molecules

Article
Nutritional and Therapeutic Properties of Fermented Camel
Milk Fortified with Red Chenopodium quinoa Flour on
Hypercholesterolemia Rats
Mohamed Saleh Al-Anazi 1 , Khaled Meghawry El-Zahar 1,2, * and Nourhan Abdel-Hamid Rabie 2

1 Department of Food Science and Human Nutrition, College of Agriculture and Veterinary Medicine,
Qassim University, Buraydah 51452, Saudi Arabia
2 Food Science Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt
* Correspondence: [email protected] or [email protected]; Tel.: +966-(0)563-974-612;
Fax: +996-163016228

Abstract: Quinoa is a nutrient-dense food that lowers chronic disease risk. This study evaluated
the physicochemical and sensory qualities of fermented camel milk with 1, 2, 3, and 4% quinoa.
The results showed that improvement in camel’s milk increased the total solids, protein, ash, fiber,
phenolic content, and antioxidant activity more effectively. Fermented camel milk with 3% of
quinoa flour exhibited the highest sensory characteristics compared to other treatments. Fermented
camel milk enriched with 3% red quinoa flour was studied in obese rats. Forty male Wistar rats
were separated into five groups: the first group served as a normal control, while groups 2–4
were fed a high-fat, high-cholesterol (HF)-diet and given 2 mL/day of fermented milk and quinoa
aqueous extract. Blood glucose, malondialdehyde (MDA), low-density lipoprotein (LDL), cholesterol,
triglyceride, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP),
creatinine, and urea levels decreased dramatically in comparison to the positive control group, while
Citation: Al-Anazi, M.S.; El-Zahar,
high-density lipoprotein (HDL), albumin, and total protein concentrations increased significantly.
K.M.; Rabie, N.A.-H. Nutritional and Fortified fermented camel milk decreased the number of giant adipocytes while increasing the
Therapeutic Properties of Fermented number of tiny adipocytes in the body. The results showed that the liver and renal functions
Camel Milk Fortified with Red of hypercholesterolemic rats were enhanced by consuming fermented milk and quinoa. These
Chenopodium quinoa Flour on results demonstrated the ability of quinoa and camel milk to protect rats from oxidative stress and
Hypercholesterolemia Rats. Molecules hyperlipidemia. Further studies are needed to clarify the mechanisms behind the metabolic effects of
2022, 27, 7695. https://doi.org/ fermented camel milk and quinoa.
10.3390/molecules27227695

Academic Editor: Gabriele Rocchetti Keywords: camel milk; red quinoa; oxidative stress; lipid profile; liver and kidney functions; athero-
genic index; obesity; adipocyte size
Received: 20 September 2022
Accepted: 27 October 2022
Published: 9 November 2022

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in
Mammalian milk is nutritious for new-borns. Camel (Camelus dromedarius L.) milk
published maps and institutional affil-
is especially popular in the Arabian Gulf. Camel milk differs significantly from cow’s
iations.
milk in chemical components and coagulability. Beneficial uses of camel milk in the
metabolic syndrome (which is the combination of abdominal obesity, dyslipoproteinemia,
and hypertension with peripheral insulin resistance) have frequently been postulated [1].
Copyright: © 2022 by the authors.
Short-and long-term frequent ingestion of camel milk benefits hypertension and diabetes
Licensee MDPI, Basel, Switzerland. in adults, due to the inhibition of oxidative and inflammatory stressors [2].
This article is an open access article South American quinoa (Chenopodium quinoa Willd.) is a nutrient-rich grain eaten
distributed under the terms and worldwide. It is one of the most flexible grain foods, with flour, flakes, and cereals as well
conditions of the Creative Commons as commercial or packaged products. Quinoa has a nutritional benefit that is comparable
Attribution (CC BY) license (https:// to that of meat and greater than that of other cereals. Quinoa is not extensively consumed,
creativecommons.org/licenses/by/ notwithstanding it has great nutritional values, for a variety of reasons, including high
4.0/). import prices as well as a lack of market knowledge of its advantages [3]. Quinoa’s dietary

Molecules 2022, 27, 7695. https://doi.org/10.3390/molecules27227695 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 7695 2 of 18

fiber lowers blood sugar and cholesterol levels after a diet even while controlling cholesterol
synthesis in the liver [4]. Quinoa seeds provide more essential amino acids, protein, carbo-
hydrates, and fiber than other grains. Those who chew a high-fat, high-cholesterol diet may
benefit from increasing protein consumption to prevent hyperlipidemia [5]. Quinoa seeds,
especially red seeds, have a high proportion of phenolic and flavonoid antioxidants [6].
Furthermore, because the glycosaminoglycan portion of this dietary fiber contains emulsi-
fying and stabilizing qualities, it has great potential for future use in the food industry [7].
Quinoa seeds can be used as an alternate source of nutrients, especially protein, while
enhancing polyphenol, antioxidant, and microbial activity [8].
However, information in the literature on camel milk structure clearly varies, depend-
ing on variables, such as the geographical origin of the milk studied, race, temporary or
physiological variants, keeping (especially feeding) conditions, animal health status or bio-
logical stage, and analytical weaknesses [9]. Camel milk contains all the essential nutrients
on which lactic acid bacteria can readily act and produce many biofunctional components
that give health benefits to the consumer. Additionally, an increase in the concentration of
amino acids, fatty acids, and organic acids was observed after the fermentation of camel
milk [10]. Hailu et al. [11] provided some information on camel milk proteins, focusing on
major differences in protein composition and molecular characteristics.
The literature describes the biofunctionalities of camel milk and its protein hydrolysates,
such as antioxidants, antidiabetic, anticancer, angiotensin-converting enzyme inhibitory, an-
timicrobial, anti-inflammatory, hepatoprotective, antiradical, antiallergic, and anti-autistic
effects. Some micronutrients, antioxidant enzymes, and protective proteins are biologically
significant [12]. Potential health benefits include inhibition of hypertension-conversing
enzymes, antimicrobial and antioxidant characteristics, and antidiabetic activities. Hy-
perlipidemia and hypercholesterolemia may be responsible for modifying and increasing
LDL, free radicals, and lipid peroxidation components, which are the main risk factors
for heart disease [13]. The bioactive peptides, such as ACE-inhibitory peptides and an-
tioxidative peptides, were also derived from fermented camel milk, and the comparative
study also showed the maximum ACE-inhibitory in fermented camel milk rather than
bovine milk [14,15]. Moslehishad et al. [14] demonstrated that fermentation of camel milk
with single strains of lactic acid bacteria (LAB) generated antioxidant activity in the whey
fractions. Lactic acid bacteria produce peptides from milk proteins during fermentation
and provide several health benefits to consumers.
Radical scavenging is the main mechanism by which antioxidants act in foods. Due to
the adverse effects of current cardiovascular disease treatments, such as gastrointestinal
difficulties, hypertension, excitability of liver enzymes, and liver failure, as well as their
exorbitant price, they are not a simple response [16]. Lactic acid bacteria have been shown
in experimental animals and humans to reduce blood cholesterol levels. Its ability to
decrease cholesterol is probably due to its lack of binding to bile salts by certain bacteria
strains producing the enzyme bile salt hydrolase [17,18]. The bacteria could secrete bile salt
hydrolase, which could successfully reduce cholesterol levels by increasing the release of
bile salts and then reducing absorption in the intestine [19,20]. The purpose of this study is
to determine the nutritional and therapeutic effects of fermented camel milk supplemented
with red quinoa flour on sensory properties, lipid profile, liver and kidney functions,
atherogenic index, and histopathological characteristics in hypercholesterolemic rats.

2. Results and Discussion

2.1. Chemical Composition and Phytochemical Properties of Red Quinoa Seed
Chemical compositions and phytochemical capabilities of quinoa seeds are presented
in Table 1a–c. Red quinoa moisture, crude protein, dietary fiber, ash, crude fat, and
carbohydrate content were 10.22%, 15.03%, 8.8%, 3.22%, 5.2%, and 57.6%, respectively
(Table 1a). These findings are consistent with the data obtained in previous studies by
Navruz-Varli et al. and Diaz-Valenica et al. [21,22], who detected that the fat, protein,
ash, and dietary fiber contents of red quinoa were 7.33, 14.0, 2.51, and 14.30 g/100 g,
Molecules 2022, 27, 7695 3 of 18

respectively. Ca, P, Mg, Na, and K were the most abundant minerals found in red quinoa
flour, with concentrations of (148.7, 383.9, 246.9, 12.2, and 926.7 mg/100 g−1 ), respectively
(Table 1b). In addition, the concentrations of Fe, Mn, Cu, and Zn were found to be 13.2, 10,
5.1, and 4.4 mg/100 g−1 , respectively, which is comparable with the findings of Abugoch-
James et al. [23]. These findings are in accordance with those obtained by Diaz-Valencia
et al. [22], who discovered that red quinoa contains 699, 578, 73, 17.5, 243, 5.63, 3.70, and
0.47 mg/100 g−1 , of K, P, Ca, Na, Mg, Fe, Zn, and Cu, respectively.

Table 1. (a). Chemical composition and energy value of red quinoa seed. (b). Content of chosen
mineral in red quinoa seed. (c). Yield extract, polyphenol compounds and total antioxidant capacity
in red quinoa seed.

(a)
Chemical Composition Chemical Composition (g/100g) on Dry Weight
Protein (%) 15.0 ± 0.24
Fiber (%) 8.8 ± 0.05
Ash (%) 3.2 ± 0.2
Carbohydrates (%) 57.58 ± 0.21
Fat (%) 5.2 ± 0.14
Moisture (10) 10.22 ± 0.21
Energy (kcal/100g) 352.12 ± 0.7
(b)
Chemical Composition Eliminates Instead of Mineral (mg/100 g)
Calcium 148.7 ± 1.2
Phosphorous 383.9 ± 2.3
Magnesium 246.9 ± 2.2
Sodium 12.2 ± 0.2
Potassium 926.7 ± 4
Iron 13.2 ± 0.3
Manganese 10.1 ± 0.24
Copper 5.1 ± 0.2
Zinc 4.4 ± 0.16
(c)
Phytochemical Properties
Yield g/100 g DW 16.86 ± 1.5
Vitamin C (mg/100 g DW) 16.4 ± 0.24
Phenolic compounds (mg GAE/100 g DW) 488.32 ± 2.91
Total Flavonoid (mg CE/100 g DW) 367.11 ± 4.22
DPPH* Inhibition (%) 47.0 ± 2.4
Data are presented as the mean ± SD (n = 3/group). GAE: gallic acid equivalents; CE: catechin equivalents; DW:
dry weight. The mean and standard deviation (n = 3) are reported.

Total phenolic compounds, total flavonoids, and vitamin C were all found in higher
concentrations in the red quinoa seeds (488.7, 367.1, and 16.4 mg/100g DW) respectively.
Red quinoa seeds showed higher levels of total phenolic compounds, total flavonoids,
and vitamin C (488.7, 367.1, and 16.4 mg/100g DW), respectively (Table 1c). Furthermore,
quinoa’s antioxidant activity and radical scavenging activity are strongly linked to the
presence of phenolic components [24,25]. Sampaio et al. [26] found that quinoa seeds have
stronger antioxidant potential than entire grains (wheat, barley, millet, rice, and buckwheat),
indicating that quinoa contains potent free radical scavenging components. Therefore,
quinoa appears to be a promising candidate for the development of new treatments and
therapeutic options for disorders related to oxidative stress.
Molecules 2022, 27, 7695 4 of 18

2.2. Gross Chemical Composition and Sensory Evaluation of Different Fermented Milks
The pH and titratable acidity values in camel milk were found in the range of 6.22
to 6.56 and 0.13 to 0.25% w/v, respectively. The percentages of fat, protein, ash, lactose,
dry matter content, and the density of the camel’s milk samples were as follows: fat 3.28,
protein 3.10, ash 0.83, lactose 5.23, and total solids 11.83, respectively (data not shown).
The results showed that the fat content in fermented milk increased slightly in com-
parison with fresh camel milk. The amount of lactic acid produced increased with a
concomitant drop in pH with an increase in fermentation time. However, the combination
of L. bulgaricus and S. thermophilus (1:1) resulted in a lower pH and higher acidity. The
total protein decrease in fermented milk samples occurs as a result of protein hydrolysis
by the starter culture. Protein degradation in all treatments increased due to the limited
hydrolysis of milk proteins by lactic acid bacteria. Fermented milk was produced by using
a traditional starter culture and then evaluated for organoleptic quality and consumer
acceptance. The ideal fortifying ratio for camel milk with red quinoa was (3% v/w), which
was used in the biological study to estimate the impact of red quinoa, as well as camel
milk, on the blood lipid profile in hyperlipidemic rats. The amount of lactic acid produced
increased with a concomitant drop in pH with an increase in fermentation time. The pH
of fermented camel milk containing red quinoa seed flour (Cam_Q4 , Cam_Q3 , Cam_Q2
and Cam_Q1 , respectively) was higher than that of camel milk (Table 2a). Furthermore,
fermented camel milk fortified with quinoa flour exhibited a higher acidity than fermented
camel milk. These findings are consistent with the results acquired by Al-Harbi et al. and
El-Zahar et al. [27,28], which indicated a rise in acidity when stored at the refrigerator
temperature. The protein, fat, fiber, and ash content of the fermented milk rapidly increased
when the replacement ratio was increased as a result of fortifying camel milk with quinoa
seed flour by approximately 8.8%, 10%, 46%, and 18.7%, respectively. This could be due
to the higher protein and carbohydrate content of quinoa compared to camel milk [22].
Protein hydrolysis by starting culture resulted in a decrease in total protein in fermented
milk samples. These findings are consistent with those reported by Omar et al. [29], who
discovered that partially replacing camel milk with preserved skim milk increased protein,
ash, and carbohydrate content in the bio-yogurt produced. It was also found that adding
barley bran to yogurt increased the protein, ash, and carbohydrate content [30]. Adding
more quinoa flour to fermented camel milk increases the amount of total phenolic compo-
nents compared to the control. The total phenolic content and radical scavenging activity
of fermented camel milk were increased in camel milk fortified with varying percentages
of oat, kiwi, and avocado [24,31].
The mean values of sensory evaluation scores are shown in Table 2b. Fermented
camel milk had lower sensory scores for body and texture than fermented camel milk
enriched with quinoa (Cam_Q3 , Cam_Q2 , Cam_Q4 and Cam_Q1 , respectively). The overall
acceptance scores of the sensory attributes revealed that the fermented milk with 3% of
red quinoa was acceptable, followed by the fermented milk fortified with 2%, while the
fermented milk fortified with 4% was the lowest ranked. The data in Table 2 shows that the
fat and protein content, as well as the overall sensory quality of the camel’s milk fermented
with 3% of quinoa flour (Cam_Q3 ), were significantly higher (p ≤ 0.05) than that of the
fermented camel milk (Cam_T). High-saturated fatty acid content, salts, weak lipolysis,
and poor protein structure can all contribute to a low sensory rating of fermented camel
milk [32]. Improvements in the sensory quality of fermented milk were mostly due to
the development of starter cultures throughout the fermentation or storage stage. The
findings of this investigation were in accordance with those of [28,29,31]. Curti et al. [33],
found that the addition of higher concentrations of quinoa flour into yogurts formulations
resulted in reduction in acceptability of aroma and flavor. Although the colour of the final
product was darkness and thus not so appealing but, sensory colour values of Kishk-soup
samples containing quinoa seeds were not significantly different and most of the soups
samples were accepted in position of sensory colour values and have the potential to be
well received by consumers.
Molecules 2022, 27, 7695 5 of 18

Table 2. (a). Physicochemical of different fermented milks fortified with red quinoa seeds. (b). Sensory
evaluation of different fermented milks fortified with red quinoa seeds.

(a)
Fermented Milk Types
Attribute
Cam_T Cam_Q1 Cam_Q2 Cam_Q3 Cam_Q4
Total Solids (%) 14.60 e ± 0.55 15.30 d ± 0.48 16.14 c ± 0.68 17.02 b ± 0.88 17.98 a ± 0.78
Protein (%) 5.02 c ± 0.16 5.14 bc ± 0.20 5.26 b ± 0. 24 5.36 ab ± 0. 22 5.44 a ± 0.24
Fat (%) 4.04 a ± 0.12 4.18 a ± 0.16 4.24 a ± 0.14 4.30 a ± 0.16 4.38 a ± 0.18
Ash (%) 0.96 a ± 0.09 1.00 a ± 0.07 1.05 a ± 0.10 1.09 a ± 0.08 1.14 a ± 0.05
Fiber (%) 0.00 0.12 d ± 0.02 0.23 c ± 0.03 0.34 b ± 0.02 0.46 a ± 0.03
Acidity (%) 0.78 c ± 0.04 0.82 b ± 0.03 0.88 ab ± 0.06 0.90 a ± 0.02 0.92 a ± 0.02
pH 4.80 a ± 0.08 4.76 b ± 0.06 4.70 c ± 0.09 4.66 d ± 0.06 4.62 e ±0.05
(b)
Fermented Milk Types
Sensory Properties
Cam_T Cam_Q1 Cam_Q2 Cam_Q3 Cam_Q4
Flavor (30) 21.18 e ± 1.2 25.30 c ± 1.4 26.60 b ± 1.3 27.10 a ± 1.1 24.60 d ± 1.3
Color (10) 30.20 e ± 4.5 32.24 d ± 3.7 34.2 b ± 4.6 35.40 a ± 4.92 36.20 c ± 3.8
Body and Texture (40) 8.2 d ± 0.3 8.6 a ± 0.4 8.4 b ± 0.4 8.3 c ± 0.6 7.9 e ± 0.5
Acidity (10) 8.82 c ± 0.2 8.93 bc ± 0.2 9.02 b ± 0.2 9.17 a ± 0.1 8.58 d ± 0.2
Overall acceptability (10) 9.01 bc ± 0.1 9.11 b ± 0.1 9.14 ab ± 0.2 9.25 a ± 0.1 8.85 c ± 0.2
Total (100) 77.41 d ± 3.12 84.18 c ± 4.22 87.36 b ± 3.70 89.22 a ± 3.82 86.13 ab ± 3.70
Data are presented as the mean ± SD (n = 3/group). Different superscript letters (a to e) within the same
raw showed significant differences among the groups (p ≤ 0.05). Fermented camel milk as a control (Cam_T),
Fermented camel milk fortified with 1% red quinoa seed flour (Cam_Q1 ), Fermented camel milk fortified with 2%
red quinoa seed powder (Cam_Q2 ), Fermented camel milk fortified with 3% red quinoa seed flour (Cam_Q3 ) and
Fermented camel milk fortified with 4% red quinoa seed flour (Cam_Q4 ).

2.3. Effect of Fermented Milk on the Body Weight Gain in Rats

The effect of oral administration of different fermented milks (2 mL/day) on rat
weight given a HF-diet was demonstrated in Figure 1. It was shown that fermented milk
contributed to a drop in blood fat levels and relative weight in rats. The initial body weights
of all rat groups were significantly higher when compared to the negative group. Whereas
the final body weights of all rat groups were not significantly different, with the exception
of the positive control group, which was significantly higher (p ≤ 0.05). Furthermore, these
findings are in accordance with Halaby et al. [34], who discovered that a diet fortified
with 30% and 40% quinoa seed powder can improve body weight gain, feed consumption,
and feed efficiency ratio. Our findings are corroborating those previously released reports
by [28,29]. It may be concluded that the addition of fermented camel milk fortified with
red quinoa seed flour in the rats’ diet resulted in a significant increase (p ≤ 0.05) in the final
weight and body weight compared with the other groups.
Data are presented as the mean ± SD (n = 3/group). NC) nontreated nonhyperc-
holesterolemic rats (negative control), PC) hypercholesterolemic rats (positive control),
Cam_T) hypercholesterolemic rats treated with fermented camel milk, Cam_Q3 ) hyperc-
holesterolemic rats treated with fermented camel milk fortified with 3% red quinoa seed
flour and EX_Q3 ) aqueous solution of red quinoa seeds flour.
Molecules 2022, 27, 7695 6 of 18
Molecules 2022, 27, x FOR PEER REVIEW 6 of 19

Figure 1. Effect of different fermented camel milks fortified with red quinoa seed powder on body
Figure 1. Effect of different fermented camel milks fortified with red quinoa seed powder on body
weight gain and food intake in hypercholesterolemic rats.
weight gain and food intake in hypercholesterolemic rats.
Data are presented as the mean ± SD (n = 3/group). NC) nontreated nonhypercholes-
2.4. Effect of Fermented Camel Milk on Fasting Serum Insulin and Glucose Levels in Obese Rats
terolemic rats (negative control), PC) hypercholesterolemic rats (positive control), Cam_T)
Untreated rats feeding
hypercholesterolemic on HF-diet
rats treated had a significant
with fermented camel milk,increase
Cam_Q3)(phypercholester-
< 0.05) in fasting
serum
olemic rats treated with fermented camel milk fortified with 3% red quinoa seed flour andwhen
insulin and glucose levels by 13.3 mlU/L and 149.2 mg/dL, respectively,
compared to the solution
EX_Q3) aqueous normal ofgroup rats (8.4
red quinoa mlU/L
seeds flour. and 66.9 mg/dL). Animal protection
with fermented camel milk and quinoa seed significantly lowered serum insulin and
2.4. Effect
glucose of Fermented
levels Camel Milk
when compared on Fasting
to the positiveSerum Insulin
control and Glucose
group (FigureLevels in Obese Ratsblood
2). Additionally,
insulinUntreated
and glucoserats levels
feedingwere normalized
on HF-diet in rats fedincrease
had a significant fermented camel
(p < 0.05) in milk.
fastingAn increase
serum
in insulin
muscleand triglyceride content
glucose levels by 13.3and visceral
mlU/L fat deposition
and 149.2 contribute
mg/dL, respectively, when to compared
hyperlipidemia
to
andthehyperinsulinemia,
normal group rats (8.4 mlU/L
and theand 66.9 mg/dL).
HF-diet Animaltoprotection
is reported promotewith fermented
insulin camel [28].
resistance
Themilk and quinoa
HF-diet seed significantly
increased body weight lowered serum hyperinsulinemia,
and caused insulin and glucose levels
whichwhenwas com-
linked to
pared to the positive
hyperglycemia control group (Figure
and hyperlipidemia, 2). Additionally,
according to our studyblood insulinThis
results. and glucose
could be levels
because
were normalized
Molecules 2022, 27, x FOR PEER REVIEW
quinoa in rats
seeds contain fed phenolic
more fermented compounds
camel milk. An increase
and fibers,inwhich
musclestimulate
triglyceride con-
7 of 19
pancreatic
tenttoand
cells visceral
release fat deposition
insulin [34]. contribute to hyperlipidemia and hyperinsulinemia, and the
HF-diet is reported to promote insulin resistance [28]. The HF-diet increased body weight
and caused hyperinsulinemia, which was linked to hyperglycemia and hyperlipidemia, ac-
cording to our study results. This could be because quinoa seeds contain more phenolic
compounds and fibers, which stimulate pancreatic cells to release insulin [34].

Figure 2. Effect of fermented camel milk fortified with red quinoa seed powder on fasting serum
Figure Effect
2. and
insulin of fermented
glucose camel milk fortifiedrats.
levels in hypercholesterolemic with red quinoa seed powder on fasting serum
insulin and glucose levels in hypercholesterolemic rats.
Data are presented as the mean ± SD (n = 3/group). NC) nontreated nonhypercholes-
terolemic rats (negative control), PC) hypercholesterolemic rats (positive control), Cam_T)
hypercholesterolemic rats treated with fermented camel milk, Cam_Q3) hypercholester-
olemic rats treated with fermented camel milk fortified with 3% red quinoa seed flour and
EX_Q3) aqueous solution of red quinoa seeds flour.
Molecules 2022, 27, 7695 7 of 18

Data are presented as the mean ± SD (n = 3/group). NC) nontreated nonhyperc-

holesterolemic rats (negative control), PC) hypercholesterolemic rats (positive control),
Cam_T) hypercholesterolemic rats treated with fermented camel milk, Cam_Q3 ) hyperc-
holesterolemic rats treated with fermented camel milk fortified with 3% red quinoa seed
flour and EX_Q3 ) aqueous solution of red quinoa seeds flour.

2.5. Effect of Fermented Camel Milk on Fasting Serum Insulin and Glucose Levels in Obese Rats
Serum concentrations of triglyceride (TG), total cholesterol (TC), LDL and very low-
density lipoprotein (VLDL) were all significantly reduced, while HDL was significantly
increased, when red quinoa seed was supplemented with fermented camel milk and given
to rats that had been subjected to oxidative stress. Table 3 reveals that TC values in the
positive control group (154 mg/dL) rose significantly compared with the negative control
group (64 mg/dL), whereas TC levels were 105, 96, and 99 mg/dL, respectively, in rat
doses with Cam_T, Cam_Q3 , and EX_Q3 . A rise in HDL levels is a useful indicator for anti-
hypercholesterolemia drugs. These grains’ dietary fiber can inhibit cholesterol absorption
and bind to bile acid, enhancing cholesterol catabolism in the colon and creating short-chain
fatty acids, reducing liver cholesterol synthesis [4,35]. Foucault et al. [36], possessed the
capability of quinoa to reduce serum glucose, TG, and LDL levels in rats fed a fructose-
enriched diet, consequently preventing the deleterious effects of fructose on HDL. Quinoa
may guard against peroxidation by raising antioxidant capacity and reducing oxidative
damage in rat plasma and tissues. Generally, feeding with fermented camels fortified with
red quinoa significantly reduced LDL levels (41.2 mg/dL) compared to the positive control
group (98.5 mg/dL). The LDL value was reduced from (53, 41, and 49.2 mg/dL) in the
Cam_T, Cam_Q3 , and EX_Q3 groups, respectively. This investigation corroborates the
prior findings [27,28]. Fermented camel may decrease cholesterol and triglyceride levels by
many proposed mechanisms: (1) inhibition of liver cholesterol synthesis and distribution
of cholesterol from the blood circulation to the liver, (2) intestinal bacteria may inhibit
the absorption of cholesterol, and (3) milk fermented by lactic acid strains may inhibit
cholesterol synthesis enzymes and decrease cholesterol levels [37,38]. Moreover, bioactive
peptides derived from camel milk proteins may reduce cholesterol through the interaction
between bioactive peptides and cholesterol, and the presence of orotic-acid in camel milk,
which is thought to be responsible for lowering cholesterol levels in rats [28].

Table 3. Effect of fermented camel milk fortified with red quinoa seed flour on the lipid profile and
atherogenic index in hypercholesterolemic rats.

Groups TG mg/dL TC mg/dL HDL mg/dL LDL mg/dL VLDL mg/dL

d d a
37.2 ± 0.9 e
14.5 ± 1.3 13.2 c ± 0.1
NC 65.9 ± 1.1 64.9 ± 2.7
PC 146.3 a ± 5.7 152.4 a ± 6.2 24.5 d ± 0.8 98.5 a ± 5.8 29.3 a ± 1.1
Cam_T 97.7 b ± 2.3 105.1 b ± 5.6 32.6 b ± 0.8 53.0 b ± 3.4 19.5 b ± 0.6
Cam_Q3 93.6 c ± 1.9 96.1 c ± 4.6 36.2 ab ± 0.9 41.2 d ± 2.7 18.7 bc ± 0.5
EX_Q3 96.4 b ± 2.1 99.3 bc ± 5.1 30.5 cd ± 0.8 49.3 c ± 2.9 19.3 b ± 0.6
Data are presented as the mean ± SD (n = 3/group). Different superscript letters (a to e) within the same raw
showed significant differences among the groups (p ≤ 0.05). (NC) Nontreated nonhypercholesterolemic rats
(negative control), (PC) hypercholesterolemic rats (positive control), (Cam_T) hypercholesterolemic rats treated
with fermented camel milk, and (Cam_Q3 ) hypercholesterolemic rats treated with fermented camel milk fortified
with 3% red quinoa seed flour and (EX_Q3 ) aqueous extract of red quinoa seeds flour.

2.6. Effect of Fermented Camel Milk Fortified with Red Quinoa on Serum AST, ALT, ALP, Liver,
and Kidney Functions in Obese Rats
Hypercholesterolemia has been known to disturb the oxidant-pro-oxidant balance
and reduce the efficacy of the antioxidant protection system, leading to tissue damage and
frequently correlated with the evolution and development of atherogenesis [39]. Lipid
peroxidation is an oxidative modification of polyunsaturated fatty acids in the cell layers
that produces several degeneration products. Certain products cause great disturbance
to cell elements and may prevent cell replication and cell endurance. The activities of
AST, ALP, and ALT were evaluated in blood serum as liver function indicators. As shown
Molecules 2022, 27, 7695 8 of 18

in Table 4, the impact of the treatments is described above on the levels of AST, ALT,
and ALP. Generally, the negative control group showed significantly lower serum AST,
ALT, and ALP levels (25, 105, and 52 U/L) in contrast to the hypercholesterolemia rat
group (59, 180, and 115 U/L). Overall, given hypercholesterolemic rats, fermented milk
fortified with red quinoa reduced the AST, ALP, and ALT levels compared to the positive
control group, where AST levels decreased from (80, 73, and 84.3 mg/dL) in the Cam_T,
Cam_Q3 , and EX_Q3 groups, respectively. The presence of ascorbic acid and phenolic
components, as well as flavonoids known as hepatoprotective factors, may explain the
liver’s activity [35]. The high content of camel milk antioxidants, which preserve the plasma
membrane of hepatocytes and protect them from rupture and exit of the cytosol loaded
with these enzymes, can be attributed to a decrease in transaminase enzymes and the
recovery of hepatocytes for some of their vital functions [28]. Rats that fed on fermented
camel milk containing 3% red quinoa flour improved, which may be due to the chemical
structure of quinoa seed, which has antihepatotoxic and antioxidant activities [5,6]. Table 4,
shows that there are significant changes in either albumin or total protein due to feeding
on various fermented milks, with albumin values ranging from 3.7 to 4.15 g/dL, and
total protein from 7.3 to 7.9 g/dL, respectively. Albumin values increased by 3.8, 4.15,
and 3.7 mg/dL in treated rats with different treatments (Cam_T, Cam_Q3 , and EX_Q3 ),
respectively. Lactic acid’s ability to control blood lipid alterations could explain the rise
in total protein and albumin levels. This reduces liver tissue damage caused by high
blood lipids and cholesterol. The results indicated that fermented camel milk containing
probiotic bacteria increased protein and total albumin, and that the effect was related to the
starting culture [27]. Urea and creatinine values have risen significantly (p ≤ 0.05) in rats
feed with HF-diet (40.9 and 2.4 mg/dL) compared to the negative control group (17.5 and
0.89 mg/dL) respectively. Generally, ingesting fermented milk containing both probiotics
and traditional fermented milk was effective at lowering creatinine and urea levels [26].
Martinez et al. [40], found that a large increase in creatinine concentration in blood serum
in rats fed an HF diet raises the risk of renal injury.

Table 4. Effect of different fermented milks on albumin, protein, serum enzyme activities of liver, and
kidney markers in hypercholesterolemic rats.

ALP ALT AST Total Protein Albumin Urea Creatinine

Groups
(U/L) (U/L) (U/L) (g/dL) (g/dL) (mg/dL) (mg/dL)
NC 105.2 b ± 7.1 25.2 d ± 1.74 52.3 d ± 5.4 7.3 b ± 0.1 3.85 a ± 0.04 17.54 d ± 0.1 0.89 e ± 0.3
PC 180.4 a ± 9.4 58.8 a ± 3.72 115.2 a ± 6.9 5.5 c ± 0.11 2.85 c ± 0.01 40.89 a ± 0.3 2.4 a ± 0.12
Cam_T 141.5 c ± 6.8 36.4 c ± 1.80 79.8 b ± 5.12 7.3 bc ± 0.12 3.81 ab ± 0.02 22.1 b ± 0.13 1.96 b ± 0.5
Cam_Q3 128.6 d ± 5.7 33.8 c ± 3.18 73.2 c ± 2.7 7.6 b ± 0.09 4.15 a ± 0.03 19.75 c ± 0.9 1. 5 d ± 0.11
EX_Q3 135.6 cd ± 5.7 41.7 b ± 1.2 84.3 b ± 5.2 7.9 a ± 0.08 3.70 b ± 0.04 23.1 b ± 0.19 1.75 c ± 0.1
Data are presented as the mean ± SD (n = 3/group). Different superscript letters (a–d) within the same raw
showed significant differences among the groups (p ≤ 0.05). (NC) Nontreated nonhypercholesterolemic rats
(negative control), (PC) hypercholesterolemic rats (positive control), (Cam_T) hypercholesterolemic rats treated
with fermented camel milk, (Cam_Q3) hypercholesterolemic rats treated with fermented camel milk fortified with
3% red quinoa seed flour and (EX_Q3) aqueous extract of red quinoa seeds.

The improvement in liver function may result from feeding groups of rats, either
conventional fermented milk or probiotics, and this was confirmed by the histological
examination of the liver (data not shown). At the end of the experiment, hepatic tissue
appeared almost natural without congestion or cellular infiltration. In the kidney, creatinine
is distilled by the glomerulus and excreted by the tubules, and only free creatinine appears
in the blood serum. The reason for enhanced serum enzymes and renal functions in HF-
diet-induced liver and renal injury by camel milk may be due to the prevention of lactic
acid and antioxidant compound abilities.
Molecules 2022, 27, 7695 9 of 18

2.7. Effect of Fermented Camel Milk Fortified with Red Quinoa Seed Flour on the Activity of Serum
MDA, GSH, SOD and TAC Level Enzymes in Hypercholesterolemic Rats
The data illustrated in Table 5, show that the obese rats had significantly lower serum
glutathione peroxidase (GSH-px) and superoxide dismutase (SOD) levels as well as reduced
MDA value compared to the control rat group. Hypercholesterolemic rats that were fed
on fermented camel milk, fermented camel milk fortified with red quinoa, and aqueous
extract of red quinoa seeds had significantly higher serum GSH-px and SOD levels and
lower serum MDA levels compared with the positive control group. These advancements
demonstrated that the fermented camel milk had antioxidative and health benefits for the
liver recovering from HF-diet injury. These enzymes rose considerably, suggesting camel
milk and red quinoa have anti-hepatotoxic and antioxidant effects [41]. Ascorbic acid and
the phenolic components present in camel milk and quinoa may be explained by their
ability to scavenge free radicals and active oxygen species [42]. Quinoa supplementation
lowered plasma malondialdehyde levels and improved antioxidant enzyme activity in
oxidative stress-induced rats [43].

Table 5. Effect of fermented camel milk fortified with red quinoa seed flour on antioxidant markers
in hypercholesterolemic rats.

MDA TAC SOD GSH-px

Groups
(µmol/L) (µmol/L) (U/g Hb) (U/g Hb)
NC 1.27 d ± 0.05 780 a ± 14 5.34 a ± 0.85 22.14 a ± 0.88
PC 2.13 a ± 0.07 550 e ± 21 3.28 d ± 0.40 15.12 d ± 0.76
Cam_T 1.56 b ± 0.03 655 d ± 18 4.08 c ± 0.55 17.28 c ± 0.85
Cam_Q3 1.32 c ± 0.02 775 b ± 16 4.60 b ± 0.68 18.22 b ± 0.80
EX_Q3 1.30 cd ± 0.01 735 c ± 15 3.98 c ± 0.55 16.82 c ± 0.85
Data are presented as the mean ± SD (n = 3/group). Different superscript letters (a–e) within the same raw
showed significant differences among the groups (p ≤ 0.05). (NC) nontreated nonhypercholesterolemic rats
(negative control), (PC) hypercholesterolemic rats (positive control), (Cam_T) hypercholesterolemic rats treated
with fermented camel milk, (Cam_Q3) hypercholesterolemic rats treated with fermented camel milk fortified with
3% red quinoa seed flour and (EX_Q3) aqueous extract of red quinoa seeds.

Polyphenols and flavonoids contained in red quinoa seeds may be helpful in decreas-
ing oxidative stress and liver inflammation as bioactive substances. Li et al. [43], explored
the potential of quinoa to decrease peroxidation and maintain antioxidant enzyme activity,
as well as its capacity to control lipid oxidation hydroxyl radicals. Another suggested mech-
anism is quinoa seed’s potential to raise GSH-px and thus rectify cardiac cells’ inadequate
thiol state.

2.8. Effect of Fermented Camel Milk Fortified with Red Quinoa on Adipose Tissue Weight and
Adipocyte Volume in Hypercholesterolemic Rats
Liver and white adipose tissue weight, and adipocyte cell size are presented in Table 6
and Figure 3a–d. The results showed a significant difference between the NC and PC
groups in the weight of adipose tissue and adipocyte size. There were no significant
differences between the NC (Figure 3), Cam_T (Figure 3c), Cam_Q3 (Figure 3b), or EX_Q3
(Table 6) groups in either adipose tissue weight or adipocyte cell size, even though rats
in these groups were fed on a HF-diet (Figure 3b). Thus, all of these treatments (Cam_T,
Cam_Q3 , and EX_Q3 ) had a significant effect in decreasing adipose tissue weight and
adipocyte size (p ≤ 0.05). The combined effect of camel milk and quinoa seed flour on
obesity in a high-fat-fed rat model was examined for the first time in the present study.
These beneficial effects were associated with a significant reduction in the body weight
gain of the rats (Figure 2). In contrast to the rats’ feed with HF-diet, the fermented camel
milk and fermented camel milk fortified with quinoa seed flour significantly reduced the
adipocyte size in rats, as shown in Table 6. The reduction in adipocyte size was a result of
the significant reduction in the adipose tissue mass of the experimental groups compared
 ipocyte size (p ≤ 0.05). The combined effect of camel milk and quinoa seed flour on obesity
in a high-fat-fed rat model was examined for the first time in the present study. These
beneficial effects were associated with a significant reduction in the body weight gain of
the rats (Figure 2). In contrast to the rats’ feed with HF-diet, the fermented camel milk and
fermented camel milk fortified with quinoa seed flour significantly reduced the adipocyte
Molecules 2022, 27, 7695 size in rats, as shown in Table 6. The reduction in adipocyte size was a result of the signif-10 of 18

icant reduction in the adipose tissue mass of the experimental groups compared to the
positive control group (Table 6). These results are in line with other studies that have re-
to the positive
ported control
a reduction group (Table
in adipocyte size6).[44],
These
andresults aretissue
adipose in linemass
with[45].
other studies that have
reported a reduction in adipocyte size [44], and adipose tissue mass [45].
Table 6. Effect of fermented camel milk fortified with red quinoa seed flour on liver weight, adipose
tissue,
Table 6.and fat cell
Effect volume incamel
of fermented hypercholesterolemic
milk fortified with rats.
red quinoa seed flour on liver weight, adipose
tissue, and fat cell volume in hypercholesterolemic rats.
Groups Liver (g) White Adipose Tissue (g) Adipocyte Size (μm2 × 103)
NC
Groups 8.59 (g)± 0.2 White Adipose
Liver
b 1.66 dTissue
± 0.2 (g) Adipocyte3.1 Size±(µm
d 0.4 2 × 103 )
PC 9.99 ± 0.3
a 5.45 ± 0.6
a 10.96 a ± 0.7
b ± 0.2 d ± 0.2 d
NC
Cam_T 8.597.9 cd ± 0.8 1.66 2.72 b ± 0.5 6.3 b±± 0.4
3.1 0.4
PC 9.99 a ±c 0.3 5.45 a ± c0.6 10.96 ac ± 0.7
Cam_Q3 8.1 ± 0.3
cd 2.3 ± 0.4
b 4.55
b ± 0.2
Cam_T 7.9 ±d 0.8 2.72 ± b0.5 6.3 bc± 0.4
EX_Q3 7.5 ± 0.8 2.62 ± 0.5 5.9 ± 0.4
Cam_Q3 8.1 c ± 0.3 2.3 c ± 0.4 4.55 c ± 0.2
Data are presented
EX_Q3 as the dmean
7.5 ± 0.8 ± SD (n = 3/group). b Different
2.62 ± 0.5 superscript letters
5.9 (a
bc to
± d)
0.4within the
same raw showed significant differences among the groups (p ≤ 0.05). (NC) nontreated nonhyper-
Data are presented as the mean ± SD (n = 3/group). Different superscript letters (a to d) within the same raw
cholesterolemic
showed significant rats (negative
differences control),
among (PC) hypercholesterolemic
the groups rats (positive
(p ≤ 0.05). (NC) nontreated control), (Cam_T)
nonhypercholesterolemic rats
hypercholesterolemic rats treated with fermented
(negative control), (PC) hypercholesterolemic camel
rats (positive milk,(Cam_T)
control), (Cam_Q3) hypercholesterolemic
hypercholesterolemic rats
rats treated
with fermented
treated camel milk,camel
with fermented (Cam_Q3)milkhypercholesterolemic
fortified with 3%rats redtreated
quinoawith fermented
seed flour andcamel milk fortified
(EX_Q3) with
aqueous
3% red quinoa
extract of red seed
quinoaflour and (EX_Q3) aqueous extract of red quinoa seeds.
seeds.

Molecules 2022, 27, x FOR PEER REVIEW 11 of 19

(a) (b)

(c) (d)
Figure
Figure 3.
3. Effect
Effect of fermented camel
of fermented camelmilk
milkeither
eitheralone
aloneororfortified
fortifiedwith
with3%3%
of of quinoa
quinoa flour
flour on on adipo-
adipocyte
cyte cells. Adipocytes in paraffin sections: (scale bar, 50 μm, magnification, 100×). (a) Negative con-
cells. Adipocytes in paraffin sections: (scale bar, 50 µm, magnification, 100×). (a) Negative control
trol group. (b) Positive control group. (c) Rats treated with fermented camel milk. (d) Rats treated
group. (b) Positive control group. (c) Rats treated with fermented camel milk. (d) Rats treated with
with fermented camel milk fortified with 3% quinoa flour.
fermented camel milk fortified with 3% quinoa flour.
2.9.
2.9. Histological
Histological Changes
Changes in
in Experimental
Experimental Rats
Rats Fed
Fed on
on Fermented
Fermented Camel
Camel Milk
Milk Fortified
Fortified with
with
Red Quinoa
The
The normal
normal histological
histological structure
structure ofof the
the central
central vein
vein and
and surrounding
surrounding hepatocytes
hepatocytes in in
the
the parenchyma
parenchyma was was observed
observed in in histological
histological analyses
analyses of of liver
liver sections
sections from
from rats
rats fed
fed on
on aa
standard diet
standard diet (Figure
(Figure4). 4).Arterial
Arterialdegradation
degradationwas wasdetected
detectedin the cytoplasm
in the cytoplasm of hepatocytes
of hepato-
in theinhypercholesteremia
cytes the hypercholesteremia rat group, as well
rat group, as as pyknosis
well in thein
as pyknosis nucleus of several
the nucleus other
of several
hepatic
other cells and
hepatic cellsinflammatory
and inflammatorycell infiltration in the in
cell infiltration portal area. Furthermore,
the portal the portal
area. Furthermore, the
area showed
portal hyperplasia,
area showed newly developed
hyperplasia, bile ducts,bile
newly developed andducts,
portaland
veinportal
dilatation
vein(Figure 4b).
dilatation
The current
(Figure study
4b). The findings
current arefindings
study consistent arewith those of
consistent Saleem
with thoseetofal. [46], who
Saleem et al.found that
[46], who
members
found thatofmembers
the Chenopodiaceae family, havefamily,
of the Chenopodiaceae significant
have hepatoprotective and antioxidant
significant hepatoprotective and
activity due to high concentrations of phytochemical compounds
antioxidant activity due to high concentrations of phytochemical compounds such as flavonoids
such as andfla-
phenolicand
vonoids acids (Figureacids
phenolic 4c,d).(Figure
Saleem4c,d).
et al.Saleem
[46], discovered
et al. [46], that Chenopodiaceae
discovered family
that Chenopodi-
members
aceae exhibit
family strongexhibit
members cardiostrong
protective
cardio and antioxidant
protective andeffects due toeffects
antioxidant high quantities
due to high of
quantities of phyto-components,
phyto-constituent’s constituent’s such as flavonoids
components, such and phenolic acids.
as flavonoids These findings
and phenolic acids.
These findings support previous findings [2,31,41] indicating quinoa seeds can improve
liver function in rats on a high-cholesterol diet due to their high bioactive component con-
tent. The nephrons and tubules in the cortex of rats given a standard diet (negative group)
revealed no histopathological alterations, and their histological structure was normal (Fig-
Molecules 2022, 27, 7695 11 of 18

support previous findings [2,31,41] indicating quinoa seeds can improve liver function
in rats on a high-cholesterol diet due to their high bioactive component content. The
nephrons and tubules in the cortex of rats given a standard diet (negative group) revealed
no histopathological alterations, and their histological structure was normal (Figure 4a).
The animals fed an HF-diet, exhibited deterioration in the tubular lining epithelium as
well as necrobiosis alteration in a distributed manner. The histopathological diagnosis
revealed severe hepatotoxicity in HF-diet-fed rats. The present results agree with a previous
Molecules 2022, 27, x FOR PEER REVIEW 12 of 19
report [47]. The biomarkers in the serum further confirmed the histopathological evaluation
(Figure 4a–d).

(a) (b)

(c) (d)
Figure4.4.Effect
Figure Effectofoffermented
fermentedcamel
camel milk
milk fortified
fortified with
with red
red quinoa
quinoa seed
seed flour
flour ononhepatic
hepatictissue
tissueininin
hypercholesterolemic rats. (a) Negative control group (Representative photomicrograph of rat liver
in hypercholesterolemic rats. (a) Negative control group (Representative photomicrograph of rat
showing normal histo-morphological structures including central vein (star), hepatic cords (thick
liver showing normal histo-morphological structures including central vein (star), hepatic cords
arrow) and sinusoids (thin arrows). H&E stain magnification ×100). (b) Positive control group (Rep-
(thick arrow) photomicrograph
resentative and sinusoids (thinofarrows).
rat liver H&E
showingstainmild congested ×
magnification 100).vessels
blood (b) Positive control group
and sinusoids (star),
(Representative photomicrograph of rat liver showing mild congested blood vessels
mild Kupffer cells hyperplasia (small arrow) besides mild lymphocytic infiltrations (arrows). and sinusoids
H and
(star), mild
E stain Kupffer cells×200).
magnification hyperplasia
(c) Rats(small
treated arrow) besides mild
with fermented lymphocytic
camel infiltrations (arrows).
milk (Representative photo-mi-
Hcrograph of rat
and E stain liver showing×slight
magnification 200). portal
(c) Ratsfibrosis among
treated withproliferated and new
fermented camel formed
milk bile ductless
(Representative
beside peri-arteryofinflammatory
photo-micrograph cells slight
rat liver showing aggregations mainlyamong
portal fibrosis lymphocytes (arrow).
proliferated andHnew
andformed
E stain bile
mag-
nification
ductless ×200).
beside (d) Rats treated
peri-artery with fermented
inflammatory camel milkmainly
cells aggregations fortified with 3% of (arrow).
lymphocytes quinoa flour (Rep-
H and E
resentative
stain photomicrograph
magnification of rat
×200). (d) Rats liver with
treated showing nearlycamel
fermented normalmilkhepatic lobules
fortified withwith
3% ofmild con-
quinoa
gested central vein (star). H and E stain magnification ×100).
flour (Representative photomicrograph of rat liver showing nearly normal hepatic lobules with mild
congested central vein (star). H and E stain magnification ×100).

The glomerular tufts demonstrated atrophy, edema, and degeneration in the corti-
comedullary lining tubular epithelium as well as congestion in the cortical blood vessels
(Figure 5b). Furthermore, no histopathological alterations were observed in the kidneys of
rats exposed to fermented camel milk and red quinoa seed flour, as shown in Figure 5c,d.
Gentamicin GM-induced renal histological anomalies, such as degeneration of glomeruli
and tubules, were suppressed by camel milk and showed better progress. The present
study confirms that pre-treatment with camel milk attenuates GM unwanted, induced renal
dysfunction, and cellular damage [48]. Certain quinoa vitamins’ bioactive constituents are
(a) (b)
important because they act as antioxidants in kidney cell membranes, such as selenium,
 hypercholesterolemic rats. (a) Negative control group (Representative photomicrograph of rat liver
showing normal histo-morphological structures including central vein (star), hepatic cords (thick
arrow) and sinusoids (thin arrows). H&E stain magnification ×100). (b) Positive control group (Rep-
resentative photomicrograph of rat liver showing mild congested blood vessels and sinusoids (star),
mild Kupffer cells hyperplasia (small arrow) besides mild lymphocytic infiltrations (arrows). H and
Molecules 2022, 27, 7695 E stain magnification ×200). (c) Rats treated with fermented camel milk (Representative photo-mi-
12 of 18
crograph of rat liver showing slight portal fibrosis among proliferated and new formed bile ductless
beside peri-artery inflammatory cells aggregations mainly lymphocytes (arrow). H and E stain mag-
nification ×200). (d) Rats treated with fermented camel milk fortified with 3% of quinoa flour (Rep-
magnesium, phyto-sterols, of
resentative photomicrograph folic
rat acid, and tocopherols,
liver showing which
nearly normal are hypothesized
hepatic to have
lobules with mild con-
antioxidant
gested centralanti-carcinogenic
vein (star). H and qualities or operate ×100).
E stain magnification as anti-inflammatory agents.

(a) (b)

(c) (d)
Figure 5.
Figure 5. Effect
Effect of
of fermented
fermented camel
camel milk
milk fortified
fortified with
with red
red quinoa
quinoa seed
seed powder
powder onon the
the histological
histological
examination of the kidneys in hypercholesterolemic rats. (a) Negative control group (Representative
examination of the kidneys in hypercholesterolemic rats. (a) Negative control group (Representative
photomicrograph of rat kidney showing normal histological architectures including glomeruli (stars)
and tubules (arrows). H and E stain magnification ×100). (b) Positive control group (Representative
photomicrograph of rat kidney showing renal cortex with areas of large eosinophilic casts (stars)
beside degenerated tubules (small arrows) and partial shrunken glomeruli (arrow) with widening of
bowman’s spaces. H and E stain magnification ×100). (c) Rats treated with fermented camel milk
(Representative photomicrograph of rat kidney showing apparently normal glomeruli and tubules
except mild congested large interstitial renal blood vessels (star) beside degenerated proximal tubules
(arrow). H and E stain magnification ×200). (d) Rats treated with fermented camel milk fortified
with 3% quinoa flour (Representative photomicrograph of rat kidney showing marked interstitial
lymphocytic infiltrations (star) besides nearly normal glomeruli. H and E stain magnification ×200).

3. Material and Methods

3.1. Sample Collection
Fresh camel milk was obtained from the frame of College of Agriculture and Veterinary
Medicine, Qassim University, KSA. Dried red quinoa seeds were obtained from a local
market in Buraidah city, KSA, and subjected to grinding to obtain a fine powder.

3.2. Chemicals
Gallic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), other chemicals, and reagents
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Kits for determination of
serum albumin, total protein, TG, TC, HDL, ALT, AST, GSH-px, GSH-Rd, SOD, urea and
creatinine were purchased from Human, Gesellschaft für Biochemical and Diagnostica
mbH, Wiesbaden, Germany.

3.3. Experimental Animals

Forty male Wistar rats (160 ± 10 g) were purchased from the College of Pharmacy,
Qassim University, and divided randomly into five groups (8 rats/group). Under a 12-h
light/dark cycle, the rats were housed in a specific pathogen-free environment at 21 ± 1 ◦ C
Molecules 2022, 27, 7695 13 of 18

and 40–60% humidity. Rats were fed on a basal diet during the experimental period
according to AIN-93 guidelines [49].

3.4. High Fat, High Cholesterol Diet (HF-Diet)

High-fat and high-cholesterol diet was prepared, containing (67 g standard diet, 31.70 g
animal fat, 1% pure cholesterol, and 0.30% bile’s acid) [50].

3.5. Red Quinoa Flour Preparation and Aquoes Extract

Quinoa flour was prepared according to [23] with some modifications to remove
saponins. Whole seeds were washed twice with cold water then seeds were soaked in
the alkaline solution for 20 min, and then rinsed with 1% citric acid solution for 10 min.
The cleaned seeds were washed with water until there was no foam as an indication of
saponins removal from the seeds hull. Later saponins-free seeds were overnight oven-dried
at 45 ± 1 ◦ C. During drying treatment, the seeds were spread in a thin layer to avoid
germination process and any further contamination. Finally, treated whole seeds were
ground into flour using blender with miller (Hanabishi-HHANDBL10-3in1) and kept at
5◦ C for further analyses. The aqueous extract was prepared according to [51]. 50 g of
red quinoa flour was mixed with 1000 mL distillate water (1:20 w/v) in 2L-glass beaker,
and stirred for 60 min at room temperature. The extract was filtered through filter paper
(Wattman No.1). The aqueous extract was stored at (5 ± 1 ◦ C) for further analyses.

3.6. Starter Cultures and Fermented Milk Manufacture

The lyophilized Yo-flex culture (YC-x11) was obtained from Christian Hansen (Copen-
hagen, Denmark), contains a mixed strain of S. thermophilus and L. bulgaricus at the ratio (1:1).
Different fermented milk was prepared according to the methods described by Tamime
and Robinson [52]. Briefly, raw camel milk was heated at 90 ◦ C/5 min then cooled to 42 ◦ C.
Quinoa seed powder was added at the rate of 1, 2, 3, and 4% immediately after incubation
with 2% activated starter culture to avoid the quick reduction of pH and transferred into
100 mL plastic containers, lightly sealed, and incubated at 42 ◦ C until the complete curd
formation, then stored at refrigerator (5 ± 2 ◦ C) for 24 h. The fermentation of yogurt was
stopped when the pH value reached 4.6 as shown in Figure 6.

3.7. Organoleptic Properties

Sensory studies were conducted with 20 students and lecturers of the Food Science and
Human Nutrition department, College of Agriculture and Veterinary Medicine, Qassim
University. Panellists were requested to evaluate the color (10 points), flavor (30 points),
acidity (10 points), body and texture (40 points), and overall acceptability (10 points)
according the scoring described by Tamime and Robinson [52].

3.8. Chemical Analysis

The chemical analysis was conducted on different fermented milk and red quinoa flour
samples to determine the pH, titratable acidity, ash, protein, fiber, moisture, fat, vitamin C,
and mineral content according to AOAC, [53]. The total polyphenolic content of quinoa
seeds was measured by Folin-Ciocalteu assay using spectrophotometer (Secomam, France)
based on the Singleton et al. [54] assay. The total flavonoid content of quinoa seeds was
determined by the aluminium chloride colorimetric method as described by Seasotiya
et al. [55]. The antioxidant activity was evaluated by the DPPH assay [56]. The scavenging
antioxidant activity percentage was determined as follows:

AOA(%) = 1 − Abssample − Absblank /Abscontrol × 100 (1)

Total cholesterol, HDL, and triglyceride levels in serum were estimated by the method
of [56]. Liver enzymes (ALP, ALT, and AST), serum albumin, and total protein were
estimated as described by [57]. Urea and creatinine were determined as indicators of
kidney function [58]. Insulin and blood glucose were determined in human blood samples
 Molecules 2022, 27, x FOR PEER REVIEW 15 of 19

3.11. Histopathological Examination

Molecules 2022, 27, 7695 14 of 18
Autopsy samples were taken from the liver and kidney of the sacrificed rats and fixed
in 10% formalin saline solution for ten hours at least, then washed in tap water for 12 h.
Tissue specimens were cleared in xylene and embedded in paraffin. The obtained tissue
according to the
sections were method
collected on of
theThomas et al.
glass slides and [59].
dyedLipid
with peroxides
hematoxylinwere
and determined
eosin stain forin serum
ashistopathological
malondialdehyde examination by the light
MDA according to microscope
Namıduru[61].et al. [60].

Figure 6.
Figure 6. Basic
Basic process
processfor
formanufacturing
manufacturingfermented camel
fermented fortified
camel with red
fortified quinoa
with four. four.
red quinoa

3.12.
3.9. Statistical Analysis
Experimental Design
All experiments
This study was as well as related
conducted analysis
with the resultsofwere
approval performedCommittee
the National in triplicate of
andBioethics
are presented as mean ± standard deviation. A variance analysis (two-way ANOVA)
NCBE at King Abdul-Aziz City for Science and Technology, KACST, KSA, Review Board with
a significance
Number: threshold
10024640, of p ≤date:
(Expiry 0.05 was used to2024).
8 August defineRats
the differences between
were randomly groups.into five
divided
Statistica 12.5 software was used to conduct the analysis (Stat Soft Inc., Tulsa, OK, USA).
groups (n = 8). The first group served as normal control (NC) without further treatment.
The other rats were switched to a HF-diet for 6 weeks and were subdivided into subgroups
as follows: group (2) continued on the HF-diet without treatment and was labelled as a
positive control (PC). Groups 3 to 5 were treated orally by intestinal tube with 2 mL/day
of either fermented camel milk with traditional culture (Came_T), fermented camel milk
fortified with 3% of red quinoa flour (Came_Q3 ), or red quinoa aqueous extract (Ex_Q3 ).
Serum samples were obtained after the slaughter of rats at the end of the experiment by
centrifuging the collected blood at 3000 rpm/10 min and stored at −20 ◦ C until analysis.

3.10. Measurement of Adipocyte Size

The volumes of adipocytes are measured according to the method of Althwab et al. [45],
in visceral fat surrounding the kidney, such as mesenteric, retroperitoneal, and epididymal
white adipose tissue. The adipose tissues were rinsed with saline, and fixed in neutral 10%
formalin solution, then embedded in paraffin, cut into 10 mm sections and stained with
Molecules 2022, 27, 7695 15 of 18

hematoxylin. Then the cell volumes were measured using the US National Institutes of
Health image software (100 cells/mouse).

3.11. Histopathological Examination

Autopsy samples were taken from the liver and kidney of the sacrificed rats and fixed
in 10% formalin saline solution for ten hours at least, then washed in tap water for 12 h.
Tissue specimens were cleared in xylene and embedded in paraffin. The obtained tissue
sections were collected on the glass slides and dyed with hematoxylin and eosin stain for
histopathological examination by the light microscope [61].

3.12. Statistical Analysis

All experiments as well as related analysis results were performed in triplicate and
are presented as mean ± standard deviation. A variance analysis (two-way ANOVA) with
a significance threshold of p ≤ 0.05 was used to define the differences between groups.
Statistica 12.5 software was used to conduct the analysis (Stat Soft Inc., Tulsa, OK, USA).

4. Conclusions
In conclusion, red quinoa seed and camel milk can be used as effective food supple-
ments for preventing overweight and obesity by lowering weight gain, adipocyte size,
and serum levels of glucose and lipids. The fortification of camel milk with red quinoa
seed powder improved the chemical, antioxidant, rheological, and sensory properties
of fermented camel milk, and these improvements were proportional to the fortification
up to 3%, which added nutritive and healthy benefits to the resultant fermented camel
milk. Consumption of fermented camel milk containing 3% red quinoa seed flour in the
hypercholesterolemic rat group caused a significant decrease in the levels of blood glucose,
MDA, LDL, TC, TG, AST, ALT, ALP, creatinine, and urea and increased HDL, total protein,
and albumin in comparison to hypercholesterolemic rats. Consequently, quinoa can be
addressed as a substitute source of nutrients and especially protein, for people with dietary
protein deficiency.

Author Contributions: M.S.A.-A.: Data curation, Investigation, Methodology, Software, Visualiza-

tion. K.M.E.-Z.: Conceptualization, Data curation, Formal analysis, Project administration, Validation,
Writing—review and editing. N.A.-H.R.: Formal analysis, Investigation, Methodology, Visualization,
Writing—original draft. All authors have read and agreed to the published version of the manuscript.
Funding: Researchers would like to thank the Deanship of Scientific Research, Qassim University for
funding the publication of this project.
Institutional Review Board Statement: This study was conducted with the approval of the approval
of the National Committee of Bioethics (NCBE) at King Abdul-Aziz City for Science and Technology
(KACST), KSA, Review Board Number: 10024640, (Expiry date: 8 August 2024).
Informed Consent Statement: Not applicable.
Data Availability Statement: The authors declare the availability of data and material; they also
declare the data is transparent for this manuscript.
Conflicts of Interest: The authors declare that they have no conflict of interest with the contents
of this article. The authors are not part of any associations or commercial relationships that might
represent conflicts of interest in the writing of this study.
Sample Availability: Not applicable.

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