Post on 20-Jan-2021
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
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
Wild speciesCarduus argyroa Biv.
Onopordum illyricum L.
Ptilostemon casabonae (L.) GreuterLocal traditions
Galactites tomentosa Moench.
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
Carduus nutanssubsp.macrocephalus
EXPERIMENTAL PROCEDURES
Plant material
Carduus argyroa Biv.
Carduus pycnocephalusL.
Carduus cephalantus Viv.
subsp.macrocephalus(Desf.) Nyman
Ptilostemon casabonae(L.) Greuter
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
EXPERIMENTAL PROCEDURES
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
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
Università degli Studi di Torino
Dipartimento di Scienza e
Tecnologia del farmaco
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
EXPERIMENTAL PROCEDURES
Biomolecular analysis
DNA extraction
PCR
(DNA barcoding genes)
Gel
electrophoresis
SequencingLaboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
RESULTS
Species:Carduus argyroa Biv.
Carduus pycnocephalus L.
Carduus cephalantus Viv.
Carduus nutans subsp. macrocephalus (Desf.) Nyman
Ptilostemon casabonae (L.) Greuter
Carduus Spp.
Ptilostemon casabonae (L.) Greuter
Chemical
analysis
Biomolecular
analysis(BARCODING)
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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.
• 3,5 Dicaffeoylquinic Ac.
• 4,5 Dicaffeoylquinic Ac.
Caffeoylquinic Acids derivates
Laboratory of Economic and Pharmaceutical Botany
Department of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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
Luteolin 7-O-glucoside
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
Luteolin 7-O-glucoside
1,5 Dicaff. Acid+Kaempferol 3-O-gluc
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
1,5 Dicaff. Acid+Kaempferol 3-O-gluc
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
Laboratory of Economic and Pharmaceutical Botany
Department of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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.
Carduus pycnocephalus L.
Carduus cephalantus Viv.
Carduus nutans subsp. macrocephalus (Desf.) Nyman
Ptilostemon casabonae (L.) Greuter
P.casabonae
Carduus
P.casabonae
C.argyroa
C.nutans
C.nutans
C.argyroa
Principal component analysisOnly Carduus species
SpeciesCarduus argyroa Biv.
Carduus pycnocephalus L.
Carduus cephalantus Viv.
Carduus nutans subsp. macrocephalus (Desf.) Nyman
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
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
Electropherogram
Sequencing
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
Alignment of the
forward and reverse
sequences
Laboratory of Economic and Pharmaceutical Botany
Department of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
Alignment of the
sequences belonging
to each individuals
�CONSENSUS
sequence
UPGMA tree
Laboratory of Economic and Pharmaceutical Botany
Department of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
Biomolecular markers
• Sequences
• Discrimination between species
Further genes analysis
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
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.
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)
FUTURE PERSPECTIVES
• Run further statistical analysis and experiments
• �• Biological assays � test the extracts to verify the
presence of biological activities
Laboratory of Economic and Pharmaceutical BotanyDepartment of Life and Environment Sciences
Botany and Botanical Garden Division
University of Cagliari, Italy (UNICA)