Effect-Directed Discovery of Bioactive Compounds Followed by Highly

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Effect-directed discovery of bioactive compounds followed by highly targeted characterization, isolation and identification, exemplarily shown for Solidago virgaurea Ágnes M. Móricz, Péter G. Ott, Tim T. Häbe, András Darcsi, Andrea Böszörmenyi, Ágnes Alberti, Daniel Kruzselyi, Péter Csontos, Szabolcs Béni, and Gertrud Elisabeth Morlock Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02007 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 25, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

1

Effect-directed discovery of bioactive compounds followed by

2

highly targeted characterization, isolation and identification,

3

exemplarily shown for Solidago virgaurea

4 5

Ágnes M. Móricz1,2*, Péter G. Ott1, Tim T. Häbe2, András Darcsi3, Andrea Böszörményi3, Ágnes

6

Alberti3, Dániel Krüzselyi1, Péter Csontos4, Szabolcs Béni3, Gertrud E. Morlock2

7 8 9

1

10 11

Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Herman O. Str. 15, 1022 Budapest, Hungary

2

Interdisciplinary Research Center (IFZ) and Institute of Nutritional Science, Department of Food

12

Sciences, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen,

13

Germany

14

3

15 16 17

Department of Pharmacognosy, Faculty of Pharmacy, Semmelweis University, Üllői Str. 26, 1085 Budapest, Hungary

4

Institute for Soil Sciences and Agricultural Chemistry, Centre for Agricultural Research, Hungarian Academy of Sciences, Herman O. Str. 15, 1022 Budapest, Hungary

18 19 20

*Corresponding author. Tel.: (0036) 14877515; Fax: (0036) 14877555; E-mail address:

21

[email protected] (Á.M. Móricz)

22

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23

Abstract

24

A non-targeted, effect-directed screening (bioprofiling) and a subsequent highly targeted

25

characterization of antibacterial compounds from plant matrices is demonstrated on the example

26

of Solidago virgaurea root extracts. The procedure comprises high-performance thin-layer

27

chromatography (HPTLC) coupled with six bacterial bioassays including two plant pathogens, a

28

radical scavenging assay, an acetylcholinesterase assay as well as in situ and ex situ mass

29

spectrometric analyses. In situ mass spectra were directly recorded from the adsorbent using the

30

Direct Analysis in Real Time interface (HPTLC-DART-MS), whereas ex situ mass spectra were

31

recorded using an elution head-based interface (HPTLC-ESI-MS). For further bioassay-guided

32

isolation of the main antimicrobial compounds, flash chromatographic fractionation and semi-

33

preparative high-performance liquid chromatographic purification were used and nuclear

34

magnetic resonance data allowed the identification of the unknown antimicrobial compounds as

35

2Z,8Z- and 2E,8Z-matricaria esters. The discovered antibacterial activity was confirmed and

36

specified by a luminometric assay and as minimal inhibitory concentration in the liquid phase.

37 38

Keywords

39

Direct bioautography; Effect-directed profiling; EDA; HPTLC; Bioassay; DART; Mass

40

spectrometry; Solidago virgaurea

41


Page 2 of 37

Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


42

Introduction

43

The frequently used microbial inhibition assays, like dilution or diffusion methods, provide an

44

estimate of the antimicrobial potential of a sample as a whole, but cannot define the single active

45

compounds in a complex sample material. Moreover, the poor solubility and slow diffusion

46

restrict the sample throughput.1,2 There are not such limits in direct bioautography (DB).3-7 It is

47

the combination of planar chromatography with biological assays, in which the planar

48

chromatogram is immersed into a respective cell suspension. This makes a direct interaction

49

possible between separated compounds and cells. Therefore, the influence of the separated

50

compounds on the viability and metabolic activity of the test microorganism is easily observed.

51

Consequently, DB is simpler and faster than the dilution or diffusion methods (0.5–2.5 h versus

52

1–3 days) and allows the detection of single active compounds. Such a non-targeted screening

53

can lead to the identification of known and especially unknown compounds. High-performance

54

thin-layer chromatography (HPTLC) hyphenated with effect-directed assays (effect-directed

55

analysis, EDA) and mass spectrometry (MS) provides a streamlined way for the discovery of

56

single bioactive compounds and their further targeted characterization.5,8 For example,

57

compounds of interest can be characterized by Direct Analysis in Real Time (DART)-MS.9,10

58

Substance identification can be supported by the use of standards, if available11-15, or by

59

recording mass spectra with electron ionization (EI) and comparing with spectra from libraries of

60

volatile substances.16-18 In other cases, bioactive compounds were tentatively assigned by liquid

61

chromatography (LC)-MS/MS or remained unknown.19-22 Fulfilling the requirements of a bio-

62

monitoring system, DB enables the bioassay-guided isolation of unknown antimicrobial

63

components and their structure elucidation by nuclear magnetic resonance (NMR) spectroscopy.5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

64

European goldenrod (Solidago virgaurea L.) is a herbaceous perennial plant of the family

65

Asteraceae, with erect stems that grow to heights of 60–100 cm, yellow flowers and a branching

66

underground rhizome. It has a considerably wide native distribution range that includes most of

67

Europe as well as North Africa and Northern, Central, and Southwestern Asia. European

68

goldenrod is a safe and gentle remedy for a number of disorders.23,24 In traditional medicine, the

69

plant is used as a mouth rinse to treat inflammation of the mouth and throat, as a valuable

70

astringent remedy treating wounds and bleeding. Using internally, it is recommended for treating

71

urinary tract, nephritis, cystitis and bladder dysfunction, and it is often found in teas to support

72

passing kidney stones. Alcoholic extracts of the plant contained numerous constituents including

73

diterpenoids,25,26 saponins,27 phenols,28 polyacetylenes,29 and flavonoids,24,30 all compounds

74

causing multiple biological effects. Antimicrobial, amoebicidal, antimycotic, anti-inflammatory,

75

analgesic, sedative, hypotensive and anticancer activities for extracts were reported.30-34

76

The aim of this paper was to demonstrate a systematic EDA procedure for screening,

77

characterization, isolation and identification of unknown antibacterial and free radical scavenging

78

(antioxidant) compounds as well as acetylcholinesterase inhibitors from the ethanolic extract of

79

Solidago virgaurea roots. For this purpose, planar chromatography was coupled with bioassays

80

of six bacterial species (including two plant pathogenic strains), a radical scavenging assay, an

81

acetylcholinesterase assay and various MS methods. Additionally, forced-flow column

82

chromatographic fractionation and purification as well as NMR were applied for the isolation and

83

further identification of unknown bioactive compounds.

84


Page 4 of 37

Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


85

Experimental section

86

Materials. HPTLC plates and HPTLC foils silica gel 60 F254, methanol (MS grade) and

87

ammonium formate were obtained from Merck Millipore, Darmstadt, Germany. Isopropyl

88

acetate, methanol, acetylcholinesterase lyophilisate (from Electrophorus electricus), Fast Blue

89

Salt B, neomycin, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH*, 97%), and proteose peptone

90

were from Sigma-Aldrich (Steinheim, Germany or Budapest, Hungary). Acetonitrile (gradient

91

grade) was supplied by Fisher Scientific, Pittsburg, PA, USA. Deuterated methanol (CD3OD,

92

99.8 atom% D) for NMR was purchased from VWR, Budapest, Hungary. All other solvents

93

applied for HPTLC were from Reanal (Budapest, Hungary), who also supplied formic acid,

94

glucose and potassium dihydrogen phosphate (KH2PO4), or Carl Roth (Karlsruhe, Germany). The

95

latter manufacturer supplied also bovine serum albumin, 3-(4,5-dimethylthiazol-2-yl)-2,5-

96

diphenyltetrazolium bromide (MTT) and tris(hydroxymethyl)aminomethane. α-Naphthyl acetate

97

was obtained from Panreac, Barcelona, Spain. Yeast extract was from Scharlau, Barcelona,

98

Spain. Pure water was produced by a Merck Millipore Direct-Q 3 UV system. The bacterial

99

strains used in the bioassays were marine bacterium Aliivibrio fischeri (DSM-7151, Leibniz

100

Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Berlin, Germany), soil

101

bacteria Bacillus subtilis subsp. spizizenii (DSM 618, Merck Millipore) and Bacillus subtilis

102

strain F1276,35 Arabidopsis pathogen Pseudomonas syringae pv. maculicola (Jun Fan, John Innes

103

Center, Department of Disease and Stress Biology, Norwich, UK),36 paprika pathogen

104

Xanthomonas euvesicatoria (Hungarian paprika isolate, János Szarka, Primordium Kft.,

105

Budapest, Hungary), and probiotic bacterium Lactobacillus plantarum (ATCC 8014).

106

Sample preparation. Roots of full flowered Solidago virgaurea L. (European goldenrod or

107

woundwort) were collected in a sessile oak-Turkey oak wood in the Buda hills in Hungary in



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

108

June 2014. The roots were dried at 25 °C in the dark and pulverized in a coffee grinder (Bosch

109

MKM6000, Stuttgart, Germany). The milled roots (3 g) were macerated for 24 h with 20 mL of

110

ethanol in a glass bottle. The supernatant with 12.1 mg/mL dry weight was directly used for flash

111

chromatography. For HPTLC, the crude extract was diluted 1:10 with ethanol.

112

Plate pretreatment for MS. For HPTLC-MS, HPTLC plates were pre-chromatographed with

113

methanol – water (4:1, v/v) and dried at 100 °C for 20 min.37 For HPTLC-DART-MS, HPTLC

114

plates were cut with the smartCUT Plate Cutter (CAMAG, Muttenz, Switzerland) as 2 cm wide

115

and 10 cm long plate strips.38

116

HPTLC-UV/Vis/FLD. The root extracts were separated in a Twin Trough Chamber (20 cm × 10

117

cm, CAMAG) either with n-hexane – isopropyl acetate – acetic acid (83:14:3, v/v) on HPTLC

118

plates or with n-hexane – acetone (85:15, v/v) on HPTLC foils. 1–10 µL of the sample were

119

sprayed as 7 mm bands onto the plate by an automatic TLC sampler ATS4 or a Linomat IV (both

120

CAMAG) with 8 mm distance from the bottom. After development the plate was dried in a cold

121

air stream and cut with a blade or smartCUT Plate Cutter (CAMAG). The parallel tracks were

122

used for EDA and MS. Zones were derivatized by immersion (TLC Immersion Device,

123

CAMAG) into the vanillin-sulphuric acid reagent (mixture of 40 mg vanillin, 10 mL ethanol and

124

200 µL concentrated sulphuric acid), followed by heating at 110 °C for 5 min and documented at

125

white light illumination (transmittance mode). The chromatograms at UV 254 nm, UV 366 nm as

126

well as before and after derivatization were documented using the DigiStore 2 or TLC Visualizer

127

Documentation System (both CAMAG) or a digital camera (Cybershot DSC-HX60, Sony, Neu-

128

Isenburg, Germany). Densitometry and spectra recording (200–450 nm) were carried out by

129

absorbance measurement using the TLC Scanner 3 (CAMAG). Data were processed and

130

evaluated by winCATS software, version 1.4.7.2008 (CAMAG).


Page 6 of 37

Page 7 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


131

Prior to EDA, the chromatograms were neutralized, i. e., residual acetic acid traces from the

132

mobile phase was eliminated, in a Twin Trough Chamber (CAMAG) with potassium hydroxide

133

in the second trough for 4 h. Then, the plates were placed in a cold air stream for 20 min to

134

remove the excess of potassium hydroxide adsorbed from the gas phase.

135

Radical scavenging assay. For the detection of free radical scavengers (antioxidants), the

136

HPTLC plates were dipped into a 0.02% methanolic DPPH* solution. Radical scavenging

137

compounds appeared as bright yellow zones against a purple background and were documented

138

with the TLC Visualizer at white light illumination (CAMAG, transmittance mode).

139

Enzymatic assay. Acetylcholinesterase inhibitors were detected by the method of Akkad and

140

Schwack39 modified by Hage and Morlock40 and originally based on Marston et al.41 The enzyme

141

solution was prepared by mixing 666 units of acetylcholinesterase, 100 mg bovine serum albumin

142

and 100 mL TRIS buffer (0.05 M, pH 7.8) and kept frozen until use. The substrate solution was

143

freshly prepared by dissolving 25 mg α-naphthyl acetate and 50 mg Fast Blue salt B in 90 mL

144

ethanol – water, 1:2 (v/v). The plate was dipped into the enzyme solution for 5 s, followed by

145

incubation in a vapor chamber at 37 °C for 25 min. Then, the chromatogram was immersed in the

146

substrate solution. In the presence of active enzyme α-naphthol is produced,immediately forming

147

a violet adduct with Fast Blue salt B. Therefore, enzyme inhibitors appear as bright spots against

148

a colorful background. The chromatogram was documented with the TLC Visualizer at white

149

light illumination (CAMAG, reflectance mode).

150

Direct bioautography. B. subtilis F1276, X. euvesicatoria and P. maculicola cells were grown

151

according to Móricz et al..4,9 B. subtilis spizizenii cell suspension was prepared according to

152

Jamshidi-Aidji and Morlock.42 A. fischeri was cultivated as described by Krüger et al..43 L.

153

plantarum cells were grown in culture medium containing 5 g/L peptone from meat, 20 g/L yeast



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

154

extract, 10 g/L glucose and 2 g/L potassium dihydrogen phosphate at 28 °C on an orbital shaker

155

with the speed of 130 rpm to reach the late exponential phase (5x108 cells/mL, OD600 = 1.2).

156

Chromatograms were immersed into one of the cell suspensions using a home-made cassette5 or a

157

TLC Immersion Device (CAMAG). When working with luminescent bacteria (A. fischeri and P.

158

maculicola), the bioautograms were documented under a glass plate allowing sufficient humid air

159

above the layer using either the BioLuminizer (CAMAG) at an exposure time of 50 s (A. fischeri)

160

or the cooled low-light camera IS-4000 (Alpha Innotech, San Leandro, CA, USA) at an exposure

161

time of 15 min (P. maculicola). Dark spots against a luminous background indicate the inhibitory

162

activity. ImageJ (NIH, Bethesda, MA, USA) processing and analysis program was applied for

163

image evaluation. As for B. subtilis F1276, X. euvesicatoria and L. plantarum bacterial strains,

164

after 2 h incubation time in a vapor chamber at 28 °C, the zones with an antibacterial effect were

165

visualized by dipping the bioautograms into an aqueous solution of MTT vital dye (1 mg/mL) for

166

1 s and dried at 60 °C for 5 min. Metabolically active cells reduce the yellow MTT to a bluish

167

formazan, revealing the inhibition zones as bright spots against a bluish background, which was

168

documented by a Sony Cybershot DSC-HX60 digital camera. The same procedure was used for

169

B. subtilis spizizenii (DSM 618), except for incubation at 37 °C, staining with buffered MTT

170

solution (0.2% phosphate-buffered saline, pH 7.5) and documentation with TLC Visualizer. The

171

B. subtilis spizizenii bioautograms were scanned at 546 nm using the mercury lamp of the TLC

172

scanner 3 (CAMAG).

173

Microdilution test. The minimal inhibitory concentration (MIC) of the S. virgaurea extract and

174

its isolated component dissolved in ethanol was determined against B. subtilis and X.

175

euvesicatoria by a broth microdilution method. The experiments were made in triplicate.

176

Neomycin (1 mg/mL in ethanol, MIC = 16 µg/mL) was used as positive and ethanol as a negative

177

control. Ethanolic two-fold dilution series of the samples were prepared and 5 µL of each was


Page 8 of 37

Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


178

mixed with 140 µL of cell suspension (105 CFU/mL) in 96-well sterile microtiter plates to give a

179

final concentration of 5.4–690 µg/mL. MIC values were obtained by adding 35 µL of yellow

180

MTT dye solution (0.5 mg/mL) after incubation at 28 °C for 16 h. The MICs were determined as

181

the lowest concentrations of tested samples that completely inhibited the bacterial growth, which

182

was indicated by the absence of the formation of the bluish formazan.

183

Luminometric antibacterial assay. Luminometric measurement was performed by mixing 1 µL

184

of the samples (10 mg/mL in ethanol) with 50 µL of luminescent bacterial cell suspension of

185

about 5×108 (P. maculicola) and 107 (A. fischeri) cells/mL, in an Eppendorf tube. The tube was

186

put into a luminometer (Model 2020n; Turner Biosystems, Sunnyvale, CA) to measure the

187

intensity of the emitted light corresponding to the viability. The experiments were made in

188

triplicate.

189

HPTLC-ESI-MS. The HPTLC-ESI-MS system consisted of a HPLC pump (HP 1100, Agilent,

190

Waldbronn, Germany), the TLC-MS Interface with oval elution head (4 mm × 2 mm; CAMAG),

191

a C18 SecurityGuard cartridge (Phenomex, Torrace, CA, USA) and a single quadrupole MS

192

equipped with an ESI ion source (expression CMS, Advion, Ithaca, NY, USA). The bioactive as

193

well as background zones at a similar hRF were eluted using methanol – 10 mmol (pH 4)

194

ammonium formate buffer (95:5, v/v) at a flow rate of 0.1 mL/min and directly led into the MS

195

system. Mass spectrometer settings for positive/negative ionization mode were: ESI voltage

196

+4000/-3000 V, capillary voltage +100/-170 V, source voltage offset 10/25 V (span 0 V),

197

nebulizer gas pressure 60 psi, temperatures of the capillary and the source gas 250 °C and

198

desolvation gas flow rate 4 L/min. Total ion current (TIC) chronograms were recorded in full

199

scan mode in a range of m/z 50–800. Data acquisition, processing and evaluation were carried out

200

with Mass Express 2.0.45.4 and Data Express 2.0.50.9 (both from Advion).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

201

HPTLC-DART-MS. A modified HPTLC-DART-MS system for precise HPTLC scanning38

202

coupled to the amaZon ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany)

203

was used according to Móricz et al..9 Gas flow and gas temperature were set to 3.0 L/min at

204

500 oC and the grid voltage to 50 V for DART scanning in positive and negative ionization mode.

205

The enhanced resolution mode was used with an ICC target of 200,000 for the acquisition of the

206

TIC chromatograms in a range of m/z 70–1000 with averages set to 5 spectra and rolling averages

207

set to 2 cts. The TIC, extracted ion chromatograms (EIC) and mass spectra for substance zones of

208

interest were extracted and processed (gauss smoothing algorithm width: 1.32 s) with

209

DataAnalysis version 4.0 (Bruker Daltonics).

210

Solid phase micro extraction-gas chromatography (SPME-GC)-EI-MS. 100 µL of root

211

extract or 0.5 mL of the active main compound (the HPTLC band was scraped off and eluted

212

with 0.5 mL of ethanol) was transferred into 20 mL headspace vials sealed with a

213

silicon/polytetrafluoroethylene septum for the subsequent SPME-GC-EI-MS analysis. Static

214

headspace (sHS)-SPME was carried out with a CTC Combi PAL (CTC Analytics, Zwingen,

215

Switzerland) using a polydimethylsiloxane/divinylbenzene SPME fiber (fiber diameter 65 µm,

216

StableFlex, Supelco, Bellefonte, PA, USA). After an incubation period of 5 min at 100 °C,

217

extraction was performed by exposing the fiber into the headspace for 10 min at 100 °C. Then,

218

the fiber was transferred to the injector port of the GC-MS (6890N/5973N, Agilent, Santa Clara,

219

CA, USA) and desorbed for 1 min at 250 °C. The SPME fiber was cleaned and conditioned in the

220

Fiber Bakeout Station (CTC Analytics) using pure nitrogen atmosphere at 250 °C for 15 min

221

after desorption. The GC oven containing an HP-5MS capillary column (30 m × 250 µm ×

222

0.25 µm, Agilent) was programmed to increase from 60 °C (3 min isothermal) to 250 °C at

223

8 °C/min (1 min isothermal). High purity helium (6.0) was used as carrier gas at 1.0 mL/min

224

(37 cm/s) in a constant flow mode. The injector temperature was 250 °C and the split ratio was


Page 11 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


225

1:50. The MS was equipped with an EI source (70 eV), a quadrupole mass analyzer and was

226

operated in full scan mode (41–500 amu at 3.2 scan/s). Data evaluation was performed with MSD

227

ChemStation D.02.00.275 (Agilent) and NIST 2.0 spectral library.

228

Flash chromatography. A low pressure flash chromatograph (Macherey-Nagel, Düren,

229

Germany) was used to fractionate 8 mL of crude extract by utilizing a column (Macherey-Nagel,

230

3 cm inner diameter (ID), 12 cm length) filled in-house with preparative silica gel (Sigma-

231

Aldrich, St. Louis, MO; No. 60752, high-purity grade, 60 Å pore size, 230–400 mesh particle

232

size) and cyclohexane – acetone 9:1 (v/v) as mobile phase accelerated by nitrogen gas pressure.

233

The extract was dried onto 5 g of silica gel and layered to the top of the column. Moreover, 1 cm

234

of sand was applied to the bottom and top of the stationary phase. The eluate was collected in 8

235

fractions of 15–16 mL each.

236

HPLC-DAD-ESI-MS and semi-preparative HPLC. HPLC-MS analysis of the main

237

antibacterial flash chromatography fraction was performed on a Shimadzu LC-MS-2020

238

equipped with an SPD-M20A UV/VIS photodiode array detector and a single quadrupole mass

239

analyzer with an electrospray ionization (ESI) interface (Shimadzu, Kyoto, Japan). Data

240

acquisition and processing were accomplished using Shimadzu LabSolution software (Ver. 5.72).

241

Chromatographic separation was performed in the isocratic mode at 35 °C, on a Gemini C18

242

column (250 mm length, 4.6 mm ID, 5 µm particle size) purchased from Phenomenex (Torrance,

243

CA, USA). Eluent A was 5% aqueous acetonitrile with 0.05% formic acid and eluent B was

244

acetonitrile with 0.05% formic acid. The mobile phase was 50% B and the flow rate was set at

245

1.0 mL/min. The injection volume was 1 µL. ESI conditions were as follows: temperature:

246

applied voltage 4.5 kV, desorption liquid temperature 250 oC, heat block temperature 400 oC,

247

nebulizer gas (nitrogen) flow rate 1.5 L/min, drying gas flow rate 15 L/min. Full mass scan

248

spectra were recorded in the positive ionization mode over the range of m/z 50–800.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

249

Analytical HPLC was scaled up by increasing the diameter of the column (Gemini C18, 250 mm

250

length, 10 mm ID, 10 µm particle size, Phenomenex) and the mobile phase flow rate to 6

251

mL/min. The injection volume was 100 µL, and the appropriate fractions were collected based on

252

the chromatogram at UV 210 nm. It was repeated five-times; the combined fractions were dried

253

by the use of a rotary evaporator (Büchi Rotavapor R-134, Flawil, Switzerland) at 40 oC.

254

HPLC-ESI-Q-TOF. Accurate mass analyses were performed using an Agilent 1200 HPLC

255

system hyphenated with an Agilent 6520 quadrupole time of flight (Q-TOF) mass spectrometer

256

(Agilent Technologies, Santa Clara, CA, USA) equipped with an ESI ion source (dual electro-

257

spray). An isocratic mobile phase of 0.1% aqueous formic acid – acetonitrile (50:50, v/v) was

258

used at a flow rate of 0.5 mL/min. The ESI ion source (dual electrospray) was operated in

259

positive ionization mode (fragmentor voltage: 70 V, capillary voltage: 3500 V). Nitrogen was

260

applied as drying gas at the temperature of 325 oC, at a flow rate of 5 L/min, the nebulizer

261

pressure was 30 psi. Full scan mass spectra were recorded in the range of m/z 100–1000. For

262

collision induced dissociation (CID) high purity nitrogen was used as collision gas, the collision

263

energy varied between 10 and 30 eV. Product ion mass spectra were recorded in positive ion

264

mode in the range of m/z 25–500.

265

NMR. All NMR experiments were carried out on a 600 MHz Varian DDR NMR spectrometer

266

equipped with a 5 mm inverse-detection gradient (IDPFG) probe head. Standard pulse sequences

267

and processing routines available in VnmrJ 3.2 C/Chempack 5.1 were used for structure

268

identifications. The complete resonance assignments were established from direct 1H–13C, long-

269

range 1H–13C, and scalar spin-spin connectivities using 1D 1H, 13C,1H–1H gCOSY, 1H–1H

270

NOESY,

271

respectively. The probe temperature was maintained at 298 K and standard 5 mm NMR tubes

1

H–13C gHSQCAD (J = 140 Hz), 1H–13C gHMBCAD (J = 8 Hz) experiments,


Page 13 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


272

were used. The 1H chemical shifts were referenced to the applied NMR solvent CD3OD (δ

273

(CD2HOD) = 3.31 ppm) and 13C chemical shifts were referenced to 49.00 ppm.

274 275

Results and discussions

276

Bioprofiling by HPTLC-UV/Vis/FLD-effect directed detection. Constituents of S. virgaurea

277

root extract were separated on HPTLC using a mobile phase composed of 14% isopropyl acetate

278

and 3% acetic acid in n-hexane and subsequently tested in situ in the adsorbent bed for

279

antibacterial, free radical scavenging (antioxidant) and acetylcholinesterase inhibitory effects.

280

This non-targeted screening procedure revealed four active zones (Figure 1 a-g) and one of them,

281

at hRF 66 (Sv1), proved to be active in all assays. The compound at hRF 77 (Sv2) exhibited

282

antibacterial effect only. Beyond inhibition of A. fischeri, zones at hRF 35 (Sv3) and hRF 43 (Sv4)

283

showed also free radical scavenging (antioxidant) and acetylcholinesterase inhibitory activity,

284

respectively. Of all the bioassays, the A. fischeri bioassay turned out to be the most suitable one

285

for a quick first bioprofiling. Prior to the bioassay, the neutralization of acidic or alkaline residual

286

mobile phase traces in the layer was a crucial step to provide an appropriate ambience for

287

enzymatic or microbial activity. The elimination of the acetic acid with potassium hydroxide

288

worked perfectly for B. subtilis and A. fischeri strains as well as for acetylcholinesterase.

289

However, certain plant pathogens were found to accommodate this neutralization process less (X.

290

euvesicatoria) or not at all (P. maculicola). Therefore, an acid-free eluent (15% acetone in n-

291

hexane) was developed and used for further DB assays. This mobile phase mixture was more

292

apolar compared to the one used in Figure 1, and thus, the upper chromatogram region containing

293

the antimicrobial compounds of Figure 1 was spread in Figure 2. The corresponding zones

294

obtained from the acidic and the acid-free developments were determined by 2D-HPTLC (Figure



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

295

S-1), so the antimicrobial compounds at hRF 24, 49 and 69 were assigned as Sv3, Sv1 and Sv2,

296

respectively (Figure 2). Sv1 and Sv2 inhibited all tested bacteria, while Sv3 was only active

297

against Gram negative bacteria (A. fischeri, X. euvesicatoria and P. maculicola). After the

298

bioactivity profiling, the discovered effective zones were further characterized by various

299

absorbance measurements (Figure 1 h-l). All antibacterial zones could be localized in the UV 254

300

nm image or densitograms at 240 nm, or at 500 nm after derivatization with the vanillin sulphuric

301

acid reagent (Figure 1 a, h and i). The UV spectra of the four target compounds were also

302

recorded (Figure 1 l).

303

Characterization of unknown antimicrobials by HPTLC-MS. Full scan mass spectra were

304

recorded to assign the mass signals of the compounds corresponding to the four bioactive zones.

305

HPTLC-ESI-MS, performed via the elution-based TLC-MS Interface in the positive ionization

306

mode, provided characteristic mass spectra (Figure 3). ESI is a soft ionization mode that typically

307

results in molecular, adduct and/or dimer ions. For the zone Sv4, one intense mass signal was

308

obtained and assigned to be the protonated molecule at m/z 281 [M+H]+, while its deprotonated

309

molecular ion at m/z 279 [M-H]- was also detected in the negative ionization mode (Figure S-2).

310

The mass spectrum of the zone Sv3 showed several mass signals, probably caused by coeluting

311

compounds. However, a clear base peak was evident at m/z 295 [M+H]+. For Sv1 and Sv2, very

312

similar mass spectra were recorded with the following assignments (m/z): 175 [M+H]+, 197

313

[M+Na]+, 215 [M+H2O+Na]+, 229 [M+CH3OH+Na]+, 371 [2M+Na]+. The similar mass spectra

314

that were rich in adduct ions supported the idea that Sv1 and Sv2 are isomers and probably

315

contain double or triple bonds or an aromatic part. No fragmentation was observed with the ESI

316

settings chosen.

317

HPTLC-DART-MS was also employed for the characterization of the bioactive zones. This

318

desorption-based ionization technique under ambient conditions was applicable for scanning a


Page 15 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


319

whole track by automated moving of the track underneath the excited gas beam. Only two

320

bioactive chromatographic spots were clearly detectable due to the lower sensitivity and

321

discriminative feature of DART. The two major inhibition zones Sv1 and Sv2 of the A. fischeri

322

bioassay were observable in the TIC and EIC (m/z 175) and their mass spectra showed the

323

protonated molecule at m/z 175 [M+H]+ (Figure 4). DART is a soft ionization technique and due

324

to the absence of solvents and high desorption temperatures, sodium or solvent adducts or dimers

325

were not observed if compared to the respective ESI mass spectra (Figure 4).

326

Confirmation by SPME-GC-EI-MS. SPME-GC-EI-MS investigation of the crude ethanol

327

extract showed four volatile compounds in the TIC chromatogram at 16.7, 16.8, 16.9 and 17.3

328

min with identical mass spectra (Figure S-3). Therefore, the presence of four geometric isomers

329

was suggested in the ratio 62:32.6:1.7:3.7, respectively, and thus, at least two double bonds were

330

assumed to be present in the molecule. The molecule ion of the four compounds m/z 174

331

corresponded to the bioactive zones Sv1 and Sv2 in the HPTLC bioautogram. The NIST library

332

identified all of them as 4-methoxy-1-naphthol at a given low hit probability (73%). Using

333

specific or selective derivatization reagents in HPTLC, a functional group in a supposed structure

334

can easily be confirmed. However, Sv1 and Sv2 were not detectable by HPTLC using

335

derivatization reagents for the detection of 1-naphthol (sodium hydroxide reagent44) and aromatic

336

hydrocarbons (formaldehyde sulphuric acid reagent44). For this reason, the GC-EI-MS

337

identification was denied, as the structure could not be confirmed by the derivatization reagents.

338

The impression that Sv1 and Sv2 were geometric isomers was also confirmed by 2D-HPTLC

339

using n-hexane – acetone 85:15 (v/v) in both dimensions (Figure S-4). After the second

340

development the transformation of Sv1 to Sv2 was observed, and vice versa.

341

Fractionation, scale up and isolation. Normal phase fractionation and orthogonal reversed-

342

phase purification were involved in the bioassay-guided isolation of the bioactive isomers Sv1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

343

and Sv2. The crude extract was fractionated by flash chromatography. The eight collected

344

fractions were subjected to DB (B. subtilis) to determine that the 4th and 5th fractions contained

345

both Sv1 and Sv2, whereby Sv1 was present at a higher amount (Figure S-5). The two fractions

346

were combined and concentrated. For the separation and detection of Sv1 and Sv2, an analytical

347

HPLC-DAD-ESI-MS method was developed (Figure 5). Although the UV spectra of Sv1 and

348

Sv2 showed high similarity, their ESI-MS spectra – also here, the presence of the molecular ion

349

at m/z 175 [M+H]+ was evident as for HPTLC-ESI-MS shown before – differed by the formation

350

of a characteristic acetonitrile adduct at m/z 216 [M+C2H3N+H]+ solely in the case of Sv2. The

351

analytical separation was scaled up to a semi-preparative one by changing the column diameter

352

(4.6 to 10 mm), the particle size (5 to 10 µm) of the column packing, the flow rate (1 to 6

353

mL/min) and the injected volume (1 to 100 µL). The resolution achieved was suited for

354

compound isolation, and thus, fractionation (Figure 5b). The semi-preparative isolation was

355

performed five times. The yields of the combined fractions were 11.1 mg Sv1 and 1.8 mg Sv2,

356

both being yellow oils. Sv1 and Sv2 could transform into each other and this alteration was

357

inevitable during the isolation process, especially caused by the 210 nm UV light used for

358

detection. Thus, it was not unexpected that both isolated compounds Sv1 and Sv2 contained also

359

the other form as a side component (about 23%). For this reason, only Sv1 was investigated by

360

2D-NMR, which allowed the identification of both isomers.

361

Antibacterial activity of Sv1. Liquid-phase antimicrobial assays were employed for the

362

confirmation and comparison of the antibacterial activity of the crude extract and Sv1. The

363

respective MIC, the lowest concentration that prevents bacterial growth, was determined for B.

364

subtilis and X. euvesicatoria. Both ethanol extract and Sv1 inhibited the studied bacterial strains.

365

For B. subtilis the MIC concentrations of the extract and Sv1 were 172 and 86 µg/mL,

366

respectively. For X. euvesicatoria they were found to be 86 and 34 µg/mL, respectively. The


Page 17 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


367

activity of Sv1 was determined as bacteriostatic because live B. subtilis and X. euvesicatoria cells

368

were found in the cell suspensions treated with the highest concentration of Sv1 (1.8 mg/mL),

369

which could multiply on an agar plate. Luminometric measurements also proved the antibacterial

370

activity of both extract and Sv1 against A. fischeri and P. maculicola (Figure S-6). However, their

371

antibacterial effect was less pronounced and shorter in duration for P. maculicola as compared to

372

A. fischeri.

373

Identification by NMR. NMR assignments of Sv1 and Sv2 allowed their identification as 2Z,8Z-

374

matricaria ester (methyl (2Z,8Z)-deca-2,8-diene-4,6-diynoate) and 2E,8Z-matricaria ester,

375

respectively (Figure S-7, Table S-1). The core structure contains two double bonds that explains

376

the presence of four compounds with the same GC-EI-MS spectra. Most probably the two minor

377

peaks belonged to the (E,E) and (Z,E) isomers.

378

The structural identification was further confirmed by HRMS analysis. The accurate mass of the

379

native matricaria ester corresponded to a molecular formula of C11H10O2 (experimental mass: m/z

380

174.0679, calculated mass: m/z 174.0681, score: 95.67%, mass deviation 1.13 ppm). The accurate

381

mass found for the [M+H]+ ion of matricaria ester was m/z 175.0754. The MS2 spectrum with the

382

proposed fragmentation pattern is shown in Figure S-8.

383

The polyacetylene (Z,Z)-matricaria ester had been reported to be present in S. viragurea root and

384

shoot29 and in essential oils obtained from other Asteraceae species.45-48 In some oils its

385

geometric isomers were also detected.45,46 In a previous study using agar disc diffusion and broth

386

dilution methods and various microorganisms (other than those used in the present study), the

387

essential oil of Conyza canadensis including 88.2% (Z,Z)-matricaria ester did not show any

388

antibacterial effect against Gram-positive Enterococcus faecalis, Staphylococcus aureus,

389

Streptococcus pyogenes and Gram-negative Escherichia coli and Pseudomonas aeruginosa

390

bacteria, but was found to be antifungal.45 However, (Z,Z)- and (E,Z)-matricaria esters showed



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

391

inhibitory activity against Mycobacterium tuberculosis and M. avium, using a radiorespirometric

392

bioassay.49 (Z,Z)-Matricaria ester can also inhibit melanin generation; therefore, it can be

393

effectively used as cosmetic ingredient for skin whitening.50

394 395

Conclusions

396

A bioassay-guided approach for screening natural products was successfully utilized for the

397

determination of bioactive components of European goldenrod root extract. This rapid, effective

398

and purposive methodology included a non-targeted bioprofiling by HPTLC-UV/Vis/FLD-EDA

399

using various (bio)assays for the detection of separated compounds with the desired effect. The

400

discovered bioactive compounds were characterized and isolated by subsequent highly-targeted

401

methods towards substance identification. The combination of all these different LC, meaning

402

HPLC and HPTLC, GC, EDA, MS and NMR methods clearly demonstrated the benefit for

403

screening, characterization, isolation and identification of antibacterial and free radical

404

scavenging (antioxidant) compounds as well as acetylcholinesterase inhibitors. Via this analytical

405

toolbox, it was possible to trace and identify different isomers of matricaria esters in the Solidago

406

virgaurea root extract.

407 408

ASSOCIATED CONTENT

409

Supporting Information

410

The Supporting Information is available free of charge on the ACS Publications website at DOI:

411

This material is available free of charge via the Internet

412

Figures S-1 to S-6 show the 2D-HPTLC separation for compound assignment, mass spectrum of

413

the Sv4 zone in the negative ionization mode, SPME-GC-EI-MS TIC chromatogram of European


at http://pubs.acs.org.

Page 19 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


414

goldenrod root extract and mass spectrum of Sv1, 2D-HPTLC separation for isomer proof

415

(artefact), HPTLC separation of the flash chromatography fractions and luminometric detection

416

of the inhibiting activity in the liquid phase. Table S-1 demonstrates the NMR spectral data and

417

structure of Sv1 and Sv2.

418 419

AUTHOR INFORMATION

420

Corresponding Author

421

*E-mail: [email protected], Tel.: 0036-14877515; Fax: 0036-14877555.

422

Notes

423

The authors declare no competing financial interest.

424

Acknowledgements

425

Á.M. Móricz thanks for the DAAD scholarship (A/13/03577) that allowed her to stay at JLU

426

Giessen and for the Bolyai grant (BO/00543/12). The authors thank Stephanie Krüger, Maryam

427

Jamshidi-Aidji and Salim Hage, all JLU Giessen, for their support using A. fischeri, B. subtilis

428

spizizenii and acetylcholinesterase enzyme assays, respectively,. The authors would like to thank

429

the laboratory of the National Institute of Pharmacy and Nutrition (OGYEI) to access the Q-TOF

430

MS instrument. This work was partially supported also by OTKA PD83487, K101271 and

431

PD109373 grants.

432



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

433

References

434

(1) Di, L.; Kerns, E.H. In: Solvent Systems and Their Selection in Pharmaceutics and

435

Biopharmaceutics; Augustijns, P.; Brewster, M., Eds.; Springer: New York, 2007; pp 111-136.

436

(2) Cos, P.; Vlietinck, A. J.; Berghe, D. V.; Maes, L. J. Ethnopharmacol. 2006, 106, 290-302.

437

(3) Choma, I. M.; Grzelak, E. M. J. Chromatogr. A 2011, 1218, 2684-2691.

438

(4) Móricz, Á. M.; Ott, P. G. J. AOAC. Int. 2015, 98, 850-856.

439

(5) Móricz, Á. M.; Ott, G. P. In Forced Flow Layer Chromatography; Tyihák, E., Ed.; Elsevier:

440

Amsterdam, 2016; pp 347-395.

441

(6) Morlock, G. ACS Symp. Ser. 2013, 1185, 101-121.

442

(7) Morlock, G.; Schwack, W. J. Chromatogr. A 2010, 1217, 6600–6609.

443

(8) Morlock, G.; Schwack, W. TrAC, Trends Anal. Chem. 2010, 29, 1157−1171.

444

(9) Móricz, Á. M.; Häbe, T. T.; Böszörményi, A.; Ott, P. G.; Morlock, G. E. J. Chromatogr. A

445

2015, 1422, 310-317.

446

(10) Häbe, T. T.; Morlock, G. E. Rapid Commun. Mass Spectrom. 2016, 30, 321-332.

447

(11) Taha, M. N.; Krawinkel, M. B.; Morlock, G. E. J. Chromatogr. A 2015, 1394, 137-147.

448

(12) Krüzselyi, D.; Nagy, R.; Ott, P. G.; Móricz, Á. M. J. Chromatogr. Sci. 2016, 54, 1084-1089.

449

(13) Pepeljnjak, S.; Kosalec, I. FEMS. Microbiol. Lett. 2004, 240, 111-116.

450

(14) Klöppel, A.; Grasse, W.; Brümmer, F.; Morlock, G.E. J. Planar Chromatogr. 2008, 21, 431–

451

436.

452

(15) Chen, Y.; Morlock, G. E. Chromatographia 2015, 79, 89-96.

453

(16) Móricz, Á. M.; Szarka, S.; Ott, P. G.; Héthelyi, É. B.; Szőke, É.; Tyihák, E. Med. Chem.

454

2012, 8, 85-94.


Page 21 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


455

(17) Chintaluri, A.K.; Komarraju, A.L.; Chintaluri, V.K.; Vemulapalli, B. J. Essent. Oil Res.

456

2015, 27, 9-16.

457

(18) Móricz, Á. M.; Horváth, G.; Ott, P. G. J. Planar Chromatogr. 2015, 28, 173–177.

458

(19) Móricz, Á. M.; Ott, P. G.; Alberti, Á.; Böszörményi, A.; Lemberkovics, É.; Szőke, É.; Kéry,

459

Á.; Mincsovics, E. J. AOAC. Int. 2013, 96, 1214-1221.

460

(20) Jesionek, W.; Móricz, Á. M.; Alberti, Á.; Ott, P. G.; Kocsis, B.; Horváth, G.; Choma, I. M. J.

461

AOAC. Int. 2015, 98, 1013-1020.

462

(21) Jesionek, W.; Fornal, E.; Majer-Dziedzic, B.; Móricz, Á. M.; Nowicky, W.; Choma, I. M. J.

463

Chromatogr. A 2016, 1429, 340-347.

464

(22) Móricz, Á. M.; Fornal, E.; Jesionek, W.; Majer-Dziedzic, B.; Choma, I. M.

465

Chromatographia 2015, 78, 707-716.

466

(23) Chevallier, A. The Encyclopedia of Medicinal Plants. Dorling Kindersley: London, 1996.

467

(24) Tyszkiewicz, E. W. Assessment report on Solidago Virgaurea L., herba. European

468

Medicines

469

http://www.emea.europa.eu

470

(25) Goswami, A.; Barua, R.N.; Sharma, R.P.; Baruah, J.N.; Kaulanthaivel, P. Phytochemistry

471

1984, 23, 837-841.

472

(26) Starks, C. M.; Williams, R. B.; Goering, M. G.; O'Neil-Johnson, M.; Norman, V. L.; Hu, J.

473

F.; Garo, E.; Hough, G. W.; Rice, S. M.; Eldridge, G. R. Phytochemistry 2010, 71, 104-109.

474

(27) Bader, G.; Wray, V.; Just, U.; Hiller, K. Phytochemistry 1998, 49, 153-156.

475

(28) Jaiswal, R.; Kiprotich, J.; Kuhnert, N. Phytochemistry 2011, 72, 781-790.

476

(29) Lam, J. Phytochemistry 1971, 10, 647–653.

477

(30) Derda, M.; Hadas, E.; Thiem, B. Parasitol. Res. 2009, 104, 705-708.

478

(31) Demir, H.; Açık, L.; Bali, E. B.; Koç, L. Y.; Kaynak, G. Afr. J. Biotechnol. 2009, 8, 274-279.

Agency. Evaluation

of

medicines

for

human use. EMEA/HMPC,


2008,


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

479

(32) Thiem, B.; Goslinska, O. Fitoterapia 2002, 73, 514-516.

480

(33) Bader, G.; Wray, V.; Hiller, K. Planta. Med. 1995, 61, 158-161.

481

(34) Gross, S. C.; Goodarzi, G.; Watabe, M.; Bandyopadhyay, S.; Pai, S. K.; Watabe, K. Nutr.

482

Cancer 2002, 43, 76-81.

483

(35) Wang, P.Z.; Doi, R.H. J. Biol. Chem. 1984, 259, 8619–8625.

484

(36) Fan, J.; Crooks, C.; Lamb, C. Plant J. 2008, 53, 393–399.

485

(37) Morlock, G. E. J. Liq. Chromatogr. Relat. Technol. 2014, 37, 2892-2914.

486

(38) Häbe, T. T.; Morlock, G. E. Rapid Commun. Mass Spectrom. 2015, 29, 474–484.

487

(39) Akkad, R.; Schwack, W. J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 2010, 878,

488

1337-1345.

489

(40) Hage, S.; Morlock, G. E. In: Poster at International Symposium for HPTLC, Lyon, 2014.

490

(41) Marston, A.; Kissling, J.; Hostettmann, K. Phytochem. Anal. 2002, 13, 51–54.

491

(42) Jamshidi-Aidji, M.; Morlock, G. E. J. Chromatogr. A 2015, 1420, 110-118.

492

(43) Krüger, S.; Urmann, O.; Morlock, G. E. J. Chromatogr. A 2013, 1289, 105-118.

493

(44) Jork, H.; Funk, W.; Fischer, W.; Wimmer, H. Thin-Layer Chromatography - Reagents and

494

Detection Methods, VCH: Weinheim, Germany, 1990.

495

(45) Veres, K.; Csupor-Löffler, B.; Lazar, A.; Hohmann, J. ScientificWorldJournal 2012, 2012,

496

489646.

497

(46) Nazaruk, J.; Kalemba, D. Molecules 2009, 14, 2458-2465.

498

(47) Mabrouk, S.; Salah, K. B.; Elaissi, A.; Jlaiel, L.; Jannet, H. B.; Aouni, M.; Harzallah-Skhiri,

499

F. Chem. Biodivers. 2013, 10, 209-223.

500

(48) Bar, B.; Schultze, W. Planta. Med. 1996, 62, 332-335.

501

(49) Lu, T.; Cantrell, C. L.; Robbs, S. L.; Franzblau, S. G.; Fischer, N. H. Planta. Med. 1998, 64,

502

665-667.


Page 23 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


503

(50) Luo, L. H.; Kim, H. J.; Nguyen, D. H.; Lee, H. B.; Lee, N. H.; Kim, E. K. Biol. Pharm. Bull.

504

2009, 32, 1091-1094.

505



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

506

List of figures

507

Figure 1. HPTLC chromatograms (a-g) of bioactive components in European goldenrod root

508

extract after chromatography with n-hexane – isopropyl acetate – acetic acid, documented (a) at

509

UV 254 nm, (b) 365 nm and (c) under white light illumination after derivatization with vanillin

510

sulphuric acid reagent as well as bioautograms using (d) B. subtilis spizizenii and (e) A. fischeri

511

bioassays, and effect-directed detections with the (f) DPPH* assay and (g) acetylcholinesterase

512

assay, densitograms (h-k) recorded (h) at 240 nm, (i) at 500 nm after derivatization with vanillin

513

sulphuric acid reagent, (j) at 546 nm after B. subtilis spizizenii bioassay, and (k) biodensitogram

514

of the dark inhibition zones of (e), and (l) respective UV spectra (200-480 nm).

515 516

Figure 2. HPTLC chromatograms of antibacterial components in European goldenrod root

517

extract after chromatography with n-hexane – acetone (less polar compared to Figure 1),

518

documented (a) at UV 254 nm, (b) 365 nm and (c) under white light illumination after

519

derivatization with vanillin sulphuric acid reagent as well as bioautograms using (d) B. subtilis

520

F1276, (e) X. euvesicatoria, (f) L. plantarum and (g) P. maculicola bioassays.

521 522

Figure 3. HPTLC-ESI+-MS full scan spectra (m/z 100-500) of bioactive zones of European

523

goldenrod root extract, obtained via the elution head-based TLC-MS Interface and background

524

subtraction.

525 526

Figure 4. Scanning HPTLC-DART-MS of (a) the major bioactive zones according to the A.

527

fischeri bioassay (b) detected via TIC and EIC (m/z 175) in the positive ionization mode and

528

documented at 366 nm as well as the mass spectra of (c) Sv1 and (d) Sv2.


Page 25 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


529 530

Figure 5. UV chromatograms at 210 nm obtained by (a) analytical HPLC-DAD-MS and (b)

531

semi-preparative HPLC-DAD of the flash chromatographic bioactive fraction of European

532

goldenrod root as well as (c and d) UV spectra and (e and f) ESI-MS spectra of Sv1 and Sv2,

533

respectively.

534



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

535

Figure 1

536 537


Page 26 of 37

Page 27 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

538


Figure 2

539 540



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

541

Figure 3

542 543


Page 28 of 37

Page 29 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

544


Figure 4

545 546



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

547

Figure 5

548 549


Page 30 of 37

Page 31 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

550


For TOC only

551



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. HPTLC chromatograms (a-g) of bioactive components in European goldenrod root extract after chromatography with n-hexane – isopropyl acetate – acetic acid, documented (a) at UV 254 nm, (b) 365 nm and (c) under white light illumination after derivatization with vanillin sulphuric acid reagent as well as bioautograms using (d) B. subtilis spizizenii and (e) A. fischeri bioassays, and effect-directed detections with the (f) DPPH* assay and (g) acetylcholinesterase assay, densitograms (h-k) recorded (h) at 240 nm, (i) at 500 nm after derivatization with vanillin sulphuric acid reagent, (j) at 546 nm after B. subtilis spizizenii bioassay, and (k) biodensitogram of the dark inhibition zones of (e), and (l) respective UV spectra (200-480 nm). Figure 1 180x54mm (300 x 300 DPI)


Page 32 of 37

Page 33 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. HPTLC chromatograms of antibacterial components in European goldenrod root extract after chromatography with n-hexane – acetone (less polar compared to Figure 1), documented (a) at UV 254 nm, (b) 365 nm and (c) under white light illumination after derivatization with vanillin sulphuric acid reagent as well as bioautograms using (d) B. subtilis F1276, (e) X. euvesicatoria, (f) L. plantarum and (g) P. maculicola bioassays Figure 2 90x58mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. HPTLC-ESI+-MS full scan spectra (m/z 100-500) of bioactive zones of European goldenrod root extract, obtained via the elution head-based TLC-MS Interface and background subtraction. Figure 3 170x111mm (300 x 300 DPI)


Page 34 of 37

Page 35 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Scanning HPTLC-DART-MS of (a) the major bioactive zones according to the A. fischeri bioassay (b) detected via TIC and EIC (m/z 175) in the positive ionization mode and documented at 366 nm as well as the mass spectra of (c) Sv1 and (d) Sv2. Figure 4 90x98mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. UV chromatograms at 210 nm obtained by (a) analytical HPLC-DAD-MS and (b) semi-preparative HPLC-DAD of the flash chromatographic bioactive fraction of European goldenrod root as well as (c and d) UV spectra and (e and f) ESI-MS spectra of Sv1 and Sv2, respectively. Figure 5 90x135mm (300 x 300 DPI)


Page 36 of 37

Page 37 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


graphical abstract for TOC 175x81mm (150 x 150 DPI)


Effect-Directed Discovery of Bioactive Compounds Followed by Highly

Recommend Documents