Food Anal.

Methods
DOI 10.1007/s12161-009-9106-z

Nutritional Quality of Edible Parts of Moringa oleifera

Dalia I. Sánchez-Machado & José A. Núñez-Gastélum &
Cuauhtémoc Reyes-Moreno &
Benjamin Ramírez-Wong & Jaime López-Cervantes

Received: 6 June 2009 / Accepted: 20 August 2009

# Springer Science + Business Media, LLC 2009

Abstract This study was carried out in order to compare Introduction

the biochemical characteristics from three edible parts of
the multipurpose tree Moringa oleifera such as the leaves, The Moringaceae family consists of 12–14 species which
flowers, and immature pods. On average, the three most belong to a single genus called Moringa. All of the species
abundant amino acids were glutamic acid, arginine, and are native of North India, where they have been introduced
aspartic acid. The fatty acids present at the highest content to all warm regions of the world. Fast growing and not very
were linolenic acid (C18:3ω3), palmitic acid (C16:0), linoleic demanding as to climate and soil quality, Moringa oleifera
acid (C18:2ω6), and oleic acid (C18:1ω9). The chemical is the most common species cultivated throughout the
composition (of dry weight) ranged from 19.34% to 22.42% tropical regions of the world (Tsaknis et al. 1999; Vlahov et
for protein, 1.28% to 4.96% for lipids, 7.62% to 14.60% for al. 2002; Manzoor et al. 2007; Muluvi et al. 1999).
ash, and 30.97% to 46.78% for dietary fiber. M. oleifera is a The leaves, flowers, immature pods (which are called long
nonconventional plant with substantial nutritional value. green pods), and roots are edible. Due to the flavor of its root,
it is known as the horseradish tree. When the fruit is ripe, it
Keywords Moringa oleifera . Amino Acids . Fatty Acids . turns brown and contains 10–12 seeds that, when fried, have a
Chemical Composition peanut flavor. The tender pods, chopped or cooked, can be
used in different and delicious dishes. The leaves and green
seeds have a similar flavor to that of asparagus. In some parts
of the world, these parts are eaten as fresh vegetables and can
be frozen or canned. Combined with curries, the parts of the
plant are commonly prepared with chicken or sea food as a
soup (Anwar and Bhanger 2003).
D. I. Sánchez-Machado : J. A. Núñez-Gastélum :
J. López-Cervantes (*) In many reports, the M. oleifera is described as an
Departamento de Biotecnología y Ciencias Alimentarias, ornamental plant with medicinal, therapeutic, or healing
Instituto Tecnológico de Sonora, properties. The leaves are used for fever, indigestion, or
5 de Febrero 818 Sur,
eyes treatments. The anti fungi activity of the extract has
CP 85000 Cd. Obregón, Sonora, México
e-mail: [email protected] been investigated and possesses a protective action to the
toxic effects of arsenic. This plant has been recommended
C. Reyes-Moreno as a feedstuff for milk-producing cattle or for Nile tilapia,
Facultad de Ciencias Químico-Biológicas,
and as a supplementary feed for goats and lambs. The oil
Universidad Autónoma de Sinaloa,
Cd. Universitaria, Av. de las Américas y Josefa Ortiz S/N, from the seed is useful as vegetable oil and for the
CP 80000 Culiacán, Sinaloa, México production of soap and cosmetics. In addition, this seed
oil possesses resistance to oxidative degradation and fuel
B. Ramírez-Wong
properties. The protein press cake obtained after oil
Departamento de Investigación y Posgrado en Alimentos,
Universidad de Sonora, extraction has one of the best natural coagulants that is
CP 83142 Hermosillo, Sonora, México effectively utilized for treatment and purification of highly
 Food Anal. Methods

turbid water (Makkar and Becker 1996; Chuang et al. 2007; Ammonium monohydrogen phosphate, (NH4)2HPO4,
Gupta et al. 2007; Reyes et al. 2006; Richter et al. 2003; stock solution (2 M), used for preparation of HPLC eluents,
Ndemanisho et al. 2006; Rashid et al. 2008; Ben Salem and was adjusted to pH 6.5 with ammonium dihydrogen
Makkar 2009; Kwaambwa and Maikokera 2007). phosphate, NH4H2PO4. FMOC-Cl was dissolved in aceto-
Most of the research on the chemical characteristics of this nitrile at 4 mg/ml. A borate buffer was prepared from a
plant and its products is mainly focused on the oil from the 250 mM boric acid solution adjusted to pH 8.5 with 1 M
seed with very little focus on the rest of the plant. The study of sodium hydroxide solution prepared from sodium pellets.
oil extraction and physicochemical properties (using solvents The alkaline cleavage reagent was prepared daily in
and enzymatic methods; Abdulkarim et al. 2005), oxidative 1,000 μl batches by mixing 680 μl of 850 mM sodium
stability, fatty acids composition, steroids content and hydroxide solution with 300 μl of 500 mM hydroxylamine
tocopherol (Tsaknis et al. 1999; Anwar and Bhanger 2003; hydrochloride solution and 20 μl of 2-(methylthio)ethanol.
Anwar et al. 2007), characterization of the triacylglycerols The quench reagent was acetonitrile–acetic acid (8:2, v/v).
present in oil (Vlahov et al. 2002), and the elemental analysis
and seed proximal (Anhwange et al. 2004) are currently in Samples Preparation
process. Although the profile of the amino acid content in the
leaves has been documented in different research works The site of the collection of samples of the M. oleifera was a
(Makkar and Becker 1996; Sena et al. 1998; Freiberger et al. field of about 40 ha located in the State of Sonora in
1998), the entire characterization of the M. oleifera plant has Northwest Mexico. Samples (the young leaves, flowers, and
not yet been reported. In a previous research, as a part of a immature pods of 2 weeks old) were manually collected
nutritional study, this research group has quantified the from six different trees during November of 2006, and
content of alpha- and gamma-tocopherol in leaves, flowers, transported in plastic bags at room temperature. These parts
and immature pods from M. oleifera (Sánchez-Machado et al. were oven-dried at 60°C for 8 h, and then were stored in
2006). The objective of this study was to determine the plastic bags in a dark room. All samples were ground and
chemical composition and the profile of amino acids and fatty passed through a mesh sieve (60 meshes) to be analyzed.
acids of leaves, flowers, and immature pods from M. oleifera.
Chemical Composition

Materials and Methods The total content of protein was calculated by multiplying
the total nitrogen (Kjeldahl method) by 6.25 (Association of
Chemicals Official Analytical Chemists (AOAC) 1984). The content of
total dietary fiber was quantified following the gravimetric-
High-performance liquid chromatography (HPLC)-grade enzymatic method, with residual protein and residual ash
methanol and acetonitrile were obtained from EMD correction (Sánchez-Machado et al. 2004a). The ash content
Chemicals and the selenium reagent mixture and the was calculated by incineration of the samples at 525 °C for
chloroform were obtained from Merck (Dramstadt, 5 h to a constant weight (Association of Official Analytical
Germany). Glacial acetic acid, boric acid, anhydrous Chemists (AOAC) 1984). To evaluate the total lipid content,
ammonium monohydrogen phosphate, anhydrous dihydrogen the lipid was extracted from samples with 2:1 chloroform-
phosphate, sodium hydroxide, ethylenediaminetetraacetic methanol (Sánchez-Machado et al. 2004b; Berdeaux et al.
acid, sulphuric acid, ethanol, and acetone reagent grade were 2009). Specifically, 0.5 g of dry sample was extracted with
purchased from Productos Químicos Monterrey (Monterrey, 3 ml of solvent mixture, and after 2 min in a vortex mixer,
Nuevo Leon, Mexico). The anhydrous sodium sulfate was the content was filtered through Whatman No. 41. The
obtained from Fluka (USA). Amino acid standard, toluene, operation was repeated once more with residue obtained.
potassium carbonate, acid hydroxylamine hydrochloride, 9- The two extracts were combined and concentrated to dryness
fluorenylmethyl chloroformate (FMOC-Cl), and 2-(methyl- under nitrogen, and the weight of the resulting residue was
thio)-ethanol, and the enzymes heat-stable alpha-amylase, taken as the total lipid content of the sample. Nonstructural
protease, and amyloglucosidase were purchased from Sigma carbohydrates were calculated by difference ð100 @
(St. Louis, Missouri, USA). Fatty acids standard FAME Mix ðprotein þ lipid þ ash þ dietary fiberÞÞ. All samples were
C8-C22 and PUFA No. 3 were obtained from Supelco analyzed in duplicate.
(Bellefonte, Pennsylvania, USA). All reagents were analyti-
cally grade, unless otherwise noted. All aqueous solutions Determination of Amino Acids Composition
were prepared with ultrapure water purified with the NANO-
pure Diamond UV system (Barnstead International, Dubuque, Sixteen amino acids were determined in the samples,
Iowa, USA). aspartic acid, glutamic acid, serine, histidine, glycine,
Food Anal. Methods

threonine, alanine, proline, tyrosine, arginine, valine, a wide concentration range in concordance with the level of
methionine, isoleucine, leucine, phenylalanine, and lysine. each amino acid found in the samples that were analyzed.
All samples were analyzed in duplicate. To determine the
total content of the amino acids, an acid hydrolysis was Determination of Fatty Acids Composition
performed on the proteins (50 mg of the sample was added
with 10 ml of HC1 6 N and was heated at 110°C for 24 h). The fatty acid profile was determined by gas chromatography
The hydrolyzed product was vacuumed and filtered, and in accordance with the procedure of (Sánchez-Machado et al.
then diluted at 250 ml in a volumetric flask (Sánchez- 2004b) with minimal modifications. Specifically, 0.5 g dry
Machado et al. 2003). A 300 μl was oven dried at 60°C all sample was weighed in a tube with a screw cap and was
night, and the residue was diluted in 300 μl of a borate treated with 2 ml of toluene and 3 ml HCl methanolic 5%
buffer through agitation (15 s). (v/v). This mixture was vortexed and placed into a water bath
Then, derivatization and quantification by chromatogra- for 2 h. After the samples had cooled to room temperature,
phy of liquids of the amino acids were performed according 3 ml K2CO3 6% and 2 ml of toluene was added and
to the procedures presented by López-Cervantes et al. followed by agitation in the vortex. The samples were then
(2006). A 300 μl of the amino acid standard solution or of centrifuged for 5 min at 2,400 rpm (Compact II Centrifuge,
the prepared sample was deposited in a vial with the Clay Adams, USA), and the organic phase was separated
capacity of 1.5 ml and then 300 μl of FMOC reagent was and dried with Na2SO4 anhydride. The organic phase (1 ml)
added and vortexed for 90 s. The cleavage reagent was was filtered with a 0.45 µm membrane. A 3 µl of this sample
added (180 μl), and the tubes were vortexed for 15 s and solution was injected into the gas chromatography system.
then left for 5 min at room temperature. A 420 μl of quench All samples were analyzed in duplicate.
reagent was then added. The resulting solution was The equipment used was a gas chromatograph with a flame
vortexed for 15 s and filtered with a membrane 0.45 μm. ionization detector, with a capillary column CP-Sil 5 CB
A 5 μl sample of this solution was injected onto the column (15 m×0.25 mm i.d., thickness of 0.25 μm) and CP-8410
of the HPLC system. autoinjector, all from Varian Inc. (Palo Alto, California, USA).
The HPLC system (GBC, Dandenong, Australia) was The injection volume was 3 μl (at 250°C), the carrier gas was
equipped with an autoinjector LC 1650, an online solvent helium (1 ml/min), and the detector temperature was held
degasser LC 1460, a system controller with WinChrom for constant at 270°C. The column temperature was held at 110°C
chromatography data analysis, a pump LC 1150, a column for 1 min and then was increased to 240°C at a rate of 3°C/min,
oven LC 1150, a 20 μl injection loop (Rheodyne, Cotati, which was maintained for 15 min. Peak identifications were
CA, USA), and a fluorescence detector LC 5100. Chro- based on comparison of retention times with the standards, and
matographic analysis was performed using an analytical the area of the peaks was quantified using the software Galaxie
scale (250×4.6 mm i.d.) SGE Hypersil ODS C18 column Workstation (Varian Inc., Palo Alto, California, USA). The
with a particle size of 5 μm (SGE, Dandenong, Australia). relative amount of each fatty acid (percent of total fatty acids)
HPLC conditions were as follows: mobile phase A, 30 mM was quantified by integrating the area under the peak and
ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol- dividing the result by the total area for all fatty acids.
water; mobile phase B, 15:85 (v/v) methanol-water; mobile
phase C, 90:10 (v/v) acetonitrile-water. The elution gradient Statistical Analyses
(min/A%/B%/C%) was: 0/24:63:13, 32/11:43:46, 34/
11:43:46, 34.05/0:0:100, 36.5/0:0:100, 36.55/24:63:13, 50/ Data was processed with the statistics package SPSS 12.0 for
24:63:13. The flow rate was constant at 1.1 ml/min and the Windows (SPSS Inc., Chicago, IL, USA) which analyzed
column maintained at 38°C. The detection was by the average and the standard deviation of repetitions.
fluorescence using the wavelengths of excitation and
emission at 270 and 316 nm, respectively. The total time
between injections was 50 min. Results and Discussion
The HPLC method allows simultaneous analysis of 16
amino acids, and the different amino acids were identified The M. oleifera has been planted and naturalized in many
by comparison with retention times for amino acid stock areas around the world. Now, this plant has been introduced
solutions. For determination of retention times, the refer- as a potential crop for arid regions to be utilized in the
ence standards were injected both individually and as a nutritional programs of the rural population. This research
mixture. The concentration of each amino acid was is directed at obtaining the nutritional value of the leaves,
obtained by direct interpolation of the peak area in the flowers, and immature pods to promote their incorporation
correspondent linear calibration curve (peak area vs. into the population's diet, as any other vegetable. There are
concentration). These calibration curves were obtained over no available reports on the nutritional composition or the profile
 Food Anal. Methods

of the amino acids and fatty acids of the flowers and immature The immature pods possessed the lowest lipid values, which
pods of this plant. Therefore, this study will be the first is contrary to the values expected in brown and dry seeds of
nutritional documentation of these parts of the M. oleifera. mature pods (38–42%) (Anwar and Bhanger 2003).
As shown in Table 1, the dietary fiber content in the
Chemical Composition immature pods (46.78%) is obviously the highest of all the
samples; this value was approximately double that of
The chemical composition of the leaves, the flowers, and the the leaves and the flower content. Another finding, of the
immature pods from M. oleifera are shown in Table 1. The present study, is that the content of dietary fiber in immature
highest protein, ash, and lipid concentrations were found in pods is similar to that reported in seaweed (Sánchez-
the leaf samples (22.42%, 14.60%, and 4.96%, respectively). Machado et al. 2004a). The nonstructural carbohydrates
In general, the chemical composition of the plant depends on contents, which ranged from 24.98% in immature pods to
the edible parts that were analyzed. 36.04% in flowers, were calculated by difference. The values
The protein content for leaves (22.42%) is similar to that obtained for flowers and leaves are within normal range for
presented by Makkar and Becker, (1996), Sena et al. (1998) most green vegetables, additionally, nonstructural carbohy-
of 25.10% and 22.90%, respectively; while it differs from drates as such glucose, fructose, sucrose, and starch can
that obtained by Reyes et al. (2006) (17.80%), and Gidamis reach concentrations of 300 to 400 mg/g dry weight by
et al. (2003; 33.82%). The leaves that show a higher biomass deposition in growth zones (Gidamis et al. 2003).
content of protein compared with the chard (Beta vulgaris)
contain only 2.90% protein, (Macías et al. 2003) and for Amino Acids Profile
some edible seaweeds, the content is around 5% and 13%
(Sánchez-Machado et al. 2004b). There is little research The amino acid profile of the edible parts of the M. oleifera
about the protein content in the immature pods; Gidamis et (Table 2) shows that the total amino acid concentrations are
al. (2003) reported 20.66%, similar to that reported in this in the range of 74.5 to 172.7 mg/g (dry matter) for flowers
study. However, the protein content of the flowers and and leaves, respectively. The observation, of the total amino
immature pods contrast with a report by Tacon (1989) for acid content, revealed that the essential amino acids
the pea pods (Pisum sativum, 10.79%). A profile of 30.07% represented 44% of leaves, 30% of the immature pods and
and 33.26% of protein has been reported for mature seeds 31% of the flowers. The parts of the plant with the highest
from M. oleifera cultivated in Pakistan and Brazil, total content of amino acid were the leaves, which are
respectively (Manzoor et al. 2007; Oliveira et al. 1999). double that of the flowers. The amino acids which were
As different studies have reported (Makkar and Becker concentrated in a higher proportion in the different samples
1996; Reyes et al. 2006; Gidamis et al. 2003), the leaves are glutamic acid, arginine, and aspartic acid; while the
show lower ash values than the results in this research lowest concentrations are methionine and tyrosine.
(14.60%; Table 1), even though the results are still higher The amino acid profile in M. oleifera leaves were tested
than those reported for other edible plants as the chard in previous studies (Makkar and Becker 1996; Sena et al.
(2.00%) (Macías et al. 2003), with the exception of edible 1998; Freiberger et al. 1998). The composition of amino
seaweed, which is higher than 20% in ash value (Sánchez- acids as aspartic acid, glutamic acid, histidine, threonine,
Machado et al. 2004b). The differences observed among the alanine, tyrosine, arginine, methionine, phenylalanine, and
diverse studies in leaves can be attributed to the influence lysine, show a variation to the published data by at least one
of the weather conditions in the region. of the mentioned researchers. All the parts of this plant
In keeping with the results, the total lipid content of edible contain high percentages of essential amino acids except for
portions from M. oleifera is less than 4.96% (Table 1). The methionine, commonly deficient in green leaves. It could be
average lipid content in the analyzed leaves and immature possible that the variations in the amino acid composition
pods is similar to the quantity reported by Makkar and of the leaves are influenced by the quality of the protein and
Becker (1996; 5.40%), and by Gidamis et al. (2003; 2.54%). the origin of the plant (cultivated or wild).

Table 1 Chemical composition (percent on dry weight basis) of the edible parts of M. oleifera
Sample Protein Ash Lipids Dietary fiber Nonstructural carbohydratesa

Leaves 22.42±1.4 14.60±0.9 4.96±0.1 30.97±1.0 27.05

Immature pods 19.34±0.2 7.62±0.3 1.28±0.1 46.78±2.2 24.98
Flowers 18.92±1.3 9.68±0.7 2.91±0.3 32.45±0.5 36.04

Mean value ± standard deviation (n=3, by duplicate)

a
Calculated by difference
Food Anal. Methods

Table 2 Amino acids content (milligram per gram of dry weight oleifera is shown in Table 3. In leaves, linolenic acid
basis) of the edible parts of M. oleifera
(C18:3ω3) was found in high quantity followed by palmitic
Amino acid Leaves Immature pods Flowers acid (C16:0), of which both represent 80% of the total fatty
acids, similar to that presented by Freiberger et al. (1998),
Aspartate 15.8±1.5 7.4±0.3 12.3±0.9 Sena et al. (1998). Caprylic acid (C8:0), lauric acid (C12:0),
Glutamate 17.1±1.4 14.6±2.3 17.0±2.2 and arachidonic acid (C20:4ω6) were the fatty acids in a
Serine 9.4±0.5 7.5±1.8 7.5±0.4 smaller percentage. Flowers and immature pods show
Histidinea 7.0±0.4 2.0±0.3 3.1±0.4 similar amounts of palmitic acid, linoleic acid (C18:2ω6),
Glycine 10.3±0.7 4.3±0.5 6.5±0.3 linolenic acid and oleic acid (C18:1ω9), representing 90%
Threoninea 7.9±0.4 3.3±0.5 5.4±0.2 of the total fatty acids. All the edible parts of M. oleifera
Alanine 12.5±0.6 4.2±0.7 8.1±0.5 showed the presence of linoleic acid and linolenic acid,
Proline 12.4±0.9 4.0±0.6 6.6±0.5 considered essential fatty acids, and arachidonic acid
Tyrosinea 4.8±0.9 0.4±0.1 0.4±0.1 (C20:4ω6) precursor of biologically active eicosanoids
Arginine 12.2±0.8 8.1±2.5 20.1±1.2 (Sánchez-Machado et al. 2004b). Saturated and unsaturated
Valinea 11.3±1.1 4.3±1.0 6.4±0.6 fatty acids showed a relation of 1:2 in flowers and
Methioninea 1.4±0.3 0.9±0.2 1.0±0.2 immature pods. Polyunsaturated fatty acids were found in
Isoleucinea 8.9±0.7 3.1±0.4 5.2±0.5 a ratio of 2:1 with respect to the monounsaturated fatty
Leucinea 17.5±0.2 5.6±0.5 8.7±0.9 acids in flowers and immature pods. In the leaves, the
Phenylalaninea 8.9±0.3 2.3±0.4 3.8±0.5 presence of polyunsaturated fatty acids was very high in
Lysinea 15.3±0.6 2.5±0.6 4.6±0.5 comparison to the monounsaturated fatty acids.
Total 172.7±3.2 74.5±4.3 116.7±3.2
Essentials 83±1.8 24.4±1.5 38.6±1.5
Conclusion
Mean values±standard deviation (n=4, by duplicate)
a
Essential amino acid M. oleifera parts show that could be successfully utilized as
food for human consumption. The leaves and flowers are a
Fatty Acids Content protein source with an adequate profile of amino acids and
ash, while the immature pods show a high content of dietary
In this work, 14 fatty acids were identified in all samples. fiber and low lipid content. Unsaturated and essential fatty
The fatty acid composition of the three parts analyzed of M. acids are present in all samples. The collection and

Table 3 Fatty acid composition

(percent of total fatty acids) of Fatty acid Leaves Immature pods Flowers
the edible parts of M. oleifera
C8:0 0.05±0.01 0.24±0.06 0.09±0.03
C12:0 0.12±0.02 0.11±0.02 0.09±0.01
C14:0 0.96±0.18 0.26±0.04 0.59±0.03
C14:1w5 0.53±0.11 0.15±0.03 1.96±0.22
C16:0 23.28±1.38 26.48±0.5 23.43±0.84
C16:1w7 0.43±0.07 nd nd
C18:0 4.08±0.15 3.10±0.13 4.52±0.09
C18:1w7 1.15±0.16 1.00±0.2 2.00±0.21
C18:1w9 5.12±0.18 17.00±1.32 21.55±0.97
C18:2w6 6.11±0.83 23.50±1.01 18.96±0.58
C18:3w3 56.87±1.57 26.15±0.74 23.01±0.38
C20:0 0.72±0.08 0.39±0.02 0.98±0.08
C20:1w9 nd 0.24±0.03 0.75±0.08
C20:4w6 0.21±0.02 0.24±0.03 0.19±0.03
C22:0 0.70±0.06 1.06±0.10 2.06±0.19
Saturated FAs 29.89±1.40 31.64±0.53 31.76±0.87
Monounsaturated FAs 7.23±0.27 18.39±1.33 26.26±1.01
Mean values ± standard devia- Polyunsaturated FAs 63.19±1.77 49.89±1.25 42.16±0.69
tion (n=4, by duplicate)
Unsaturated FAs 70.42±1.79 62.28±1.83 68.42±1.23
FA fatty acid, nd not detected
 Food Anal. Methods

consumption of all the parts of the plant could have financial Kwaambwa HM, Maikokera R (2007) Colloids Surf B 60:213
López-Cervantes J, Sánchez-Machado DI, Rosas-Rodríguez JA (2006)
and social benefits for the population of countries where this
J Chromatogr A 1105:106
tree is being cultivated, due to its fast growth and high Macías CS, Montenegro MA, Arregui T, Sánchez PMI, Nazareno MA,
tolerance of the different weather conditions. López MB (2003) Ciênc Tecnol Aliment 23:33
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Acknowledgements This research was financed under project no. Manzoor M, Anwar F, Iqbal T, Bhanger MI (2007) J Am Oil Chem
SON-2004-C03-016 with Mixed Funds of the Government of the Soc 84:413
State of Sonora and the National Council for Science and Technology Muluvi GM, Sprent JI, Soranzo N et al (1999) Mol Ecol 8:463
(FOMIX-CONACYT). Michael A. Bryan, MBA and Native English Ndemanisho EE, Kimoro BN, Mtengeti EJ, Muhikambele VRM
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(1999) J Sci Food Agric 79:815
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