d (2.2 Hz) 110.0 methyl group at the C1 position of hyperpolyphyllirin is 1 N9 unusual in hyperforins. Some major signals in the HNMR a Shikimic acid CH2, 7a 2.18 dd (18, 32.0 spectrum of the H. polyphyllum extract, due to fatty acids 5 Hz) appearing at d 0.90 (terminal CH3 groups), 1.29 and CH2, 7b 2.68 dd (18, 32.0 1.33 ppm (inner-chain CH2 groups), could not be assigned 5 Hz) to individual fatty acids. In the hydroxy methine region b CH, 3 6.78 137.3 (4.0–5.5 ppm), a-glucose, b-glucose and fructose are the a Resonances assigned based on H. inodorum spectra most abundant sugars in all extracts examined. They were b Unresolved signals identified from anomeric signals at d 5.11, 4.47 and 4.02, 123 A comparison of MS and NMR metabolomics 585 respectively (Fig. 3c). In the aromatics region and 1.60) and fatty acids (d 0.90 and 1.29) contributing (5.5–9.0 ppm), flavonoids were detected though at low positively to PC1. The second group is characterized by levels (Fig. 3d). Some aglycon resonances for quercetin resonances appearing at d 2.18 and 2.68 for shikimic acid conjugates are readily assigned due to their chemical shifts, and at d 6.70 for chlorogenic acid (Supplementary Fig. 8b), together with their splitting and couplings patterns both contributing negatively to PC1. Shikimic acid (Table 2). In particular, the doublet resonances at 6.20 (Fig. 1), which plays a key role in the biosynthesis of many (2.2 Hz) and 6.36 (2.2 Hz) were assigned to H6 and H8 of important natural products including aromatic amino acids quercetin and its conjugates (Bilia et al. 2001). These data and phenylpropanoids (Payne and Edmonds 2005), has the were confirmed through HSQC experiments by connec- strongest impact on species discrimination (Supplementary tivities with the carbon resonances at 99.7 and 94.6 ppm, Fig. 8b). These results are in agreement with TP content, respectively. The proton signals of ring B of quercetin revealing that in H. perforatum, H. androsaemum, H. ko- derivatives (6.6–7.1 ppm) overlapped with the aromatic uytchense and H. inodorum, phenolics are more enriched proton signals of chlorogenic acid. Chlorogenic acid, pre- (Supplementary Table 3). Shikimic acid was not detected viously reported in Hypericum, was assigned by compari- using LC–MS, whereas caffeoylquinic acids co-eluted at a son with spectra of an authentic sample based on the very short rt (rt = 2.27–2.58 min, Table 1) close to the characteristic signals of the trans double bond protons at solvent front, thwarting its accurate quantification. The 6.28 and 7.56 ppm with a large coupling constant of 16 Hz. slight differences in the score plots derived from data of These protons show connectivities in the HSQC spectrum both techniques could be ascribed to absence of mass with the 13C resonances at 115.5 and 147.3 ppm, respec- signals for fatty acids in the MS loading plots (Fig. 2b), tively. Shikimic acid presents another organic acid present which were found to be discriminating for Hypericum in most Hypericum extracts. It is evident from its charac- species segregation in NMR plots (Supplementary Fig. 8b). teristic H7a and H7b (2.18 and 2.68 ppm) proton signals While score plots in PCA were similar for MS and NMR (Supplementary Fig. 7) showing HMBC correlations to C3. datasets, the associated loading plots reveal differences in Furthermore, H5 and H6 (3.63 and 3.98 ppm) were the types of metabolites contributing to the species segre- assigned by 1H–1H COSY correlation. The assignments of gation. It should be noted that signals for sugars detected the shikimic acid signals (Table 2) were also supported by by NMR (Fig. 3) do not contribute to segregation in PCA comparison with spectra of an authentic sample. None of loading plots along PC1 in the NMR dataset, suggestive the extracts showed unambiguous signals characteristic for that sugars are present at comparable levels in all Hyperi- naphthodianthrones (hypericins), likely because of the low cum species. abundance, hiding signals under other ones in the region. In addition to the clear visual differences between the species 3.7 Quantification of hyperforins and phenolics 1H NMR spectra (data not shown), for nonbiased inter- in Hypericum extracts pretation of the results, the samples were analyzed using PCA. To confirm the results above, i.e. that discrimination between species is dominantly related to hyperforins and 3.6 Comparison of 1H NMR and LC–MS multivariate shikimic acid levels, quantifications were attempted in 1 PCA analysis flower extracts using H NMR signals (CH3-15 for hyperforins), and (CH2-7 for shikimic acid). For the quanti- We compared the performance of PCA from LC–MS and fication to be optimal, however, full relaxation of the 1H NMR spectra of extracts to better evaluate the classi- protons of targeted signals and the internal standard fication potential of both technologies. When compared to (HMDS) has to be achieved. For this purpose an acquisi- the PC plot obtained from LC–MS data (Fig. 2a), it is tion time of 2.7 s and a rather large relaxation delay time of apparent that the NMR results (Supplementary Fig. 8a) 17.3 s was used, as the longest relaxation times were 4.5 s were in general agreement. In both score plots, H. poly- for the HMDS protons. The biological variance within each phyllum and H. tetrapterum were plotted on the right side of the examined species was assessed accordingly by (positive score values) from the other species, which comparing the metabolites of the flower extracts of each mostly gave negative score values. In contrast, and con- cultivar, as shown in Supplementary Table 3. Based on trary to LC–MS, H. kouytchense and H. undulatum were NMR quantification, it was revealed that H. perforatum has switched in position. The separation observed in PCA can the highest content of hyperforins followed by H. poly- be explained in terms of the identified compounds, using phyllum. It should be noted that although H. tetrapterum the loading plots for PC1 exposing the most discriminatory shows strong signals for phloroglucinols, absolute quanti- signals. Two major groups stand out in this plot. The first fication was hindered due to uncertainty in its chemical corresponds to the chemical shifts of hyperforins (d 1.05 structure. In agreement with PCA results, H. 123 586 A. Porzel et al. androsaemum, H. inodorum and H. kouytchenese were it cannot be excluded that other unidentified constituents found to be more enriched in shikimic acid, with the may act additively or synergistically with hypericins and highest content found in H. androsaemum. Positive rela- hyperforins, or even may eventually be more relevant for tionship appears to exist between shikimic acid levels and the cytotoxic effects of Hypericum extracts. total polyphenol (TP) content for most species. Species enriched in shikimic acid show higher amounts in phenolics, i.e. H. androsaemum; a similar trend was observed for 4 Concluding remarks species low in shikimic acid, i.e. H. undulatum (Supple- mentary Table 3). The customary method for quantifying To the best of our knowledge, this study provides the first hyperforins and flavonoids in Hypericum is HPLC, which comparative metabolomic approach to reveal for compo- needs calibration and is more laborious, error prone and sitional differences among Hypericum species. NMR and time-consuming than NMR. We could show that 1HNMR, MS techniques coupled with multivariate data analyses without the need of any chromatography step, is an were used and compared to obtain the experimental results, appropriate tool for profiling as well as structural elucida- and interesting and meaningful differences between the tion of hyperforins and related main constituents in Hy- various species and detection methods were identified. pericum flower extracts. While the NMR approach allows for an easy quantification of hyperforin contained in Hypericum extracts as major 3.8 Cytotoxic effect in relation to naphthodianthrones component, the high sensitivity of MS was more suitable content for metabolites with low abundance, such as naphthodianthrones. PCA consistently grouped and separated spe- Evidence in the literature point towards a marked cytotoxic cies, independent of whether NMR chemical shifts or effect for the Hypericum major secondary metabolites, rt/mass signal pairs (LC–MS) were used as data basis, including hyperforin (Schempp et al. 2002a). Our objective suggesting its validity for the classification of Hypericum was to investigate the cytotoxic potential of extracts species. Additionally, absolute quantifications of shikimic derived from species with different chemical composition acid and hyperforin using NMR (Supplementary Table 3) as revealed from our chemical profiling. Methanol extracts confirmed the loading plot results derived from an NMR prepared from the seven species were tested for growth dataset (Supplementary Fig. 8b). The fact that H. poly- inhibition effects against (mutated androgen dependent) phyllum, H. perforatum, and H. tetrapterum not only prostate (PC3) and (androgen independent) colon (HT29) cluster in HCA analysis, but also exhibited the strongest cancer cell lines at concentrations up to 100 lg/mL. Hy- cytotoxic effect, suggest that bioactivity might be corre- perforin and hypericin were used as references at doses of lated with gross metabolic profiling, which also can be 5, 10, 50 and 100 lg/mL. Hypericin and hyperforin stan- correlated to phylogeny. Although our selection of genetic dards exhibit significant activity with slightly lower IC50 sources does not cover a worldwide selection of the Hy- values for hyperforin (PC3, 20 lg/mL = 37 lM; HT29, pericum species studied, our approach is certainly feasible 15.5 lg/mL = 29 lM) than for hypericin (PC3, 24.2 lg/ for analysing other species from such further sources. The mL = 47 lM; HT29, 19.5 lg/mL = 39 lM). Naphtho- comparative metabolomics approach developed in this dianthrones enriched extracts prepared from H. polyphyl- work can be further applied for investigating effects of lum, H. perforatum and H. tetrapterum (Supplementary other factors such as organ type, storage, harvesting time, Fig. 2), showed growth inhibition against HT29 cell line or seasonal variation on secondary metabolite composition. with IC50 values of 20, 33, 49 lg/mL, respectively. Whereas only H. perforatum and H. tetrapterum, inhibited Acknowledgments Mohamed A. Farag thanks the Alexander von PC3 cell line growth with IC values of 51 and 54.0 lg/ Humboldt-Foundation, Germany for financial support. We also thank 50 Christoph Bo¨ttcher for assistance with the UPLC–MS. We are mL, respectively. Other Hypericum species showed no grateful to Steffen Neumann and Tilo Lu¨bken for providing R scripts growth inhibition effect up to a dose of 100 lg/mL. These for NMR and MS data analysis. findings verify that hypericins constitute an indispensable part of Hypericum anti-cancer efficacy, and suggest that bioactivity may be predicted by either gross metabolic profiling or chemometric methods. It should be noted that References flavonoids present in Hypericum extracts are unlikely to mediate the reported cytotoxic effect, considering that in H. Adam, P., Arigoni, D., Bacher, A., & Eisenreich, W. (2002). polyphyllum, H. perforatum and H. tetrapterum, flavonoids Biosynthesis of hyperforin in Hypericum perforatum. Journal of Medicinal Chemistry, 45(21), 4786–4793. were present at lower levels compared with other species Agnolet, S., Jaroszewski, J. W., Verpoorte, R., & Staerk, D. (2010). like H. kouytchense (Supplementary Fig. 3). Nevertheless, H-1 NMR-based metabolomics combined with HPLC–PDA– 123 A comparison of MS and NMR metabolomics 587

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