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Article
Ameliorative Effect of Graviola (Annona muricata) on
Mono Sodium Glutamate-Induced Hepatic Injury in
Rats: Antioxidant, Apoptotic, Anti-inflammatory,
Lipogenesis Markers, and Histopathological Studies
Mustafa Shukry 1, * , Ahmed M. El-Shehawi 2,3 , Wafaa M. El-Kholy 4, Rasha A. Elsisy 5,
Hazem S. Hamoda 6, Hossam G. Tohamy 7, Mohamed M. Abumandour 8and Foad A. Farrag 9
1Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University,
33511 Kafrelsheikh, Egypt
2Department of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
elshehawi@hotmail.com
3Department of Genetics, Faculty of Agriculture, Alexandria University, 21527 Alexandria, Egypt
4Department of Zoology, Faculty of Science, Mansoura University, 35516 Mansoura, Egypt;
wafaa_elkholy2002@yahoo.com
5Department of Anatomy, Faculty of Medicine, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt;
Rasha_2002@yahoo.com
6Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Aswan University,
81528 Aswan, Egypt; Hamouda2000@yahoo.com
7Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, 22785 Alexandria, Egypt;
hossam.gafar@yahoo.com
8Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Alexandria University,
22785 Alexandria, Egypt; m.abumandour@yahoo.com
9Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Kafrelsheikh University,
33511 Kafrelsheikh, Egypt; foad.farrag@yahoo.com
*Correspondence: mostafa.ataa@vet.kfs.edu.eg
Received: 29 September 2020; Accepted: 25 October 2020; Published: 30 October 2020
Simple Summary:
Food additives, especially monosodium glutamate (MSG), induces serious liver
disorders. This study premeditated to investigate the effect of Graviola extract (GE) on hepatic and
cellular alterations induced by MSG. Our result revealed that GE administration normalized the
oxidative stress markers, as well as the proinflammatory cytokines, in addition to downregulation
of the inducible nitric oxide synthase (iNOS) and FAS, hepatic fatty acid synthase, and led to the
upregulation of the silent information regulator protein one (SIRT1) gene. This is the first report
investigating the intracellular pathway and mechanism of Graviola extract’s action in alleviating the
MSG supplementation injuries.
Abstract:
Monosodium glutamate (MSG) is a widely used food additive, and there is a trepidation
that MSG plays a critical role in multiple hepatic disorders. This study was planned to investigate
Graviola extract (GE) effects on hepatic and cellular alterations induced by MSG. Fifty Wistar rats were
randomly allocated into five groups: control (received normal saline), Graviola (
received 200 mg/kg
body weight), MSG (received 2.4 gm MSG/kg, 15% of Lethal dose (LD
50
) of MSG), Graviola +
monosodium glutamate (MSG +GE; received GE, 200 mg/kg/day and MSG 2.4 gm/kg body weight
(BW) for the next four weeks), and monosodium glutamate +Graviola (received MSG only (2.4 gm/kg
BW) daily for four weeks, then concomitant with Graviola (200 mg/kg BW) daily for the next four
weeks. MSG and GR were administered orally for eight weeks. Our results showed that MSG
caused a significant increase in oxidative stress markers malondialdehyde (MDA), reactive oxygen
species (ROS), nitric oxide (NO), hydrogen peroxide
(
H
2
O
2)
, proinflammatory cytokines interleukin
Animals 2020,10, 1996; doi:10.3390/ani10111996 www.mdpi.com/journal/animals
Animals 2020,10, 1996 2 of 19
6 (IL-6) level, a tumor protein (P53), hepatic cellular damage, as well as proapoptotic markers
caspase-3, and B-cell lymphoma 2 (BCL-2)-like protein 4 (Bax). A significant decrease in superoxide
dismutase (SOD), catalase (CAT), glutathione S transferase (GST), reduced glutathione (GSH),
and an antiapoptotic agent B-cell lymphoma 2 (BCl-2) was observed. The detected MSG effects
were normalized by Graviola administration, either a prophylactic or protecting dose. Besides,
Graviola reduced the expression of inducible nitric oxide synthase (iNOS) and hepatic fatty acid
synthase (FAS) and led to the upregulation of the silent information regulator protein one gene
expression gene (SIRT1).In conclusion, the results suggest that Gaviola’s interrelated antiapoptotic,
antioxidant, and anti-inflammatory properties are potential mechanisms to enhance hepatic deficits
and protect the liver. Graviola can, therefore, be considered a promising hepatoprotective supplement.
Additionally, further human clinical trials are also necessary to validate the present research.
Keywords: Graviola; liver; monosodium glutamate; antioxidant; apoptosis; iNOS; SIRT1
1. Introduction
Many synthetic contaminants, such as industrial toxins and food additives, have been implicated
in adverse effects. Some food additives act as either preservatives or palatability enhancers. One such
food additive is monosodium glutamate (MSG). MSG is one of these food additives that is openly
used as a flavor enhancer. It is glutamic acid salt [
1
]. It is documented that rats administrated MSG
encountered many disorders such as gonadal dysfunction, an increase in stomach cancer incidence,
brain damage, learning difficulty, and depletion in some neurotransmitters in the hypothalamus
region [
2
]. The extravagant administration of MSG was shown to cause liver and kidney damage [
3
],
as well as oxidative stress in the tissue, with degenerative changes in hepatocytes [
4
]. MSG enhances
meals’ palatability and significantly improves the appetite center and, consequently, increases body
weight [
5
]. Although MSG enhances flavor stimulation and boosts appetite, it is considered toxic to
humans and experimental animals [
6
]. In normal conditions, glutamate has a very low acute toxicity;
the oral dose lethal to LD
50
in rats and mice is
∼
15,000–18,000 mg/kg body weight, respectively [
7
].
Besides, an oral gavage dose of MSG for a dose level of 0.8, 1.6, and 2.4 gm/kg body weight (BW)/day
for 30 and 40 days, respectively, presumably suppresses the female’s reproductive system in rats [
8
].
In the intoxicated model, MSG rats, a daily dose of MSG (4 g/kg orally) was given for seven days,
which induced kidney injury in rats [
9
]. According to the last update of the Joint Food and agriculture
organization (FAO)/WHO Expert Committee on Food Additives (JECFA), the U.S. Food and Drug
Administration (FDA), and the European Food Safety Association (EFSA), the acceptable daily intake
of MSG to humans is 30 mg/kg/day [
10
]. The acceptable daily intake (ADI) is the maximum amount of
a chemical that can be ingested daily over a lifetime with no appreciable health risk and is based on the
highest intake that does not give rise to observable adverse effects.
The liver is the body’s most prominent glandular tissue, and it has a significant role in body
metabolism. It has a wide range of functions, including the storage of glucose, plasma protein
synthesis, and bile production [
11
]. The liver may be susceptible to harm due to toxic substances,
because it is involved in these diversified functions. Appropriate alternative therapies should
be sought to increase the treatment’s efficacy by recognizing the pathophysiological processes
responsible for creating hepatic injury. The disparity between the generation of ROS and antioxidants
leads to free radical species disruption to oxidative substances in cells, including proteins, lipids,
or nucleic acids [
12
]. Supplementations with natural antioxidants have been shown to improve
the body’s efficiency in stressful conditions [
13
]. Natural medicines have traditionally been used
to cure several diseases. Muricata Annona L. (Graviola), a member of the Annonaceae family,
is a perennial tree species used as herbal medicine. The plant has various pharmacological
functions, including cytotoxicity, antimicrobial, and wound care [
14
]. It has antidiabetic and
Animals 2020,10, 1996 3 of 19
hypolipidemic [
15
], antiarthritic [
16
], antinociceptive [
17
], hepatoprotective and bilirubin-lowering [
18
],
powerful antioxidants activities [
19
], antihypertensive [
20
], anticancer [
21
], gastroprotective [
22
],
and anti-inflammatory and anticonvulsant [
23
] effects. The most useful aspects of this tree are Graviola
leaves. It has active ingredients, bulatacin, asimisin, and squamosin, which contain acetogenins [
24
].
The presence of secondary metabolites like tannins, steroids, and cardiac glycoses was revealed by a
chemical screening of Graviola extract [
25
]. The current research was carried out to study Graviola’s
prophylactic and protective effects on rats’ MSG-induced hepatic injuries.
2. Materials and Methods
2.1. Chemicals
All chemicals and kits used were purchased from standard confirmed companies and were of
the highest grade. MSG (<99%) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Complete RNA extraction and SYBR Green Master Mix kits were purchased from (QIAGEN, Hilden,
Germany). Graviola dry extract
®
was imported from Origini Naturali Company (Quarrata, Pistoia, Italy).
2.2. Ethics Statement
The study was approved with the NIH Guide for the Treatment and Use of Animals from the
University of Kafrelsheikh, Egypt, Faculty of Veterinary Ethics Committee. All procedures were taken
during the process to alleviate animal suffering. Egypt KSU/VetMed-2018-/1155.
2.3. Experimental Design
Fifty Wistar male rats ten weeks of age average (179
±
1 g) were purchased from the Egyptian
Institute for Vaccine and Serological Production, Helwan, Egypt and were housed in the animal house
of the Department of Physiology, Faculty of Veterinary, Kafrelsheikh University. They were maintained
under standard laboratory conditions with a 12 h light/dark cycle and free access to food pellets and
tap water ad libitum. After two weeks of acclimatization, the rats were randomly allocated into
five groups as seen in Figure 1: control (received normal saline (0.9%) with the same procedure and
volume as Graviola-treated groups, orally once daily for eight weeks), Graviola (orally administrated
Graviola extract (GE) at 200 mg/kg BW daily for 8 weeks), and Graviola dry extract was dissolved
in normal saline (0.9%) and administered daily with 200 mg extract/kg BW using gavage needles.
The selected dose of aqueous Graviola extract (GE) was according to the previous study of [
26
,
27
],
MSG (orally administrated 15% of LD
50
of MSG, 2.4 gm/kg daily for 8 weeks) according to [
8
],
Graviola +monosodium glutamate (MSG +GR; received GE, 200 mg/kg/day once daily orally four
weeks, then concomitant with MSG 2.4 gm/kg BW for next four weeks), and monosodium glutamate +
Graviola (orally administered MSG only (2.4 gm/kg BW) daily for four weeks, then concomitant with
GE (200 mg/kg BW) daily for next four weeks. All treatments were initiated and continued for eight
weeks with a basal diet [28,29]. The body weight and food intake were daily recorded.
2.4. Sampling
Rats at the end of the experimental period (eight weeks) were anesthetized and sacrificed
for sampling and consequent analysis by intravenous sodium pentobarbital injection (30 mg/kg).
Blood samples were collected part in centrifuge clean glass tubes, left to clot, and centrifuged at
4000 rpm for 15 min. The clear, not hemolyzed sera were quickly removed and put in labeled Eppendorf
tubes; the sera were frozen at
−
20
◦
C for further biochemical analysis. The other part was collected in
sterilized Ethylenediaminetetraacetic acid (EDTA) tubes (Al-Gomhuria Chemical Company, Cairo,
Egypt) for hematological analysis. Liver samples (n =6 per group) from each rat were removed,
weighed, and homogenized in cold phosphate-buffered saline (PBS). At 4
◦
C, the homogenates were
centrifuged for 10 min at 3000
×
g. For biochemical assays, the collected supernatants were stored at
−
20
◦
C. The remaining liver specimen was held frozen at
−
80
◦
C to study gene expression and other
Animals 2020,10, 1996 4 of 19
biochemical assays. Different samples of the liver tissue were stored in neutral formalin (10%) for
histopathological studies.
Animals 2020, 10, x 4 of 21
Figure 1. Time scheme of the experiment. MSG: monosodium glutamate. GR: Graviola.
2.4. Sampling
Rats at the end of the experimental period (eight weeks) were anesthetized and sacrificed for
sampling and consequent analysis by intravenous sodium pentobarbital injection (30 mg/kg). Blood
samples were collected part in centrifuge clean glass tubes, left to clot, and centrifuged at 4000 rpm
for 15 min. The clear, not hemolyzed sera were quickly removed and put in labeled Eppendorf tubes;
the sera were frozen at −20 °C for further biochemical analysis. The other part was collected in
sterilized Ethylenediaminetetraacetic acid (EDTA) tubes (Al-Gomhuria Chemical Company, Cairo,
Egypt) for hematological analysis. Liver samples (n = 6 per group) from each rat were removed,
weighed, and homogenized in cold phosphate-buffered saline (PBS). At 4 °C, the homogenates were
centrifuged for 10 min at 3000× g. For biochemical assays, the collected supernatants were stored at
−20 °C. The remaining liver specimen was held frozen at −80 °C to study gene expression and other
biochemical assays. Different samples of the liver tissue were stored in neutral formalin (10%) for
histopathological studies.
2.5. Biochemical and Hematological Analysis
Materials and services in this section were obtained from Bio-Diagnostic Co. Dokki, Giza, Egypt,
unless otherwise indicated. The automatic measurement of hematology parameters was performed
using an Avantor Performance Materials Inc. Business, Center Valley (USA), H32 VET 3-Part
differential analyzer of hematology. Serum total cholesterol and triglyceride concentration were
estimated according to the method of [30,31], respectively. Serum high-density lipoprotein (HDL-C)
concentration was assayed using the colorimetric enzymatic method of [32]. Serum low-density
lipoprotein (LDL-C) concentration was calculated according to the equation described by [33]. Serum
Aspartate Aminotransferase (AST) and Alanine aminotransferase (ALT) activities were measured
according to the colorimetric kit technique using Diamond Diagnostics co., Cairo, Egypt [34]. Serum
Alkaline phosphatase (ALP) activities were measured according to the method described by [35]. The
serum gamma-glutamyltransferase (GGT) activity was determined according to the process of [36].
The total bilirubin level in the serum was determined using kits purchased from Diamond
Diagnostics, Co., Cairo, Egypt and was measured by a colorimetric method [37]. Serum total protein
Figure 1. Time scheme of the experiment. MSG: monosodium glutamate. GR: Graviola.
2.5. Biochemical and Hematological Analysis
Materials and services in this section were obtained from Bio-Diagnostic Co. Dokki, Giza, Egypt,
unless otherwise indicated. The automatic measurement of hematology parameters was performed
using an Avantor Performance Materials Inc. Business, Center Valley (USA), H32 VET 3-Part differential
analyzer of hematology. Serum total cholesterol and triglyceride concentration were estimated
according to the method of [
30
,
31
], respectively. Serum high-density lipoprotein (HDL-C) concentration
was assayed using the colorimetric enzymatic method of [
32
]. Serum low-density lipoprotein
(LDL-C) concentration was calculated according to the equation described by [
33
]. Serum Aspartate
Aminotransferase (AST) and Alanine aminotransferase (ALT) activities were measured according
to the colorimetric kit technique using Diamond Diagnostics co., Cairo, Egypt [
34
]. Serum Alkaline
phosphatase (ALP) activities were measured according to the method described by [
35
]. The serum
gamma-glutamyltransferase (GGT) activity was determined according to the process of [
36
]. The total
bilirubin level in the serum was determined using kits purchased from Diamond Diagnostics, Co.,
Cairo, Egypt and was measured by a colorimetric method [
37
]. Serum total protein (TP) contents
were estimated, as described by [
38
]. Serum albumin (Alb) content was determined according to [
39
],
using a kit obtained from Diamond Diagnostics, co., Cairo, Egypt.
2.6. Analysis of the Antioxidant Status in Hepatic Tissues
The liver homogenate malondialdehyde (MDA) content was determined according to the
colorimetric technique of [
40
], liver nitric oxide (NO) activity was estimated by the method of [
41
],
liver homogenate H
2
O
2
activity was assayed by the method of [
42
], and liver homogenates glutathione
(GSH) and glutathione S transferase (GST) according to [
43
,
44
], respectively. Liver superoxide dismutase
(SOD) and catalase (CAT) activity were estimated according to [
45
,
46
], respectively. All assays were
evaluated using kits from Biodiagnostic Co. Dokki, Giza, Egypt.
Animals 2020,10, 1996 5 of 19
2.7. Flowcytometric Analysis for B-Cell Lymphoma 2 (BCL-2), P53, BCL-2-like Protein 4 (Bax), and Caspase-3
The BCL-2, P53, Bax, and caspase-3 were assessed using a flow cytometer instrument (San Jose,
CA, USA) in the Mansoura Children Hospital FACS calibur flow cytometer (Becton Dickinson, San Jose,
CA, USA) [
47
], in which samples from the liver were prepared according to the method done by [
48
],
with some modifications. In brief, the tissue’s fresh specimens were washed with isotonic tris EDTA
buffer, centrifuged at 1800 rpm for 10 min, and then, the supernatant was removed. The liver samples
were suspended in phosphate-buffered saline (PBS) with bovine serum albumin (BSA) divided into
aliquots, fixed in ice-cold 96–100% ethanol stored at 4
◦
C for analyses. For Bcl-2, Bax, and Tumor protein
p53 (P53), anti-B-cell lymphoma 2 (Bcl2), anti-Bax, and anti-P53 (Santa Cruz Biotechnology, Inc., Dallas,
TX, USA.) were added to the PBS/BSA buffer and incubated for 30 min (diluted at 1:100), centrifuged at
400×gfor 5 min for resuspending in 0.5% paraformaldehyde in PBS/BSA, and analyzed using a flow
cytometer. Caspase-3 was done by using the following antibodies (Fluorescein isothiocyanate (FITC)
rabbit anti-active caspase-3 (1:500) (CPP32, Yama, Apopain; BD Bioscience)). The analysis of the flow
cytometer was carried out by using BD Accuri
™
C6 (BD Biosciences, San Jose, CA, USA) with the Cell
Quest Pro software (Becton Dickinson, San Jose, CA, USA) for data acquisition and analysis [49].
2.8. Determination of Hepatic Reactive Oxygen Species (ROS) and Interleukin 6 (IL-6) Content Using ELISA
ROS content was determined using the ROS kit from AMSBIO Co., Milton, UK, according to [
50
].
The IL-6 levels in the tissue were estimated quantitatively using a RAT ELISA kit from Ray Biotech, Inc.
(Norcross, GA, USA).
2.9. Gene Expression Analysis
In 1 mL QIAzol (79306, QIAGEN Inc., Valencia, CA, USA), the total RNA content was extracted
with chloroform from liver tissue. The corresponding cDNA was synthesized with RevertAid Premium
reverse transcriptase (EP0733, Thermo Fisher Scientific, Darmstadt, Germany). Amplification curves
and cycle threshold (CT) values were developed with Stratagene MX3005P software. The CTs of each
sample were compared to the positive control group by the "alternative to CT" approach to estimate
the RNA differences in the sample gene expressions. Primer sequence and information for relevant
genes are summarized in Table 1.
Table 1. Primers sequences.
Genes 50–30Primer Sequence Accession Number References
FAS F: CCTGGACAACATGGTAGCTGC NM 017332.1 [51]
R: GCAGTGCCTTCCTTGAGAACAG
SIRT1
F: TGA CTT CAG ATC AAG AGA TGG
TAT TTA TG NM 001372090 [52]
R: TGG CTT GAG GAT CTG GGA GAT
iNOS F: GGATATCTTCGGTGCGGTCTT S71597 [53]
R: CTGTAACTCTTCTGGGTGTCAGA
GAPDH F: TCAAGAAGGTGGTGAAGCAG NM 017008.4 [54]
R: AGGTGGAAGAATGGGAGTTG
SIRT1, silent information regulator protein one gene expression; iNOS, inducible nitric oxide synthase; FAS, fatty acid
synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; and GGT, gamma-glutamyltransferase.
2.10. Histopathological Studies
Liver tissues were accurately fixed in a neutral formalin solution (10%). They were dehydrated
in an ascending series of ethanol, were cleared in xylene, were embedded in paraffin wax, and were
sectioned at 5–7
µ
m by microtome and were stained with eosin and hematoxylin. The stained
Animals 2020,10, 1996 6 of 19
sections were examined and were photographed under a light microscope to detect histopathological
changes [55].
2.11. Statistical Analysis
All data were expressed as means
±
SE using one-way ANOVA, followed by Tukey’s multi-range,
post-hoc check using SPSS software, version 20.0 (SPSS Inc., Chicago, IL, USA). Repeated measures
ANOVA was used for determining a change in body weight at different intervals. The differences were
statistically significant at p≤0.05.
3. Results
3.1. Body Weight
At the end of the experiment (56 days), body weight was found to increase significantly in the
MSG-treated group compared to the control group (p<0.05). There was a significant decrease in
body weight in rats orally administered MSG +GE and GE +MSG compared to the MSG-treated
group.Concerning control one, there were no significant shifts with the Graviola treatment. (Figure 2).
Animals 2020, 10, x 7 of 21
Figure 2. Body weight in control and differently treated rats. Data presented as means ± SEM for six
rats in each group and % of change. Different superscript letters (a, b and c) indicate significant
differences. The significant change at p ≤ 0.05. (N = 6).
3.2. Hematological and Biochemical Findings
As shown in Table 2, the obtained result demonstrated that Hemoglobin (Hb), Red blood cells
(RBCs), White blood cells (WBCs), Packed cell volume (PCV%), and platelets significantly decreased
in the MSG-treated group compared to the control and Graviola groups. Graviola-treated groups,
either protected or prophylactic, showed a significant increase in Hb, RBCs, WBCs, PCV%, and
platelets related to the MSG-treated group.
The level of total cholesterol, triglyceride, and Low-density lipoprotein-cholesterol (LDL-C) and
the liver enzymes Alanine aminotransferase (ALT). Aspartate aminotransferase (AST), Alkaline
phosphatase (ALP).and gamma-glutamyltransferase (GGT) and the total bilirubin in the MSG-treated
group were higher than that of the control group and Graviola group. In the same context, the
Graviola-treated groups, either prophylactic or treated, showed significant decreases in these
parameters. On the contrary, the levels of HDL-c, albumin, and total protein were significantly
decreased (p < 0.05) in MSG-treated rats, which dramatically improved with Graviola treatment, as
described in Table 3.
Table 2. Effects of Graviola and monosodium glutamate (MSG) on the hematological parameters.
Parameters Control Graviola MSG Graviola + MSG MSG + Graviola
Hb(g/L) 154.6 ± 1.35 a 159.2 ± 0.45 a 85 ± 0.64 c 115.6 ± 0.78 b 105.4 ± 0.62 b
RBCs (1012/L) 9.22 ± 0.56 a 9.27 ± 0.49 a 4.06 ± 0.26 c 6.00 ± 0.30 b 5.93 ± 0.46 b
PCV(L/L) 0.510 ± 0.01 a 0.525 ± 0.01 a 0.280 ± 0.01 c 0.381 ± 0.06 b 0.347 ± 0.07 b
WBCs (109/L) 9.50 ± 0.31 a 10.20 ± 0.33 a 5.45 ± 0.32 c 7.25 ± 0.31 b 7.07 ± 0.34 b
Lymphocyte% 73.12 ± 1.15 a 74.20±1.10 a 64.92 ± 1.19 c 67.00 ± 1.7 b 68.00 ± 1.7 b
Neutrophil% 18.0 ± 1.3 a 18.1 ± 1.9 a 7.0 ± 1.1 c 16.4 ± 1.8 b 16.9 ± 1.1 b
Platelets (109/L) 755.9 ± 35.38 a 764.3 ± 40.90 a 269.6 ± 25.23 c 458.4 ± 46.40 b 443.3 ± 44.20 b
Data presented as means ± SEM for six rats in each group and % of change. The significant change
was at p ≤ 0.05. Different superscript letters (a, b and c) indicate significant differences in the same
column (N = 6). Hemoglobin (Hb), Red blood cells (RBCs), Packed cell volume (PCV), White blood
cells (WBCs).
Figure 2.
Body weight in control and differently treated rats. Data presented as means
±
SEM for
six rats in each group and % of change. Different superscript letters (a, b and c) indicate significant
differences. The significant change at p≤0.05. (n=6).
3.2. Hematological and Biochemical Findings
As shown in Table 2, the obtained result demonstrated that Hemoglobin (Hb), Red blood cells
(RBCs), White blood cells (WBCs), Packed cell volume (PCV%), and platelets significantly decreased
in the MSG-treated group compared to the control and Graviola groups. Graviola-treated groups,
either protected or prophylactic, showed a significant increase in Hb, RBCs, WBCs, PCV%, and platelets
related to the MSG-treated group.
The level of total cholesterol, triglyceride, and Low-density lipoprotein-cholesterol (LDL-C)
and the liver enzymes Alanine aminotransferase (ALT). Aspartate aminotransferase (AST),
Alkaline phosphatase (ALP).and gamma-glutamyltransferase (GGT) and the total bilirubin in the
MSG-treated group were higher than that of the control group and Graviola group. In the same
context, the Graviola-treated groups, either prophylactic or treated, showed significant decreases in
these parameters. On the contrary, the levels of HDL-c, albumin, and total protein were significantly
Animals 2020,10, 1996 7 of 19
decreased (p<0.05) in MSG-treated rats, which dramatically improved with Graviola treatment,
as described in Table 3.
Table 2. Effects of Graviola and monosodium glutamate (MSG) on the hematological parameters.
Parameters Control Graviola MSG Graviola +MSG MSG +Graviola
Hb(g/L) 154.6 ±1.35 a159.2 ±0.45 a85 ±0.64 c115.6 ±0.78 b105.4 ±0.62 b
RBCs (1012/L) 9.22 ±0.56 a9.27 ±0.49 a4.06 ±0.26 c6.00 ±0.30 b5.93 ±0.46 b
PCV(L/L) 0.510 ±0.01 a0.525 ±0.01 a0.280 ±0.01 c0.381 ±0.06 b0.347 ±0.07 b
WBCs (109/L) 9.50 ±0.31 a10.20 ±0.33 a5.45 ±0.32 c7.25 ±0.31 b7.07 ±0.34 b
Lymphocyte% 73.12 ±1.15 a74.20±1.10 a64.92 ±1.19 c67.00 ±1.7 b68.00 ±1.7 b
Neutrophil% 18.0 ±1.3 a18.1 ±1.9 a7.0 ±1.1 c16.4 ±1.8 b16.9 ±1.1 b
Platelets (10
9
/L)
755.9 ±35.38 a764.3 ±40.90 a269.6 ±25.23 c458.4 ±46.40 b443.3 ±44.20 b
Data presented as means
±
SEM for six rats in each group and % of change. The significant change was at p
≤
0.05.
Different superscript letters (a, b and c) indicate significant differences in the same column (n=6). Hemoglobin (Hb),
Red blood cells (RBCs), Packed cell volume (PCV), White blood cells (WBCs).
Table 3. Effect of Graviola and MSG on the serum biochemical parameters.
Parameters Control Graviola MSG Graviola +MSG MSG +Graviola
Total cholesterol (mmol/L) 2.8 ±0.06 c2.76 ±0.01 c3.25 ±0.1 a3.06 ±0.1 b3.09 ±0.2 b
Triglycerides (mmol/L) 1.10 ±0.03 c1.07 ±0.02 c1.46 ±0.04 a1.29 ±0.01 b1.32 ±0.02 b
LDL-C (mmol/L) 0.85 ±0.01 c0.87 ±0.02 c1.88 ±0.07 a1.46 ±0.06 b1.43 ±0.0.04 b
HDL-C (mmol/L) 1.30 ±0.06 a1.32 ±0.01 a0.796 ±0.02 c1.15 ±0.02 b1.097 ±0.02 b
ALT (µkat/L) 0.51 ±0.008 c0.48 ±0.01 c0.99 ±0.03 a0.73 ±0.01 b0.75 ±0.01 b
AST (µkat/L) 0.46 ±0.008 c0.49 ±0.006 c1.1 ±0.08 a0.72 ±0.01 b0.79 ±0.009 b
ALP (µkat/L) 2.19 ±0.14 c2.16 ±0.02 c2.83 ±0.02 a2.50 ±0.06 b2.48 ±0.02 b
GGT (µkat/L) 0.336 ±0.005 c0.334 ±0.008 c0.475 ±0.003 a0.416 ±0.004 b0.423 ±0.004 b
TB (µmol/L) 7.70 ±0.21 c6.84 ±0.18 c21.72 ±0.79 a13.0 ±0.64 b13.17 ±0.26 b
Albumin (g/L) 38 ±0.64 a38.5 ±0.87 a20 ±0.85 c32 ±1.25 b30 ±1.31 a,b
Total proteins (g/L) 125.90 ±0.65 a127.90 ±0.86 a84.2 ±1.49 c103 ±2.17 a,b 100.00 ±2.10 a,b
Data presented as means
±
SEM for (6) rats in each group and % of change. The significant change was
at
p≤0.05
. Different superscript letters (a, b and c) indicate significant differences in the same column
(
n=6
). Serum gamma-glutamyltransferase (GGT). High-density lipoproteins (HDL-C). Low-density lipoproteins
(LDL-C). Serum total bilirubin (TB). Alanine aminotransferase (ALT). Aspartate aminotransferase (AST),
Alkaline phosphatase (ALP).
3.3. Hepatic Antioxidant Status
We examined the effects of the Graviola treatment on the MSG-induced hepatic injury. MSG’s
toxicity hepatic toxicity through ROS formation encourages us to study Graviola’s antioxidant activity
concerning MSG hepatic toxicity. The obtained data showed a significant decrease in hepatic GSH, GST,
SOD, and CAT levels in the MSG group compared to the control group and Graviola group (
p<0.05
),
while the oral administration of GE to rat groups (MSG +GR and GR +MSG) resulted in a significant
increase in GSH content than the MSG group, as shown in Table 4.
Table 4. Effects of Graviola and MSG extract on liver oxidative status of different rat groups.
Parameters Control Graviola MSG Graviola +MSG MSG +Graviola
MDA (nmol/g) 685.8 ±36.30 c678.2 ±36.31 c1216 ±18.01 a800.4 ±18.88 b815.8 ±18.80 b
NO (µmol/g) 18.23 ±0.52 c18.11 ±0.50 c35.48 ±0.54 a25.00 ±0.50 b26.10 ±0.55 b
H2O2(mM/g) 1.32 ±0.10 c1.28 ±0.12 c4.83 ±0.15 a3.30c ±0.10 b3.37 ±0.11 b
SOD (U/g) 92.09 ±2.77 a95.31 ±2.40 a59.28 ±2.56 c79.48 ±2.66 b75.84 ±2.54 b
CAT (U/g) 189.5 ±4.40 a192.0 ±2.89 a128.5 ±4.06 d170.6 ±4.66 c165.4 ±4.54 b
GST (U/g) 5.40 ±0.57 a5.87 ±0.57 a1.25 ±0.22 c3.24 ±0.40 b3.06 ±0.24 b
GSH (mmol/g) 5.50 ±0.24 a5.54 ±0.29 a2.86 ±0.17 c4.20 ±0.16 b4.00 ±0.15 b
Data presented as means
±
SEM for six rats in each group and % of change. The significant change at
p≤0.05
(
n=6
).
Different superscript letters (a, b, c and d) indicatesignificant differences in the same column. Malondialdehyde (MDA),
nitric oxide (NO), glutathione (GSH), glutathione S transferase (GST), superoxide dismutase (SOD), and catalase (CAT).
Animals 2020,10, 1996 8 of 19
We showed a significant increase in MDA, H
2
O
2
, NO, and ROS concentrations in the MSG
group compared to our data’s control group. Furthermore, the oral administration of GE (200 mg/kg
BW) to the rat groups (MSG +Graviola and Graviola +MSG) resulted in a significant decrease in
MDA concentration compared to the MSG group. Additionally, GE’s administration only showed no
substantial change than the control group, as shown in Table 4.
3.4. Effect of Graviola on MSG-Induced Liver Cell Apoptosis
Flow cytometric analysis was conducted to examine Graviola’s antiapoptotic effects on apoptosis
in liver cells induced by MSG. The p53, caspase-3, and Bax levels of the MSG group were significantly
higher than the control group (p<0.05). Besides, the oral administration of GE (200 mg/kg BW) to
the rat groups MSG +GE and GE +MSG resulted in a significant decrease in their expression levels
compared to the MSG group. Additionally, GE administration showed no statistically significant
difference from the control group, as shown in Figure 3. Conversely, the Graviola-treated group
showed normalization to the BCL-2 level, which was significantly decreased by MSG administration.
Please see the Figures S1–S4.
Animals 2020, 10, x 9 of 21
Table 4. Effects of Graviola and MSG extract on liver oxidative status of different rat groups.
Parameters Control Graviola MSG Graviola + MSG MSG + Graviola
MDA (nmol/g) 685.8 ± 36.30
c
678.2 ± 36.31
c
1216 ± 18.01
a
800.4 ± 18.88
b
815.8 ± 18.80
b
NO (μ mol/g) 18.23 ± 0.52
c
18.11 ± 0.50
c
35.48 ± 0.54
a
25.00 ± 0.50
b
26.10 ± 0.55
b
H
2
O
2
(mM/g) 1.32 ± 0.10
c
1.28 ± 0.12
c
4.83 ± 0.15
a
3.30c ± 0.10
b
3.37 ± 0.11
b
SOD (U/g) 92.09 ± 2.77
a
95.31 ± 2.40
a
59.28 ± 2.56
c
79.48 ± 2.66
b
75.84 ± 2.54
b
CAT (U/g) 189.5 ± 4.40
a
192.0 ± 2.89
a
128.5 ± 4.06
d
170.6 ± 4.66
c
165.4 ± 4.54
b
GST (U/g) 5.40 ± 0.57
a
5.87 ± 0.57
a
1.25 ± 0.22
c
3.24 ± 0.40
b
3.06 ± 0.24
b
GSH (mmol/g) 5.50 ± 0.24
a
5.54 ± 0.29
a
2.86 ± 0.17
c
4.20 ± 0.16
b
4.00 ± 0.15
b
Data presented as means ± SEM for six rats in each group and % of change. The significant change at
p ≤ 0.05 (N = 6).
Different superscript letters (a, b and c) indicate significant differences in the same
column. Malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH), glutathione S transferase
(GST), superoxide dismutase (SOD), and catalase (CAT).
3.4. Effect of Graviola on MSG-Induced Liver Cell Apoptosis
Flow cytometric analysis was conducted to examine Graviola’s antiapoptotic effects on
apoptosis in liver cells induced by MSG. The p53, caspase-3, and Bax levels of the MSG group were
significantly higher than the control group (p < 0.05). Besides, the oral administration of GE (200
mg/kg BW) to the rat groups MSG + GE and GE + MSG resulted in a significant decrease in their
expression levels compared to the MSG group. Additionally, GE administration showed no
statistically significant difference from the control group, as shown in Figure 3. Conversely, the
Graviola-treated group showed normalization to the BCL-2 level, which was significantly decreased
by MSG administration. Please see the Figures S1–S4.
Figure 3. Flowcytometric analysis of the hepatic level of proapoptotic protein BCL-2-like protein 4
(Bax) (A), antiapoptotic protein B-cell lymphoma 2 (Bcl-2) (B), P53 (C), and caspase-3 (D) of different
treated groups. Data are presented as means ± SE for six rats in each group and % of change.
Different
superscript letters (a, b and c) indicate significant differences. The significant change was at p ≤ 0.05.
3.5. Effect of Graviola on the Levels of ROS and IL-6
Figure 3.
Flowcytometric analysis of the hepatic level of proapoptotic protein BCL-2-like protein 4
(Bax) (
A
), antiapoptotic protein B-cell lymphoma 2 (Bcl-2) (
B
), P53 (
C
), and caspase-3 (
D
) of different
treated groups. Data are presented as means
±
SE for six rats in each group and % of change.
Different superscript letters (a, b and c) indicate significant differences. The significant change was at
p≤0.05.
3.5. Effect of Graviola on the Levels of ROS and IL-6
There was a significant increase (p<0.05) in ROS and IL-6 levels in the MSG group compared
to the control group. Additionally, both MSG +GE and GE +MSG showed a significant decrease in
ROS and IL-6 levels, whereas the Graviola administration alone had no significant difference from the
control group, as shown in Figure 4.
Animals 2020,10, 1996 9 of 19
Animals 2020, 10, x 10 of 21
There was a significant increase (p < 0.05) in ROS and IL-6 levels in the MSG group compared to
the control group. Additionally, both MSG + GE and GE + MSG showed a significant decrease in ROS
and IL-6 levels, whereas the Graviola administration alone had no significant difference from the
control group, as shown in Figure 4.
Figure 4. Hepatic ROS level (A) and interleukin 6 (IL-6) (B) of different treated groups. Data are
presented as means ± SEM for six rats in each group and % of change.
Different superscript letters (a,
b and c) indicate significant differences. The significant change was at p ≤ 0.05.
3.6. Effect of Graviola on the Histopathological Alteration Induced by MSG in Liver
As shown in Figure 5, the control showed normal hepatocytes arranged in cords around the
central vein. The Graviola-treated group alone, showing normal hepatocytes arranged in cords
separated by blood sinusoid MSG, showed periportal hepatic necrosis associated with mononuclear
cells infiltration and hepatic vacuolation, (arrowhead) single-cell necrosis, and a loss of cellular
details and nuclei of some hepatocytes. The Graviola + MSG (prophylactic group) showed a few
pyknotic nuclei of hepatocytes and a mild degree of hepatocyte degeneration. In the MSG + Graviola,
limited centrilobular hepatic vacuolation and mononuclear cell infiltration were observed.
Figure 4.
Hepatic ROS level (
A
) and interleukin 6 (IL-6) (
B
) of different treated groups. Data are
presented as means
±
SEM for six rats in each group and % of change. Different superscript letters
(a, b and c) indicate significant differences. The significant change was at p≤0.05.
3.6. Effect of Graviola on the Histopathological Alteration Induced by MSG in Liver
As shown in Figure 5, the control showed normal hepatocytes arranged in cords around the central
vein. The Graviola-treated group alone, showing normal hepatocytes arranged in cords separated by
blood sinusoid MSG, showed periportal hepatic necrosis associated with mononuclear cells infiltration
and hepatic vacuolation, (arrowhead) single-cell necrosis, and a loss of cellular details and nuclei
of some hepatocytes. The Graviola +MSG (prophylactic group) showed a few pyknotic nuclei of
hepatocytes and a mild degree of hepatocyte degeneration. In the MSG +Graviola, limited centrilobular
hepatic vacuolation and mononuclear cell infiltration were observed.
Animals 2020, 10, x 11 of 21
Figure 5. Photomicrograph of the liver of the control and differently treated groups of animals. (A)
Control showing normal hepatocytes (arrow) arranged in cords around the central vein (arrowhead).
(B) Graviola is showing normal hepatocytes arranged in cords separated by blood sinusoids (arrow).
(C) MSG showing periportal hepatic necrosis (astars) associated with mononuclear cell infiltration
(arrowhead). (D) MSG is showing single-cell necrosis (arrow) with a loss of cellular details and nuclei
of some hepatocytes (arrowhead). (E) Graviola + MSG is showing a few pyknotic nuclei (arrow) and
a mild degree of hepatocyte degeneration (arrowhead). (F) MSG + Graviola showing limited
centrilobular hepatic vacuolation (arrow) and mononuclear cell infiltration (arrowhead). Stained with
hematoxylin and eosin (H & E), scale bar = 50 μm.
3.7. Effect of Graviola on Silent Information Regulator Protein One (SIRT1), Fatty Acid Synthase (FAS),
and Inducible Nitric Oxide Synthase (iNOS) Gene Expression
As shown in Figure 6, the relative ratio of hepatic SIRT1 gene expression was significantly
reduced in the MSG-treated group, and the comparable rate of SIRT1 gene expression was improved
considerably in Graviola-treated rats. FAS gene expression with MSG treatment was upregulated
Figure 5. Cont.
Animals 2020,10, 1996 10 of 19
Animals 2020, 10, x 11 of 21
Figure 5. Photomicrograph of the liver of the control and differently treated groups of animals. (A)
Control showing normal hepatocytes (arrow) arranged in cords around the central vein (arrowhead).
(B) Graviola is showing normal hepatocytes arranged in cords separated by blood sinusoids (arrow).
(C) MSG showing periportal hepatic necrosis (astars) associated with mononuclear cell infiltration
(arrowhead). (D) MSG is showing single-cell necrosis (arrow) with a loss of cellular details and nuclei
of some hepatocytes (arrowhead). (E) Graviola + MSG is showing a few pyknotic nuclei (arrow) and
a mild degree of hepatocyte degeneration (arrowhead). (F) MSG + Graviola showing limited
centrilobular hepatic vacuolation (arrow) and mononuclear cell infiltration (arrowhead). Stained with
hematoxylin and eosin (H & E), scale bar = 50 μm.
3.7. Effect of Graviola on Silent Information Regulator Protein One (SIRT1), Fatty Acid Synthase (FAS),
and Inducible Nitric Oxide Synthase (iNOS) Gene Expression
As shown in Figure 6, the relative ratio of hepatic SIRT1 gene expression was significantly
reduced in the MSG-treated group, and the comparable rate of SIRT1 gene expression was improved
considerably in Graviola-treated rats. FAS gene expression with MSG treatment was upregulated
Figure 5.
Photomicrograph of the liver of the control and differently treated groups of animals.
(
A
) Control showing normal hepatocytes (arrow) arranged in cords around the central vein (arrowhead).
(
B
) Graviola is showing normal hepatocytes arranged in cords separated by blood sinusoids (arrow).
(
C
) MSG showing periportal hepatic necrosis (astars) associated with mononuclear cell infiltration
(arrowhead). (
D
) MSG is showing single-cell necrosis (arrow) with a loss of cellular details and nuclei
of some hepatocytes (arrowhead). (
E
) Graviola +MSG is showing a few pyknotic nuclei (arrow)
and a mild degree of hepatocyte degeneration (arrowhead). (
F
) MSG +Graviola showing limited
centrilobular hepatic vacuolation (arrow) and mononuclear cell infiltration (arrowhead). Stained with
hematoxylin and eosin (H & E), scale bar =50 µm.
3.7. Effect of Graviola on Silent Information Regulator Protein One (SIRT1), Fatty Acid Synthase (FAS),
and Inducible Nitric Oxide Synthase (iNOS) Gene Expression
As shown in Figure 6, the relative ratio of hepatic SIRT1 gene expression was significantly
reduced in the MSG-treated group, and the comparable rate of SIRT1 gene expression was improved
considerably in Graviola-treated rats. FAS gene expression with MSG treatment was upregulated
considerably, showing significant downregulation with the Graviola treatment. Compared to the
control group, the relative expression of the hepatic iNOS in rats treated with MSG gene expressions
were markedly upregulated and significantly downregulated in rats treated with Graviola compared
to rats treated with MSG.
Animals 2020, 10, x 12 of 21
considerably, showing significant downregulation with the Graviola treatment. Compared to the
control group, the relative expression of the hepatic iNOS in rats treated with MSG gene expressions
were markedly upregulated and significantly downregulated in rats treated with Graviola compared
to rats treated with MSG.
Figure 6. Expression of fold changes of hepatic SIRT1, silent information regulator protein one gene
expression; FAS, fatty acid synthase; and iNOS, inducible nitric oxide synthase. Data were analyzed
with one-way ANOVA, followed by Tukey’s multiple comparison test. Different superscript letters
(a, b and c) indicate significant differences at p < 0.05. Error bars represent mean ± SEM, N = 6.
4. Discussion
Currently, there is a substantial increase in the use of food additives. Organic compounds are
deliberately added to food in small quantities during the production process to improve the
organoleptic quality of foods, such as flavor, color, taste, appearance, and texture [3]. Food additives
can instantly be harmful or be long-lasting if they are continuously consumed. Immediate effects may
include headaches, changes in energy levels, mental concentration and behavior changes, and
immune response [56]. One of those food additives widely used as an enhancer of flavor is
monosodium glutamate (MSG). It is a glutamic acid salt amino acid [1]. This encourages us to search
for a natural food additive, such as Graviola, that could offset the oxidative and inflammatory
processes induced by MSG in hepatic tissues. Our data revealed that MSG causes a significant
increase in body weight. This increase may be attributed to the fact that MSG can improve foods’
palatability by having a favorable effect on the appetite center [57] and enhancing the chemosensory
perception [58]. Increased IL-6, resistin, and tumor necrosis factors in adipose tissues are the primary
effects of MSG on body weight. The elevated serum levels of resistin and insulin can deteriorate the
visceral adipose tissue [59]. Additionally, the ingestion of MSG has a local effect. It activates the celiac
and gastric branches of the vagus nerve when found in the gastrointestinal tract, causing the
activation of limbic, hypothalamus, insular cortex and nucleus tracts, and solitary tracts, which eat
many foods [60]. An oral administration of Graviola normalized the body weight, and these findings
are in agreement with [61–63]. The decrease in triglyceride and overall cholesterol is attributed to
this, since Graviola has a hypolipidemic effect and hypolipidemic agents such as tannins, which
reduce the cholesterol absorption and, consequently, reduce the body weight gain [64,65]. The lipid
profile, including total cholesterol (TC), triglyceride (TG), and LDL-C, increased significantly in the
serum of rats administered MSG. At the same time, the HDL-C content was reduced, as shown in
Table 3; this result was inconsistence with [66,67], who reported that MSG could increase the activity
of coenzyme A (HMG CoA) reductase, 3-hydroxyl-3-methylglutaryl, the limiting factor of cholesterol
biosynthesis, which results in increased cholesterol synthesis and hyperlipidemia, with increased
Figure 6.
Expression of fold changes of hepatic SIRT1, silent information regulator protein one gene
expression; FAS, fatty acid synthase; and iNOS, inducible nitric oxide synthase. Data were analyzed
with one-way ANOVA, followed by Tukey’s multiple comparison test. Different superscript letters
(a, b and c) indicate significant differences at p<0.05. Error bars represent mean ±SEM, n=6.
4. Discussion
Currently, there is a substantial increase in the use of food additives. Organic compounds are
deliberately added to food in small quantities during the production process to improve the organoleptic
Animals 2020,10, 1996 11 of 19
quality of foods, such as flavor, color, taste, appearance, and texture [
3
]. Food additives can instantly be
harmful or be long-lasting if they are continuously consumed. Immediate effects may include headaches,
changes in energy levels, mental concentration and behavior changes, and immune response [
56
].
One of those food additives widely used as an enhancer of flavor is monosodium glutamate (MSG). It is
a glutamic acid salt amino acid [
1
]. This encourages us to search for a natural food additive, such as
Graviola, that could offset the oxidative and inflammatory processes induced by MSG in hepatic
tissues. Our data revealed that MSG causes a significant increase in body weight. This increase may
be attributed to the fact that MSG can improve foods’ palatability by having a favorable effect on the
appetite center [
57
] and enhancing the chemosensory perception [
58
]. Increased IL-6, resistin, and tumor
necrosis factors in adipose tissues are the primary effects of MSG on body weight. The elevated serum
levels of resistin and insulin can deteriorate the visceral adipose tissue [
59
]. Additionally, the ingestion
of MSG has a local effect. It activates the celiac and gastric branches of the vagus nerve when found in
the gastrointestinal tract, causing the activation of limbic, hypothalamus, insular cortex and nucleus
tracts, and solitary tracts, which eat many foods [
60
]. An oral administration of Graviola normalized
the body weight, and these findings are in agreement with [
61
–
63
]. The decrease in triglyceride and
overall cholesterol is attributed to this, since Graviola has a hypolipidemic effect and hypolipidemic
agents such as tannins, which reduce the cholesterol absorption and, consequently, reduce the body
weight gain [
64
,
65
]. The lipid profile, including total cholesterol (TC), triglyceride (TG), and LDL-C,
increased significantly in the serum of rats administered MSG. At the same time, the HDL-C content
was reduced, as shown in Table 3; this result was inconsistence with [
66
,
67
], who reported that
MSG could increase the activity of coenzyme A (HMG CoA) reductase, 3-hydroxyl-3-methylglutaryl,
the limiting factor of cholesterol biosynthesis, which results in increased cholesterol synthesis and
hyperlipidemia, with increased serum TG and TC, shifting the glucose metabolism towards lipogenesis.
The prophylactic and protective roles of orally administered Graviola in normalized parameters of the
lipid profile, as shown in Table 3, could be attributed to the involvement of hypolipidemic agents in the
GE [62,63]. The hypolipidemic and antioxidant effects of Graviola due to the presence of agents such
as tannins and other polyphenolic compounds cause decreasing cholesterol absorption by deactivating
coenzyme-A (HMG-CoA) reductase hydroxymethylglutaryl [
65
]. Our findings showed that MSG
can significantly increase the liver enzymes ALT, AST, ALP, and GGT due to the cytotoxic effect of
MSG, which resulted in damage to liver cells and canaculae and the release of these enzymes in the
circulation [
68
]. Moreover, MSG toxicity creates ammonium ions that cause hepatic toxicity through
the formation of ROS that react with polyunsaturated fatty acids contained within cell membranes that
cause plasma and mitochondrial membranes to deteriorate with the release of hepatic enzymes [
69
] via
preserving the structural integrity of the hepatic cell membrane or regenerating damaged liver cells [
70
].
Graviola preserves and prevents the leakage of the intracellular enzyme70 liver injury triggered by
MSG Graviola [
71
], which supports our finding of the protective role of Graviola. Our research
reaffirmed that MSG has harmful effects on hematological parameters, with characteristic leukopenia
consistent with [
72
,
73
] attributed to RBCs’ short half-life due to the hemolytic effect of MSG. This may
be due to the atrophy induced by MSG in the gastric mucosa (gastritis) as the L-form of glutamic acid
is acidic, resulting in a reduction in intrinsic factor synthesis leading to vitamin B12 malabsorption,
which is the main cause of anemia [
74
]. MSG caused leukopenia, which was in the same line as [
75
],
who attributed this effect to the immune-suppressant effect of MSG due to MSG’s hazardous effects
on the thymus and spleen. Graviola significantly cured the adverse impact of MSG. The obtained
result was in the same line with [
76
], who attributed this finding to Graviola’s ability to restore body
fluids and stimulate erythropoietin [
77
]. Orally administered MSG led to increased oxidative stress
markers, such as MDA, ROS, NO, and H
2
O
2
, and decreased SOD, CAT, GST, and GSH that supported
this finding by [
9
,
68
,
78
–
81
] as a result of the exhaustion of SOD and accumulation of H
2
O
2
as a
result of ROS formation as a result of MSG. Besides [
9
], this effect is revealed to MSG lipogenesis
characters that consume nicotinamide adenine dinucleotide (NAD) +hydrogen (H) (NADH). Similarly,
the conversion of the majority of GSH in the liver to glutathione disulfide (GSSG) by the glutathione
Animals 2020,10, 1996 12 of 19
reductase enzyme to protect the liver cells from toxic material damage decreased the GSH level.
The elevated levels of MDA and NO return to the difficulty of glutamate transportation across the
cell membrane, which initiate lipid peroxidation (LPO) and alter the cell redox state [
82
], leading to
membrane damage [
83
]. Graviola administration normalized the oxidant status of the liver cells,
this result being in harmony with [
43
] due to Graviola antioxidant activity [
84
]. Graviola has a
protective role against free radicals (OH) and H
2
O
2
[
85
]. Therefore, it stopped the elevation of LPO [
86
]
and converted the ROS to nontoxic or dangerous goods [
87
]. Graviola possesses potent antioxidant
properties due to the presence of acetogenins, which can play an essential and significant role in
free radical scavenging [
85
]. The IL-6 proinflammatory protein is one of the families of cytokines
that help organisms react to infectious agents and increases the development of Interleukin-6 (IL6)
inflammation boost in the MSG community due to chronic inflammation leading to overexpression of
the IL6 mRNA gene [
88
]. Our results showed that the Graviola extract normalized the level of IL6 that
is inconsistent with [
89
] due to the presence of anti-inflammatory agents in Graviola extracts, such as
alkaloids, saponins, flavonoids, and tannins, which inhibit prostaglandin synthesis [
90
,
91
]. This study
shows that the administration of MSG led to significant increases in P53, caspase-3, and apoptotic
(Bax) proteins and a substantial reduction in antiapoptotic (Bcl-2) proteins. This was in the same line
with [
92
,
93
], who explained that glutamate-induced the Ca2
+
influx and destruction of the internal
mitochondrial membrane potential, resulting in the unregulated mitochondrial permeability of the
pores to apoptotic markers [
94
]. Graviola was found to decrease the higher levels of Bax, caspase 3,
and P53 and significantly increase Bcl2 in MSG-treated rats. This finding was in agreement with [
95
]
on the overexpression of bcl2, which prevents DNA fragmentation due to its antioxidant activity and
blocks the cytochrome C release and mitochondrial permeability.
In terms of the current histopathological findings, normal hepatocytes arranged in regulated
cords around the central vein and GE-treated group, and periportal hepatic necrosis associated with
mononuclear cell infiltration, hepatic vacuolation was seen in MSG-treated rats. Such findings are
comparable to [
96
–
98
] findings. Due to MSG, therefore, the cell is not able and cannot repair the
damage entirely due to excess glutamine. Vesicular degeneration and necrosis are expected to occur in
hepatic tissue [
99
]. Increased central adiposity and the gene expression of white adipose tissue can
also cause fatty liver damage caused by hepatic exacerbation.
Furthermore, MSG has been shown to cause oxidative stress and hepatotoxicity [
100
].
The vacuolization of hepatocytes has been described as a ballooning degeneration. It has been
interpreted as a cellular defense mechanism for harmful substances, which collects and prevents
interference with the biological acting elements. Additionally, the high level of MDA caused by the
LPO effect of MSG may lead to hepatic necrosis [
68
]. On the other hand, the administration of GE had
a therapeutic effect on the liver architecture for rats before and after MSG treatment. Such findings
are comparable to those of [
101
]. The involvement inhibits cyclo-oxygenase 2 of tannins in GE and
acetogenin [102,103].
Sirt1 is a Sirtuin family prototype. It is a crucial metabolism regulator involved in cell metabolism,
fat utilization and insulin tolerance, cell division and senescence, metabolic stress, and disease [
104
].
Liver Sirt1 appears to be playing a significant function in the regulation of homeostasis. However,
fatty acid beta-oxidation and gluconeogenesis are reduced by its depletion. The upregulation
expression of SIRT1 with Graviola could be due to its antioxidant action, which could explain
Graviola’s anti-inflammatory pathway as a regulated SIRT1 activation of various factors, including the
transcription factor nuclear factor kappa B (NF-
κ
B) [
105
]; therefore, it could regulate the inflammation
process. On the contrary, FAS gene expression was significantly upregulated with MSG treatment,
which showed a marked downregulation with the Graviola treatment. This result was supported
by [
106
] by finding that MSG rats are dyslipidemic, counteracting the stimulated liver lipogenic process
by the high expression of their master regulator genes, Sterol regulatory element-binding transcription
factor 1 (SREBP1c) target genes and fatty acid synthase (FAS) [
107
]. They showed that MSG rats
developed an insulin-resistant state and increased oxidative stress, and severe liver injury characterized
Animals 2020,10, 1996 13 of 19
by inflammation and metabolic signs involved lipogenesis. This supports our obtained results for
the body weight. Conversely, the NO levels in the MSG rats were also boosted in conjunction with a
substantial increase in gene expression of the iNOS (the source of NO) in the current study. NO and
iNOS perform complicated roles in the development of hepatic injury. The relationship between NO
and hepatic injury includes cytotoxicity, the inflammatory response, and metabolic energy abnormality.
An extreme extracellular expression of iNOS induced an outrageous leak of NO to cells with developed
adverse effects [
108
]. Therefore, the hepatic expression of iNOS in different models for liver injury is
essential for hepatic repair [
109
]. As shown in Figure 7, Graviola supplementation can overcome the
monosodium glutamate-induced hepatic injury through many intarcellular pathways.
Animals 2020, 10, x 15 of 21
Figure 7. Graphical summary showing the effect of Graviola extract on MSG-induced hepatic injuries.
5. Conclusions
Food additives, especially monosodium glutamate, induce hepatic injury in rats. Graviola
supplementations overcame these alterations by modulating liver apoptosis markers and enhancing
the hepatic antioxidant status, which was accompanied by a reduction in inflammatory markers and
cellular apoptosis. Additionally, by modulating the lipogenesis gene, Graviola improved the
transcriptomic effect induced by monosodium glutamate. Clinical human trials are required to
validate the animal studies to qualify this effect found in hepatic rats.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Bax
flowcytometry, Figure S2: Bcl2 flowcytometry, Figure S3: Caspase 3 flowcytometry, Figure S4: P53
flowcytometry.
Author Contributions: Conceptualization, M.S., and A.M.E.-S.; methodology W.M.E.-K., M.S., R.A.E., and
H.S.H.; software, H.G.T., M.S., M.M.A., and W.M.E.-K.; validation, H.G.T. and M.M.A.; formal analysis, F.A.F.,
M.S., W.M.E.-K., and A.M.E.-S.; investigation, M.S., F.A.F., and M.M.A.; resources, H.G.T.; funding acquisition;
A.M.E.-S.; data curation, M.S.; writing—original draft preparation, M.S.; writing—review and editing, M.S.,
F.A.F., H.S.H., and F.A.F.; visualization, W.M.E.-K., M.S., and F.A.F.; and supervision, M.S. and F.A.F. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors express many thanks to the Central Lab, Faculty of Veterinary Medicine,
Kafrelsheikh University, Kafrelsheikh, Egypt. This work was funded by the Taif University Researchers
Supporting Project number TURSP-2020/75, Taif University, Taif, Saudi Arabia.
Conflicts of Interest: No conflicts of interest are declared by the authors.
Figure 7.
Graphical summary showing the effect of Graviola extract on MSG-induced hepatic injuries.
5. Conclusions
Food additives, especially monosodium glutamate, induce hepatic injury in rats.
Graviola supplementations overcame these alterations by modulating liver apoptosis markers and
enhancing the hepatic antioxidant status, which was accompanied by a reduction in inflammatory
markers and cellular apoptosis. Additionally, by modulating the lipogenesis gene, Graviola improved the
transcriptomic effect induced by monosodium glutamate. Clinical human trials are required to validate the
animal studies to qualify this effect found in hepatic rats.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2076-2615/10/11/1996/s1,
Figure S1: Bax flowcytometry, Figure S2: Bcl2 flowcytometry, Figure S3: Caspase 3 flowcytometry, Figure S4:
P53 flowcytometry.
Author Contributions:
Conceptualization, M.S., and A.M.E.-S.; methodology W.M.E.-K., M.S., R.A.E., and H.S.H.;
software, H.G.T., M.S., M.M.A., and W.M.E.-K.; validation, H.G.T. and M.M.A.; formal analysis, F.A.F., M.S.,
W.M.E.-K., and A.M.E.-S.; investigation, M.S., F.A.F., and M.M.A.; resources, H.G.T.; funding acquisition; A.M.E.-S.;
data curation, M.S.; writing—original draft preparation, M.S.; writing—review and editing, M.S., F.A.F., H.S.H.,
and F.A.F.; visualization, W.M.E.-K., M.S., and F.A.F.; and supervision, M.S. and F.A.F. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
The authors express many thanks to the Central Lab, Faculty of Veterinary Medicine,
Kafrelsheikh University, Kafrelsheikh, Egypt. This work was funded by the Taif University Researchers Supporting
Project number TURSP-2020/75, Taif University, Taif, Saudi Arabia.
Animals 2020,10, 1996 14 of 19
Conflicts of Interest: No conflicts of interest are declared by the authors.
Abbreviation
ALB Serum Albumin
BAX BCL-2-like protein 4
BCL2 B-cell lymphoma 2
CAT Catalase
FAS Fatty acid synthase
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
GSH Reduced glutathione
GST Glutathione S transferase
GGT Serum gamma-glutamyl transferase
H2O2Hydrogen peroxide
HDL-C High-density Lipoprotein
IL-6 Interleukin 6
NOS Nitric oxide synthase
iNOS inducible nitric oxide synthase
LDL-C Low-Density Lipoprotein
MDA Malondialdehyde
MSG Monosodium glutamate
NO Nitric oxide
ROS Reactive oxygen species
SIRT1 Silent information regulator protein one gene expression
SOD Superoxide dismutase
TB Serum total bilirubin
TP Serum Total Protein
p53 Tumor protein
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