Overview Phytochemistry and Bioactive Natural Products … · Chemical Diversity of Caralluma...

Conf Hygia, Paris 7 novembre 2012

Phytochemistry and Bioactive Natural Products

NOUVELLES MÉTHODOLOGIES ANALYTIQUES POUR LA

DÉCOUVERTE DE PRODUITS BIOACTIFS NATURELS

J.L. WolfenderSchool of Pharmaceutical Sciences,

University of Geneva, Geneva, Switzerland

Overview• Boactive natural products

– Analytical and biological challenges– Bioactivity guided fractionation

• Analytical Platform– UHPLC-TOF-MS– CapNMR

• Applications– Miniaturized drug discovery– Stress induction of NPs– Metabolomics

• Conclusions and perspectives

Procedure for obtaining the active principles from plants

pure constituent(s)

extraction

extract(s)

separation

toxicology

structure modification

bio-assay

bio-assaybio-assay

synthesis

structure elucidation

fractionsmedicinal plants

Bioactivity guided fractionation

Spectroscopicaldata on-line:- LC/UV- LC/MS- LC/NMR

µg

Spectroscopical data off-line:- UV- MS- NMR- IR ...

mgType of phytochemical analysis: targeted vs profiling

Sensitivity

Dynamic range

Res

olut

ion

LC-NMR 10-6 mol

LC-UV 10-9 mol

GC-MS 10-12 mol

LC-MS 10-15 mol

LC-LIF 10-19 mol

CE-LIF 10-22 molSumner et al. Phytochemistry 2003, 62, 817-

836.

Conférence hygia JL Wolfender UNIGE

Profiling an extract

NMR

LC

MS

10’s cpds

1000’s cpds

100’s cpds

NMR is like your mother - it always tells

you the truth.

Ian Wilson SGMS 2011

MS is like your lover: It always

tells you what you want to hear - and

it always lies to you.

Ian Wilson SGMS 2011

Advantages of hyphenation for profiling extracts

iMAO activity profile of S milthiorizza

Potterat, O.; Hamburger, M. Curr. Org. Chem. 2006, 10, 899-920.

bioactivityadditional structuralinformation

UV

NMR

150 200 250 300 350 400 450 500 550 6000

20

40

60

80

100 485.3

427.2293.2 465.2497.5361.2551.4

MS

METHOD TRANSFER

C15H10O5

STRUCTURALINFORMATION

A

B

D

E

C

F

FRACTIONCOLLECTION,

DRYING

BIOASSAYON

ALIQUOTS

StrategyID of bioactive NP’s

LC-MS profiling

LC-microisolation

LC biological profiling

localisation of bioactive cpds

absolute quantification

De novo bioactive metabolite ID

dereplication

UPLC-TOF-MS and CapNMR technological platform

m/z < 5 ppmparticles

1.7 μm

HIGH RESOLUTION LCHIGH RESOLUTION MS

UPLC TOF-MS

sample limited applications

HIGHLY SENSITIVE NMR

Capillary NMR


Ultra High Pressure Liquid Chromatography(UHPLC)

5 μm Analytical Particles

60 μm Human Hair (very fine hair)

1.7 μm ACQUITY UPLC Particles

Optimal Particle Size distribution

for Maximum Efficiency at a given Pressure H

igh

pres

sure

: ca

100

0 ba

rHPLC UHPLC

Column Xbridge BEH C18150 x 4.6 mm I.D., 5 µm5-40% ACN in 60 min at 1 mL/minDetection neg ESI-TOF-MS

ESI-TOF-MS -Column Acquity BEH C18150 x 2.1 mm I.D., 1.7 µm5-40% ACN in 60 min at 0.35 mL/min.

ESI-TOF-MS -

time ÷ 9same selectivitysame resolution

Column Acquity BEH C18150 x 2.1 mm I.D., 1.7 µm5-40% ACN in 60 min at 0.35 mL/min.

ESI-TOF-MS -

44.0 min

ESI-TOF-MS -

Why UHPLC

Guillarme D., Nguyen D.T.T., Rudaz S. and Veuthey J.L.: Eur J Pharm Biopharm. 2007, 66, 475‐482 + 2008, 68, 430‐440.

same timesame selectivityresolution ↑↑

Column Acquity BEH C1850 x 2.1 mm I.D., 1.7 µm5-40% ACN in 6.76 min at 0.6 mL/min.

ESI-TOF-MS -

5.0 min

HR MS Analyzers: time of flight TOF

R 20’000

TOF-MS Arabidopsis thaliana

m/z100 200 300 400 500 600 700 800 900 1000

%

0

100 595.171

596.174

[M+H]+591.1153

C30H23O13

NP dereplication based on UHPLC-TOF-MS

retention time

m/z

250,000 NPs

Cross search withchemotaxonomical

information

logP

CwH

xOyN

z

UPLC TOF-MS

few hits

Partial / full identificationEl

emen

t num

ber r

estr

ictio

nLe

wis

and

sen

io c

heck

Isot

ope

patte

rn fi

lterin

gH

/C &

NO

PSra

tio c

heck

Elem

ent r

atio

chec

k


2 4 6 8 10 12 14 16 18 20 22 24 26 28

%

0

100 TOF MS ES-

BPI1.58e5

Chemical Diversity of Caralluma sinaica EtOH Extract

• C. Sinaica (100 mg/kg) decreased plasma

glucose level to normal

• Antidiabetic activity comparable to Glibenclamide

C. sinaica

NMR profiling of Caralluma sinaica EtOH Extract

2 4 6 8 10 12 14 16 18 20 22 24 26 28

%

0

100 TOF MS ES-

BPI1.58e5

%

100101008_PE_SM_MSMS_neg 1: TOF MS E

B1.33

7.76593.1519

1.10377.0854

4.01485.2236

3.51485.2246

2.97323.1340

2.14385.1335

5.87531.2448

5.02365.1453

6.81447.0916

21.38491.2442

17.951021.4984

15.76847.4100

10.06978.4679

12.59329.2326

10.45915.3986

12.51329.2324

12.70329.2329

13.82574.3763

12.82329.2325

13.95594.3442

14.21849.3920

17.39991.4902

20.65989.5110

27.96355.1586

24.73553.2599

22.801083.5134

24.32553.2593

23.941051.5291

26.27623.4169

26.05354.3000

25.05609.4001

27.78327.2904

27.30637.4330


On line molecular formula assignement of more than 100 peaks with MW > 500

2 4 6 8 10 12 14 16 18 20 22 24 26 28

%

0

100 TOF MS ES-

BPI1.58e5

%

100101008_PE_SM_MSMS_neg 1: TOF MS E

B1.33

7.76593.1519

1.10377.0854

4.01485.2236

3.51485.2246

2.97323.1340

2.14385.1335

5.87531.2448

5.02365.1453

6.81447.0916

21.38491.2442

17.951021.4984

15.76847.4100

10.06978.4679

12.59329.2326

10.45915.3986

12.51329.2324

12.70329.2329

13.82574.3763

12.82329.2325

13.95594.3442

14.21849.3920

17.39991.4902

20.65989.5110

27.96355.1586

24.73553.2599

22.801083.5134

24.32553.2593

23.941051.5291

26.27623.4169

26.05354.3000

25.05609.4001

27.78327.2904

27.30637.4330


On line molecular formula assignement of more than 100 peaks with MW > 500

m/z300 500 1000

%

0

100TOF MSMS817.42ES-

1.92e3

817.4209655.3727

493.3173 MS/MS

%

0

100TOF MSMS979.47ES-

2.87e3

979.4763817.4243

655.3674300 500 1000

Putative structure

identical skeleton


Pregnanes from C. sinaica

Compound 4.2

Compound 4.6a

Compound 4.6b

Compound 5.9

Compound 5.13

Compound 1.1

Compound 3.6

Compound 4.3

Compound 4.5

Compound 4.6

Compound 4.7

Compound B7

HPLC-NMR

?

Report

Crude Samples/Mixtures

LCPurification

At-line CapNMR analysis

NMR

CapNMR Detection

On-line

MSDAD/ELSDHigh Sensitivity Detection

bioassay

Prep Stage

vacuumcentrifuge

Mass-directed

fractionation

SPE

On-flow

At-line

5 mm std. NMR tube

270 lit.(~17 mm)

5 mm Rf Coil

Cap-NMR microcoil flow probe

1.5 lit.(~1 mm)

5 lit.

Solenoidal flow probe

50-200 µl

A) Saddle-type rf coil

A

flowcell

ConventionalLC/NMR flow probe

Sample volume:150 x smaller than standard NMR tubes10-40 x smaller than LC/NMR

m/z < 5 ppm

particles

1.7 μm

HIGH RESOLUTION LCHIGH RESOLUTION MS

UPLC TOF-MS

LOD 1 µg

HIGHLY SENSITIVE NMR

Capillary NMR

LC-MS microfractionation

μg

mg

F2 (ppm)

7.37.47.57.67.77.87.98.08.1

F1

(ppm)7.3

7.4

7.5

7.6

7.7

7.8

7.9

8.0

8.1

Cap NMR spectra


LC-MS micro-fractionationfor CapNMR

Injection 50 mgColumn Xbridde(250 x 9 mm i.d.)Flow 2 ml/minFractions 1 ml/min

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

0

R T : 3 3 .2 3 - 4 2 .7 6 S M : 5 B

3 4 3 5 3 6 3 7 3 8 3 9 4 0 4 1 4 2T im e ( m i n )

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Rel

ativ

e Ab

unda

nce

3 3 .6 2

3 3 .6 9

3 7 .9 43 8 .2 5

3 8 .9 3

3 7 .7 0

3 7 .4 0

3 9 .1 7

3 9 .8 5

4 0 .0 5

4 2 .1 34 2 .0 33 4 .4 8

4 1 .1 14 0 .4 63 6 .2 43 6 .1 4

3 5 .3 6

N L :1 .5 9 E 7B a s e P e a k M S w 3 h 8 0 m g 2

C44C45C46C47C48

Samples drying by speed-vac

Samples drying by speed-vac

Dissolution with 6.5 µl of deuterated solvent

Injection5 µlCapNMR

1H CapNMR of adjacent microfractions

C44

C45

C46

C47

Each fraction 1ml = 30 sec of peak elution

RT:21,21 - 25,16SM:3B

21,5 22,0 22,5 23,0 23,5 24,0 24,5 25,0Time (min)

2

4

6

8

uAU

20406080

100

20406080

100

20406080

100

20406080

100

20406080

100m/z 755

m/z 477

m/z 739

m/z 445

m/z 369

UV trace

Single ion traces(LC/ES-MS neg. ions)

O

OH

OO

OH

O

OO

OH

OHHO

CH3

OH

OHHO

O

O

HO

OH

OHH3C

Automated CapNMR analysis of micro fractions Miniaturized hit discovery platform

uninjured control

injured control

effect of a fraction

Zebrafish assay

CapNMR

DereplicationIdentification of active compoundsAbsolute quantitative measurement

Bioassays compatible with

96 well plates

mg of extracts

µg of microfractions

C44

C45

C46

C47

CapNMR spectra of microfractions

8 7 6 5 4 3 2 ppm


behavioral assays toxicological assays physiological assays developmental assays

proconvulsantphenotype

anti-angiogenicphenotype

anti-inflammatoryphenotype teratogenic phenotype

cont

rol

activ

es

hepatotoxicphenotype

Zebrafish as a Technology Platform for Natural Product Discovery

Collaboration with Dr A. Crawford

Zebrafish bioassay-guided microfractionation antiinflammatory and antiangiogenic activities R.viscosa

Rhynchosia viscosa

Zebrafish antianiogenic assay


Rapid and rational microfrationation

optimisation


gradienttransfert



bioassay

Zebrafish bioassay-guided microfractionation antiinflammatory and antiangiogenic activities R.viscosa

012345678f1 (ppm)

**

*

**

*

H-2H-2'H-6'

H-3'H-5'

H-8 H-6

HDO MeOD acetone

012345678f1 (ppm)

HDO MeOD acetone

H-2

H-7'H-4'

H-6'H-8 H-3'

H-6

H-1'

*

** * **

*

*

*

a

b

CapNMR™ 3%

97%

1.5 mL

5 mL

O

O

OH

OH OHgenistein

O

O

OH

OH

O

OH

CH3 CH3

sophoraisoflavone A

lycoisoflavone A

O

O

OH

OH

OH

OH

CH3

CH3

Injured control

genistein

Microgram-scale, in vivo natural product discovery using zebrafish bioassays

for the search of anticonvulsants

Solanum torvum L., Solanaceae

Medicinal plant of the Philippine Recorded in 19th c. and 20th c. Philippine pharmacopeia texts as an anticonvulsant used by locals


Zebrafish as a model for high-throughput analysis of behavior

control pentylenetetrazol-treated

Pro-convulsant


F1

F2

F3

F4

F5

F6

F7

F8

UPLC-TOF-MS profile of fraction 7

UV 254 nm

Micro fractionation of the extract

0

20

40

60

80

100

120

140

160

0 10 20 30 40 50 60 70 80 90 100

Abs

Time (min)

Gilson fractions

2D map of fraction 7 UPLC-TOF-MS profile

CapNMR spectra of the most active microfraction

C38H64O13

Ca 200 µg

ICAR 2010

I. Long distance inhibition

A B

III. Contact inhibition

A D

II. Zone line

C D

IV. Overgrowth

A E

Induction of bioactive cpds from combative fungi

I. Interactions between fungi species

A C

Fungal Co-culture Concept

pheromones

interspecies crosstalk

symbiotic metabolites

Defense moleculesDefense molecules

K. Scherlach, Org. Biomol. Chem. 2009

Trichophyton rubrumVs

Bionectria ochroleuca

Botryosphaeria obtusavs

Eutypa lata


Medium throughput micro co-culture production

8 ml

Sin 150 Sin 128

Sin 141 Sin 128

H

2 ml

Sin 134 Sin 141 Sin 150

Sin 128 Sin 128 Sin 128

D

120 ml

Strain A

Strain B

A x B

8 ml

Sin 150 Sin 128

Sin 141 Sin 128

H

0

2

4

6

8

10

12

1 ml 2 ml 3 ml 4 ml

New10 X

Num

bre

of in

duce

dco

mpo

unds

4 dpi

Culture volume

2 ml

2 dpi 4 dpi 7 dpi 9 dpi

Sin 134

Sin 141

Sin 150

Culture duration

Medium throughput micro co-culture production

A B A x B1

A B A x B2

A B A x B3

Antifungal bioassay

Induction of bioactivityby co-culture

Study mycoalexins in zone lines of grapevine fungal pathogens

Pure strain of Botryosphaeria obtusa

Confrontationzone

Pure strain ofEutypa lata

• Extraction : chloroforme/methanol/water(64/36/8, v/v/v)

Slice of a vine stock having the ESCA

apoplectic disease

Differential analysis by UPLC-TOF-MS

Botryosphaeria obtusa

Eutypa lata

Confrontation


Up scaling of the LC separation to 40 Petri dishes

….

LC-MS microfractionation of the barrage zone

0 10 20 30 40 50 60 70 Time (min)

100

0

%

A

142-3

ppm1234567

2+3m/z 209

1234567 ppm

1m/z 193

blan

k

conf

ront

atio

n

phytotoxicity

12+3

Diff: 16 Da

Barragezone

Semi-prep LC-MSESI NI

2 3O-methylmellein

1

MPLC-UV-ELSD configuration for large scale isolation of NPs

UV detector

Fraction Collector

Binary-Pump

MPL

C-C

olum

n

Solvents

Injector

LSD detector

Split-valve

mV

0100

200

300

400

2060

Heu

res

80

Col 13

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80

%

0

100 1.73785.43

1.45785.44

0.16191.060.79477.110.70463.09 1.18947.49

1.52771.42

3.95769.44

2.27785.431.81771.42

2.07785.44

4.77981.58

Compounds of interest

UHPLC-MS 5 min

MPL

C (1

0g)

80 h

ours

Col 13

Time0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40

%

0

100

0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40

%

0

100120514_SC_Storvum_MPLC_80_neg 1: TOF MS ES-

771.41 0.20Da8.15e3

1.52771.41

120514_SC_Storvum_MPLC_71_neg 1: TOF MS ES- 785.43 0.20Da

8.27e31.47

785.43

Col 13

Time0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40

%

0

100

0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40

%

0

100120514_SC_Storvum_MPLC_80_neg 1: TOF MS ES-

771.41 0.20Da8.15e3

1.52771.41

120514_SC_Storvum_MPLC_71_neg 1: TOF MS ES- 785.43 0.20Da

8.27e31.47

785.43

TOF-MSC39H64O13

Rel

ated

isom

ers

MPLC x UHPLC for fractionation

& monitoring

UPLC-TOF MS and CapNMRfor revealing the richness of plant metabolome

UPLC-ES-TOF-MS NI

> 250 peaksdetected

BPI tracesN= ca 70000

417.176

m/z200 400 600 800 1000

%

0

100 417.1760

C19H29O11

TOF-MS

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5Chemical Shift (ppm)

0.01

0.02

0.03

0.04

0.05

0.06

Nor

mal

ized CapNMR

LC-NMR 10-6 mol

LC-UV 10-9 mol

GC-MS 10-12 mol

LC-MS 10-15 mol

LC-LIF 10-19 mol

CE-LIF 10-23 mol

sensitivities

Sumner et al. Phytochemistry 2003, 62, 817-836.

deeper metabolome

studies


Latest technological advances for natural products: evolution or revolutionevolution revolution

techniques– HPLC UHPLC – MS HRMS– NMR microNMR– Innovative bioassays

approaches– Profiling metabolomics– Bioactivity guided fractionation

– Systems biology

Acknowledgments

P.EugsterN. BohniS. ChallalG. GlauserE. GrataDr. K. NdjokoDr. S. BertrandDr. G Marti

Prof. J.L VeutheyDr S RudazDr D. GuillarmeDr J. Boccard

Prof. E.E. Farmer

Prof. M. CuendetDr. P ChristenDr. E. Ferreira-Queiroz

Prof. Vanderlan Bolzani

A. Crawford

Agroscope changin

Dr C. GindroDr. S Schuerch

Dr. S M Almassarani

Acknowledgments

Thank you for your attention

[email protected]


Overview Phytochemistry and Bioactive Natural Products … · Chemical Diversity of Caralluma...

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