International Journal of Food Sciences and Nutrition

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Nutritive evaluation of a non-conventional leafy

vegetable (Pereskia aculeata Miller)

Cristina Y. Takeiti, Graziella C. Antonio, Eliana M. P. Motta, Fernanda P.

Collares-Queiroz & Kil J. Park

To cite this article: Cristina Y. Takeiti, Graziella C. Antonio, Eliana M. P. Motta, Fernanda P.
Collares-Queiroz & Kil J. Park (2009) Nutritive evaluation of a non-conventional leafy vegetable
(Pereskia�aculeata Miller), International Journal of Food Sciences and Nutrition, 60:sup1, 148-160,
DOI: 10.1080/09637480802534509

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International Journal of Food Sciences and Nutrition,
August 2009; 60(S1): 148160

Nutritive evaluation of a non-conventional leafy

vegetable (Pereskia aculeata Miller)

CRISTINA Y. TAKEITI1, GRAZIELLA C. ANTONIO1,

ELIANA M. P. MOTTA1, FERNANDA P. COLLARES-QUEIROZ2 &
KIL J. PARK1
1
School of Agricultural Engineering, University of Campinas (UNICAMP), Campinas, Sao
Paulo, Brazil, and 2School of Chemical Engineering, University of Campinas (UNICAMP),
Campinas, Sao Paulo, Brazil

Abstract
Pereskia aculeata Miller is a native cactus that can be found in Brazil and is called ‘ora-pro-nobis’
(OPN). Many people from poor communities consume the dark green leaves of OPN as a
vegetable. The objective of the present work was to evaluate the nutritional components in
terms of proximate composition, minerals, vitamins, protein content and their in vitro protein
digestibility. OPN leaves showed remarkable levels of total dietary fiber (39.1% dry basis),
minerals (calcium, magnesium, manganese and zinc) and vitamins (vitamin A, vitamin C and
folic acid). Among amino acids, tryptophan was the most abundant (20.5% of the total amino
acids) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed small peptides,
inferior to 6.5 kDa, and four major bands (61 kDa, 53 kDa, 33 kDa, and 15 kDa). The protein
digestibility corrected amino acid score showed the lowest value of sulfur-amino acids (Met

Cys). OPN leaves could be considered a good source of minerals, vitamins and amino acids, and
may serve as a potential functional ingredient.

Keywords: Chemical composition, dietary fiber, minerals, vitamins, amino acids, protein
digestibility

Introduction
Pereskia aculeata Miller is a native cactus that can be found in American tropics, such
as the southern region of the United States (Florida), and in Brazil. In Brazil, this
cactacea is known as ‘ora-pro-nobis’ (OPN) and is widely distributed between the
states of Bahia and Rio Grande do Sul. Typical cacti are green, leafless stem-succulent
plants covered in numerous spines. However, members of the genus Pereskia are
broad-leaved trees and shrubs. They are clearly members of the cactus family, due to
the presence of spine-bearing areoles, floral cups with leaf-bearing nodes and
numerous perianth segments (Butterworth and Wallace 2005). This plant is
characterized by indigenous and non-seasonal development. Many people consume
OPN’s dark green leaves as a vegetable in underdeveloped areas (Dayrell and Vieira
1977). In addition, P. aculeata exhibits cheerful flowers that attract a lot of bees, which

Correspondence: Kil Jin Park, School of Agricultural Engineering, University of Campinas (UNICAMP),
C.P. 6011, 13083-875, Campinas, Brazil. Tel: 55 19 3521 1076. Fax: 55 19 3521 1010.
E-mail: kil@agr.unicamp.br

ISSN 0963-7486 print/ISSN 1465-3478 online # 2009 Informa UK Ltd

DOI: 10.1080/09637480802534509
 Nutritive evaluation of native cactus 149

in turn recommends them as honey producers. The protein content of the leaves was
reported as high (Almeida-Filho and Cambraia 1974; Albuquerque et al. 1991) when
compared with other vegetables, such as common corn (710% w/w) or beans
(1820% w/w). The levels of essential amino acids, except methionine, were found to
be higher than the minimum amount recommended by the FAO as necessary for adult
human consumption (Dayrell and Vieira 1977) and were also found to present an
excellent amino acid profile (Albuquerque et al. 1991). An important result found was
that lysine constitutes 56% w/w of the total protein content, and, considering its
essentiality in animal nutrition and limitation in the cereal grain, this protein could
complement lysine-deficient cereal-based foods. The fiber content is also elevated
according to Almeida-Filho and Cambraia (1974), Dayrell and Vieira (1977) and
Albuquerque et al. (1991), but these studies neither identified the dietary fiber in
terms of soluble and insoluble fractions nor determined the masses of the polypeptides
to correlate their digestibility and their biological relevance.
The ash content of P. aculeata are reported as approximately 14% and 22% by
Almeida-Filho and Cambraia (1974) and by Albuquerque et al. (1991), respectively,
and calcium was the major component found, followed by phosphorous and manganese.
Indigenous vegetables represent inexpensive, high-quality nutrition sources for the
underprivileged population, especially where malnutrition is widespread (Odhav et al.
2007; Bhat and Sridhar 2008), and therefore P. aculeata is agriculturally, economic-
ally and technologically important as a non-conventional food source that can be
studied more accurately considering that Raju et al. (2007) reported a high content of
carotenoids in green leafy vegetables, including b-carotenes and a-carotenes, lutein,
and zeaxanthin. In addition, it is known that vitamin A deficiency and age-related
macular degeneration are due to inadequacy of provitamin A and macular pigment in
the diet, and are accepted as serious public health problems among children and
adults in developing countries. The objective of this work was to evaluate the
nutritional component in terms of proximate composition, minerals, vitamins,
protein content and their in vitro protein digestibility. These results will permit
evaluating this novel resource, allowing their application in foods and improving the
nutritional value.

Materials and methods

Plant material
Fresh, green, tender leaves of OPN were harvested in a greenhouse located at
Serra da Cantareira, Mairiporã City, Sao Paulo, Brazil (23819?12ƒS latitude and
46835?18ƒWGr longitude). After harvesting, they were washed under running water to
remove stems and foreign materials. In order to preserve their biological protein value,
the leaves were drained, cut and frozen at 188C and were subsequently freeze-dried
in equipment model Modulyo (Edwards, Sussex, UK) at 508C and 13.3 Pa for
72 h. The dried leaves were ground into a fine powder (PL) in a domestic blender and
stored at room temperature in a desiccator for determination of the proximate
composition, amino acid composition, molecular weight profile and in vitro protein
digestibility. To prevent the possible spoil, fresh harvested leaves were collected
without stems, washed with water, drained and ground for 2 min in a blender, and
were immediately submitted to mineral, vitamin and dietary fiber analysis.
150 C. Y. Takeiti et al.

Proximate composition
The moisture content of the PL was determined gravimetrically in a vacuum oven at
708C and 3,333 Pa until it reached a constant weight. The levels of crude proteins
(determined by the Kjeldhal method, N 6.25), lipids (Soxlet method), crude fibers
and ash were determined in accordance with the standard methods of the AACC
(1995). All analyses were conducted in triplicate. The enzymaticgravimetric method
proposed by Prosky et al. (1984) and Horwitz (2005) was used to determine total
dietary fiber. The total dietary fiber was calculated as the sum of soluble dietary fiber
and insoluble dietary fiber after correcting for ash and undigested protein. Dietary
fiber was expressed as grams per 100 g sample on a wet weight basis.

Mineral content
The levels of mineral elements (calcium, magnesium, potassium, phosphorus,
manganese, zinc, iron, boron and copper) of the fresh harvested leaves were
determined according to Sarruge and Haag (1974), using a flame analyzer and
an atomic absorption spectrophotometer (model 3110; Perkin Elmer, Waltham,
Massachusetts, USA) after ash content determination by incinerating at 5508C for
2 h. Boron was determined by a colorimetric method using a digital spectro-
photometer (model B 34211; Micronal, Sao Paulo, Brazil).

Amino acid composition

The amino acid composition of OPN leaves was determined, as described by White
et al. (1986), using an automated amino acid analyzer (model PCX3100 Post-Column
Reaction Module; Pickering Laboratories, Mountain View, California, USA) with a
Spectra System UV2000 detector and a Spectra System P4000 injector pump
(Mountain View, California, USA). A known quantity of PL was hydrolyzed with 6 N
HCl at 1108C for 24 h. After cooling, HCl was eliminated in a rotary evaporator (Büchi
Labortechnik AG, Postfach, Switzerland) and the derivatization was realized using a
methanol
MiliQ water
triethylamine solution. The amino acid profile, except for
tryptophan, was determined using a C18 column, 3.9 mm (inner diameter)150 mm
(length) (Nova Pak† ; Waters, Milford, Massachusetts, USA) and 20 ml injection
volume. Standard amino acids (Pierce Biotechnology, Inc., Rockford, Illinois, USA)
were used to construct a calibration curve. The tryptophan content was determined,
according to Spies (1967), using a DU 640 spectrophotometer (Beckman Coulter,
Fullerton, California, USA). The essential aminoacids (EAA) scores are the result of
dividing grammes of EEA in 100 g test protein by grammes of EEA in 100 g FAO/WHO
(1990) reference pattern for children between 2 and 5 years old, employing the formula
(essential amino acid [EAA] score) presented in Equation (1):
EAA score  100[g EAA in 100 g protein test]=[g EAA in 100 g FAO=WHO
reference pattern (1990)] (1)

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis

The molecular weight profile for the protein fractions of OPN leaves and di-
gested protein were determined using sodium dodecyl sulfate-polyacrylamide gel
 Nutritive evaluation of native cactus 151

electrophoresis (SDS-PAGE). A known quantity of PL (10 mg protein/ml) was added

to the buffer containing 0.0625 M TrisHCl, pH 6.8, 2% (w/v) SDS and 5% (v/v) b-
mercaptoethanol. Two different kinds of systems to reveal protein fractions were used.
First, the SDS-PAGE/glycine system (Laemmli 1970), with 12% resolving gel and 4%
stacking gel was used for the OPN sample. The gel thickness was 0.75 mm. The gels
were run in MiniProtean-3 Cells (Bio-Rad Laboratories, Hercules, California, USA).
The conditions were set at 120 V constant, and gels were run for 90 min. The
molecular weights of the protein were determined using Bio-Rad 16-0304 standards
containing the following: 97.4 kDa, 66.2 kDa, 45 kDa, 31 kDa, 21.5 kDa, and
14.4 kDa. Second, the SDS-PAGE/tricine system (Schägger and Jagow 1987), with
15% resolving gel (total concentration of 15% with a cross-linking of 4%), 10% spacer
gel (10% total concentration; 3% cross-linking) and 4% stacking gel (4% total
concentration; 3% cross-linking) was used for OPN-digested sample. The gel
thickness was 1.5 mm. The gels were run in MiniProtean-3 Cells (Bio-Rad
Laboratories). The conditions were set at 75 V constant, and gels were run for 3
4 h. The molecular weights of the protein were determined using Sigma-Aldrich
M3546 standards (St Louis, MO, USA) containing the following: 26.6 kDa, 17 kDa,
14.4 kDa, 6.5 kDa, and 3.5 kDa.
The estimated molecular weights, as well as the relative content, were performed
using a JX 330 densitometer (Sharp Electronics Co., Mahwah, New Jersey, USA) in
combination with Image Master Software (Amersham Pharmacia, Little Chalfont,
Buckinghamshire, UK).

In vitro protein digestibility

In vitro protein digestibility was determined by the method of Akeson and Stahmann
(1964). The PL (500 mg) was digested with swine pepsin (P7012; Sigma-Aldrich) at
an enzyme/substrate ratio (w/w) of 1:16.6, in triplicate. The suspension was incubated
at 378C for 3 h in a shaker water bath. The pH of the suspension was adjusted to 7 and
then was digested once more with swine pancreatin (P1625; Sigma-Aldrich) at an
enzyme/substrate ratio (w/w) of 1:12.5, at 378C for 24 h. The reaction was stopped
with Trichloroacetic acid (TCA) 30%, and the TCA-soluble nitrogen in the
supernatants was determined by the micro-Kjeldhal method using a conversion factor
of 6.25. In vitro protein digestibility was calculated according to Equation (2):

In vitro protein digestibility (%)

100[protein in digest]=[protein in OPN leaves] (2)

The protein digestibility corrected amino acid score (PDCAAS) of EAA was
calculated based on EAA requirements for children between 2 and 5 years old (FAO/
WHO 1990), as in Equation (3):

PDCAAS (%)([amino acid content in food protein]

[true digestibility])=[amino acid content of reference pattern] (3)

The digestion process was also analyzed by SDS-PAGE systems, as described

before.
152 C. Y. Takeiti et al.

Vitamin contents
Vitamin levels were measured using fresh OPN leaves that were collected and
immediately submitted to analysis. b-Carotene was extracted with acetone and ether,
separated by open column, as previously reported (Carvalho et al. 1992) and
evaluated by high-performance liquid chromatography on a chromatographer with a
RP-18 (5 mm) packed column (ID 4.6 mm 250 mm) and a visual spectro-
photometric detector (454 nm). Quantification was carried out by external standard,
and the purity of the standard was checked before use. The content of vitamin C
(ascorbic acid) was determined using the titration method (Ranganna 1977). HPO3
solution (3%) was used for titration of ascorbic acid after a quantitative reduction of
2.6-dichlorophenol-indophenol dyestuff by ascorbic acid. Folic acid was determined,
according to Catharino (2000). Briefly, the folic acid present in the fresh OPN leaves
was extracted by potassium hydroxide (0.1 mol/l) with ultrasonic vibration, and the
clean up of the extract was carried out by the addition of trichloroacetic acid (350ml).
In the chromatographic process, a Microsorb C18 column (ODS-2, 5 mm, 150 mm 
4.6 mm) with a Bondesil C18 (5 mm, 10 mm4.6 mm) guard column (Varian Inc.,
Palo Alto, California, USA) was used. Gradient elution was performed with acetate
buffer (0,166 mol/l) and acetonitrile (90:10 v/v), changing to acetonitrile and acetate
buffer (76:24 v/v) after 8.5 min. The column re-equilibration time before each new
injection was 5 min. Detection was obtained with an ultraviolet detector (290 nm) and
quantified using external standards (F-7876; Sigma-Aldrich).

Statistical analysis
Experimental data were analyzed by the Tukey Test, using the computer statistical
program Statistica 5.0 (StatSoft, Tulsa, Oklahoma, USA). Least-significant differ-
ences were computed at P 50.05.

Results and discussion

General composition
Table I presents the general composition of OPN leaves. Considering they are fresh,
green leaves, the moisture content is expected to be high (89.5%). However, the
freeze-drying process reduced the moisture content to 1.70%. Total protein, lipid, ash
and total fiber contents are similar to those related by Almeida-Filho and Cambraia
(1974) and Albuquerque et al. (1991): 2528% (dry basis) for total protein, 6.36.8%
(dry basis) for lipid fraction, 14.220.1% (dry basis) for ash and 7.79.1% (dry basis)
for total fiber, respectively.
The total dietary fiber of OPN leaves (3.8 g/100 g) is considered low when
compared with sweet potato leaves (5.9 g/100 g) but is comparable to spinach (3.5 g/
100g), which are both known as vegetables with high dietary fiber content. In
advanced countries where dietary fiber intake is insufficient in common dietary life,
their importance is highlighted in connection with diabetes, hyperlipidemia and colon
cancer (Ishida et al. 2000). The OPN leaves showed high soluble dietary fiber content
(5.2 g/100 g), similar to results related by Ishida et al. (2000) who studied two
different kinds of sweet potato leaves (6.8% and 5.8%). Thus, OPN leaves should also
be evaluated regarding physiological action of dietary fibers.
 Nutritive evaluation of native cactus 153
Table I. General composition of OPN leaves (g/100 g).

Spinacia I. batatas poir

P. aculeata L. synanthera oleracea Lepidium (sweet potato
(OPN leaves)a (chomte)b (spinach)b sativum (cress)b leaves)c

Moisture, fresh 89.590.2 82.5 90.7 89.4 87.1

leaves
Total proteind 28.490.4 (3.1)e 6.3 3.2 2.6 3.8
Lipids 4.190.3 (0.4)e 0.4 0.3 0.7 0.3
Ash 16.190.1 (1.7)e 1.7 0.7 0.7 1.9
Crude fiber 9.890.2 (1.0e 2.8 0.6 1.1 
Soluble dietary 5.2 (0.590.02)e    6.8 (0.9)e
fiber
Insoluble dietary 33.9 (3.390.07)e    39.1 (5.1)e
fiber
Total dietary fiber 39.1 (3.8 90.06)e  3.5  45.9 (5.9)e
a
Values are means in triplicate determinations. Values expressed on a dry basis, except for moisture.
b
Values refer to g/100 g raw leaves according to Salazar et al. (2006).
c
Values refer to g/100 g raw leaves according to Ishida et al. (2000).
d
6.25N g/100 g.
e
Values in parentheses are related to fresh green leaves.

Mineral content
Table II presents the mineral analysis, and indicates a high concentration of calcium
(3,420 mg/100 g), followed by magnesium (1,900 mg/100 g). Almeida-Filho and
Cambraia (1974) also reported similar results (3,400 mg/100 g and 1,500 mg/100 g,
respectively). According to Salazar et al. (2006) the high level of calcium is very
interesting, since calcium intake in rural communities is low, where animal milks are
scarce or not habitually consumed; in these cases, contributions of non-milk foods to
calcium intake could be valuable, considering that women living in theses areas have
elevated requirements because of repeated pregnancies and long lactation periods.

Table II. Mineral contents in fresh green leaves (mg/100 g).

Calcium Magnesium Potassium Phosphorus Manganese Zinc Iron Boron Copper

P. aculeata 3,420 1,900 1,632 156 46.4 26.7 14.2 5.55 1.4
(OPN
leaves)a
L. 252 75.2 417 47.3 1.5 0.5 1.9  0.4
synanthera
(chomte)b
S. oleracea 106 62 662 51  0.2 3.1  0.2
(spinach)b
L. sativum 81 27 606 76   1.3  0.1
(cress)b
I. batatas 187 79 639 68  0.8 5.4  0.4
(sweet potato
leaves)c
a
Values are expressed in triplicate determinations (wet weight basis).
b
Values refer to mg/100 g raw leaves according to Salazar et al. (2006).
c
Values refer to mg/100 g raw leaves according to Ishida et al. (2000).
154 C. Y. Takeiti et al.

Considering microelements, OPN leaves are rich sources of manganese (46.4 mg/
100 g), zinc (26.71 mg/100 g) and iron (14.18 mg/100 g). Manganese was identified
as a constituent of mitochondrial glutamine synthetase, pyruvate carboxylase and
mitochondrial superoxide dismutase, a primary enzyme in the anti-oxidative defense
system, while magnesium and zinc are known to prevent cardiomyopathy, muscle
degeneration, growth retardation, alopecia, dermatitis, immunologic dysfunction,
gonadal atrophy, impaired spermatogenesis, congenital malformations and bleeding
disorders (Chaturvedi et al. 2004).
The iron content (14.18 mg/100 g) is considered high when compared with that
reported for spinach (3.1 mg/100 g), widely considered a vegetable with a high content
of iron, and levels of OPN leaves are also superior to sweet potato leaves (5.43 mg/
100 g) (Ishida et al. 2000). However, the iron value in foods should not be evaluated
by iron content alone because the intestinal absorption rates of heme-iron and non-
heme-iron are different. In addition, 90% of the iron intake is non-heme-iron.
Furthermore, Bothwell et al. (1989) reported that 1 mg/day iron is suitable for adult
humans to maintain the daily balance of intake and excretion, and that the iron
absorption rate increases with vitamin C intake. Thus, the intake of iron from dark
green vegetables is also meaningful and OPN leaves could be considered a source of
iron.
Considering the recommended dietary allowance for minerals for adults (calcium,
1000 mg/day; copper, 900 mg/day; zinc, 10 mg/day; magnesium, 400 mg/day; man-
ganese, 7 mg/day; and iron, 8 mg/day), the OPN leaves provide high levels compared
with recommended dietary allowances for calcium, copper, zinc, magnesium,
manganese and iron. These results point out that this unexploited plant is a good
source of minerals.

Amino acid composition and evaluation

The amino acid composition (g/100 g dry weight) of OPN leaves and the EAA score
are presented in Table III. The FAO/WHO suggested requirements of essential amino
acids for 2-year-old to 5-year-old children are also included (FAO/WHO 1990). The
protein content (i.e. the sum of individual amino acids) was 26.96 g/100 g dry weight.
Among EAAs, the most abundant was tryptophan (5.52 g/100 g dry weight),
contributing 20.46% to the total amino acid content; and among non-essential amino
acids, glutamic acid showed the highest content (2.67 g/100 g dry weight), comprising
9.9% of the total amino acid content. Amino acids serve as precursors for the
synthesis of different neurotransmitters. This is the case particularly for tryptophan,
the limiting precursor of serotonin that plays an important role in the regulation of
sleep (Minet-Ringuet et al. 2004).
With regard to the nutritional quality of OPN leaves, the amino acid content was
evaluated by comparing the percentages of the essential amino acids with those of a
FAO/WHO standard protein (1990), as shown in Table III. The OPN leaves test
results had only two scores that fell below 100%, which were for two limiting amino
acids, lysine and sulfur-containing amino acids (Met
Cys). Similar results were also
reported by Ishida et al. (2000), Ayaz et al. (2006) and Wang et al. (2008), who
investigated the amino acid composition. In comparison, our results were analogous
to those reported by Almeida-Filho and Cambraia (1974) and Albuquerque et al.
(1991) for OPN leaves, except for the methionine content, which showed a low value
 Nutritive evaluation of native cactus 155
Table III. Amino acid composition, EAA score, FAO/WHO (1990) recommended allowances for pre-
school children (25 years old), in vitro protein digestibility and PDCAAS of OPN leaves.

g/100 g FAO/WHO PDCAAS

Amino acid (dry basis)a % of total EAA scoreb (1990) (%)c

Essential
Arginine 1.4490.02 5.32
Histidine 0.5990.01 2.17 114 1.9 86.50
Isoleucine 1.0790.01 3.95 141 2.8 107.0
Leucine 2.0090.02 7.40 112 6.6 85.0
Lysine 1.4390.05 5.29 91 5.8 69.1
Methionine 0.2390.01 0.85
Phenylalanine 1.2790.01 4.71
Threonine 1.0090.01 3.71 109 3.4 82.7
Valine 1.2890.01 4.75 136 3.5 103.2
Tryptophan 5.5290.19 20.46 1,860 1.1 1,411.7
S subtotal 15.83 59.61 181 32.8
Non-essential
Aspartic acid 1.7190.22 6.32
Serine 1.0090.01 3.71
Glutamic acid 2.6790.03 9.90
Proline 1.1190.01 4.10
Cystine 0.3590.02 1.28
Glycine 1.3190.01 4.86
Alanine 1.3690.01 5.04
Tyrosine 1.2190.03 4.49
S subtotal 11.13 39.7
Total sulfur amino acids 0.58 2.13 85 2.5 64.5
(Met
Cys)
Total aromatic amino acids 2.48 9.2 146 6.3 110.8
(Phe
Tyr)
In vitro protein digestibility 75.9090.83
(%)
a
Values are means of duplicate determinations.
b
EAA score100[mg EAA in 100 mg test protein]/[mg EAA in 100 mg FAO/WHO (1990) reference
pattern].
c
See Equation (3).

(0.23) when compared with these researches (1.7 and 2.2 g/100 g dry weight,
respectively). Infants have very critical nutritional requirements due to rapid growth
and immaturity of gastrointestinal function, and nine amino acids have been
identified as essential for infants: Thr, Val, Leu, Ile, Lys, Trp, Phe, Met and His.
Except lysine and sulfur-containing amino acids, other essential amino acids are
sufficient for the FAO/WHO suggested requirements for 2-year-old to 5-year-old
children, and the application of OPN leaves in food products could be considered.

SDS-PAGE analysis
The SDS-PAGE profiles of protein constituents present in OPN leaves in the presence
of b-mercaptoethanol as well as the in vitro protein digestibility profile are shown in
Figure 1. The estimated molecular weight and relative contents of different subunits
are summarized in Table IV.
156 C. Y. Takeiti et al.

Figure 1. (a) SDS-PAGE profiles of OPN leaf proteins (system a). MW, molecular weight markers (a, b, c,
d, e and f correspond to 97.4 kDa, 66.2 kDa, 45 kDa, 31 kDa, 21.5 kDa and 14.4 kDa, respectively). Lanes
1 and 2, duplicate of analysis. AS and BS, acidic and basic subunits of soy glycinin; b, corresponding minor
subunit of soy b-conglycinin. (b) SDS-PAGE profiles of OPN leaf proteins and OPN leaf proteins after in
vitro protein digestibility (HOPN) (system b). MW, molecular weight markers (a, b, c, d and e correspond to
26.6 kDa, 17 kDa, 14.4 kDa, 6.5 kDa and 3.5 kDa, respectively). Lane 1, OPN leaf proteins; lanes 2 and 3,
duplicate of OPN proteins after in vitro protein digestibility (HOPN).

In Figure 1a, four major kinds of protein constituents are shown before digestion,
located at 61 kDa, 53 kDa, 33 kDa and 15 kDa. In addition, small peptides can
be seen below 6.5 kDa, which comprise 6% of the total protein constituents
(Table IV).

Table IV. Molecular weights and relative content of the major subunits of OPN leaf proteins.

Molecular weight (Da) Relative content (%)

97,400 2.0
61,000 (b)a 23.0
53,000 10.0
46,000 3.7
41,000 3.7
38,500 5.5
33,000 (AS)a 12.0
28,000 1.8
25,500 2.2
19,000 5.0
16,500 7.0
15,000 (BS)a 18
6,000b 6.0
a
These bands correspond to 7 and 11-S soy protein fractions.
b
Estimate value obtained using 15% separating gel.
 Nutritive evaluation of native cactus 157

The bands located at 61 kDa, 33 kDa, and 15k Da (Table IV) correspond to the
b-subunit of soy b-conglycinin (7S globulin, 60 kDa), acidic polypeptide (AS, 34 kDa)
and basic polypeptide (BS, 18 kDa) of soy glycinin (11S globulin), respectively.
However, after in vitro protein digestibility (Figure 1b), the bands higher than
26.6 kDa disappeared, and a band located at 14 kDa can be observed as well as
absence of peptides below 6.5 kDa, which indicate intensive hydrolysis and suggests
the formation of free amino acids.
The presence of the band at 14.4 kDa after in vitro enzymatic hydrolysis could
be explained by differences of polypeptide structures; that is, the basic polypeptide
is more hydrophobic and, therefore more compact, which becomes less acces-
sible to proteolysis. This behavior, also reported by Wang et al. (2008), led to
investigation of protein isolates and their hydrolysates. These authors also related
the decrease of intensity bands with the increase of hydrolysis degree. The relative
resistance of proteins to proteolysis is attributed to the compactness of the tertiary
structure by disulfide interactions, which protect the peptide bonds against
enzymatic attack.

In vitro protein digestibility

The in vitro protein digestibility of hydrolyzed OPN leaves and the PDCAAS are also
shown in Table III. The digestibility of OPN leaves was 75.9%, considered a low result
when compared with Almeida-Filho and Cambraia (1974) for two different kinds of
OPN (90.2% and 79.5%). This behavior could be explained by SDS-PAGE analysis,
where the presence of a band at 14 kDa after hydrolysis (Figure 1b) can be observed.
Considering the PDCAAS, the lowest value for sulfur-amino acids (Met
Cys)
corroborates with our previous findings in the SDS-PAGE analysis, where the
presence of the band at 14.4 kDa after hydrolysis could be due to a dense tertiary
structure via disulfide bonds that make proteolysis difficult and, therefore,
impair in vitro protein digestibility. In general, mild thermal treatment applied
on proteins enhances their digestibility, due to partial denaturation of polypeptide
chains, which become more susceptible to enzymatic attack (Takeiti et al. 2004).
For this reason, the OPN leaves’ flour obtained by convective drying using mild
conditions could show an improved digestibility when compared with freeze-dried
leaves.

Vitamin contents
Vitamin contents of the fresh OPN leaves and other leafy vegetables are presented in
Table V. The b-carotene content in 100 g fresh leaves was 4.2 mg, and this value is
close to that reported by Lisiewska et al. (2006) for dill leaves (Anethum graveolens L.)
(3.95 mg/100 g). However, the b-carotene content of OPN is much higher than chomte
leaves (Lycianthes synanthera, 0.31 mg/100 g) and sweet potato leaves (Ipomoea batatas,
0.40 mg/100 g). Ishida et al. (2000) reported that most of the vitamin contents
(vitamin A, vitamin B2, vitamin C and vitamin E) tend to be higher in the leaves than
in other parts of the vegetable. Raju et al. (2007) also stated that carotenoids found in
green leafy vegetables are consumed frequently in popular Indian medicine and less
commonly used for nutritional purposes, which is due to lack of awareness of their
nutritional importance.
158 C. Y. Takeiti et al.
Table V. Vitamin content of fresh leaves.

b-carotene Vitamin A Vitamin C Folic acid

(mg/100 g) (IU/100 g) (mg/100 g) (mg/100 g)

P. aculeata (OPN leaves)a 4.290.2 (43.1) 2333 185.8914.3 19.3

L. synanthera (chomte)b 0.31  22.3 
S. oleracea (spinach)b 4.86  51 
L. sativum (cress)b 5.58  69 
I. batatas (sweet potato 0.40  62.7 
leaves)c
A. graveolens L. (dill)d 3.95  186 
X. sagittifolium S. (taioba)e   100-230 
a
Values in parenthesis are expressed on a dry basis.
b
Values refer to mg/100 g raw leaves according to Salazar et al. (2006).
c
Values refer to mg/100 g raw leaves according to Ishida et al. (2000).
d
Values refer to mg/100 g raw leaves according to Lisiewska et al. (2006).
e
Values refer to mg/100 g raw leaves according to Martins et al. (2006).

The vitamin C content in fresh OPN leaves was 185.8 mg in 100g fresh leaves. This
result was similar to the 186 mg in dill leaves (Lisiewska et al. 2006) and was
also comparable with taioba (Xanthosoma sagittifolium Schott) leaves, obtained by
conventional, organic and natural cultivation according to Martins et al. (2006),
which showed a range between 100 and 230 mg/100 g. According to Lisiewska et al.
(2006), depending on the usable part, the content of vitamin C varied and the younger
plants exhibit the higher vitamin C content. According to Martins et al. (2006),
vitamin C losses due to the dehydration process (608C/12 h) were not significant and
presented low values for leaves obtained by natural and organic harvest. These authors
pointed out that taioba flour could be an important source of minerals and vitamin C.
Nevertheless, sweet potatoes, chomte and mustard leaves showed low values of vitamin
C (62.7 mg/100 g, 22.3 mg/100 g and 38.6 mg/100 g, respectively) when compared
with OPN leaves (Table V).
Folic acid is a water-soluble vitamin, acting as a coenzyme in many single-carbon
transfer reactions in the synthesis of DNA, RNA and protein components. The folic
acid content of the OPN leaves was 19.3 mg/100 g (fresh weight), a high result when
compared with some selected Fijian green leafy vegetables, such as bele (Abelmoschus
manihot, 0.13 mg/100 g) or Chinese cabbage (0.065 mg/100 g), according to Devi
et al. (2008). The result indicate that green vegetables are a significant source of folic
acid, which depends on the source of the food (leafy vegetables, roots and tubers,
fruits or animal food products) and on environmental aspects; for example,
geographical and geological conditions, seasons or climate.
Epidemiological studies have shown that folic acid supplementation can also
significantly reduce hematological diseases, neurological and neuropsychiatric dis-
orders and different forms of cancer (Giovanucci 2002). Gliszczynska-Swwiglo (2007)
demonstrated antioxidant activities of folates comparable with those of vitamin C and
E, which suggest protective effect of folates in the degenerative diseases. These
antioxidant activities may become important in vivo considering nutritional supple-
mentation and fortification of food with folic acid. Taking into account vitamin
contents in fresh OPN leaves, future research will contemplate thermal treatment
effect on vitamin levels and bioavailability studies.
 Nutritive evaluation of native cactus 159

Conclusions
OPN leaves showed a high level of total dietary fiber, as well as considerable amounts
of minerals (calcium, magnesium, manganese and zinc). In addition, vitamin levels
(vitamin A, vitamin C and folic acid) of fresh leaves are remarkable. Among essential
amino acids, tryptophan was the most abundant. Considering the EAA score, lysine
and sulfur-amino acids (Met
Cys) were limited. SDS-PAGE analysis showed small
peptides inferior to 6.5 kDa and four major bands at 61 kDa, 53 kDa, 33 kDa, and
15 kDa. The protein digestibility corrected amino acid score demonstrated that the
lowest value was for sulfur-amino acids (Met
Cys), which corroborates with our
previous findings in SDS-PAGE analysis. This study revealed nutritional benefits of
OPN leaves taking into consideration the minerals, dietary fiber, vitamins and
essential amino acids. These potential nutritional features of OPN leaves’ powder
should be exploited and used as a functional ingredient.

Acknowledgements
The authors thank FAPESP (Process n. 06/57880-9), UNICAMP and Mr Rogelio
R. Dosouto for donation of OPN leaves.

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This paper was first published online on iFirst on 22 May 2009.