Valorizzazione di specie vegetali eduli spontanee e coltivate … · 2016. 1. 22. · 51_PM432...

Università degli studi di Cagliari

CORSO DI DOTTORATO IN SCIENZE E TECNOLOGIE DELLA TERRA E DELL'AMBIENTE

INDIRIZZO BOTANICA AMBIENTALE E APPLICATA

Ciclo XXIX

Valorizzazione di specie vegetali eduli spontanee e coltivate attraverso sistemi di caratterizzazione chimica e molecolare a

garanzia di qualità

DOTTORANDA: Dott.ssa Arianna Marengo TUTOR: Prof. Andrea Maxia

CO-TUTOR: Prof.ssa Patrizia Rubiolo,

Prof.ssa Cinzia Bertea

First PhD year

• Literature research on Sardinian traditions

• Collection of plant species

• Validation of chemical and biomolecular

techniques

Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences

Botany and Botanical Garden Division

University of Cagliari, Italy (UNICA)

Asteraceae family

CARDUEAE TRIBE

Carlininae

CarduinaeCarduinae

Carlininae

(del Bosque et al., 2014)

Echinopsinae

Centaureinae

Sardinia � ~ 50 species




Echinopsinae

Centaureinae

(del Bosque et al., 2014)

(Pignatti, 1982) CardopatiinaeCardopatiinae

Cynara cardunculus L.

•Component of the mediterranean diet

•Nutritional and pharmacological properties

Silybum marianum (L.) Silybum marianum (L.)

Gaertn.

•Native plant from the Mediterranean area

•Leaves and flowers used as vegetable

•Active compounds: silymarin




Wild speciesCarduus argyroa Biv.

Onopordum illyricum L.

Ptilostemon casabonae (L.) GreuterLocal traditions

Galactites tomentosa Moench.




AIM OF THE WORK

Investigation on different edible species that are

consumed in the Sardinian tradition

Chemical

analysis

Biomolecular

analysis(BARCODING)

Some species has been poorly investigatedLaboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences



Carduus nutanssubsp.macrocephalus

EXPERIMENTAL PROCEDURES

Plant material

Carduus argyroa Biv.

Carduus pycnocephalusL.

Carduus cephalantus Viv.

subsp.macrocephalus(Desf.) Nyman

Ptilostemon casabonae(L.) Greuter





Chemical analysis

Photodiode detector SPD-M20A coupled to a

triple quadrupole Shimadzu LCMS-8040

system and an electrospray ionization (ESI)

Apigenin 7-O-glucoside

� 432 g/mol

Absorption spectrum

Mass spectrometry

source




Università degli Studi di Torino

Dipartimento di Scienza e

Tecnologia del farmaco





Biomolecular analysis

DNA extraction

PCR

(DNA barcoding genes)

Gel

electrophoresis

SequencingLaboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences



RESULTS

Species:Carduus argyroa Biv.

Carduus pycnocephalus L.


Carduus nutans subsp. macrocephalus (Desf.) Nyman

Ptilostemon casabonae (L.) Greuter

Carduus Spp.


Chemical

analysis

Biomolecular

analysis(BARCODING)




CHEMICAL ANALYSIS

Carduus argyroa

80

90

100

110

120

mAU254nm,4nm

24P

M_

448

36+

37_

PM

516+

448

43_

PM

446

51P

M_

432

Retention time

UV spectra

Mass analysis

Product Ion Scan

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 min-10

0

10

20

30

40

50

60

70

1_

PM

354

2_

PM

354

18_

PM

580

20_

PM

580

22_

464

36+

37_

PM

516+

448

39_

PM

592

41_

PM

432

61_

PM

286

71_

PM

270

76_

PM

286

77_

PM

330+

300

80_

PM

243




CHEMICAL ANALYSIS

Carduus argyroa

80

90

100

110

120

mAU254nm,4nm

Lu

teo

lin

3-O

-glu

cosi

de

aci

d+

Kaem

pfe

rol

3-O

-glu

cosi

de

43_

PM

446

51P

M_

432

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 min-10

0

10

20

30

40

50

60

70

Neo

chlo

rog

enic

aci

d

Ch

loro

gen

icaci

d

18_

PM

580

20_

PM

580

Qu

erce

tin

3-O

-glu

cosi

de

1,5

Dic

aff

eoyl

qu

inic

aci

d+

Kaem

pfe

rol

39_

PM

592

Ap

igen

in3-

O-g

luco

sid

e

Lu

teo

lin

Ap

igen

in

Kaem

pfe

rol

77_

PM

330+

300

80_

PM

243




Identified compounds• Apigenin

• Apigenin 7-O-glucoside

• Diosmin

• Kaempferol

• Kaempferol 3-O-glucoside

• Kaempferol 3-O-rutinoside

• Luteolin

• Luteolin 7-O-glucoside

O-glycosides of

Apigenin, Kaempferol,

Luteolin, and Quercetin

Flavonoids• Luteolin 7-O-glucoside

• Neochlorogenic Acid

• Quercetin 3-O-glucoside

• Rutin

• Chlorogenic Acid

• 1,5 Dicaffeoylquinic Ac.



Caffeoylquinic Acids derivates

Laboratory of Economic and Pharmaceutical Botany

Department of Life and Environment Sciences



Flavonoids

Card

uu

sn

utan

ssu

bsp

. macro

ceph

alus

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

min

-10 0 10

20

30

40

50

60

70

80

90

100

110

120 m

AU

254n

m,4

nm

Neochlorogenic acid

Chlorogenic acid

18_PM580 20_PM580

Quercetin 3-O-glucoside

Luteolin 7-O-glucoside

1,5 Dicaff. acid+ Kaempferol 3-O-gluc.

39_PM592

Apigenin 7-O-glucoside43_PM446

51PM_432

Luteolin

Apigenin

Kaempferol77_PM330+300

80_PM243

Card

uu

sarg

yroa

mA

U

254n

m,4

nm

Lab

ora

tory

of E

con

om

ica

nd

Ph

arm

ace

utica

lB

ota

ny

De

pa

rtme

nt

of Life

an

d E

nviro

nm

en

t Scie

nce

s

Bo

tan

ya

nd

Bo

tan

ical

Ga

rde

n D

ivisio

n

Un

ive

rsity o

f Ca

glia

ri, Italy

(UN

ICA

)

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

min

-30

-20

-10 0 10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

254n

m,4

nm

Neochlorogenic acid

Chlorogenic acid

17aPM_610

18_PM580

Rutin


Kaempferol 3-O-rutinoside

1,5 Dicaff. Acid+Kaempferol 3-O-gluc

39_PM592

Apigenin 7-O-glucoside

43_PM446

51_PM432

Luteolin

ApigeninKaempferol

77_PM330+300

80_PM243

Card

uu

scep

halan

tus

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

min

0 25

50

75

100

125

150

175

200

225

250

mA

U

254n

m,4

nm

Neochlorogenic acid

Chlorogenic acid

23_PM462



3,4 Dicaff. acid

42_PM59444_PM59446_PM608

4,5 Dicaff. acid

51_PM432

Luteolin

ApigeninKaempferol77_PM330+300

80_PM243

Card

uu

sp

ycno

ceph

alus

mA

U

254n

m,4

nm

gluc

Lab

ora

tory

of E

con

om

ica

nd

Ph

arm

ace

utica

lB

ota

ny

De

pa

rtme

nt

of Life

an

d E

nviro

nm

en

t Scie

nce

s

Bo

tan

ya

nd

Bo

tan

ical

Ga

rde

n D

ivisio

n

Un

ive

rsity o

f Ca

glia

ri, Italy

(UN

ICA

)

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

min

-25 0 25

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

Neochlorogenic acid

Chlorogenic acid

17_PM610Rutin

Quercetin 3-O-glucoside23PM_462

Luteolin 7-O-glucosideKaempferol 3-O-rutinoside


3,5 Dicaff. acid39_PM592 42_PM594

43_PM44644_PM594Diosmin

4,5 Dicaff acid

51_PM432

Luteolin

Apigenin

Kaempferol

77_PM330+300

80_PM243

Ptilostemon casabonae

60

70

80

90

100

110

120

130

140

150

160

170

mAU

254nm,4nm

Neo

chlo

rog

enic

aci

d

Ru

tin

glu

cosi

de

Kaem

pfe

rol

3-O

-ru

tin

osi

de

1,5

Dic

aff

aci

d+

Kaem

pfe

rol

3-O

-glu

c

3,5

Dic

aff

aci

dA

pig

enin

7-O

-glu

cosi

de

47_

PM

616

55_

PM

616

58_

PM

716





5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 min

-10

0

10

20

30

40

50

60

Neo

chlo

rog

eni

Ch

loro

gen

icaci

d

Qu

erce

tin

3-O

-glu

cosi

de

Kaem

pfe

rol

Ap

igen

in43_

PM

446

4,5

Dic

aff

aci

d

Lu

teo

lin

Ap

igen

in

Kaem

pfe

rol

80_

PM

243

Principal component analysis

SpeciesCarduus argyroa Biv.





P.casabonae

Carduus

P.casabonae

C.argyroa

C.nutans

C.nutans

C.argyroa

Principal component analysisOnly Carduus species

SpeciesCarduus argyroa Biv.




C.nutans

C.argyroa

C.pycnoc.

C.cephal.

C.nutans

C.argyroa C.pycnoc.

C.cephal.

• Metabolite profile

• Identification of some compounds

• Statistical analysis � differences between• Statistical analysis � differences between

species

Further statistical studies




BIOMOLECULAR ANALYSIS

ITS gene

Species1. Carduus argyroa Biv.1. Carduus argyroa Biv.

2. Carduus pycnocephalus L.

3. Carduus cephalantus Viv.

4. Carduus nutans subsp. macrocephalus (Desf.) Nyman

5. Ptilostemon casabonae (L.) Greuter




Electropherogram

Sequencing




Alignment of the

forward and reverse

sequences





Alignment of the

sequences belonging

to each individuals

�CONSENSUS

sequence

UPGMA tree





Biomolecular markers

• Sequences

• Discrimination between species

Further genes analysis




CONCLUSIONS

Identification of closely related thistle species in

the genus Carduus as well as Ptilostemon

This may suggest that both chemical and genetic

differences are present between the considered

species.




FUTURE PERSPECTIVES

• Run further statistical analysis and experiments

• �• Biological assays � test the extracts to verify the

presence of biological activities




Valorizzazione di specie vegetali eduli spontanee e coltivate … · 2016. 1. 22. · 51_PM432...

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Transcript of Valorizzazione di specie vegetali eduli spontanee e coltivate … · 2016. 1. 22. · 51_PM432...