Accepted Article PROF.

AMIRA DARWISH (Orcid ID : 0000-0003-3586-1575)

Article type : Research

Effect of germination on the nutritional profile of quinoa (Cheopodium quinoa Willd.) seeds
and its anti-anemic potential in Sprague-Dawley male albino rats
Amira M.G. Darwish1*, Huda A.O. Al- Jumayi2 and Hassan A. Elhendy3
1 Food Technology Department, Arid Lands Cultivation Research Institute (ALCRI), City of
Scientific Research and Technological Applications (SRTACity), 21934 Alexandria, Egypt
2 Nutrition and Food Science Department, Faculty of Science, Taif University, Kingdom of Saudi Arabia
3Home Economics Department, Faculty of Agriculture, Alexandria University, Egypt

Running title: Influence of quinoa seeds germination on anemic albino rats

*Corresponding author: (Amira M.G. Darwish, Ph.D.)

Department of Food Technology, Arid Lands Cultivation Research Institute (ALCRI), City of
Scientific Research and Technological Applications (SRTA-City), Universities and Research
Center District, New Borg El Arab, P.O. Box 21934, Alexandria, Egypt.
Tel.: +2 0122 44 96 333 Fax (+203) 4593423
ORCID https://orcid.org/0000-0003-3586-1575
E-mail addresses:
Amira M.G. Darwish: amiragdarwish@yahoo.com
Huda A.O. Al- Jumayi: drhuda2008@gmail.com
Hassan A. Elhendy: elhendy99@yahoo.com

This article has been accepted for publication and undergone full peer review but has not been
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differences between this version and the Version of Record. Please cite this article as doi:
10.1002/CCHE.10366
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Accepted Article
Abstract
Background and objectives: Iron-deficiency anemia (IDA) is the most common form of
nutritional anemia in both developed and developing countries. We aimed to evaluate the
germination impact on the nutritional profile of quinoa seeds and the anti-anemic potentials of
quinoa fortified phytogenic diet in treatment of iron deficiency anemia-induced albino rats.
Findings: The results revealed that quinoa seeds are considered a good source of fiber, protein
(43.08 and 30.62% of DV) and vitamin C (three-fold of daily requirement). Germination of the
seeds increased iron, calcium and zinc contents by 39.43, 49.04 and 20.25%, vitamin C and
carotenoids by 32.17 and 26.02%, respectively, and decreased anti-nutritional factors saponin,
phytic acid and tannins by 59.60, 50.0 and 11.32 %, respectively. The biological assay indicated
that fortification with 10% quinoa sprouts (GII6) can be recommended as the best treatment to
restore body weight, organs weights, serum profile (protein, ferritin, iron and zinc) and blood cell
counts (RBCs, WBCs, Hb and platelets), as reflected in red cell indices (Hct, MCV, MCH and
MCHC) of IDA induced rats being at optimum levels, which were comparable to the healthy rats
group (GI).
Conclusion: Increased antioxidants, vitamins, minerals content and bioavailability of nutritional
compounds due to decreased anti-nutritional factors content in the germinated sprouts resulted in
anti-anemic health potentials in treated rats.
Significance and novelty: Evidence indicates that germinated quinoa seeds are superior in
nutrients compared to the ungerminated seeds due to the activation of endogenous enzymes that
degrade anti-nutritional factors, elevate mineral bioavailability and antioxidant properties.
Trial registration number: AU08190625330; Date of registration: 25-06-2019
Key words: Quinoa seeds/ sprouts; Iron Deficiency Anemia (IDA); Sprague-Dawley male albino
rats; Anti-nutritional factors; Mineral bioavailability; Red cell indices.
1. Introduction
Iron deficiency (IDA) is in the top 20 risk factors for the global distribution of disease burden
(Hegde, Rich, & Gayomali, 2006; Mousa, Saleh, Higazi, & Ali, 2016). WHO is working with the
Egyptian government to address major challenges due to the prevalence of iron deficiency anemia
among 40% of children between 2 to 5 years, which may increase to 51% in rural children. Similar

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 prevalence was reported among women of reproductive age and in pregnancy, in addition to being
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associated with other diseases (Abdel-Rasoul, Elgendy, & Abd Elrazek, 2017; Rezk, Marawan,
Dawood, Masood, & Abo-Elnasr, 2015; Simbrunner et al., 2020; WHO, 2010). Iron deficiency has
adverse consequences for health as a major contributor to maternal and fetal morbidity and
mortality, affects productivity, work capacity, cognitive development, resistance to infection and
pregnancy in women (Helmy, Elkhouly, & Ghalab, 2018; Means, 2020).
Zinc is significant to cellular growth and differentiation; about one third of the world's population
is listed at high risk especially those living in low income countries. The effects of zinc deficiency
are impaired growth, increased morbidity and mortality, dysfunction of the immune system,
adverse pregnancy outcomes and abnormal neurobehavioral development (Maggini, Pierre, &
Calder, 2018; Palanog et al., 2019).
Quinoa (Chenopodium quinoa Willd.) is considered as one of the oldest crops in America; it was
cultivated in Peru prior to 5000 BC and was used prior to 3000 BC. Quinoa is classified as a
pseudo-cereal, it is "one of the grains of the 21st century" due to its ability to endure extreme
environmental conditions, in addition to its nutritional and biological properties, which made it an
option to increase food security worldwide (FAO, 2011). It is gluten-free and a good source of
high quality protein, fiber, carbohydrates, vitamins, minerals, phytochemicals and bioactive
peptides. The anti-nutritional factors present in quinoa, such as saponins, tannins and phytic acids,
can decrease the bioavailability due to forming insoluble complexes with minerals, such as zinc
and iron, which can be alleviated by germination (Desai, Desai, & Kaur, 2009; Jarvis et al., 2017;
Vilcacundo & Ledesma, 2017). Saponins consist of a sugar moiety usually containing glucose,
galactose, glucuronic acid, xylose, rhamnose or methylpentose, glycosidically linked to a
hydrophobic aglycone (sapogenin) which may be triterpenoid or steroid in nature (Desai et al.,
2009). El Hazzam et al., (2020) reported that quinoa seed bitterness is essentially due to the
presence of quinoside A, while others related bitter tasting to surface activity, which can cause
intensive foaming in aqueous solutions (Schoenlechner, Siebenhandl, & Bergho, 2008).
Germination is a widely practiced method to improve the nutritional value of seeds. It incorporates
a series of events that begins with the uptake of water by the quiescent dry seed and terminates
with the elongation of the embryo axis. The subsequent mobilization of the major storage reserves
is associated with the growth of the seedling (Benincasa, Falcinelli, Lutts, Stagnari, & Galieni,
2019). Germination has been considered a cheap and effective method to enhance the antioxidant
capacity, increase the bioavailability of essential minerals and vitamins (Nkhata, Ayua, Kamau, &

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 Shingiro, 2018). In recent years, quinoa sprouts have become widely popular because of their
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various health benefits and they have been thoroughly investigated as means of delivering
nutrients (FDA, 2006).
This study was carried out to investigate the impact of germination of quinoa seeds on their
chemical composition, minerals, vitamin C, antioxidants and anti-nutritional factors. As well, a
biological assessment was conducted to monitor the influences of the addition of quinoa seeds/
sprouts (at 5% and 10%) in a fortified phytogenic diet, on iron deficiency anemia induced male
Sprague-Dawley (SD) albino rats including effects on body weight, organs weights, serum protein
and minerals content, complete blood cell count (CBC) and red cell indices. Well-designed
clinical trials and more scientific studies are needed in this area of research.
2. Materials and methods
2.1. Materials
White organic quinoa seeds (Chenopodium quinoa Willd.) (Quinua Real, Bolivia), were obtained
from the local market of Jeddah, Saudi Arabia.
http://www.theplantlist.org/tpl/search?q=Chenopodium+quinoa+&_csv=on.
2.2. Quinoa sprouts germination
Quinoa seeds were cleaned, freed of broken particles and foreign materials, and then soaked in
distilled water at 25oC/12 h, with a ratio of 1:5 seeds to water (w/v). The non-imbibed soaking
water was discarded to remove anti-nutritional elements including saponins present in the external
seed layers, which are responsible for the bitter taste due to the strong binding affinity of the
aglycones to minerals (Brady, Ho, Rosen, Sang, & Karwe, 2007).
For germination, the seeds were spread on wet jute bags, covered with muslin cloth, and sprinkled
with water every 12 hours until the end of germination process (72 h). The resulting sprouts were
gathered, washed, drained, dried at 50oC/ 24 h in a mechanical convection oven to constant weight
and were milled using a laboratory grinder. The obtained flour was then kept in polyethylene bags
for analyses (Echendu, Obizoba, & Anyika, 2009).
2.3. Chemical composition of quinoa seeds and sprouts
2.3.1. Chemical composition
Moisture, crude protein, crude fiber, fat and ash contents were assessed according to (AOAC,
2016). Calculations of energy value were carried out using the Artwater factor 4 for protein, factor
9 for fat and factor 4 for carbohydrate (WHO/FAO, 2002).
2.3.2. Minerals assessment

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 Iron, calcium, and zinc were analyzed as described (Perales, Barberaä, Lagarda, & Farre, 2005),
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using a flame atomic absorption spectrophotometer (AAS; Perkine Elmer, mod. 3100 Norwalk,
USA).
2.3.3. Vitamin C content
Vitamin C levels were analyzed using 2,6-dichlorophenoIindophenoI and 2,4-
dinitrophenylhydrazine and determined by spectrophotometer (Jenway 6300, UK) (Schultze,
1947).
2.3.4. Daily Values for nutrition labeling (DV %)
Daily Values (%) for nutrition labeling were calculated based on a daily intake of 2,000 calories,
which has been established as the reference for adults and children 4 or more years of age (FDA,
2004).
2.4. Chromatographic analysis of carotenoid content
The carotenes were extracted by homogenizing twice with an acetone: hexane mixture (40:60) and
the extract was concentrated in a rotary evaporator at reduced pressure (Bucher Instruments, Fort
Lee, NJ). Carotenoids were analyzed by reverse-phase HPLC using a Waters 600 HPLC System
consist of a Waters 996 photodiode array detector operating at 450nm, 600 solvent pump, 600
controller, and a 717 autosampler injector. The quantification of carotenoids (α- and β-carotene)
was determined using retention time comparison with commercially available standards (Sigma
Aldrich, Merck, St. Louis, Mo). Storage and processing of data was carried out using Millenium
4.00 software (Waters, Stockholm, Sweden). Absorption spectra were recorded between 250 and
500 nm (Dietz, Kantha, & Erdman, 1988; Rodriguez-amaya, 1997).
2.5. Anti-nutritional factors in quinoa seeds and sprouts
The saponin content was determined using HPLC-UV according to (Kołodziej et al., 2018). The
tannin was determined using the Folin Denis spectrophotometric method as described by
(Elgailani & Ishak, 2014). Content of phytic acid was analyzed using spectrophotometric method
of (Vayupharp & Laksanalamai, 2013). Phytic acid concentration in the sample was calculated by
the below formula,
𝑃ℎ𝑦𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 𝑐𝑜𝑛𝑡𝑒𝑛𝑡(𝑔 /100𝑔) = 𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑟𝑢𝑠 𝑝ℎ𝑦𝑡𝑎𝑡𝑒 𝑋 4.97
2.6. Biological assessment of quinoa seeds/ sprouts influences on induced anemic rats
2.6.1. Animals and diet
Thirty six male Sprague-Dawley (SD) albino rats weighing 150 ±10 g were bred and maintained
under standard conditions (22°C±1°C, 12-h light/ 12-h dark cycle), fed fundamental chow (fat 2.8

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 %, protein 18.5 %, fiber 11.2 %) and water ad libitum, for one acclimatization week before
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experiments in the Experimental Animals Facility, Home Economics Department, Faculty of
Agriculture, Alexandria University, Egypt. All of the experimental procedures were performed in
accordance with the guidelines approved by Alexandria University Institutional Animal Care and
Use Committee (AlEXU-IACUC), a member of International Council for Laboratory Animal
Science (ICLAS) (permission number: AU08190625330).
The animals were randomly divided into two main groups: Group I (n=6 rats), classified as the
negative control group, which was fed fundamental diet (healthy rats), and Group II (n= 30 rats),
which was induced for iron deficiency anemia by feeding diet contained 20g/kg of body weight
tannic acid for four weeks. Group II was then randomly divided into 5 sub-groups of 6 rats each;
Group II2, positive control (+), while the other 4 sub-groups GII3, GII4, GII5 and GII6 were
treated by feeding fundamental diet supplemented with quinoa, either quinoa seeds 5% and 10%
or quinoa sprouts, 5% and 10% respectively, for two weeks (Afsana, Shiga, Ishizuka, & Hara,
2004). A flow diagram of the biological assay experimental design is detailed in Fig. (1).
2.6.2. Sampling procedures
At the end of the experimental period, final weights were recorded, and rats were sacrificed after
overnight fasting under light diethyl ether anesthesia. Body weight gain (BWG), average daily
gain weight and relative organ weights were calculated (Chapman, Castillo, & Campbell, 1959).
Blood samples, withdrawn from the veins of the eye plexus, were placed into tubes that contained
5 mL Drabkin’s solution for the determination of complete blood cell count (CBC) according to
the method of Ibrahium, (2015).
Another set of blood samples was collected from the hepatic portal vein into plain tubes,
centrifuged at 4000 rpm for 20 minutes at 4°C for serum separation for biochemical analysis
following the method described by Drury & Wallington (1980). Sample specimens of blood were
taken using micro capillary tubes and centrifuged at 5000 rpm for 5 min. The volume of blood
cells was measured by using a graded scale. Red blood cell, white blood cells, and platelets count
were measured according to Fischbach, (2015).
Serum samples were analyzed for determination of serum iron according to Wick, Pinggera, &
Lehmann (1996) and serum zinc concentrations were assessed by flame atomic absorption
technique according to Wu (2006). Total protein was determined according to Sonnenwirth &
Jarett (1980) and ferritin according to White, Kramer, Johnson, Dick, & Hamiton (1986).
2.7. Calculations of red cell indices

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 The hematocrit measures the volume of red blood cells compared to the total blood volume (red
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blood cells and plasma) (Billett, 1990). Diagnosis of anemia may be assisted by red cell indices;
the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and the mean
corpuscular hemoglobin concentration (MCHC), to define the size (MCV) and hemoglobin
content (MCH, MCHC) of red blood cells. Termed red cell indices, MCV, MCH and MCHC
values are useful in elucidating the etiology of anemia, which can be calculated from hemoglobin,
hematocrit, and red blood cell count according to (SARMA, 1990) as follows:
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑝𝑎𝑐𝑘𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 /1000 𝑚𝐿 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑
MCV = 𝑅𝑒𝑑 𝑏𝑙𝑜𝑜𝑑 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 𝑖𝑛 𝑚𝑖𝑙𝑙𝑖𝑜𝑛𝑠 /𝑚𝐿 𝑓𝐿 𝑜𝑟 μm3 (1)
𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 𝑖𝑛 𝑔 /1000 𝑚𝐿 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑
MCH = 𝑅𝑒𝑑 𝑏𝑙𝑜𝑜𝑑 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 𝑖𝑛 𝑚𝑖𝑙𝑙𝑖𝑜𝑛𝑠 /𝑚𝐿𝑝𝑔/cell (2)
𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 𝑖𝑛 𝑔 /100 𝑚𝐿 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑 𝑋 100
MCHC = 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑝𝑎𝑐𝑘𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 /100 𝑚𝐿 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑𝑔/𝑑𝐿 𝑜𝑟 % (3)

2.8. Statistical analysis:

Data were expressed as means ± standard deviation (SD). Results were analyzed by multiple
comparisons one-way analysis of variance (ANOVA) using Duncan test, IBM SPSS Statistics 23
software program where probability (p < 0.05) considered statistically significant.
3. Results and discussion
3.1. Influence of germination on chemical composition of quinoa seeds
Micro and macro elements contents of white quinoa seeds and sprouts including nutrients,
minerals (iron, calcium and zinc) and vitamin C (ascorbic acid) are given in Table (1). Results
revealed the high nutritional value of quinoa seeds, as 100 g of seeds provided energy of 371.18
Kcal, represented a good source of fiber and protein (43.08 and 30.62%) of daily requirements
respectively, with only 20.47 and 10.98% of daily values of carbs and fats. On the other hand, the
seeds can be considered a main source for vitamin C providing 3-fold of its daily requirements, as
well as iron covering more than half the daily requirements (56.78 DV%). Nutrients content of
white quinoa seeds were in agreement with USDA (USDA, 2018) and Padmashree et al., (2018).
These results supported previous studies in the area, which reported that quinoa provide more fiber
and minerals than many common grains (Abugoch James, 2009; Alvarez-Jubete, Arendt, &
Gallagher, 2009). Vilcacundo and coauthors (2017) reported that 40 g of quinoa seeds should meet
the daily recommendations (RDA) of essential nutrients, mainly vitamins, minerals and essential
amino acids (Vilcacundo & Ledesma, 2017).
Micro and macro nutrients play an important role in metabolic processes throughout the plant life
cycle (Halaby, Abdel-rahman, & Hassan, 2017); consequently their concentrations were

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 significantly affected during germination. Results revealed that germination of quinoa seeds
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significantly decreased protein content by 11% from 15.13 to 13.50 g/ 100g. The protein
mobilization during seed germination was reported as either due to degradation to smaller peptides
and amino acids or synthesis of new proteins (Ahmed, Abdel-Rahim, Abdel-Fatah, Erdmann, &
Lippmann, 1995). On the other hand, some researchers reported contra results of increased protein
content after sprouting (Padmashree et al., 2018; Shimizu & Mazzafera, 2000). The significant
decrease in carbohydrate content after germination from 61.42 to 58.69 g/ 100g can be attributed
to its utilization as a source of energy for the growth of embryo during germination process
(Ferreira et al., 2009; Vidal-Valverde et al., 2002). Similar observations were reported by (Pilco-
Quesada, Tian, Yang, Repo-Carrasco-Valencia, & Suomela, 2020). The significant increases
recorded in fat and fiber content of sprouts of 7.95 and 12.54 g/100g as compared to quinoa seeds,
which were 7.14 and 10.77 g/100g respectively, could be relative to the decreases of other
nutrients.
Fig. 2 (A and B) illustrates the influences of germination on antioxidants and anti-nutritional
factors of quinoa seeds. Dietary carotenoids are a class of phytonutrients that are thought to
provide health benefits by decreasing the risk of certain cancers and eye disease (Johnson, 2002).
Quinoa seeds were reported to possess the highest antioxidant content of 10 cereals, pseudo-
cereals and legumes (Valcárcel-Yamani & Lannes, 2012). The germination process could offer a
good strategy to improve the antioxidant activity properties of the seeds. The carotenoids content
was significantly increased by 26% in the obtained results (Fig. 2A). Carotenoids were found to
accumulate in seeds during seed germination (Smolikova, Laman, & Boriskevich, 2011).
Therefore, dried germinated quinoa seeds are recommended as a dietary ingredient in the
formulation of valuable functional foods (Carciochi, Manrique, & Dimitrov, 2014).
On the other hand, germination decreased the anti-nutritional factors, saponins, phytic acid and
tannins by 60, 50 and 11%, respectively (Fig. 2B). Despite saponins being a main cause of the
bitter taste, they are proposed to have biological effects such as antifungal, antiviral, anticancer,
diuretic, antithrombotic, hypocholesterolemic, hypoglycemic and anti-inflammatory activities
(Vega-Gálvez et al., 2010). The decrease in saponins is attributed to their leaching from quinoa
seeds during washing and soaking, as previously reported (Bhathal, Grover, & Gill, 2015).
Significant decrease in phytic acid content may be due to its break down as source of phosphorus
for utilization during germination (Padmashree et al., 2018). The decrease in tannins was due to
enzymatic changes during the germination period in seed (Megat Rusydi & Azrina, 2012).

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 Dietary minerals are essential chemical elements that play a role in regulating electrolyte balance,
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glucose homeostasis, the transmission of nerve impulses and as enzyme cofactors in the body. The
content of minerals illustrated in Table (1) showed that iron, calcium and zinc in quinoa seeds
(10.22, 32.44 and 0.79 mg/100 g, respectively) were increased by germination to 14.25, 48.35 and
0.95 mg/100g, i.e., by 39, 49 and 20%, respectively. Saponin, phytic acid and tannins are reported
to reduce the availability, solubility and functionality of minerals. Phytate chelates minerals and
decreases their bioavailability (Ekholm, Virkki, Ylinen, & Johansson, 2003). Endogenous
enzymes such as phytase accelerate phytic acid de-phosphorylation through catalysis and release
iron, calcium and zinc (Berry, Shang, Waltham Sajdak, & Zelazny, 2007). The significant
decrease in anti-nutritional factors in parallel with significant increase in carotenoids as
antioxidants by germination (Fig. 2), may explain the increased minerals content (Megat Rusydi
& Azrina, 2012).
Furthermore, vitamin C content was increased by 32% from 197.1 mg/100g to 260.5 mg/100g in
sprouts. These results are in agreement with the study of (Marton, M., Mandoki. Z., Caspo J.,
Caspo-Kiss, Marton, Mándoki, & Marton, M., Mandoki. Z., Caspo J., Caspo-Kiss, 2010), which
proposed the importance of vitamin C as an antioxidant and cell marking modulator in
physiological processes including biosynthesis, supporting phytohormone synthesis, the
establishment of the stress resistance, cell-division and the growth. The results confirm that
germination of quinoa seeds is a promising method to enhance their nutritional value (Pitzschke,
Fraundorfer, Guggemos, & Fuchs, 2015).
3.2. Influences of quinoa seeds/ sprouts diet on body weight gain
Data in Table (2) illustrate the average body weight gain of rats groups, which could be a
parameter indicating the health of the animals. Body weight gain was 156.6 g in the negative
control (GI), while it was 77.6 g for the positive control for induced anemic rats (GII2). Compared
to anemic rats (GII2), treatment with feeding 5 and 10% quinoa seeds reduced the weight loss and
increased the body weight gain to 91.8 g and 105.5 g in groups GII3 and GII4, respectively. On
the other hand, feeding the anemic rats 5 and 10% germinated quinoa (GII5 and GII6) increased
the body weight gain to 120.3 g and 126.0 g respectively. The significant increases in treated
anemic rats weights which were fed germinated quinoa could be related to the above-mentioned
nutritional profile changes in quinoa seeds brought by germination, especially enzymatic protein
breakdown and absorption along with elimination of saponins, which negatively affect
digestibility (Potter, Jimenez-Flores, Pollack, Lone, & Berber-Jimenez, 1993; Vlencia-Chamorroa,

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 2003). Anti-anemic impact of quinoa seeds and sprouts were previously documented (Ibrahium,
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2015; Manikandaselvi et al., 2015).
3.3. Relative organs weights of rats groups
Weights of organs, namely kidney, liver, spleen and heart of the rat groups, are exhibited in Table
(3). The data indicated that the organs most affected by iron deficiency anemia were kidney and
liver, which showed significant decreases in induced rat group GII2 of 37 and 24% respectively,
followed by spleen and heart with significant decreases of 15 and 14%, respectively.
Erythropoietin (EPO), also known as haematopoietin or haemopoietin, is a glycoprotein cytokine
hormone secreted mainly by the kidney in response to cellular hypoxia. EPO stimulates the bone
marrow stem cells to maintain and increase the production of red blood cells . The significant
effect of induced iron deficiency on the weight of the kidneys could be a result of the stress on
kidneys being forced to compensate by an altered mechanism to produce more RBCs (Xu, Peng,
& Ke, 2017). Furthermore, liver cells synthesis to EPO is activated when the kidneys are unable to
secrete sufficient amounts, which in turn may cause stress on liver as well affecting its weight (De
Seigneux et al. 2016).
Senescent red blood cells are engulfed by macrophages, primarily in the spleen and liver where
they are degraded and catabolized to be recycled and release the bound iron which considered the
primary source of iron (Johnson Wimbley & Graham, 2011). Since 1973, Rodvien and his
colleagues reported that the splenic changes associated with severe iron deficiency anemia
include: a) fragmentation and exaggerated phagocytosis of blood cells, b) mitochondrial changes
and c) changes associated with membranous structures and the endoplasmic reticulum(Rodvien,
Tavassoli, & Crosby, 1974). These findings may explain the significant decrease in spleen weight
reported in Table (3). Severe iron deficiency was reported to produce left ventricular dysfunction
and overt heart failure (Hegde et al., 2006). Decrease in heart weight pronounced in Table (3)
could be an alarm bell ringing for further complications.
All tested treatments, either with quinoa seeds (5 and 10%) or sprouts (5 and 10%), succeeded in
restoring organs of induced rats to become comparable to normal rats GI (negative control) with
no significant differences (p<0.5). These results supported the anti-anemic role of quinoa seeds,
which was previously reported (Bhathal et al., 2015; Navruz-Varli & Sanlier, 2016). Comparing
the four treated groups, GII3, GII4, GII5 and GII6, the most effective treatment was feeding 10%
quinoa sprouts (GII6). The increased bioavailability of iron in sprouts is hypothesized to be due to
the increased ascorbic acid and reduced phytic acid and anti-nutritional factors during sprouting;

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 these effects, together with elevated content of carotenoids, are proposed to be the main
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contributors to the anti-anemic effect of quinoa sprouts. Similar results were reported concerning
Vigna radiata sprouts (Manikandaselvi et al., 2015)
3.4. Impact of treatments on serum profile of rat groups
The impact of the treatments on the serum protein profile of the rat groups is illustrated in Fig. 3
(A and B). Total serum protein content (Fig. 3A) showed significant decrease by induction (GII2),
which was restored by feeding quinoa seeds or sprouts. Groups GII4, GII5 and GII6, which were
fed 10% quinoa seeds, 5 and 10% quinoa sprouts, respectively, reflected the restoration of total
protein to normal levels, with insignificant differences compared with healthy rats (GI). Although
there was an overall decrease in protein content of quinoa seeds with germination (Table 1),
Marton and colleagues stated that, beyond the changes in the quantity of the proteins, some were
noted to increase, and others to decrease or not to change during germination; the biological value
of the protein in sprouts increased, and greater digestibility was established in animal experiments
(Marton, M., Mandoki. Z., Caspo J., Caspo-Kiss et al., 2010).
Ferritin is a blood cell protein that contains iron, serves to store iron in tissues, and is a well-
known marker of iron deficiency anemia (Daru et al., 2017; Son et al., 2019). Ferritin
concentrations in the rat groups shown in Fig. (3B), were the lowest in induced anemic group
(GII2), and its content was enhanced in rats treated with quinoa seeds or sprouts. Germinated
quinoa 5 and 10% in groups GII5 and GII6, respectively, restored ferritin concentrations to the
normal level (GI). These results supported earlier research, which reported that high phytic acid
concentrations greatly inhibit iron bioavailability (Welch & Graham, 2004), and that germination
increased both concentration and bioavailability of minerals such as iron and zinc (Benincasa et
al., 2019).
The same hypothesis can explain the impact of treatments on serum mineral profiles of the rat
groups, which showed that concentrations of serum iron (Fig. 4A) and serum zinc (Fig. 4B)
exhibited the same pattern illustrated in ferritin concentrations Fig (3B). These results were in
agreement with the mineral concentrations in quinoa seeds and sprouts (Table 1). The
bioavailability of minerals in cereals is limited by the presence of anti-nutritional factors, which
presents challenges from a nutrition viewpoint (Das, Raychaudhuri, & Chakraborty, 2012).
3.5. Influence of quinoa seeds/ sprouts diet on complete blood count (CBC)
The complete blood count represented by red blood cells (RBCs), white blood cells (WBCs),
platelets and the protein hemoglobin (Hb) of the rat groups are shown in Table 4. The data

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 revealed that the iron deficiency anemia was reflected in all blood parameters. It was noteworthy
Accepted Article
that WBCs were the most affected parameter as they decreased in the induced rats group GII2
compared to the negative control group GI, by 50%, followed by RBCs (40%) then Hb and
platelets with 32 and 25%, respectively.
The positive control group, GII2, showed iron deficiency anemia (IDA) in all blood count
parameters. After 14 days of feeding the fundamental diet supplemented with quinoa seeds (5 and
10%) or sprouts (5 and 10%), in treated groups GII3, GII4, GII5 and GII6 the blood cell counts
were restored to be comparable to the control, especially in sprout-fed groups, GII5 and GII6. The
regeneration of blood cells could be due to the elevated ferritin, iron and zinc levels in quinoa
seeds/ sprouts treated rats, which showed the same pattern as the blood cell count (Figs. 3 and 4).
The restoration of blood cells, and especially Hb, was more pronounced in GII5 and GII6, after
feeding 5 and 10% quinoa sprouts, such that that there were no significant differences compared to
healthy rats (GI). These results support the traditional use of sprouts in the treatment of anemia in
that they increase bioavailability of minerals through reducing the anti-nutritional factors, in
agreement with previously reported results (Manikandaselvi et al., 2015).
3.6. Red blood cell indices of rat groups
Red blood cell indices of the rat groups, represented by Hct, MCV, MCH and MCHC, are reported
in Table 5. As can be seen, Hct and MCV values were decreased as a result of IDA induction in
GII2 compared to the healthy rat group (GI), and then increased by providing quinoa seeds and
sprouts. Feeding sprouts to GII5 and GII6s restored RBCs to the normal levels of the negative
control group GI. In contrast, the MCH index increased with induction in GII2 to 25.83 pg/cell
then decreased back to the normal levels when quinoa sprouts (5% or 10%) were fed (GII5 and
GII6). The hemoglobin content of red blood cells represented in the MCHC index showed an
insignificant decrease with induction (GII2), whereas the treated groups GII3, GII4 and GII5
showed lower values than the positive control GII2. The only positive effect was with 10% quinoa
sprouts (GII6), which restored the values to be comparable to healthy rats (GI). Similar results
were also reported by Manikandaselvi et al. (2015). Most clinical laboratories use the red blood
cell indices for their role in reflecting a precise vision that helps to reflect the levels of blood ratios
(Sarma, 1990). These results confirm the role of quinoa sprouts in treatment of IDA and indicate
that 10% quinoa sprouts were the optimum to restore the values of the blood indices to the healthy
levels.
4. Conclusions

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 Quinoa seeds were shown to be a good source of fiber, protein and vitamin C. Seed germination
Accepted Article
increased iron, calcium, zinc content by 39, 49 and 20%, and vitamin C and carotenoids by 32 and
26%, respectively, whereas anti-nutritional factors saponin, phytic acid and tannins were
decreased by 60, 50 and 11 %, respectively. The biological assays indicated that fortification of
feed with 10% quinoa sprouts (GII6) restored body and organs weights, serum profiles (protein,
ferritin, iron and zinc), blood cell counts (RBCs, WBCs and platelets), Hb, and red blood cell
indices (Hct, MCV, MCH and MCHC) in rats with induced iron deficiency anemia to levels
comparable to the healthy rat group (GI) after a 2-week period. Germinated quinoa seeds were
superior in nutrients compared to ungerminated seeds, which we propose is due to the activation of
endogenous enzymes that degrade anti-nutritional factors, elevate antioxidant properties, increase
vitamin C and increase the bioavailability of minerals, to exert anti-anemic effects. The results
indicated that germination is a key to improving quinoa as a complementary food and that dried
germinated quinoa seeds may be used as a dietary ingredient to improve the nutritional value of
functional foods.
Compliance with ethical standards
All institutional and national guidelines for the care and use of laboratory animals were followed.
This study protocol was reviewed and approved by Alexandria University Institutional Animal
Care and Use Committee (AlEXU-IACUC), a member of International Council for Laboratory
Animal Science (ICLAS) (permission number: AU08190625330)
Author contributions
Conceptualization, A.M.G.D., H.A.O.A. and H.A.E; Methodology, A.M.G.D., H.A.O.A. and H.A.E;
Formal Analysis, A.M.G.D., H.A.O.A. and H.A.E; Resources, A.M.G.D.; Data Curation, A.M.G.D.;
Writing – Original Draft Preparation, A.M.G.D. and H.A.O.A.; Writing – Review & Editing,
A.M.G.D., H.A.O.A. and H.A.E.
Funding
This research received no external funding.
Conflicts of Interest
The authors declare no conflict of interest.
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 Table (1): Influence of germination on quinoa seeds chemical composition
Accepted Article
Chemical composition Unit Quinoa seeds DV% Quinoa sprouts DV%
Energy Kcal 371.18a --- 360.3b ---
Nutrients
Moisture g/100g 10.77±0.51a --- 12.54±0.74c ---
Protein g/100g 15.31±2.4a 30.62 13.50±2.1b 27.00
Crude Fat g/100g 7.14±1.3a 10.98 7.95±1.5c 12.23
Crude fiber g/100g 10.77±2.7a 43.08 12.54±3.8b 50.16
Total carbohydrates g/100g 61.42±7.6c 20.47 58.69±6.4b 19.56
Ash g/100g 5.36±1.8b --- 7.32±1.3c ---
Minerals
Iron (Fe) mg/100g 10.22±2.2e 56.78 14.25±2.6b 79.17
Calcium (Ca) mg/100g 32.44±5.4f 3.24 48.35±6.2e 4.84
Zinc (Zn) mg/100g 0.79±0.06g 5.27 0.95±0.08b 6.33
Vitamin C
Ascorbic acid mg/100g 197.1±22.3a 303.23 260.5±31.4c 400.77
- Data presented (based on dry weight) are the means of duplicates ±SD
- DV is the Daily Values for nutrition labeling, calculated based on a caloric intake of 2,000
calories, for adults and children 4 years or more.
- A,b,..
Mean in the same row followed by different superscript letters in the same row differ
significantly (p<0.05)

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Accepted Article
Table (2): Body weight gain of rats groups
Groups Initial body weight Final body weight Body weight gain Average daily gain
Group I 155.0±2.70a 312.0± 1.58a 156.6± 2.70a 5.22± 0.05a
Group II2 155.4±2.60a 233.0± 2.34d 77.6± 4.04d 2.59± 0.07d
Group II3 153.8±3.49a 245.8± 1.92c 91.8± 5.10c 3.06± 0.08c
Group II4 154.9±2.13a 260.5±2.05b 105.5±1.21b 3.52±0.08c
Group II5 156.2±2.34a 276.5±3.51b 120.3±1.95b 4.01±0.04b
Group II6 153.2± .58a 279.2± 4.43b 126.0± 2.44b 4.20± 0.04b
- Group I: Negative control, Group II2: iron anemia induced group, Group II3: Quinoa seeds
5%, Group II4: Quinoa seeds 10%, Group II5: Quinoa sprouts 5% and Group II6: Quinoa
sprouts 10%
- Animals weights are represented in (g)
- Data are the means of 6 rats per group ±SD
- a,b,c,..
Means values with unlike letters in the same column are significantly different (p<0.05).

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Accepted Article
Table (3): Relative organs weight of rats groups
Groups Kidney Liver Spleen Heart
Group I 1.32 ±0.05a 3.69 ±0.10a 0.60 ±0.04a 0.65 ±0.06a
Group II2 0.83 ±0.04b 2.80 ±0.06b 0.51±0.03b 0.56 ±0.06b
Group II3 0.98 0.03ab 3.10 ±0.08a 0.56 ±0.05ab 0.60±0.04ab
Group II4 1.00 0.04ab 3.12 ±0.03a 0.58 ±0.04a 0.61±0.09ab
Group II5 1.11±0.02 a 3.25 ±0.02a 0.58 ±0.01a 0.62 ±0.05 a
Group II6 1.21±0.05 a 3.41±0.03a 0.59 ±0.01a 0.63 ±0.04a
- Group I: Negative control, Group II2: iron anemia induced group, Group II3: Quinoa seeds
5%, Group II4: Quinoa seeds 10%, Group II5: Quinoa sprouts 5% and Group II6: Quinoa
sprouts 10%
- Animals relative organs weights are represented in (g)
- Data are the means of 6 rats per group ±SD
- a,b,c,..
Means values with unlike letters in the same column are significantly different (p<0.05).

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Accepted Article

Table (4): Complete blood cell count (CBC) of rats groups

Groups RBCs WBCs Hb Platelets
(106/μL) a (103/μL) a (g/dL) (μm3) a
Group I 6.04±0.56 9.38±1.96 13.64±0.40a 438.6±4.54
Group II2 3.6±0.2c 4.66±0.82c 9.3±0.32c 330.2±4.16c
Group II3 4.36±0.11b 5.54±1.87b 11.18±0.44b 353.0±7.17b
Group II4 4.88±0.48b 6.28±1.92b 11.76±0.32b 391.0±4.63b
Group II5 5.27±0.42a 7.38±1.08a 12.25±0.25a 408.0±5.36a
Group II6 5.91±0.27a 8.49±1.19a 13.39±0.28a 427.0±4.92a
- Group I: Negative control, Group II2: iron anemia induced group, Group II3: Quinoa seeds
5%, Group II4: Quinoa seeds 10%, Group II5: Quinoa sprouts 5% and Group II6: Quinoa
sprouts 10%
- RBCs, Red Blood Cells; WBCs, White Blood Cells; Hb, Hemoglobin
- Data are the means of 6 rats per group ±SD
- a,b,c,..
Means values with unlike letters in the same column are significantly different (p<0.05).

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Accepted Article
Table (5): Red cell indices of rats groups
Groups Hct MCV MCH MCHC
(%) (μm3) a (pg/cell) (%)
Group I 41.20±0.83a 91.4±4.56 22.58±1.18a 33.11±1.18a
Group II2 29.0±1.22b 68.4±3.04c 25.83±1.06b 32.07±1.34a
Group II3 36.6±1.51ab 82.2±3.27b 25.64±0.98b 30.55±1.26b
Group II4 38.6±1.81ab 82.8±1.64b 24.10±1.27ab 30.47±1.53b
Group II5 39.82±1.08a 86.45±1.59a 23.24±0.86a 30.76±1.28b
Group II6 40.15±1.24a 89.62±1.39a 22.66±1.10a 33.35±1.07a
- Group I: Negative control, Group II2: iron anemia induced group, Group II3: Quinoa seeds
5%, Group II4: Quinoa seeds 10%, Group II5: Quinoa sprouts 5% and Group II6: Quinoa
sprouts 10%
- Hct, Hematocrit; MCV, Mean Corpuscular Volume; MCH, Mean Corpuscular Hemoglobin
and MCHC, Mean Corpuscular Hemoglobin Concentration
- Data are the means of 6 rats per group ±SD
- a,b,c,..
Means values with unlike letters in the same column are significantly different (p<0.05).

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 cche_10366_f1-4.docx

Accepted Article
Thirty six male Sprague-Dawley (SD) albino rats
(150 ±10 g)

Standard conditions (22°C±1°C, 12-h light/ 12-h dark cycle) Acclimatization

Fundamental diet (fat 2.8 %, protein 18.5 %, fiber 11.2 %) (one week)
Water ad libitum

GI Iron Deficiency Anemia

Negative Control Induced Groups
Group (GII)

Induction diet 20g/kg of body Induction

weight tannic acid (Four weeks)

Test Groups
(Two weeks)

GII2 Treated Groups

Positive Control Group
Fundamental Diet

GII3 GII4 GII5 GII6

Fundamental Diet + Fundamental Diet + Fundamental Diet + Fundamental Diet +
Quinoa Seeds 5% Quinoa Seeds 10% Quinoa Sprouts 5% Quinoa Sprouts 10%

Fig. 1. Flow diagram of biological assay experimental design

- Groups are 6 rats each

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Accepted Article 60
A
b

50 c

40
g/100g

30

20

10

0
Quinoa seeds Quinoa sprouts

Quinoa seeds Quinoa sprouts

B
3 a
2.5
g/100g

1.5
b e
1
f c
c
0.5

0
Saponin Phytic acid Tannins

Fig. 2. Influences of germination on antioxidants and anti-nutritional factors of quinoa

seeds
A: Carotenoids content in quinoa seeds and sprouts, B: Anti-nutritional factors in
quinoa seeds and sprouts (Saponin, phytic acid and tannins)
Data presented are the means of duplicates ±SD
a,b,c,..
Means values with unlike letters are significantly different (p<0.05).

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Accepted Article 7
A
a a
a
6 a
b
5
c
4
g/dl

B
250 a
a
a
b
200
c

d
150
μg/L

100

50

Fig. 3. Impact of treatments on serum protein profile of rats groups

A: Total protein concentrations in rats groups, B: Ferritin concentrations in rats groups
- Group I: Negative control, Group II2: iron anemia induced group, Group II3: Quinoa seeds
5%, Group II4: Quinoa seeds 10%, Group II5: Quinoa sprouts 5% and Group II6: Quinoa
sprouts 10%
- Data are the means of 6 rats per group ±SD
- a,b,c,..
Means values with unlike letters are significantly different (p<0.05).

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Accepted Article 4
A
a
a
3.5 b ab ab

3
2.5
μg/dl

2 c

1.5
1
0.5
0

B
180 a a
160 a
b
140 c
d
120
100
μg/dl

80
60
40
20
0

Fig. 4. Impact of treatments on serum mineral profile of rats groups

A: Iron concentrations in rats groups, B: Zinc concentrations in rats groups
- Group I: Negative control, Group II2: iron anemia induced group, Group II3: Quinoa seeds
5%, Group II4: Quinoa seeds 10%, Group II5: Quinoa sprouts 5% and Group II6: Quinoa
sprouts 10%
- Data are the means of 6 rats per group ±SD
- a,b,c,..
Means values with unlike letters are significantly different (p<0.05).

This article is protected by copyright. All rights reserved