Identification of closely related phenanthrene derivatives. The position of the substituents was solved by 2D NOESY experiment. 7 µg. 24 µg. 10 µg. 38 µg. 16 µg.
State-of-the-art hyphenated techniques LC-HRMS/MS and HPLC-DAD-MS-SPE-UV/NMR for new approaches in phytochemical exploration of orchids Veronika Cakova, Nicole Rimlinger, Cyril Antheaume, Patrice André, Frédéric Bonté and Annelise Lobstein Pharmacognosy and bioactive natural substances Faculty of pharmacy, University of Strasbourg UMR7200-Laboratoire d’innovation thérapeutique
UMR 7200
Introduction Orchidaceae family: 25.000-30.000 species The most heterogeneous family Impressive longevity and resistance to extreme life conditions Few scientific studies about: Phytochemical composition Biological activities
Objectives Discover, understand and explore orchids
Exploration of one Asian species with complex chemical composition Contribute to a general knowledge about chemical composition of orchids
UMR 7200
Aim of the present study Phytochemical study of the stem of an Aerides rosea Lodd. ex Lindl. & Paxton never described before, using the state-of-the-art hyphenated techniques
Objective: Quickly describe the chemical composition of that species Accelerate the identification of the unknown compounds
Restrictions: Aerides rosea is a rare and expensive plant material No bibliographic data Not enough quantity of the dried plant to isolate some mg of minor compounds (e. g. for NMR identification)
Strategy Using hyphenated LC-ESI-HRMS/MS and HPLC-DAD-MS-UV-SPE/NMR to obtain “on-line” a rapid information about the chemical composition of the selected species
UMR 7200
Previous investigations No bibliographic data for the species, researches extended to genus and neighboring species (same tribe or sub-tribe) Taxonomy: Aerides sp. belongs to Vandeae tribe A phenanthropyran isolated from Aerides crispum
Aeridin
Previous studies in our laboratory Phytochemical and biological studies of Vanda coerulea Griff ex. Lindl. Identification of stem-specific metabolites Five stilbenoids: flavidin, imbricatin, coelonin, methoxycoelonin and gigantol
UMR 7200
Simmler C., Antheaume C., Lobstein A.: Plos One. 2010 Oct 28;5(10):e13713
1st step: Dereplication by LC-ESI-HRMS/MS Aerides rosea crude stem extract (EtOH/water 90/10 v/v, 30 min reflux) is compared to Vanda coerulea stem crude extract by liquid chromatography DAD detection at 220 nm
Stationnary phase: Zorbax C18 50 mm x 2,1mm i.d. 1,8µ ;HPLC Agilent technologies, mass spectrometer Agilent TOF/Q-TOF Flow: 0,5 mL/min ; Injection volume: 1µL ; Mobile phase: A - water + 0,5% A.F.; B - ACN+ 0,5% A.F. Mobile phase conditions -time (min) (%A;%B): 0 (80;20), 7 (70;30), 8,5-14,5 (5;95), 15 (80;20).
Vanda coerulea crude stem extract
Flavidin
Aerides rosea crude stem extract
Unknown
UMR 7200
Imbricatin
Coelonin
Methoxycoelonin
Gigantol
1st step: Dereplication by LC-HRMS/MS MS and MS/MS spectra of compounds in a targeted elution zone are compared to spectra of the five references (coelonin, flavidin,imbricatin, methoxycoelonin and gigantol) Imbricatin
Aerides rosea crude stem extract
Methoxycoelonin
DAD detection at 220 nm
Gigantol
Unknown compounds
Coelonin
Targeted elution zone of gigantol Targeted elution zone of imbricatin Targeted elution zone of methoxycoelonin
Compound name
Targeted mass [M+H]+
z
Δ RT (min)
Collision energy (eV)
Targeted MS/MS fragments
Flavidin
241,0800
1
3,75 ± 1,5
15,000
195,0780; 224,0800; 239,0600
Coelonin
243,1000
1
4,13 ± 1,5
12,000
183,0800; 211,0750; 228,0780
Imbricatin
271,1000
1
4,196 ± 1,5
12,000
239,0685
Methoxycoelonin
273,1000
1
4,603 ± 1,5
11,000
213,0911; 241,0862
Gigantol
275,1000
1
6,308 ± 1,5
9,000
91,0547; 137,0600; 151,0762
Stationnary phase: Zorbax C18 50 mm x 2,1mm i.d. 1,8µ ;HPLC Agilent technologies, mass spectrometer Agilent TOF/Q-TOF, ionization source ESI Flow: 0,5 mL/min ; Injection volume: 1µL ; Mobile phase: A - water + 0,5% A.F.; B - ACN+ 0,5% A.F. UMR 7200 Mobile phase conditions -time (min) (%A;%B): 0 (80;20), 7 (70;30), 8,5-14,5 (5;95), 15 (80;20).
1st step: Dereplication by LC-ESI-HRMS/MS First “on-line” informations : If RT, MS and MS/MS match known compounds identified Extract
Presence of flavidin
Presence of coelonin
Presence of imbricatin
Presence of methoxycoelonin
Presence of gigantol
Aerides rosea
NO
Yes
Yes
Yes
Yes
Aerides rosea crude stem extract
Further phytochemical explorations to identify the minor unknown compounds Enriched extract (AcOEt) different techniques
fractionated
by
2nd step: identification “on-line” using hyphenated HPLC-DAD-MS-SPE-UV/NMR UMR 7200
Unknown compounds
2nd step: HPLC-DAD/MS/SPE/NMR Principle:
MS-ESI MS
100 80 60 40 1 500 1 400
20
UV
mAU
1 300 1 200
0
5%
1 100 1 000 900
100
200
300 m/z
400
500
800 700 600 500 400 300
split
200 100
0 1
2
3
4
5
6
7
8
9
NMR
10 11 12 13 14 15 16 17 18 19 20
LC 95% UV
1.0
0.5
0
200
300
nm
400
96 well-plates SPE UV check UMR 7200
500
with 5mL Capprobe
2nd step: HPLC-DAD/MS/SPE/NMR Inject as much as possible without losing MS-ESI resolution to limit multi-injection 10µL up to 100µL
MS
100 80 60 40
1 500 1 400
20
UV
mAU
1 300
0
1 200
100
200
300 m/z
400
500
1 100 1 000 900
5%
800 700 600 500 400 300
split
200 100
0 1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20
Only 5% of the chromatography goes to the MS detector (ion trap) to limit its saturation
LC
Both detection (UV or MS) can be used for “peak trapping” to SPE UV, no MS
UMR 7200
2nd step: HPLC-DAD/MS/SPE/NMR
split
95%
95% flows trapped on SPE cartridges Different SPE phases were testedchose the best cartridges to optimize the compounds retention and NMR signal to noise ratio with 5µL CapNMR probe
96 well-plates SPE “Make up pump” UV
1.0
0.5
0
UMR 7200
200
300
nm
400
500
2nd step: HPLC-DAD/MS/SPE/NMR
14,56
Enriched Aerides rosea extract (AcOEt) fractionated by solid phase extraction (SPE) and exclusion chromatography (Sephadex LH-20) 2 fractions of the unknown compounds were studied Intens. mAU
Imbricatin
2
100 mg 1 000
1 500
3
9,76 9,43 10,28
1 500
Sephadex LH-20 Fraction 36-40 at 260 nm
RT [min]
2 000
2
4
6
8
10
12
14
16
30 mg
4
15,76
mAU
1 500
18
20
22
24
26
28
30
32
34
36
38
40
16,68
0 0
5 Sephadex LH-20 Fraction 44-49 at 260 nm
3 10,27
1 000
500
0 0
RT [min] 2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
Stationnary phase: Sunfire C18 150 mm x 4,6mm i.d. 3,5µ ;HPLC Varian LC920 Flow: 1 mL/min ; Injection volume: 5 µL ; Mobile phase: A - water + 0,5% A.F.; B - ACN+ 0,5% A.F. UMR 7200 Mobile phase conditions -time (min) (%A;%B): 0 (80;20), 3 (70;30), 11 (65;35), 25 (50;50), 37-40 (0;100).
34
36
38
5 unknown compounds to identify Few quantity to isolate these compounds by traditional ways (semipreparative HPLC, TLC etc.)
2nd step: HPLC-DAD/MS/SPE/NMR HPLC gradient optimized conditions MS and UV spectra obtained from 5% of the sample injected Trapping on HLB 10x1mm SPE cartridges (UV or MS detection used)
1 2 0
5
10
3 15
20
MS positive mode UV spectrum of the compound 3 MS negative mode
Stationnary phase: Sunfire C18 150 mm x 4,6mm i.d. 3,5µ ;HPLC Agilent technologies; MS ion trap Flow: 0,8 mL/min ; Injection volume: 1µL ; Mobile phase: A - water + 0,5% A.F.; B - ACN+ 0,5% A.F. UMR 7200 Mobile phase conditions -time (min) (%A;%B): 0 (70;30), 15 (50;50), 16 (0;100).
RT min
2nd
-O-CH3
step: HPLC-DAD/MS/SPE/NMR
Compounds trapped on the cartridges are then eluted with deuterated solvents (Methanol-d4) to obtain 1D (1H) and 2D (NOESY, HMBC and HSQC) spectra 7 µg SPE push volume 120 μL to NMR probe (vs. 500 μL) Identification of closely related phenanthrene derivatives 24 µg
10 µg
38 µg
16 µg 5 µL capNMR probe UMR 7200
The position of the substituents was solved by 2D NOESY experiment
2nd step: HPLC-DAD/MS/SPE/NMR
Compound 1 5-methoxy-9,10dihydro-2,3,7phenanthrenetriol
Compound 2 3-methoxy-9,10dihydro-2,5,7phenanthrenetriol
UMR 7200
Compound 3 5-methoxyphenanthrene2,3,7-triol
Compound 4 3-methoxy-2,7-dihydroxy5H-phenanthro[4,5bcd]pyran
Compound 5 3,5dimethoxyphenanthrene2,7-diol
Conclusion HPLC-DAD-MS-SPE-UV/NMR is a fast method which can quickly identify the unknown compounds in enriched fractions
We obtained LC chromatogram, MS, UV and 1D and 2D NMR spectra ON-LINE without the necessity to isolate the compounds Spectra obtained from some µg of trapped compounds thanks to the capNMR probe Saving of the rare and expensive plant material thanks to the limited quantity needed
UMR 7200
Conclusion Thanks to this state-of-the-art hyphenated technique, we could identify 5 phenanthrene derivatives in the Aerides rosea Using the LC-ESI-HRMS/MS dereplication method we could identify 4 compounds common to the neighboring species Vanda
coerulea
TOTAL: 9 compounds identified on-line in a newly explored orchid
UMR 7200
Sincere thanks to…
UMR7200 Laboratory of pharmacognosy Annelise Lobstein Aurélie Urbain
Patrice André Frédéric Bonté Beatriz Soengas
UMR7199 Service commun d’analyse Cyril Antheaume Nicole Rimlinger Patrick Wehrung
And thank you for your attention UMR 7200
