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Nutritional value and impact of wheatgrass
juice (Green Blood Therapy) on increasing
fertility in male albino rats
Hesham A. Eissa
1
, Sherif S. Mohamed
2*
and Ahmed M. S. Hussein
1
Abstract
Background and objective: The wheatgrass juice (WGJ) contains a high concentration of vitamin C. WGJ contains
a lot of highly functional nutritive ingredients potent to unify the liver with the kidneys for detoxification of the
organs and filtration of the blood to build a strong immune system. Also, it boosts fertility and increases sexual
desire because of the high magnesium content in phytochemical pigment (chlorophyll) which boosts the production
of the enzymes that restores sex steroids.
Materials and methods: The experimental animals were divided into 3 groups of 8 rats. The first group (G1) was fed
on the standard normal diet. The same feeding was used also in the second group (G2) and third group (G3). But in
the G2, a pharmaceutical formula (contain zinc and vitamin A) was used as a food supplement to increase fertility, and
in the G3, wheatgrass juice of 11 mg/day was applied. Hunter L,a,andbvalues; glucose, fructose, and sucrose
contents; pH; total soluble solids (TSS); acidity; concentration of vitamin C and vitamin B complex contents; and
phenolic compounds of wheatgrass quality juice were measured.
Results: The juice samples showed L* value of 21.78 (as a lightness index), a* value of −7.11 (as a redness index), b*
values of 17.35 (as a yellowness index), pH (6.7), TSS (5°Bx), and acidity (0.00992%). In the same time, wheatgrass
chlorophyll represents 70% of its total chemical constituents that is an antioxidant and rebuilds the bloodstream. In
addition, it gives the juice its distinctive green color which is the major quality factor in juice products. The results
amounted to normal values of vital organs such as the liver and kidney functions in all groups. The values of aspartate
aminotransferase (AST) were 27.88 ± 2.10, 22.50 ± 4.93, and 23.25 ± 4.71 μ/ml in G1, G2, and G3, respectively. Meanwhile,
also the results of sexual hormones indicated an elevation in testosterone hormone in G3 (2.90 ± 0.26 ng/ml) than the
normal negative control (2.78 ± 0.23 ng/ml) and pharmaceutical formula positive control (2.04 ± 0.40 ng/ml). However,
follicle-stimulating hormone (FSH) decreased to 1.44 ± 0.28 IU/L and 1.45 ± 0.24 for G3 and G2, respectively, compared
to 1.65 ± 0.23 IU/L in G1.
Conclusion: The findings proved that WGJ increased fertility and promoted youthfulness, and the wheatgrass (WG)
has the potential to be used as a “functional herb”containing natural bioactive compounds.
Keywords: Wheatgrass juice, Nutritional value, Juice quality, Restores fertility, Oxidative stress
© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
* Correspondence: shereifsalah@yahoo.com
2
Nutrition and Food Science Department, National Research Centre, 33 El
Bohouth St., Cairo 12622, Egypt
Full list of author information is available at the end of the article
Bulletin of the National
Research Centr
e
Eissa et al. Bulletin of the National Research Centre (2020) 44:30
https://doi.org/10.1186/s42269-020-0272-x
Introduction
Nowadays, natural or organic bioactive compounds that
exist in herbs are considered as an “alternative”medicine.
Among herbal and natural active compounds that are
gaining scientific concept, wheatgrass (WG) as a “func-
tional food”is becoming more available and popular as a
research topic. Wheatgrass is the mature shoot of Triticu-
maestivum Linn. belonging to the family Gramineae (Rana
et al. 2011). Fortunately, wheatgrass considered a high nu-
tritional phytoactive ingredients content that boost alter-
native medicine value as well as anti-inflammatory,
antioxidant, immunomodulatory, anticarcinogenic, laxa-
tive, and anti-aging properties, and the use of WG in ath-
erosclerosis, colitis, kidney malfunctions, and swelling has
many remedies for many ailments.
Wheatgrass juice (WGJ) can be swallowed orally and as
a colon implant without any risk or hazard effects. WGJ is
rich in chlorophyll which is an important phytopigment
that has an anti-bacterial effect; meanwhile, the chloro-
phyll of wheatgrass is associated with many health benefits
and rebuilds the bloodstream. Studies revealed that ani-
mals that intake chlorophyll in the diet are free of any
toxic reaction (DeVogel et al. 2005). In the same time,
chlorophyll is a potent antioxidant with an effect on can-
cer prevention. Additionally, selenium and laetrile exist in
wheatgrass have anti-cancer activity. Selenium is incorpo-
rated as a bioactive natural element that enhances the im-
mune system and can reduce the risk factors of cancer.
Wheatgrass comprises of at least 13 vitamins that include
B12 and abscisic acid in addition to superoxide dismutase
(SOD), cytochrome oxidase, and mucopolysaccharide
(Ferruzzia and Blakesleeb 2007; Wheat and Currie 2008).
A normal person should drink 1 fl oz (30 ml) of fresh
wheatgrass juice twice a day or take 3 g of wheatgrass
powder twice a day. A little honey may be added to the
juice to enhance the taste. Alternately, approx. 50 g of
fresh wheatgrass could be chewed. Food should not be
consumed for half an hour before and after taking
wheatgrass. Wheatgrass powder may also be added to
regular food as children sometimes may avoid taking it
(Grunewald 2009). WGJ is a main source that supple-
ments a lot of nutrients such as iron, calcium, magne-
sium, amino acids, chlorophyll, bioflavonoids, phenol
compounds, and vitamins A, B, C, and E which have an
important role in the prevention of various diseases
(Peryt et al. 1992; Mujoriya et al. 2012; Sangeetha and
Baskaram 2010; Durairaj et al. 2014).
Wheatgrass can be consumed on its own or used in
combination with other juices or supplements (Eissa
et al. 2018). According to Lani Lopez, a naturopath and
the author of the “Natural Health,”detoxing and de-
creasing the oxidative stress of the body before preg-
nancy can help elevate the fertility and boost a healthy
pregnancy. Detoxification is the best process for both
men and women as a part of pre-pregnancy care, as
purifying internal organs of toxins and oxidative stress
can strengthen the body. Drink WGJ daily before preg-
nancy clean the blood from many toxins (Pannu and
Kapoor 2015). The Mayo Clinic reports stated that 15%
of couples are unable to have a child as one or both of
them suffer from fertility problems. Diet may also influ-
ence the quality of fertility status. In addition to support-
ing the health and increase body protection, wheatgrass
may be potent to boost human fertility due to getting
adequate amounts of vitamin C according to the WGJA
study defined by Fernandes Glaura (2011) who investi-
gated the vitamin C effect on hyperglycemic rats and
found that it declines the amount of abnormal sperm
and raises testosterone in blood. Also, it retrieves fertility
and induces a healthy youthfulness because of promot-
ing magnesium content from chlorophyll which is a co-
enzyme for the enzymes that rejuvenate sex hormones.
Moreover, it heightens resistance to ailments by elimin-
ating body toxins, and because of its alkaline properties,
it is best for urinal disease.
In the same time, the absorption of such nutrients
through the digestive system is necessary. Many people
with these deficiencies suffer from cavities and other
dental problems even though they brush and floss every
day. Brushing and flossing are undoubtedly vital to per-
fect dental health but cannot be effective unless mineral
and vitamin deficiencies are corrected (Sareen et al.
2014).
The objective of this research was to evaluate the nu-
tritional value and quality of WGJ to produce a healthy
with vitality function product, high-quality color, and
freshness. So, the current research studied the ability
and potentiality of WGJ as a functional juice to improve
the fertility and sexual potency in male rats. It based on
the WG plant rich in bioavailability of multi-active nu-
trients which have induced fertility and promote
youthfulness.
Material and methods
Raw materials
Wheatgrass (Triticum aestivum cv.) obtained from field
experimental in the Institute of Agronomy Crops at the
Agricultural Research Institute, Giza, Egypt. Wheatgrass
samples were used for all processing trials and were
stored at 4 °C after receipt and prepared within 24 h.
Extraction and processing of wheatgrass juice
In First, the fresh wheatgrass cleaned, soon after cutting
it on a pounding basin and crush it well. This wheatgrass
can be also crushed in the electric juicer or mixer also.
A stainless steel sieve could also be used for this pur-
pose. Second, wrap them in a clean and thin piece of
double-layer cheesecloth and strain the juice out of it to
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 2 of 11
remove the pulp and obtain the juice, and store at 4 °C
prior to processing and blends. The extraction of wheat-
grass juice will be in a greater quantity, and its effective-
ness is also strengthened.
Physico-chemical analyses
The pH of WGJ was measured using a digital pH-meter
(HANNA, HI 902 m, Germany). The percent of total sol-
uble solids (TSS), expressed as °Bx (0–32), was determined
with a hand refractometer (ATAGO, Japan). Titratable
acidity of juice samples was determined according to the
method reported by Tung-Sun et al. (1995).
Color characteristics
Color of wheatgrass juice was measured using spectro-
colorimeter (Tristimulus Color Machine) with the CIE-
LAB color space (International Commission on Illumin-
ation) as mentioned by Sapers and Douglas (1987) and
Hunter (1975). The color of fresh wheatgrass juice sam-
ples was measured using a HunterLab colorimeter
Hunter a*, b*, and L*. Parameters were measured with a
color difference meter using a spectro-colorimeter (Tri-
stimulus Color Machine) with the CIE lab color scale
(Hunter, Lab Scan XE - Reston VA, USA) in the reflec-
tion mode. The instrument was standardized each time
with a white tile of Hunter Lab Colour Standard (LX
No. 16379): X= 72.26, Y= 81.94, and Z= 88.14 (L*=
92.46, a*=−0.86, b*=−0.16). The instrument (65°/0°
geometry, D25 optical sensor, 10° observer) was cali-
brated using white and black reference tiles. The color
values were expressed as L* (lightness or brightness/
darkness), a* (redness/greenness), and b* (yellowness/
blueness). The Hue (H)*, Chroma (C)*, and browning
index (BI) were calculated according to the method of
Palou et al. (1999) as follows:
H¼ tan−1b=a
½ ð1Þ
C¼square root of a2þb2½ ð2Þ
BI ¼100 x−0:31ðÞ½10:72 ð3Þ
Where −X=(a* + 1.75 L*)/(5.645 L*+a*−3.012b*)
ΔE¼Δa2þΔb2þΔL2ðÞ1=2ð4Þ
where all values were recorded as the mean of triplicate
readings.
Vitamin C determination
Vitamin C was analyzed using the A.O.A.C. (2006)
method. Vitamin C retention was calculated using Eq.
(5).
Retention %ðÞ¼
Mg ascorbic acid=100 mL juice after treatment
Mg ascorbic acid=100 mL juice before treatment 100
ð5Þ
Sugar determination
The extraction for determination of sugar acids using
high-performance liquid chromatography (HPLC) was
achieved by stirring 3.0 g of each sample with 20 mL of
distilled water, followed by centrifugation at 10,000 g for
10 min at 30 °C. The residues were washed four times
with the same amount of water in order to remove all
sugar, and the supernatants were combined. An aliquot
of each sample was filtered through 0.22 μm Millipore
membranes.
For the determining sugars, an Agilent model 1100
Series (Agilent, USA) high-performance liquid chroma-
tography equipped with a quaternary pump, refractive
index detector, and Shim-pack SCR-101N (300 mm L. ×
7.9 mm I.D., 10 μm). The mobile phase was deionized
water, degassed under vacuum in an ultrasonic bath.
The flow rate was 0.7 mL min
−1
at a temperature of
40 °C. The quantification was achieved by comparison
with analytical curves using glucose, fructose, and su-
crose standards.
Determination of B complex vitamins
Samples were submitted to successive hydrolysis with
hydrochloric acid and enzyme hydrolysis using diastase
following a procedure described by Viñas et al. (2003).
Phenolic acid compound profile
Phenolic acids were extracted and determined according
to Kim et al. (2006).
Diet animal materials
The ingredients used for the preparation of the diet
given to the animals were purchased from the local mar-
ket. These items were corn starch, sucrose, and soybean
oil purchased fresh from specialized stores. Casein was
obtained from SiscoResearch Laboratories PVT, LTD,
India. Salts and vitamins used for the preparation of the
salt and vitamin mixtures were obtained from Merck,
Germany, and prepared according to (AIN 93) Philip
et al. (1993).
Animals used in this experiment were Sprague-Dawley
male rats, obtained from the animal house of the Na-
tional Research Centre; their body weight ranged be-
tween 90 and 110 g. Kits used for the estimation of the
analyzed parameters were obtained from Biodiagnostic,
Egypt.
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 3 of 11
Design of animal experiment
The animal experiment comprised 3 groups each of 8
rats. The 1st group (G1) was fed on the standard normal
diet. The same 2nd group (G2) and 3rd (G3) were fed
on a standard normal diet. The same 2nd group feed the
pharmaceutical formula of zinc and vitamin A calculated
according to the Institute of Medicine IOM, Food and
Nutrition Board (2001) and added to rat diet. It is often
prescribed to patient suffered from sexual dysfunction as
a dietary supplement in the form of gelatin capsules,
each contains 25 mg of zinc, vitamin A 50000u, and vita-
min E 100 mg.
The 3rd group demonstrated wheatgrass juice by gav-
age, the quantity of juice calculated the zinc content in the
juice, and zinc concentrations were given based on the
daily dietary allowance of 11 mg/day for male rats (Insti-
tute of Medicine IOM, Food and Nutrition Board 2001).
Animals were kept individually in stainless steel cages,
standard diet and distilled water were allowed ad libitum,
and the room temperature was adjusted at 25 °C. The
feeding period continued for 6 weeks. During the experi-
mental period, the bodyweight of the animals was
followed. The experimental procedure was carried out ac-
cording to the Institutional Animal Ethics Community of
the NRC, Egypt. At the end of the experimental period,
animals fasted overnight, and in the morning, blood sam-
ples were taken from each rat by open heart puncture
under light ether anesthesia. Blood samples were left to
clot at room temperature and then centrifuged at 3500
rpm and serum was separated. The serum samples were
kept in the deep freeze at –20 °C till analysis.
Aspartate aminotransferase (AST), alanine amino-
transferase (ALT), total bilirubin, and albumin were esti-
mated according to Reitman and Frankel (1957), Malloy
and Evelyn (1937), and Bartholomev and Delany (1966).
Urea, creatinine, and uric acid were determined accord-
ing to Fawcett and Soctt (1960), Bartles et al. (1972), and
Carroll et al. (1971), respectively.
Malondialdehyde was assessed in blood serum accord-
ing to the procedure of Satoh (1979), superoxide dismut-
ase (SOD) determination was based on the method
developed by McCord and Fridovich (1969), and gluta-
thione peroxidase (GPx), catalase (CAT), and reduced
glutathione (GSH) were determined according to the
methods of Weinhold et al. (1990), Aebi (1984), and Ell-
man (1959), respectively. The total cholesterol was eval-
uated by the technique described by Allain et al. (1974),
serum triglycerides were determined according to Fos-
sati and Prencipe (1982), LDL-ch was assessed according
to Levy (1981), and HDL-ch was evaluated according to
Burstein (1970).
The levels of hormones were evaluated with detection
kit according to the manufacturer’sinstructionsusing
ELISA technique: AFP, TSH, FSH, LH, E2, and
Testosterone using ELISA technique purchased from
Immunosec Corporation., USA, and Elisa Kit for Testos-
terone Testing in Rats and Mice by Rocky Mountain Diag-
nostics Inc.
Histopathological examination
After blood collection, all rats were rapidly sacrificed
and the testes of each animal were dissected, and a por-
tion of it was preserved in 10% buffered neutral formalin
and paraffin-embedded. Four sections (5 microns in
thickness) were taken from each test tissue, each section
being at a distance at least 500 μfrom the proceeding
one section was stained with hematoxylin-eosin, and the
slides were examined with a light microscope under × 20
magnification. Randomly selected fields were evaluated
for cellular and tubular structures. Degeneration in epi-
thelium and interstitial spaces were also noted according
to Ross et al. (1989).
Statistical analysis
All studied data were statistically analyzed using Co-Stat
6.303 Software Computer Program 2004 hypothesis test-
ing methods included one-way analysis of variance
(ANOVA) using Duncan Test (COSTAT-C) (1988).
Results
Physico-chemical of wheatgrass juice
It can be observed in Table 1the results of physico-
chemical parameters of pH, total soluble solids (TSS, °Bx
or %), titratable acidity, TSS/acidity ratio, and vitamin C
(mg/100 ml) in the samples obtained after the processing
of wheatgrass juice. Freshly extracted of wheatgrass juice
was subjected to various tests in order to determine its
chemical composition.
The TSS was 5°Bx. The total titratable acidity was
cleared in terms of citric acid as the percentage on a
fruit weight basis. Titratable acidity was measured as
0.00992%, and the TSS/acidity ratio was found to be in
wheatgrass juice of 504. Also, the pH value was recorded
as 6.7 as seen in Table 1.
Results in Table 1illustrated that the wheatgrass juice
contained a good value of vitamin C (0.360 mg/100 mL
juice). The good ratio of vitamin C in wheatgrass juice
Table 1 Physico-chemical of wheatgrass juice
Physico-chemical Wheatgrass juice
pH 6.7 (± 0.03)
**
TSS (%) 5.00 (± 0.05)
Titratable acidity
*
0.00992 (± 0.01)
TSS/acidity ratio 504 (± 0.06)
Vitamin C (mg/100 ml) 0.360 (± 0.01)
*
Total or titratable acidity expressed as citric acid (mg/100 g)
**
±Standard deviation (SD)/SQR
2
(n), where n=3
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 4 of 11
should be considered as a good source for such vitamin.
These results amounted to an agreement with the results
of Tung-Sun et al. (1995).
Color characteristics and parameters of wheatgrass juice
Color is a major quality factor in juice products, and a
part of the overall appearance, measurement directly in
the juice samples with a Hunter Lab Ultra Scan revealed
that color changed in some commercial juice sample
(Table 2). In this case, brightness (L* values) decreased,
redness (a* values) increased, and yellowness (b* values)
decreased. The results of periodically examined proper-
ties of the commercial wheatgrass are shown in Table 2.
Vitamin B complex contents in wheatgrass juice
There is no spotting on foods of current interest to in-
crease nutrition awareness among consumers. But,
wheatgrass juice can be easily found with nutritional
compounds to provide convenient juices, in order to
supplement vitamin in the diet and nutrition. The
amount of vitamin B3 (nicotinic) in wheatgrass juice
samples was 34.47 μg/ml (Table 3). The highest content
of vitamin B3 was found in wheatgrass juice. Among all
wheatgrass juice samples, vitamin B2 (riboflavin) was the
lowest in wheatgrass juice (1.28 μg/ml). The highest con-
tent of vitamin B1 (thiamine) was present in wheatgrass
juice which was 201.80 μg/ml.
Furthermore, pyridoxine (vitamin B6) content in
wheatgrass juice was 4.66 μg/ml. However, the levels of
the other two vitamins, B9 (folic) and B12 (cobalamin)
differed between wheatgrass juice samples being 7.40
and 28.90 μg/ml, respectively. From the aforementioned
data, it could be concluded that investigated wheatgrass
juice is considered to be rich sources of B complex vita-
mins and had a necessary bio-components that play a
great role to protect the human body.
To overcome these limitations in the determination
and extraction of B vitamins, HPLC-UV is the best
option. The accuracy of this method has been validated
by using the recovery tests. Determining the concentra-
tions of B1 (thiamine), B2 (riboflavin), B3 (nicotinamide),
B6 (pyridoxine), B9 (folic acid), and B12 (cyanocobala-
min) in fresh wheatgrass juice samples was the aim of
this method. The results are shown in Table 3. For the
spiked wheatgrass juice, samples had the valuable recov-
ery ranges from 94.5–117.3%. The concentrations of B1,
B2, B3, B6, B9, and B12 in the wheatgrass juice samples
were obtained to be 201.8, 1.28, 34.47, 4.66, 7.4, and
28.9 μg/ml, respectively, as seen in Table 3.
Phenolic acid compound profiles in wheatgrass juice
The estimation of total phenolic acids quantified in this
research varied for the different samples. No significant
difference (p< 0.05) was observed for the phenolic acids
identified. The major compounds were high in sinapic
(27.98 μg/ml) and protocatechuic (22.34 μg/ml), followed
by caffeic acid (12.04 μg/ml), rosmarinic (11.32 μg/ml),
and gallic (8.9 μg/ml), but were low in catechin (0.81 μg/
ml), ferulic (0.47 μg/ml), cinnamic (0.299 μg/ml), vanillic
(2.17 μg/ml), chlorogenic (3.3 μg/ml), p-hydroxybenzoic
(4.05 μg/ml), and p-coumaric acids (3.04 μg/ml), as seen
in Table 4.
The wheatgrass juice had widely different phenolic
contents as revealed in the data presented in Table 4.
Total phenolics measured by the Folin-Ciocalteu assay
varied 8.6-fold and 19-fold when measured by HPLC.
The wheatgrass juice not only contained the highest
standard of phenolic acids, but also contained the largest
number of individual phenolic compounds; 24 were
identified, with 11 being present in concentrations of >
1μg/ml (Table 4).
Glucose, fructose, and sucrose analysis by HPLC in
wheatgrass juice
From the result represented in Table 5, it can be defined
that HPLC is more accurate in the determination of glu-
cose, fructose, and other sugars; furthermore, it gives a
direct fast reading because it is a computerized feeding
system. Using peak areas, a linear regression line was
drawn for each sugar (glucose and fructose). The con-
tent of total sugar in juices is essentially made up of glu-
cose, fructose, and sucrose (saccharose). Contents of
Table 2 Color characteristics and parameters of wheatgrass
juice
Color characteristics Wheatgrass juice
Lvalue 21.78 (± 0.38)
avalue −7.11 (± 0.15)
bvalue 17.35 (± 0.31)
ΔE 73.54 (± 0.06)
A
420 nm
0.91 (± 0.01)
A
400 nm
0.91 (± 0.01)
C value 18.75 (± 0.34)
H value 67.73 (± 0.08)
BI value 35.40 (± 0.32)
± Standard devia tion (SD)/SQR
2
(n), where n=3
Table 3 Vitamins B complex of wheatgrass juice contents
B complex vitamins Wheatgrass juice (μg/ml)
Nicotinic acid, B3 34.480
Thiamine, B1 201.80
Pyridoxine, B6 4.660
Folic acid, B9 7.400
Riboflavin, B2 1.280
Cyanocobalamin, B12 28.900
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 5 of 11
glucose and fructose were 17.48 mg/ml and 12.10 mg/ml
in wheatgrass juice, respectively, by HPLC; glucose (Gl)
to fructose (Fr) ratios were 1.44, while according to the
HPLC determination, there is no sucrose in the wheat-
grass juice, as shown in Table 5. It is necessary to deter-
mine the contents of sucrose, glucose, and fructose, as
well as glucose to fructose ratio in wheatgrass juice. On
the basis of sucrose, glucose, and fructose contents, as
well as glucose to fructose ratio, it cannot be stated
whether the examined wheatgrass juice is authentic or
adulterated with other wheatgrass juice.
Biological and biochemical evaluation
It is clear in Table 6that the liver and kidney functions
are in normal level in all experimental rats, this vital or-
gans such as the liver and kidney that describes the tox-
icity or the safety of the WGJ in which the values of AST
27.88 ± 2.10, 22.50 ± 4.93, and 23.25 ± 4.71 μ/ml in G1,
G2, and G3, respectively, were improved than the normal
control (G1). We observed also no differences between
the results of total bilirubin and albumin 0.58 ± 0.19 and
0.60 ± 0.17 μ/ml; 0.55 ± 0.16 and 3.78 ± 0.24; and 3.74 ±
0.17 and 3.59 ± 0.21 g/dl, respectively.
Table 7presented the parameters of lipid in the blood
of rats that the lowest value of mean ± SD of triglycer-
ides, total cholesterol, and LDL cholesterol in G3
amounted to 105.00 ± 10.22, 83.75 ± 13.02, and 95.00 ±
10.33 mg/dl, respectively.
As observed in Table 8, the increased in anti-oxidative
enzyme value mean ± SD of superoxide dismutase (SOD)
as well glutathione peroxidase (GPx) reduced the gluta-
thione (GSH) and catalase (Cat) 27.88 ± 3.14 μ/mg,
163.75 ± 10.61 μ/mg, 181.25 ± 10.61 mmol/g, and 38.63 ±
2.67 U/mg, respectively.
On the other hand, wheatgrass contain many vitamins
recorded in Table 1with vitamin C value 0.358 mg/100
ml and in Table 3with vitamin B12 value 28.89 μg/ml
acts as an antioxidant, reduces oxidative stress, and also
delays aging of cells in the body that causes much illness
such as brain and heart problems. Many of the studies
showed that the water extracts of wheatgrass as WGJ are
a good source of antioxidants. Such wheatgrass juice ex-
tracts antioxidant more potential and can be used as a
dietary source for antioxidant nutrients such as polyphe-
nols as showed in Table 4, gallic acid with an altitude score
of 8.943 μg/ml, and flavonoids (Mujoriya et al. 2012).
Meanwhile, the results of sexual hormones from
Table 9indicated elevation in testosterone hormone
mean ± SD value 2.90 ± 0.26 ng/ml than normal negative
control value 2.78 ± 0.23 ng/ml and pharmaceutical for-
mula positive control value 2.04 ± 0.40 ng/ml, but
follicle-stimulating hormone (FSH) decrease in mean ±
SD value 1.44 ± 0.28 IU/L less than values 1.45 ± 0.24
and 1.65 ± 0.23 IU/L respectively.
Histopathological of testicular tissue
Figure 1revealed a normal histological appearance in
G1, G2, and G3 of the seminiferous tubules, germ cells
with sperm formation, and interstitial Leydig cells. The
testes from the groups are noticed with apparently nor-
mal seminiferous tubules (H&E × 200), and spermato-
genesis is extremely regular and highly efficient such
that in the normal adult rat (> 10 weeks old), there are
very few degenerating or depleted germ cells. But in G3,
there were no degenerating, depleting, or increasing
germ cells compared with G1 and G2.
Table 4 Phenolic acid compound profiles in wheatgrass juice
Phenolic compounds Wheatgrass juice (μg/ml)
Gallic 8.942794
Protocatechuic 22.34275
p-hydroxybenzoic 4.051335
Gentisic 1.749315
Catechin 0.808809
Chlorogenic 3.298519
Caffeic 12.0373
Syringic 0.582461
Vanillic 2.166147
Scopoletin 0.628308
Ferulic 0.468136
Sinapic 27.98077
Rutin 6.061411
p-coumaric 3.044506
Naringin 0
Hesperidin 0
Apigenin-7-glucoside 1.851254
Rosmarinic 11.31671
Cinnamic 0.298989
Luteolin 0
Naringenin 0
Apigenin 0.226837
Kaempferol 0.339542
Chrysin 1.258273
Table 5 Glucose, fructose, and sucrose analysis by HPLC in
wheatgrass juice
Sugar contents Wheatgrass juice (mg/ml)
Glucose (Gl) 17.48
Fructose (Fr) 12.10
Sucrose (Suc) 0
Gl to Fr ratio 1.44
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 6 of 11
Discussion
Exactly, results revealed the total soluble solid (TSS), the
total titratable acidity was cleared in terms of citric acid
as percentage on fresh fruit weight basis, TSS/acidity ra-
tio was found to be in wheatgrass juice, and the pH are
in line with the findings of Ashoke et al., Srivastava
et al., and Stamp and Labuza (Rathod et al. 2011; Ingle
et al. 1981; Srivastava and Rajput 2003; Rajput et al.
2004). The decline in the total titratable acidity should
lead to elevate the total sugar content of the fruits. At
the time of maturity, fruits will be having a higher
amount of titratable acidity, but as the fruits advance to-
wards ripening, the acid content will decrease. These re-
sults are in line with the findings of Ingle et al. (1981),
who observed a decrease in acidity during the ripening
of sapota fruits.
In this study, the L* value (21.78 as a lightness index),
a* value (−7.11 as a redness index), b** values (17.35 as
a yellowness index), H* (67.73), C* (18.75), browning
index (35.40, BI), ΔE (73.54), and A420 nm (0.91) as
non-enzymatic browning according to Stamp and
Labuza 1983 and Birk et al. 1998 were statistically sig-
nificant (p< 0.01) and were found in fresh wheatgrass
juice. These results are in line with the findings of Eissa
et al. (2018).
The family of water-soluble vitamins (WSVs) com-
prises nine vitamins, for example, thiamine (vitamin B1)
and riboflavin (vitamin B2) (Heudi 2012; Nohr and Bie-
salski 2009). As a result of the crucial role that vitamins
play an optimum role in disease prevention and as thera-
peutic aids, many dietary supplements (also known as
nutrition or food supplements) are available (Heudi
2012). Samples of WGJ were prepared as a procedure
specified above “Sample Preparation.”Solid-phase ex-
traction procedure allowed extracting all sex vitamins
(water-soluble B1, B2, B3, B6, B9, and B12 as well as
vitamin C), which were present in the juices. Table 1
shows a chromatogram example of wheatgrass juice
extract. The presence of analytes, in particular with real
samples, was confirmed by comparing the absorption
spectra in the range of 200–600 nm and adding standard
solutions. Signals without labels are derived from the
matrix (Płonka et al. 2012). The B vitamins are import-
ant nutrients that support carbohydrate metabolism,
promote immune system function, and induce cell
growth (Adejumo 2012).
Furthermore, it is worth noting that the major phen-
olic compound concentration was up to three times
higher. This fact could lead to an increase in juice astrin-
gency and color, as well as in juice aroma. The findings
of these last acids are in accordance with those men-
tioned by other authors (Stalmach et al. 2011; dos Santos
Lima et al. 2015); however, the phenolic acid values ob-
tained here were closely the same in the literature. Phen-
olic acids like naringin, naringenin, luteolin, and
hesperidin were not detected in wheatgrass juice, as seen
in Table 4.
In the majority of native fruit juices, the content of
saccharides is limited only to glucose, fructose, and su-
crose. Contents of these sugars as well as glucose to
fructose ratios are different for different juices; hence,
they can be the indicators of their authenticity. These
parameters are compared with standards, e.g., those spe-
cified in the Code of Practice. Deviations from accepted
norms are the indicators of non-declared juice addition
(Stój and Targoñski 2005). Kallio et al. (2000) deter-
mined higher concentrations of glucose in juices of dif-
ferent strawberry varieties—from 18.9 g/L in the juice of
Senga VP cv. in 1998 to 45.2 g/L in the juice of Polka cv.
in 1997. In juices of Senga cv., Kallio et al. (2000) found
from 18.9 to 32.2 g/L of glucose. The average content of
fructose in raspberry juice of Canby cv. was significantly
higher (28.51 g/L) than the average contents of fructose
in juices of Beskid (24.28 g/L) and Malling Seedling cv.
(22.44 g/L). Other authors found greater ranges of fruc-
tose concentrations in raspberry juices. Durst et al.
Table 6 Liver and kidney functions in rats’experimental animals
Parameter groups AST (μ/ml) ALT (μ/ml) T. bilirubin (μ/ml) Albumin (g/dl) Urea (mg/dl) Createnine (mg/dl) Uric acid (mg/dl)
G1 (−ve control) 27.88 ± 2.10 29.50 ± 4.24 0.58 ± 0.19 3.78 ± 0.24 47.75 ± 7.40 0.48 ± 0.07 2.41 ± 0.26
G2 (+ve control) 22.50 ± 4.93 19.88 ± 3.40 0.60 ± 0.17 3.74 ± 0.17 43.75 ± 3.49 0.44 ± 0.04 2.45 ± 0.16
G3 (WGJ) 23.25 ± 4.71 22.25 ± 3.28 0.55 ± 0.16 3.59 ± 0.21 45.38 ± 7.76 0.48 ± 0.07 2.63 ± 0.21
All data are presented as means ±SD. Values with different superscript letters are significantly different at p< 0.05
Table 7 The lipid profile of rat’s blood serum
Parameters groups Total cholesterol (mg/dl) Triglycerides (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl)
G1 (−ve control) 110.00 ± 13.63 111.25 ± 22.32 26.00 ± 2.27 61.75 ± 11.11
G2 (+ve control) 106.25 ± 10.61 91.88 ± 12.52 28.75 ± 2.76 59.13 ± 9.06
G3 (WGJ) 95.00 ± 10.33 83.75 ± 13.02 23.38 ± 2.67 105.00 ± 10.22
All data are presented as means ± SD
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 7 of 11
(1995) reported on the fructose content ranging from
6.391 g/L in the juice of meeker cv. to 37.047 g/L in the
juice of Heritage cv.
Green Blood Therapy is the term that is called “green
blood”of wheatgrass which normally has phytochemical
pigment chlorophyll content which accounts for about
70% of its total chemical constituents (Swati et al. 2010)
and the use of WGJ to cure many multiple diseases. In
addition, wheatgrass is called as the green blood which
is a medicinal herb; therefore, wheatgrass like all green
plants, due to the high score of chlorophyll content, is
high in oxygen content, more amount of vitamins (espe-
cially vitamin E), and antioxidants too. And also, the
WGJ contains nearly all the nutrients which the body
needs and is to be considered a complete food.
The normal liver function recorded in all groups of ex-
perimental rats with respect to the normal hepatocytes
without hepatic necrosis showed also, in the same find-
ings, the normal kidney function. Wheatgrass juice
cleanses the toxins and pollutants of the body, by many
enzymes, and amino acids play an essential role as a nat-
ural cleanser to detoxify the liver and to eliminate toxic
heavy metals from the body’s bloodstream, rid the waste
matter out of the body, and delay the aging process
(Sareen et al. 2014).
This study suggested that sterols found in WGJ
might have reduced cholesterol absorption in the in-
testine and elevated cholesterol excretion out of the
body in anti-hyperlipidemia activity of WGJ. Saponins
another highly active phytochemicals considered to
increase fecal cholesterol excretion (Matsui et al.
2006). Mainly, cholesterol in the intestine can arise
by the diet and hepatic bile salt secretions. Further,
inhibitions of cholesterol absorption from the intes-
tine also decrease the delivery of cholesterol to the
liver, meaning that its potential is thereby to reduce
serum as well as hepatic cholesterol. This action leads
to accelerate the rat in the absorption of LDL from
plasma via LDL receptors and increase the clearance
of plasma cholesterol (Patel 2004;Halaetal.2014).
Recently, it was documented that during germination,
some biologically active phytochemicals were synthesized
in the wheat sprouts (Calzuola et al. 2004). Wheat sprouts
accomplished the maximum antioxidant potentials and
antioxidant enzyme superoxide dismutase (SOD) after 7
days of plant growth. In addition to this, wheat sprout ex-
tracts such as WGJ were found to be anti-mutagenic in
the Ames test (Peryt et al. 1992) capable of inhibiting oxi-
dative DNA damage (Falcioni et al. 2002) and responsible
for metabolic deactivation of carcinogens.
Eventually, various forms of physical and psychological
stress are believed to reduce sexual functions. Many
studies have examined the relationship between stress
and sexual potency in male rats (Stefan et al. 2011).
These reports showed that the chronic psychological
and physical stress induce erectile dysfunction, which re-
sults from neurotransmission changes in various erectile
response pathways and reduced blood flow in genital or-
gans (Vijay Prasad et al. 2012). Oxidative stress has fre-
quently been involved as a leading cause of male factor
infertility too (Stefan et al. 2011).
As expected, from previous parameters, the testicular
membranes are rich in polyunsaturated fatty acids, and
thus, mainly the cells are susceptible to peroxidation in-
jury (Stefan et al. 2011). The WGJ is the richest source
of natural antioxidants that mainly protect the testicular
cells of oxidation deleterious stress and permit the test
cells in the best normal function. Also, oral zinc dose
given in natural juice form (WGJ) and in a pharmaceut-
ical formula is a rich source of zinc (93 mg per100 ml)
as founded by Eissa et al. (2018) because zinc is import-
ant for normal growth and regeneration, testicular
Table 8 The assessment oxidative enzyme of rats’blood
Parameters groups SOD (μ/ml) GPx (μ/ml) GSH (mmol/ml) MD (mmol/ml) Cat. (U/ml)
G1 (−ve control) 21.50 ± 3.46 153.75 ± 12.46 26.00 ± 2.67 161.25 ± 22.00 37.88 ± 4.67
G2 (+ve control) 26.88 ± 3.40 160.00 ± 15.12 29.38 ± 4.84 171.25 ± 12.46 39.00 ± 3.12
G3 (WGJ) 27.88 ± 3.14 163.75 ± 10.61 29.50 ± 2.67 168.75 ± 14.08 38.63 ± 2.67
All data are presented as means ± SD
Table 9 The evaluation of sexual hormone in rats’blood serum
Parameters groups FSH (IU/L) LH (IU/L) TSH () Prog. (ng/ml) Testo (ng/ml) E2 (pg/ml)
G1 (−ve control) 1.65 ± 0.23 0.89 ± 0.12 0.48 ± 0.10 0.31 ± 0.05 2.78 ± 0.23 12.15 ± 2.31
G2 (+ve control) 1.45 ± 0.24 1.29 ± 0.33 0.64 ± 0.17 0.42 ± 0.05 2.04 ± 0.40 15.73 ± 0.84
G3 (WGJ) 1.44 ± 0.28 0.74 ± 0.08 0.54 ± 0.12 0.34 ± 0.06 2.90 ± 0.26 14.93 ± 0.73
All data are presented as means ± SD
LH luteinizing hormone, FSH follicle-stimulating hormone, Prog progesterone, E2 estradiol & test testosterone
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 8 of 11
maturation, neurological function, wound healing
process, and immune-competence (European Commis-
sion 2003).
It may due to the negative feedback effect of testoster-
one on the hypothalamus which in turn causes reduction
in the secretion of FSH and LH by the gonads of the an-
terior pituitary gland (Guyton and Hall’s2006). Gonado-
tropin production is under the feedback control of sex
hormones (Ganong 2003). It exists under physiological
status in the divalent. The structure and function of cell
membranes are also affected by zinc; hence, the loss of
zinc from biological membranes can elevate their sus-
ceptibility to oxidative damage and impair their function
(O’Dell 2000).
Wheatgrass with chlorophyll content is primarily a
phytonutrient, which is rich in macro- and micronutri-
ents alongside and, at the same time, contain enzymes
that work mutually for strong immunity. It builds up
protection from diseases and detoxification of the body,
and because of its alkaline properties, it has good prop-
erties for urinal problems. WGJ returns fertility and im-
proves youthfulness because of the high magnesium
content in the chlorophyll chemical structure which
builds enzymes that get back sex hormones. Wheatgrass
helps to detoxify and clean the body by fracturing im-
pacted substances in the colon (Duke 1983).
Conclusion
The prepared wheatgrass juices as a natural juice to im-
prove a healthy life additionally resulted in a highly ac-
ceptable and nutritious ready-to-drink juice, as well as a
good level of vitamin C. The highest content of vitamin
B1 (thiamine), B3 (nicotinic acid), and B12 was found in
wheatgrass juice. Meanwhile, the major compounds of
phenolic compounds in wheatgrass juice were high in
sinapic and protocatechuic, followed by caffeic acid, ros-
marinic, and gallic. Contents of glucose and fructose were
high, while according to the HPLC determination, there is
no sucrose in the wheatgrass juice. Due to its high content
of bioactive compounds, it could be recommended for
consumption as a fresh juice blend to elevate fertility and
promote youthfulness. This juice meets the changes in the
present-day consumers’lifestyle which has led to a vital
change in the marketing trends of the food sector. It also
highly encourages the consumption of juices for their im-
portance for micronutrient and macronutrient and their
health benefits as recommended by FAO/WHO Expert
Consultation on juice.
Abbreviation
ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; B1: Thiamine;
B12: Cyanocobalamin; B2: Riboflavin; B3: Nicotinamide; B6: Pyridoxine;
B9: Folic acid; CAT: Catalase; E2: Estradiol & test testosterone; FSH: Follicle-
stimulating hormone; GPx: Glutathione peroxidase; GSH: Reduced
glutathione; HPLC: High-performance liquid chromatography; IOM: Institute
of Medicine; LH: Luteinizing hormone; Prog: Progesterone; SD: Standard
deviation; SOD: Superoxide dismutase; TSS: Total soluble solids; WG: Follicle-
stimulating hormone (FSH)\wheatgrass; WGJ: Wheatgrass juice; WSVs: Water-
soluble vitamins
Acknowledgements
The authors are grateful and thankful to the projects’department and animal
house, National Research Centre, for their help and facilities which supported
us to manage this research.
Fig. 1 Testes from G1, G2, and G3
Eissa et al. Bulletin of the National Research Centre (2020) 44:30 Page 9 of 11
Significance statement
The study represented novel properties of WGJ as well as fertility and
promotes youthfulness that can be beneficial to sexual dysfunction in males
that may occur in complications with diabetes syndrome or other diseases.
Meanwhile, many disease treatments are important topics on utilizing and
exploring combinations of WGJ with a diabetic diet system or extracted in a
pharmaceutical formulation that will be potent in sexual dysfunction in
males’drugs.
Authors’contributions
SSM made the study design, proceeded the rat experiments and biochemical
analysis, collected and analyzed the data statistically, and in the end drafted the
manuscript. HAE contributed to the preparation of the juice technology and in
writing the manuscript. AMSH participated in the HPLC studies and biochemical
investigations and helped in the writing and submission of the manuscript. Also, he
participated in the sample collection, data collection, and analysis. This work was
carried out in collaboration with all authors. This manuscript was revised and
approved by all authors.
Funding
This work was supported by the Food Industries & Nutrition Division, National
Research Centre.
Availability of data and materials
All data generated or analyzed during this study already exist in this published
article.
Ethics approval and consent to participate
The healthy male rats (albino Wistar) used were procured from the animal
house of National Research Centre, Cairo, Egypt.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Foot Technology Department, National Research Centre, 33 El Bohouth St.,
Cairo 12622, Egypt.
2
Nutrition and Food Science Department, National
Research Centre, 33 El Bohouth St., Cairo 12622, Egypt.
Received: 21 March 2019 Accepted: 16 January 2020
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