The document summarizes research conducted on phytochemical extracts from red beetroot, purple Opuntia fruits, and yellow Opuntia fruits. It describes obtaining extracts rich in betalains and polyphenols from the plant materials and evaluating the extracts' bioactivity. Specifically, the beetroot extract was purified using solid phase extraction and fractions were characterized. The purified beetroot fraction was then tested in yeast models of neurodegenerative diseases and for anti-inflammatory activity, finding potential neuroprotective effects.

1. Student: Elena Valeria Girlus
Supervisor: Regina Menezes, Ph.D, IBET/ITQB-UNL
Co-Supervisor: Ana Matias, Ph.D, IBET/ITQB-UNL
December 2015
Master in Biochemistry for Health

2. Phytochemicals
2
o natural compounds
o found in fruits, vegetables and grains
o many used as pigments
o long use in traditional medicine
o potential protective activity for human health
Angel Nivya et al. 2012; Andlauer & Fürst 2002
Phytochemical food sources
(http://www.exoduswellness.org)

3. 3
Betaxanthins (BX)
λ → 480nm
Betacyanins (BC)
λ → 540nm
o water-soluble nitrogen-containing
pigments
o stability (pH 3-7)
o bioactive natural compounds
- antiviral, antimicrobial, antioxidant and
anti-inflammatory activity
- chemopreventive
cyclo-Dopa
amino acids
Betanin Indicaxanthin
Betalains
Betalains
Delgado-Vargas et al. 2000; Slavov et al. 2013
(Nakatsuka et al. 2013)

4. (Poly)phenols
4
o natural compounds
o great structural diversity
o beneficial properties for human health against
- cardiovascular diseases
- type 2 diabetes
- chronic inflammation
- neurodegenerative diseases
(Liu 2013)
Total phenolic content of common fruits and vegetables
Flavonoid general structure
(http://www.rdchemicals.com)
Opuntia spp. fruits
Pace et al. 2014: Del Rio et al.2012

5. Neurodegenerative diseases
5
Accumulation of
abnormal proteins
Neuronal death
Chen & Zheng 2012; Narayan et al. 2014; Wyss-Coray & Mucke 2002
0
10
20
30
40
2000 2050
Cases(×106)
Year
Prevalence of neurodegenerative diseases
- Parkinson´s Disease (PD)
- Amyotrophic Lateral Sclerosis Disease (ALS)
- Huntington´s Disease (HD)
- Alzheimer´s Disease (AD)
Cellular stress
InflammationNeurodegeneration
Disrupted
homeostasis

6. Saccharomyces cerevisiae as a model organism
6
o easy genetic manipulation
o central pathways of eukaryotic cell biology
o reproduce basic processes taking place
upon accumulation of abnormal proteins in
mammalian cells
o relevant system suitable for molecule
screening
(http://www.gettyimages.pt)
Yeast cells
Khurana & Lindquist 2010; Duina et al. 2014

7. 7
Yeast models
Yeast model of neurodegenerative diseases
Yeast inflammation model of Crz1/NFAT activation
PD – Parkinson´s disease; ALS – Amyotrophic Lateral Sclerosis´s disease; HD – Huntington´s disease; AD – Alzheimer´s disease; Crz1 – Calcineurin responsive zinc finger;
NFAT - Nuclear Factor of Activated T cells; CDRE - calcineurin dependent response element
(Garcia et al. 2015, in press)

8. Objectives
8
 Obtain extracts rich in phytochemicals (betacyanins, betaxanthins and
(poly)phenols) from three natural matrixes (beetroot, purple and yellow Opuntia
spp. fruits)
 Evaluate the bioactivity of extracts rich in phytochemicals
• for neuroprotective activity against Parkinson´s, Amyotrophic Lateral Sclerosis,
Huntington´s and Alzheimer's
• for anti-inflammatory activity
1
2

9. Red beetroot extracts
9
(Swamy et al. 2014)
60 ⁰C
84 min
405 rpm
acidified H2O
n = 3
matrix/volume ratio 1:311
pH 3, 5 and 7
freeze-dried
matrix
matrix/volume ratio 1:311
pH 7.1
(no acidification)
fresh matrix
matrix/volume ratio 1:10
pH 5
freeze-dried
matrix
BC – Betacyanins; BX – Betaxanthins
0
100
200
300
400
500
3 5 7
mg.100g-1dryextract
pH
BC
BX
0
100
200
300
400
500
7,1
mg.100g-1dryextract
pH
BC
BX
0
100
200
300
400
500
5
mg.100g-1dryextract
pH
BC
BX
fresh beetroot – low
reprodutibility
(Swamy et al. 2014)
pH 5 - higher content in
betalains
larger scale – high
extraction efficiency

10. Purple Opuntia spp. extracts
10
(Prakash Maran et al. 2013)
matrix/volume ratio 1:193
405 rpm
acidified H2O
n = 3
pH 5
42 ⁰C
84 and 115 min
pH 6.9
42 ⁰C
115 min
Betaxanthins – undetectable levels
0
100
200
300
400
500
6,9
mg.100g-1dryextract
pH
0
100
200
300
400
500
5mg.100g-1dryextract pH
84 min
115 min
BC – Betacyanins; BX – Betaxanthins
**
**
**

11. Yellow Opuntia spp. extracts
11
pH 6.9
matrix/volume ratio 1:10
84 min
405 rpm
acidified H2O
n = 3
(Prakash Maran et al. 2013)
Low content in betalains
BC – Betacyanins; BX – Betaxanthins
0
5
10
15
20
25
mg.100g-1dryextract
BC
BX
60 ⁰C40 ⁰C 50 ⁰C

12. 12
BC – Betacyanins; BX – Betaxanthins
higher contents in betacyanins and
betaxanthins
0
50
100
150
200
250
300
350
400
450
mg.100g-1dryextract
BC
BX
Yellow
Opuntia spp. extract
Beetroot
extract
Purple
Opuntia spp. extract
Betalain contents of the three matrixes

13. Beetroot extract
13
Evaluate anti-inflammatory activity
using Crz1 activation yeast model
BC – Betacyanins; BX – Betaxanthins; TPC – Total Phneolic Content; GAE – Gallic Acid Equivalents; ORAC - Oxygen radical absorbance capacity; TE – Trolox Equivalents;
PD – Parkinson´s Disease; ALS – Amyotrophic Lateral Sclerosis´ Disease; HD – Huntington´s Disease; AD – Alzheimer´s Disease; Crz1 – Calcineurin responsive zinc finger
Evaluate protective activity using
yeast model for neurodegenerative
diseases:
PD, ALS, HD and AD
BC → 390 mg. 100 g-1 dry extract
BX → 301 mg. 100 g-1 dry extract
TPC → 945 mg GAE.100 g-1 dry extract
ORAC → 16519 µg TE.100 g-1 dry extract

14. 14
Yeast model of neurodegenerative diseases
Experimental procedure
24 h
Cells incubated in raffinose
media
30 ⁰C
200 rpm
24 h
Cells incubated in
glucose or
galactose media
+ extract
24 h
Growth curveON
OFF
0,000
0,100
0,200
0,300
0,400
0,500
0,600
0,700
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Control (Glucose)
Model (Glucose)
Control (Galactose)
Model (Galactose)

15. Beetroot extract
15
Sugar interference
on cells growth ?
0,000
0,100
0,200
0,300
0,400
0,500
0,600
0,700
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Control
0.4% (v/v)
3.1% (v/v)
12.5% (v/v)

16. Solid Phase Extraction (SPE)
Beetroot extract
16
Experimental Procedure:
Column C-18 (2 g/12 mL)
 Conditioning the column
- Wash the column
15 mL of 0.5% CH3COOH/ACN
- Equilibrate
15 mL of 0.5% CH3COOH/H2O
 Sample loading
 Washing
15 mL of 0.5% CH3COOH/H2O
 Elution
6 mL of 2% CH2O2/CH3OH(http://www.intechopen.com)
SPE schematic procedure
Conditioning Sample loading Washing Elution
CH3COOH – acetic acid; ACN – acetonitrile; H2O – water; CH2O2 – formic acid; CH3OH – metanol

17. 0
100
200
300
400
500
600
700
800
Beetroot
extract
Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6
mg.L-1
BC
BX
TPC
SPE beetroot fractions characterization
17
BC – Betacyanins; BX – Betaxanthins; TPC – Total Phenolic Content
purified
fraction
Sample loading Washing Elution
g.L-1

18. Bioactivity assays to evaluate potential neuroprotective activity
using the purified fraction
18
% (v/v)
purified
fraction
Phytochemicals
TPC
(µg GAE.mL-1)
Betacyanins
(µg.mL-1)
Betaxanthins
(µg.mL-1)
0.8 1.96 1.41 0.26
1.6 3.91 2.81 0.51
3.1 7.81 5.63 1.03
6.25 15.63 11.25 2.06
12.5 31.25 22.5 4.11
25 62.5 45 8.22
50 125 90 16.44
TPC – Total Phenolic Content; GAE – Gallic Acid Equivalents
Toxicity
Potential
neuroprotective
activity
%(v/v) – extract volume/(extract volume + cell volume)

19. 19
Parkinson´s Disease (PD)
3.1% and 6.25% purified fraction
tested for potential neuroprotective
effect on PD Model
0,000
0,100
0,200
0,300
0,400
0,500
0,600
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Toxicity assay
Control
3.1% (v/v)
6.25% (v/v)
12.5% (v/v)
25% (v/v)
50% (v/v)
0,000
0,100
0,200
0,300
0,400
0,500
0,600
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Protective activity
Control
PD Model
3.1% (v/v)
6.25% (v/v)

20. 20
Amyotrophic Lateral Sclerosis´ Disease (ALS)
3.1% and 6.25% purified fraction
tested for potential neuroprotective
effect on ALS Model
0,000
0,100
0,200
0,300
0,400
0,500
0,600
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Toxicity assay
Control
3.1% (v/v)
6.25% (v/v)
12.5% (v/v)
25% (v/v)
50% (v/v)
0,000
0,100
0,200
0,300
0,400
0,500
0,600
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Protective activity
Control
ALS Model
3.1% (v/v)
6.25% (v/v)

21. 21
Huntington´s (HD) and Alzheimer´s (AD) Diseases
1.6% and 3.1% purified fraction
tested for potential neuroprotective
effect on HD Model and AD Model
0,000
0,100
0,200
0,300
0,400
0,500
0,600
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Toxicity assay
Control
0.8% (v/v)
1.6% (v/v)
3.1% (v/v)
6.25% (v/v)
12.5% (v/v)
0,000
0,100
0,200
0,300
0,400
0,500
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Protective activity
Control
AD Model
1.6% (v/v)
3.1% (v/v)
0,000
0,100
0,200
0,300
0,400
0,500
0 2 4 6 8 10 12 14 16 18 20 22 24
OD600
time (h)
Protective activity
Control
HD Model
1.6% (v/v)
3.1% (v/v)

22. Anti-inflammatory activity using yeast model of
Crz1 activation
Experimental procedure
22
Overnight
Cells incubation in
synthetic complete
supplement media
Cells incubated at 30 ⁰C
200rpm
8-10 h
Cells diluted 1:10 and
incubated at 30 ⁰C
200rpm
2 h
Cells*
+ extract
measurement of β-
galactosidase activity
Cells diluted and
incubated overnight
at 30 ⁰C
200rpm
• MnCl2 inducer of Crz1 activity
• FK506 inhibitor of calcineurin and Crz1 activity

23. 0
500
1000
1500
2000
2500
3000
3500
4000
Control FK506 0.06%
(v/v)
0.31%
(v/v)
0.63%
(v/v)
1.25%
(v/v)
3.13%
(v/v)
6.25%
(v/v)
Millerunits(β-galactosidadeactivity)
Anti-inflammatory activity
Purified fraction
23
* compared to Control
TPC – Total Phenolic Content; GAE – Gallic Acid Equivalents
% (v/v)
purified
fraction
Phytochemicals
TPC
(µg GAE.mL-1)
Betacyanins
(µg.mL-1)
Betaxanthins
(µg.mL-1)
0.06 10 7.2 1.3
0.31 25 18 3.3
0.63 50 36 6.6
1.25 100 72 13.2
3.13 250 180 33
6.25 500 360 66
***
β-galactosidade activity is
reduced in the presence of
purified fraction
↓
%(v/v) – extract volume/(extract volume + cell volume)
***
***
***
***
***
attenuation of
Crz1 activation

24. 0
500
1000
1500
2000
2500
3000
3500
Control FK506 0.06%
(v/v)
0.16%
(v/v)
0.31%
(v/v)
0.62%
(v/v)
1.56%
(v/v)
3.13%
(v/v)
6.25%
(v/v)
Millerunits(β-galactosidadeactivity)
24
Anti-inflammatory activity
Betanin standard
No effect on Crz1
modulation
% (v/v)
Betanin Standard
(BC)
0.06% 0.16% 0.31% 0.62% 1.56% 0.31% 6.25%
Betacyanins (BC)
(µg.mL-1)
10 25 50 100 250 500 1000
%(v/v) – extract volume/(extract volume + cell volume)
***
* compared to Control

25. Conclusions
25
o Betalain extraction was optimized comparing with literature, for beetroot and
purple Opuntia spp. fruits
- for beetroot, pH 5 was found to be optimum
- for purple Opuntia spp. fruits, pH 6.9 allowed higher content extracted
o Beetroot richer in betalains and rich in (poly)phenols
o No protective activity observed for the purified fraction tested on the yeast
models of neurodegenerative diseases ( PD, ALS, HD, AD)
o Potential anti-inflammatory activity of purified fraction on the yeast model of
Crz1 activation
o Potential anti-inflammatory activity does not rely only on betanin

26. Future work
26
o Chemical characterization of beetroot extract
o Test (poly)phenol and betaxanthin standards to evaluate the potential bioactivity
of these compounds
o Validation on mammalian cell lines and primary cell cultures
o Bio-guided fractionation of beetroot extract
F1
F2
F3
F 2.1
F 2.2
F 2.3
.
.
.
.
.
.
…

27. Acknowledgments
27
Disease and Stress Biology group:
Ana Margarida Nunes
Ana Vanessa Oliveira
Andreia Almeida
Andreia Gomes
Carolina Jardim
Claudia Santos
Gonçalo Garcia
Inês Costa
Ines Figueira
Joana Pereira
Joel Guerra
Paula Pinto
Regina Menezes
Rita Ramos
Nutraceuticals and Delivery group:
Agostinho Alexandre
Ana Nunes
Ana Matias
Ana Roda
Anyse Pereira
Arturo Alvarez-Bautista
Catarina Duarte
Catarina Maia
Cátia Carmo
Carolina Pereira
Daniel Lopes
Inês Silva
Joana Guerreiro
Joana Poejo
Liliana Rodrigues
Luís Martins
Maria João Pereia
Rut Romero
Teresa Serra
Vanessa Gonçalves
iNOVA4Health -
UID/Multi/04462/2013

28. Thank you!
28