Ortiz D1, Pico S1, Pachón H1, Chureeporn C2, Failla M2.1 Centro
Internacional de Agricultura Tropical, CIAT, Palmira, Colombia.
2 Department of Human Nutrition, Ohio State University, Columbus
OH, USA.
Vitamin A is an essential nutrient needed in small amounts for
the normal function of the visual system, and maintenance of cell
function for growth, epithelial integrity, red blood cell
production, immunity and reproduction.
Plant foliage is a source of carotenoids and the leaves from
different crops such as cassava arewidely consumed in some regions
of Africa, Asia and South America as a source of protein, minerals,
fiber, vitamins and essential amino acids (Siqueira et al.
2007).
Foliar extracts (FE) are prepared from cooked leaves to produce
a concentrate. Although FE, likeleaves, are high in nutrients
including carotenoids (http://www.soynica.org.ni/), the efficiency
of absorption and utilization of these nutrients from such extracts
remains largely unknown.
Introduction
1. The quantity of carotenoids partitioning in micelles digested
from leaves and foliar extracts from cassava, beans and sweet
potato will differ.
2. The bioaccessibility of carotenoids in extracts from food
plants will depend in part on type of plant matrix.
Hypotheses
Materials and Methods
Cassava Gua 86 77 Palmira
Bean CAL 96 30 Darien
Sweet potato 44086 40 Palmira
Source of leaves. Leaves without evident structural, insect and
microbiological damage were harvested from the top of the plant.
Varieties of food plants from which the leaves were harvested, the
age of the plant, and the location where it was planted are listed
in Table 1.
Table 1. Crop varieties used in the study
Foliar extract preparation. Foliar extracts were prepared
according to methods developed by Association for the Promotion of
Leaf Concentrate in Nutrition (APEF)
(http://www.nutrition-luzerne.org/index.htm) and Asociación de Soya
de Nicaragua (SOYNICA) (http://www.soynica.org.ni).Extraction of
carotenoids from leaves and foliar ex tracts. Carotenoids were
extracted following the method suggested in the literature
(Rodriguez-Amaya, 2001; Rodriguez-Amaya et al. 2004), except that
0.1 g of leaves and FE samples were weighed and the carotenoid
extracts were saponified (NaOH 30% w/v in MeOH, 15 min,
37°C).Extraction of carotenoids from digesta, micelle fra ction and
quantification by HPLC. Carotenoids from digesta and the micelle
fraction were extracted (Figure 1) and quantified by HPLC as
described in Thakkar et al. 2007.
Figure 1. Graphical representation of in vitro digestion model
to assess carotenoid bioaccessibility
Pepsin
Statistical AnalysisResults from 6 independent digestions of
leaves from each plant food and FE were analyzed for statistically
significant differences using SPSS Release 15.0 for Windows (SPSS
Inc., Chicago, IL). Paired t-tests were used to test the efficiency
of micellarization (concentration of carotenoids in micelles). The
Dunnett post-hoc test was performed to compare all FE data against
alfalfa FE. For non-normalized data, the post-hoc Wilcoxontest was
used. Statistically significant differences were those with P <
0.05.
Panel I. In sweet potato, the micellarization efficiency of
carotenoids between leaves and FE was not significantly different
(P > 0.05), except for all-trans-LUT. Qualitatively, it appeared
that the micellarization efficiency of sweet potato FE for the
trans isomers of LUT and ZEA was higher than the cis isomers, and
the opposite for BC. Panel III. In bean, the micellarization
efficiency of carotenoids between leaves and FE was not
significantly different (P > 0.05). Qualitatively, the
micellarization efficiency for trans isomers of LUT and ZEA
appeared higher than for cis isomers.Panels II & IV. A higher
concentration of carotenoids in micelles was found for FE of sweet
potato (all-trans-LUT, all-trans-ZEA, all-trans-BC and 9-cis-BC)
and bean (all-trans-LUT and cis-LUT) with respect to their leaves
(P < 0.05). The micelles in leaves and FEs of sweet potato and
bean were composed mainly of all-trans-LUT (non-pro-VA) followed by
all-trans-BC.Conclusion: The bioaccessibility of carotenoids in
sweet potato FE and bean FE was higher than their leaves for
all-trans-ZEA, all-trans-BC and 9-cis-BC in sweet potato and
all-trans-LUT and cis-LUT in bean.
Left panel: The micellarization efficiency of carotenoids in
cassava, bean and sweet potato FE were not significantly different
with respect to alfalfa FE (P > 0.05) except for all-trans-BC in
cassava and sweet potato, and all-trans-ZEA in sweet potato.
Right panel: Sweet potato and bean FE had a higher concentration
in the micelle of 6 and 4 isomers, respectively, in relation to
alfalfa FE.
Conclusion: Sweet potato FE had a greater bioaccessibility than
alfalfa FE with respect to all-trans-LUT, cis-ZEA and 9-cis-BC.
Data are mean ± SD values for 6 samples independently digested.
Means without letter above the columns not differ significant and
notcommon differ significantly (P < 0.05
Hypothesis 1 confirmed: The quantity of some carotenoids
partitioning in micelles generated during simulated digestion of
leaves vs. foliar extracts from cassava, beans and sweet potato
will differ.
Hypothesis 2 confirmed: The bioaccessibility of carotenoids
during digestion of FE depends in part on the food matrix.
Conclusions
Acknowledgements
The authors acknowledge the following units at CIAT and the
AgroNatura Science Park: Nutrition Quality Laboratory, Clayuca,
Genetic Improvement of Beans Program, Fundación para la
Investigación y el DesarrolloAgrícola (FIDAR) as well as the
funding provided by the Monsanto Fund, AgroSalud (CIDA7034161) and
CIAT. The authors also thank the Department of Human Nutrition at
Ohio State University and APEF.
LUT, lutein; ZEA, zeaxanthin; BC, β-carotene; VA, vitamin A
Abbreviations
Variety Age (days) Colombian municipality
References
1. Extracto foliar. Valores nutritivos. [Consulted, 06 november
2009], in http://www.soynica.org.ni/ext_vnutri.php
2. Leaf concentration. Domestic method of making leaf
concentration. [Consulted, 06 november 2009], In
http://www.nutrition-
luzerne.org/anglais/pdf/Domesticmethod%20English.pdf
3. Rodriguez-Amaya, DB. A Guide to Carotenoid Analysis in Foods.
Washington DC: ILSI Press, 2001.
4. Rodriguez-Amaya DB. and Kimura M. HarvestPlus Handbook for
carotenoid analysis. HarvestPlus Technical Monograph 2. Washington,
DC and Cali.
International Food Policy Research Institute (IFPRI) and
International Center for Tropical Agriculture (CIAT), 2004
5. Siqueira EMdA, Arruda SF, de Vargas RM, de Souza EMT. 2007.
β-Carotene from cassava (Manihot esculenta, Crantz) leaves improves
vitamin A status in
rats. Comp Biochem Physiol Part C 146:235–40.
6. Thakkar SK, Maziya-Dixon B, Dixon AG, Failla ML. 2007.
Beta-carotene micellarization during in vitro digestion and uptake
by Caco-2 cells is directly
proportional to beta-carotene content in different genotypes of
cassava. J Nutr137:2229–33
Data are means ± SD for 6 independently digested samples. Means
without letters above the columns are not significantly different.
Presence of different letters above bars for digested leaves and FE
indicate that means are significantly different (P < 0.05).
Evaluation of carotenoid bioaccessibility in bean,
cassava,Evaluation of carotenoid bioaccessibility in bean,
cassava,
sweet potato and alfalfa foliar extractssweet potato and alfalfa
foliar extracts
α- Amylase
Synthetic Saliva
HCl 1M
Bile, CEL
Pancreatin, Lipase
NaHCO3 1M(0.22 µm filter)
Oral Digestion
Micelles
Small Intestine Digestion
Gastric Digestion
FE, vehicle: Yogurt
Centrifugation
(Homogenization)(pH = 6.8, 10 min, 370C)
(5000 g, 45 min, 40 C ) (pH = 6.5, 2 h, 370C)
(pH = 2.5, 1 h, , 370C)
FE: Foliar extract
CEL: Carboxyl ester lipase
Step 1
Filter Aqueous Fraction
BC
ZEA
LUT
Data are means ± SD for 6 independently digested samples. Means
without letters above the columns are not significantly different.
Presence of different letters above bars for digested leaves and FE
indicate that means are significantly different (P < 0.05).
a
b
a b a b
a
ba
b
a
b ab
ab
III
a
b
a b
III IV
b a
0
100
200
300
400
500
600
700
800
al l trans cis all trans cis 13-cis all trans 9-cis
LUT ZEA BC
Tota
l car
ote
no
ids
in m
ice
lle
s (µ
g/g)
Cassava Bean Sweet Potato Alfal fa
a
b
a
b
a
ab
b
b
ab
bab
b
bab
ND
ND: Not Detectable
Alfalfa
0
20
40
60
80
100
all trans ci s al l trans ci s 13-ci s al l trans 9-ci s
LUT ZEA BC
Eff
icie
ncy
of
Mic
elle
lari
zati
on
(%
)
Cassava Bean Sweet Potato Al falfa
ND
a
b
b
b
a
b
a
b
ND: Not detectable ND: Not detectable
Left panel: No significant difference was found in the
efficiency of carotenoid micellarization between leaves and FE for
any of the 7 isomers studied (P > 0.05). Qualitatively, the
efficiency of micellarization for trans isomers of LUT and ZEA
appears higher than for cis isomers, and the opposite for BC. Right
panel: The micelles of cassava leaves and FEs were composed mainly
of all-trans-LUT (non-pro-VA) followed by all-trans-BC. Conclusion:
The bioaccessibility of carotenoids (assessed by simultaneously
evaluating micellerizationefficiency and carotenoid concentration
in micelles) in cassava FE was lower than cassava leaves for
all-trans-LUT, cis-ZEA and 9-cis-BC, because the micelle carotenoid
content in the FE of these was reduced (P < 0.05)
Data are means ± SD for 6 independently digested samples. Means
without letters above the columns are not significantly different.
Presence of different letters above bars for digested leaves and FE
indicate that means are significantly different (P < 0.05).
Bioaccessibility of carotenoids in leaves and foliar extract of
cassava
Comparison of bioaccessibility of carotenoids in foliar extract
of cassava, bean and sweet potato
with respect to alfalfa foliar extract
Bioaccessibility of carotenoids in leaves and foliar extract of
sweet potato and bean
(µg/
g)
ab
