effect of selected food and medical plants and plant molecules on ovarian functions alexander v....
TRANSCRIPT
EFFECT OF SELECTED FOOD AND
MEDICAL PLANTS AND PLANT
MOLECULES ON OVARIAN FUNCTIONS
Alexander V. Sirotkin
Constantine the Philosopher University and Research Institute of Animal
Production , Nitra, Slovakia
Food
Physiological state and health
Supply with energy (calories)
Supply with structural elements (proteins, lipids, carbohydrates)
Supply with regulatory molecules (antioxidants, phytohormones etc.)
Intracellular regulators of cell proliferation, apoptosis, differentiation
Hormones
FOOD REGULATORY MOLECULES AND REPRODUCTION:
- can food plants affect reproductive functions?
- what can be endocrine and intracellular mechanisms of
such action?
- what for plant molecules are responsible for their action?
- can some food plants jeopardize or improve
reproduction?
AIMS OF THE STUDY: to examine
- the effect of some food plants (curcuma/turmeric, green tea,
rooibos, ginkgo, flaxseed, yucca) and their components -
phytoestrogens and antioxidants (resveratrol, quercetin,
daidzein, diosgenin) on ovarian cell functions
- mechanisms of action: whether these additives work
through changes in hormones release, cell proliferation,
apoptosis or response to hormonal stimulators
- application of some plants to improve reproduction
A. Species:
- pigs- rabbits
B. Objects:
- living animals- ovarian granulosa cells
MATERIAL & METHODS
Treatments with:- plant extracts (curcumin, green tea, rooibos, ginkgo, flaxseed, yucca)- plant molecules (resveratrol, quercetin, daidzein, diosgenin)- hormones (FSH, IGF-I) + plants and plant molecules
C. Manipulations:
Cells
Analysis:reproduction, proliferation, apoptosis, hormones release (RIA, Western, imunocytochemistry, RT-PCR a.o.)
Animals
MATERIAL & METHODS
CAN FOOD PLANTS (curcumin, green tea, rooibos,
flaxseed, ginkgo, yucca etc.) DIRECTLY AFFECT
OVARIAN CELL FUNCTIONS?
Curcumin reduces ovarian cell proliferation:
- accumulation of PCNA
- accumulation of PCNA mRNA
Curcumin dose added (µg/ml medium)
0 1 10 100
% o
f cel
ls c
onta
ined
PC
NA
40
42
44
46
48
50
52
54
56
*
Rel
ativ
e un
its (
to c
ontr
ol v
alue
s)
Curcumin dose added (g/ml medium)
0 10 100
0,4
0,5
0,6
0,7
0,8
0,9
1,0
1,1
1,2
*
*
Curcumin promotes ovarian cell apoptosis:
- accumulation of bax
- accumulation of bax mRNA
0 1 10 100
% o
f cel
ls c
onta
ined
bax
45
50
55
60
65
70
75
80
*
**
Curcumin dose added (µg/ml medium)
Curcumin dose added (g/ml medium)
0 10 100
Rel
ativ
e un
its (
to c
ontr
ol v
alue
s)
-2
0
2
4
6
8
10
12
14
16
*
*
Curcumin promotes the release of hormones:
- progesterone
- testosteron
0 1 10 100
0
50
100
150
200
250
300
350
Curcumin dose added (µg/ml medium)
Pro
gest
eron
e re
leas
e (n
g/10
6 cel
ls/
day)
* *
0 1 10 100
400
600
800
1000
1200
1400
Curcumin dose added (µg/ml medium)
Tes
tost
eron
e re
leas
e (p
g/1
06 c
ells
/day
)
*
Ovarian cell proliferation (accumulation of PCNA)
is inhibited with:
- rooibos
- ginkgo
- flaxseed
- green tea
0 1 10 100
20
25
30
35
40
45
*
*
*
Rooibos dose added (µg/ml)
% o
f ce
lls c
onta
ined
PC
NA
0 1 10 100
15
20
25
30
35
40
45
** *
Ginkgo dose added (µg/ml)
% o
f ce
lls c
onta
ined
PC
NA
0 1 10 10015
20
25
30
35
40
45
*
*
*
Flaxseed dose added (µg/ml)
% o
f ce
lls c
onta
ined
PC
NA
Green tea AO dose added (µg/ml medium)
0 1 10 100
% o
c ce
lls c
on
tain
ed
PC
NA
32
34
36
38
40
42
44
46
48
50
52
54
* *
*
Ovarian cell apoptosis (accumulation of bax)
is promoted with:
- rooibos
- ginkgo
- flaxseed
- green tea
0 1 10 100
30
35
40
45
50
55
*
*
*
Rooibos dose added (µg/ml)
% o
f ce
lls c
onta
ine
d b
ax
0 1 10 100
30
35
40
45
50
55
60
**
*
Ginkgo dose added (µg/ml)
% o
f ce
lls c
on
tain
ed
ba
x
0 1 10 100
32
34
36
38
40
42
44
46
48
50
**
*
Flaxseed dose added (µg/ml)
% o
f ce
lls c
onta
ined
bax
590 effect of green tea AO on apoptosis of porcine GC
Green tea AO dose added (µg/ml medium)
0 1 10 100
% o
f ce
lls c
on
tain
ed
ba
x
45
50
55
60
65
70
75
*
Ovarian cell leptin release is inhibited with:
- rooibos
- ginkgo
- flaxseed
0 1 10 100
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
Rooibos dose added (g.ml-1)
Lept
ine
rele
ase
(ng/
106 c
ells
/day
)*
*
*
0 1 10 100
0,0
0,5
1,0
1,5
2,0
2,5
Ginkgo dose added (g.ml-1)
Lept
ine
rele
ase
(ng/
106 c
ells
/day
)
*
0 1 10 100
2,0
2,5
3,0
3,5
4,0
4,5
5,0
5,5
Flaxseed dose added (g.ml-1)
Lept
ine
rele
ase
(ng/
106 c
ells
/day
)
*
Ovarian cell progesterone release
is inhibited with:
- rooibos
- ginkgo
is stimulated with:
- green tea
- yucca
0 1 10
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
2,2
2,4
2,6
Rooibos dose added (g.ml-1)
Pro
gest
eron
e re
leas
e (m
ol/l
cells
/day
)
0 1 10
0
1
2
3
4
Ginkgo dose added (g.ml-1)
Pro
gest
eron
e re
leas
e (m
ol/l
cells
/day
)
*
0 1 10 1005
10
15
20
Yucca dose added (g/ml medium)
Pro
ge
ste
ron
re
lea
se
(n
g/1
06 t
iss
ue
/da
y) *
590 Effect of green tea PP on progesterone secretion by porcine GC
0 1 10 100
60
80
100
120
140
160
180
200
220
240
260
280
Green tea PP dose added (µg/ml medium)
Pro
ge
ste
ron
e r
ele
ase
(n
g/1
06 ce
lls/d
ay)
*
FOOD PLANTS CONTAIN PHYTOESTROGENS AND
ANTIOXIDANTS - resveratrol, quercetin, diosgenin,
daidzein.
CAN THE PLANT ACTION BE EXPLAINED BY THE
PRESENCE OF THESE MOLECULES?
DO PLANT EXTRACTS AND PLANT MOLECULES
HAVE SIMILAR ACTION?
Resveratrol
- inhibits proliferation
(accumulation of PCNA)
- promotes apoptosis
(accumulation of bax)
- Inhibits release
of progesterone
Resveratrol dose added (µg.ml-1 medium)
0 1 10 100
% o
f cel
ls c
onta
ined
PC
NA
30
35
40
45
50
55
* * *
0 1 10 100
% o
f ce
lls c
onta
ined
bax
45
50
55
60
65
70
75
** *
Resveratrol dose added (µg.ml-1 medium)
Resveratrol dose added (µg.ml medium-1)
0 1 10 100
Pro
gest
ero
ne
re
lea
se (
ng.
10
-6 c
ells
.da
y-1)
20
40
60
80
100
120
* *
*
0ng/ml 100ng/ml 1000ng/ml
Quercetin
- inhibits proliferation
(accumulation of PCNA)
- promotes apoptosis
(accumulation of bax)
Quercetin dose added (g.ml-1)
0 1 10 100
% c
ells
con
tain
ed P
CN
A
20
25
30
35
40
45
50
55
* *
Quercetin dose added (g.ml-1)
0 1 10 100
% c
ells
con
tain
ed b
ax
10
20
30
40
50
60
70
*
**
Diosgenin :
- promotes proliferation
(accumulation of PCNA)
- promotes apoptosis
(accumulation of bax)
Diosgenin dose added (µg/ml)
0 1 10 100
% o
f ce
lls c
ont
ain
ing
PC
NA
30
40
50
60
70
80
90
100
A
AA
Diosgenin dose added (µg/ml)
0 1 10 100
% o
f ce
lls c
ont
ain
ing
Bax
40
50
60
70
80
90
Daidzein :
- promotes proliferation
(accumulation of PCNA)
- promotes apoptosis
(accumulation of bax)
- Inhibits release
of progesterone
Daidzein (µM)
0 1 10 100
% o
f ce
lls
con
tain
ing
PC
NA
20
30
40
50
60
70
a a
1A
Daidzein (µM)
0 1 10 100
% o
f ce
lls
con
tain
ing
bax
30
40
50
60
70
80
a
a
1B
Daidzein (µM)
0 1 10 100
P4
secr
etio
n p
g/1
06 c
ells
/day
4
6
8
10
12
aa
a
1C
CAN PLANTS AND PLANT MOLECULES AFFECT
OVARIAN RESPONSE TO HORMONAL
STIMULATORS?
Daidzein blocks the stimulatory effect of FSH on :
- proliferation
(accumulation of PCNA)
- release of progesterone
- release of leptin
FSH dose added (IU/ml medium)
0,00 0,01 0,10 1,00
P4
secr
etio
n p
g/1
06 c
ells
/day
0
5
10
15
20
25
a
b
b
FSH
FSH + daidzein
a
b
2C
FSH dose added (ng/ml medium)
0,0 0,1 1,0 10,0
Lep
tin
sec
reti
on
ng
/10
6 c
ells
/day
-5
0
5
10
15
20
25
FSH
FSH + Daidzein
a b b
a
aa
b
a
2E
Quercetin blocks the stimulatory action of FSH on ovarian
cell proliferation (accumulation of PCNA)
FSH and quercetin dose added (g.ml-1)
0 1 10 100
% c
ells
con
tain
ed P
CN
A
20
30
40
50
60
70
80
*
**
FSH
FSH and quercetin
Resveratrol blocks the stimulatory effect of FSH on the
release of:
- progesterone
- testosteron
FSH dose added (IU/ml medium)
0,00 0,01 0,10 1,00
P4
secr
etio
n n
g/1
06 c
ells
/day
20
40
60
80
100
120
FSH + PF 046
FSH
B
B
A
FSH dose added (IU/ml medium)
0,00 0,01 0,10 1,00
T s
ecr
etio
n pg
/106 c
ells
/day
0
200
400
600
800
1000
1200
BB B B
FSH + PF 046
FSH
AA
Curcumin blocks the anti-apoptotic effect of IGF-I
0 1 10 100
10
20
30
40
50
60
% o
f cel
ls c
onta
ined
bax
IGF-I dose added (ng/ml medium)
B B
IGF-I + curcumin
IGF-I alone
AB
B
Green tea blocks the stimulatory effect of IGF-I on
progesterone release
9
0 1 10 100
0
10
20
30
Pro
gest
eron
e re
leas
e (n
g/10
6 cel
ls/d
ay)
IGF-I dose added (ng/ml medium)
A AB
AB
IGF-I + green tea PP
IGF-I alone
CAN PLANT PRODUCTS BE USED FOR
IMPROVEMENT HORMONAL AND REPRODUCTIVE
STATE?
Addition of yucca can promote hormones release by
cultured rabbit ovarian tissue:
- progesterone
- testosteron
- estradiol
Feeding with yucca increases rabbit plasma level of
- oxytocin
- prostaglandin F
- prostaglandin E
Feeding with yucca increases rabbit
- conception rate
- kindling rate
0 5 20
40
50
60
70
80
90
100
110
Effect of yucca on conception rate (%) on rabbit does
Yucca dose added (g/100kg food)
Co
nc
ep
tio
n r
ate
(a
vera
ge
)
*
*
0 5 20
30
40
50
60
70
80
90
100
110
Effect of yucca on kindling rate (%) on rabbit does
Yucca dose added (g/100kg food)
Kin
dlin
g r
ate
(ave
rag
e) *
*
CONCLUSIONS:
1. Food plants curcuma/turmeric, green tea, rooibos, ginkgo,
flaxseed can directly suppress ovarian cell functions via
- inhibition of proliferation,
- promotion of apoptosis,
- change in steroid and peptide hormone release
- blockage of response to upstream hormonal
stimulators (FSH, IGF-I).
CONCLUSIONS:
2. Phytoestrogens and antioxidants resveratrol and
quercetin act similar to curcuma, green tea, rooibos,
ginkgo and flaxseed.
Can be active component of food plants?
CONCLUSIONS:
3. Phytoestrogens and antioxidants daidzein and diosgenin
have both stimulatory and inhibitory effect on ovarian cell
functions:
- promote proliferation
- promote apoptosis
- suppress progesterone release
- suppress the response to FSH
Not responsible for the main food plant effect?
CONCLUSIONS:
4. Yucca promotes
- steroid and peptide hormones release both in vivo and in
vitro
- fertility (conception and kindling rate).
Can be useful for improvement reproduction?
COLLABORATION
A.Kadasi, A. Stochmalova, A. Alexa, M. Foldesiova,
A. Balazi, P. Chrenek (Slovakia)
R. Grossmann (Germany)
J. Kotwica (Poland)
THANK YOU!
Some effects of nutrition on physiological state
Low calories diet
- delays puberty
- prolongs duration of life
- activates immune system
- prevents and inhibits
infectious diseases
depressions
metabolic disorders
reproductive disorders and infertility
cancer
Some effects of nutrition on physiological state
Phytoestrogens
-stimulate puberty
-Inhibite some metabolic (osteoporosis) and psychic
(depression, agressivity) diseases
-promote inflammation
-promote or suppress reproductive disorders (meno- and
andropause, infertility, ovulatory cycles)
-promote or suppress cancer
• Results
Results
Resveratrol dose added (µg.ml-1 medium)
0 1 10 100
% o
f cel
ls c
onta
ined
PC
NA
30
35
40
45
50
55
* * *
Inhibitedted effect of resveratrol and rapamycin on the expression of PCNA.
Rapamycin dose added (µg.ml-1 medium)
0 1 10 100
% o
f cel
ls c
onta
ined
PC
NA
25
30
35
40
45
50
55
*
*
*
RESULTS
•Results
0 1 10 100
% o
f ce
lls c
on
tain
ed b
ax
45
50
55
60
65
70
75
** *
Resveratrol dose added (µg.ml-1 medium) Rapamycin dose added (µg.ml-1 medium)
0 1 10 100
% o
f ce
lls c
onta
ined
bax
40
50
60
70
80
90
*
**
RESULTS Stimulated effect of resveratrol and
rapamycin on the expression of bax.
• Results
Rapamycin dose added (µg.ml-1 medium)
0 1 10 100
Pro
gest
ero
ne r
ele
ase
(ng.
10-6
ce
lls.d
ay-1
)
55
60
65
70
75
80
85
90
95
*
Resveratrol dose added (µg.ml medium-1)
0 1 10 100
Pro
gest
ero
ne r
elea
se (
ng.
10-6
cel
ls.d
ay-1
)
20
40
60
80
100
120
* *
*
Inhibited effect of resveratrol and rapamycin on progesterone release.
RESULTS
•ResultsStimulated effect of resveratrol
on testosterone release.
Col 1 vs Col 2
Resveratrol dose added (µg.ml-1 medium)
0 1 10 100
Tes
tost
ero
ne
re
lea
se (
pg.
10
-6 c
ells
.day
-1)
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
*
*
Col 1 vs Col 2
Rapamycin dose added (µg.ml-1 medium)
0 1 10 100
Tes
tost
eron
e re
leas
e (p
g.10
-6 c
ells
.day
-1)
200
400
600
800
Effect of rapamycin on testosterone release was not found.
RESULTS
Resveratrol :
- inhibits the release
of progesterone
- promotes the release
of testosteron
Resveratrol dose added (µg/ml medium)
0,0 0,1 1,0 10,0P
4 se
cret
ion
ng/1
06 c
ells
/day
20
30
40
50
60
70
** *
Resveratrol dose added (µg/ml medium)
0,0 0,1 1,0 10,0
T s
ecr
etio
n p
g/1
06 c
ells
/day
600
800
1000
1200
1400
1600
1800
*
Resveratrol inhibits proliferation (accumulation of
PCNA) of ovarian cells
- 36 K 0ng/ml 100ng/ml 1000ng/ml
Dávka resveratrolu
0 ng/ml 100 ng/ml 1000 ng/ml 10000 ng/ml
% b
un
iek
obsa
hu
júci
ch P
CN
A
0
1
2
3
4
5