European Review for Medical and Pharmacological Sciences 2023; 27: 9401-9412

Alanda (Ephedra foeminea) nanoparticles as

a modulator of genomic DNA degradation in mice:
new inspiration for treating Ehrlich solid
tumour-induced kidney and liver toxicity
R.M. ELGHARABAWY1, E. TOUSSON2, I.E. EL SAYED3, A.H. ABD EL-ALEIM3,
M. ELABD3, D.T. GEBREEL4, A.S. AHMED5

1
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt
2
Zoology Department, Faculty of Science, Tanta University, Tanta, Egypt
3
Chemistry Department, Faculty of Science, Menoufia University, Menoufia, Egypt
4
Medical Equipment Department, Faculty of Allied Medical Sciences, Pharos University in
Alexandria, Alexandria, Egypt
5
Hormones Department, Medical Research and Clinical Studies Institute, National Research Centre,
Giza, Egypt

Abstract. – OBJECTIVE: The limitations faced Key Words:

by conventional drug delivery systems are being Apoptosis, Renal and hepatic toxicity, Ehrlich solid
overcome through the use of rapidly evolving can- tumour, DNA damage, Ephedra foeminea.
cer nanotherapeutics. Determining the manner in
which the Ehrlich solid tumor (EST) is impacted
by the new bioactive Alanda-loaded flax seed gum
nanoparticles (Alanda NPs) functioning as an an- Introduction
ti-carcinogenic agent represents the research ob-
jective of this paper. Cancer occurs when abnormal cells grow uncon-
MATERIALS AND METHODS: Identification
of the functional groups, surface morphology,
trollably and invade surrounding tissues. Tousson et
particle size, and zeta potential were among the al1-3 noted that by instigating apoptosis, such abnor-
characterizations and preparations made for the mal and lethal growth cells are killed in the most
prepared nanoparticles. A Control group, a Flax common cancer treatments – radiation and chemo-
Seed Gum group, a raw Alanda group, an Alan- therapy. As shown by many studies4-6, the analysis
da NPs group, an EST group, and an induced of antineoplastic impacts of multiple substance com-
EST treated with Alanda NPs group comprised pounds is enabled by the use of the Ehrlich tumor as
the six groups respectively which the 60 female
mice were separated into in this in vivo study. a transplantable growth model. According to Abdel-
RESULTS: Toxicity assessments for kidneys basset et al7 and others8,9, the second most common
and liver were performed alongside the detec- type of tumor internationally is breast cancer, which
tion of total genomic DNA degradation. The ze- is the most frequent type of cancer in women.
ta potential and the particle sizes for Alanda NPs Studies outline10-12 that a spontaneous murine
were -25.60±0.38 mv and 40±0.28 nm, respec- mammary adenocarcinoma similar to breast cancer
tively, where the latter demonstrated a mono-
disperse spherical shape, per the findings. The
was the initial categorization made for the EST. Abd
use of Alanda NPs to treat EST was found to al- Eldaim et al13-15 note that oxidative stress and toxic-
leviate the DNA damage, apoptosis, and renal ity are ways EST negatively impacts the liver and
and hepatic toxicity that EST induces. Addition- kidney tissue.
ally, the activation of oxidative stress and apop- Notably, Elgharabawy et al16 state that possible
tosis causing the renal and hepatic toxicity in- cancer treatments intriguingly include plants, among
duced by EST is counteracted by the scaveng- many other sources of compounds. Several studies17-20
ing of free radicals by the Alanda NPs.
CONCLUSIONS: A high degree of safety for show that synthetic antioxidants, endogenous antiox-
effective cancer treatment was displayed by the idants, and elevated oxidative damage are probable
newly developed oral nanoparticles while also outcomes of the strong correlation between cancer and
demonstrating strong potential in vivo. mortality and morbidity of liver and kidney diseases.

Corresponding Author: Rehab Mohamed Elgharabawy, MD; e-mail: [email protected] 9401

 R.M. Elgharabawy, E. Tousson, I.E. El Sayed, A.H. Abd El-Aleim, M. Elabd, et al

Mighri et al21 and Chouikh22 identify the ori- strictions of conventional drug delivery systems
gins of the Ephedra foeminea, a low stalky Eur- that the application of cancer nanotherapeutics re-
asian shrub from the Ephedraceae lineage, as na- solves. The circulation time in the bloodstream of
tive to the entire southeast of the Mediterranean these nanoparticles is augmented via nanoparti-
and northern Israel, otherwise known in Arabic cles with optimally designed surface characteris-
as Alanda. It has been used for at least 5,000 years tics and size, thereby enhancing the bio-distribu-
by the Chinese, thereby representing one of the tion of cancer drugs. Examining how the Ehrlich
oldest drugs in the world. Some researchers23,24 solid tumor and its effects on the kidney and liver,
note that alkaloids of the ephedrine E-type, that such as DNA damage, apoptosis, and toxicity, are
function as sympathomimetics, reside within the impacted by Alanda in nano form comprises the
Alanda. The receptor binding and enantiomeric research objective of this paper.
form features alongside the individual E alkaloid
type determine the toxicological and pharmaco-
logical effects. As outlined by many studies25-28, Materials and Methods
weight reduction, anti-proliferative, anti-asthmat-
ic, antioxidant, hypoglycemic, anti-microbial, and Extraction and Purification
anti-inflammatory effects are among the activities of Flax Seed Gum
exhibited by the Ephedra. Danciu et al29 observe A local market served to provision the flax
that Ephedrine binds to adrenergic receptors and seeds. The seeds of Linum usitatissimum were
resultantly causes notable effects on the central used to extract the Flax seed gum via the aqueous
nervous system while elevating blood pressure method, with modifications noted by Neeraj et
and stimulating the heart rate. In the context of al34. The seeds were stirred by a magnetic stirrer
treating cancer, there is sparse knowledge on the for two hours at 80°C at 1,200 rpm, following the
use of Alanda. Notably, Mendelovich et al30 state soaking of the dried seeds in 10 folds of distilled
that metastasis and cancer cell invasiveness are water. A muslin cloth was used to filter the re-
correlated to the impact of Alanda on gene ex- sultant viscous solution, which then led to cen-
pression and cell cytoskeleton, thereby reflecting trifugation for 20 minutes at 4,000 rpm. Lastly,
the potential harm of using Alanda to treat cancer. a lyophilizer was used to dry the cream-colored
Severe neurological and cardiovascular system precipitates of gum that were separated by filtra-
toxicity has also been linked to ephedrine alka- tion.
loid components that reside within Alanda. As
Jaradat et al31 highlight, the USA chose to ban the Extraction and Purification of Ephedra
use of the plant after severe issues causing death A mechanical miller was utilized to grind five
and toxic effects occurred. grams of a specifically chosen aerial portion of
The development of new drug carrier designs the plant. The produced powder had 50 ml of 5%
for particular applications could advance signifi- vinegar, otherwise called acetic acid, incorporat-
cantly via natural polymeric-based nanomateri- ed into it. The sample was then incubated over-
als. Moreover, as Zhao et al32 observe, substantial night at 30°C with automatic shaking at 160 rpm.
immunological responses with optimal biologi- Next, the supernatant was decanted after the mix-
cal safety can be induced by such nanoparticles ture was centrifuged at 3,000 rpm for 20 minutes.
that possess therapeutic characteristics. A natural Then, incubation for 30 minutes at room tempera-
heterogeneous polysaccharide is found in Linium ture occurred after the dried sample had 20 ml of
usitatissimum L., otherwise known as Flax seed vinegar added to it. In line with previous stud-
gum. The presence of groups that can be altered ies30,35, the mixture was homogenized by vortex
for functionalization and biocompatibility are after 10 ml of distilled water was added and then
among the most salient advantages of utilizing a syringe 0.45 μm Teflon filter was used to filter
polysaccharides. Mittal et al33 determine that the solution.
risks linked to toxic drugs are mitigated by Flax
seed gum which is also utilized more commonly Preparation of Ephedra-loaded
as stabilizers, suspending agents, carriers, bind- Nanoparticles
ers, and disintegrating agents. In accordance with Madani et al36, the mod-
Low therapeutic indices, deficient water solu- ified oil in water (O/W) single emulsion solvent
bility, nonspecific targeting and bio-distribution, evaporation method was used to prepare the
and poor oral bioavailability are among the re- nanoparticles. A homogenous solution was at-

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 Alanda (Ephedra foeminea) nanoparticles as a modulator of genomic DNA degradation in mice

tained by using a magnetic stirrer for four hours procured for this paper. Relative stickiness con-
at 40°C to stir the surfactant arising from the ditions consisting of 12 hours of light and dim
aqueous phase consisting of 10% concentration cycles respectively, alongside room tempera-
(90 ml) of Flax seed gum. The aqueous phases tures between 22 and 25°C housed the mice that
subjected to non-stop magnetic stirring at room were all randomized.
temperature for 30 minutes had the organic phase
comprising 10 ml of Ephedra extraction added to Animal groups and experimental designs
it in order to generate O/W emulsion type. Then, The mice were separated into six groups (Gp1-
a sonication water bath was used for 15 minutes Gp6) as demonstrated below:
to diminish the size of the nanoparticles, thereby – Gp1: the Control Gp of untreated mice.
sonicating the emulsion. The organic solvent was – Gp2: The mice orally received 25 mg/kg bw/
evaporated by using a magnetic stirrer at room 2day of Flax seed gum by stomach tube for
temperature for three hours at 1,200 rpm to stir roughly 15 days in this Flax Seed Gum Gp
the formed O/W emulsion. Ultracentrifugation (FSG).
at room temperature for 15 minutes at 4,000 rpm – Gp3: The mice orally received 25 mg/kg bw/
was used to recover the nanoparticles that were 2day of the powder from the areal parts of the
then washed with deionized water twice. Alanda via stomach tube for roughly 15 days
in the raw Alanda Gp (Alanda).
Characterization of Nanoparticles – Gp4: The mice orally received 25 mg/kg bw/
2day of Alanda NPs via stomach tube for
Scanning electron microscopy (SEM) roughly 15 days in the Alanda nanoparticles
Scanning electron microscopy was used to Gp (Alanda NPs).
observe the surface morphology and diameter of – Gp5: In line with Aldubayan et al10, the mice
the Ephedra nanoparticles. The Sem Afore (4.01 were infused hypodermically with roughly
demo, JEOI, Finland) software was used to ran- 2,500,000 cells of EAC per mouse debilitated
domly choose 30 nanoparticles and identify the in buffer saline to start EST in the Ehrlich sol-
diameter of the nanoparticles. id tumor Gp (EST) mice.
– Gp6: Treatment with only Alanda nanoparti-
Transmission electron microscopy (TEM ( cles (Alanda NPs) for 15 days follows the hy-
A transmission electron microscope (JEOL podermic infusion of 2,500,000 cells of EAC
JEM-1400 120kV; Jeol Ltd., Tokyo, Japan) was per mouse to commence the tumor (EST),
used to identify the morphology and size of the which is then left for roughly 15 days in the
Ephedra-loaded nanoparticles. EST+Alanda NPs Gp.
Fourier transform infrared spectroscopy (FTIR) Blood and Serum Samples
The potential interactions of Flax seed gum Clotting was enabled by the exclusion of anti-
and Ephedra were analyzed by monitoring the coagulant substances in the sterile and dry tube,
FTIR spectra for Ephedra-loaded nanoparticles which aseptically collected the blood samples via
and the raw material of Ephedra. venipuncture. Centrifugation at 3,000 rpm for 10
minutes followed clot formation, which occurred
Zeta Potential after the blood samples were left alone at 4°C for
The degree of stability in aqueous nanoparti- 30 minutes. Up until assessments of certain blood
cle dispersion is regulated via the zeta potential parameters were made, temperatures of -18°C
that represents a surface charge measurement. were used to store the collected serum.
Zeta potential values, whether negative or posi-
tive, should be high in order to avert aggregation Evaluation of Serum Liver Function Tests
and ensure particle stability. The Zetasizer Nano In line with Reitman and Frankel37 the serums
ZS (Malvern Instruments, Malvern, Worcester- aspartate transaminase (AST) and alanine trans-
shire, UK) was used to evaluate the zeta potential. aminase (ALT) were assessed. The estimation of
alkaline phosphatase and albumin concentration
Animals activity in sera followed the example of separate
The animal house colony Egypt vaccine academic works38,39. Conversely, the evaluation of
company was the source of the 40 Swiss albi- the serum total protein level adhered to the work
no female mice, weighing between 20 and 25 g, of another research paper40.

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Electrolytes and Kidney Functions Results

Biomarker
The estimation of urea occurred in accordance Scanning Electron Microscopy (SEM)
with a certain investigation41. Diamond, based in The surface morphology and shape of the Ephe-
Egypt, provided a commercial kit that was used dra nanoparticles loaded with flax seed extract
to evaluate the creatinine concentration in serum. were validated via the use of SEM. The shape of
Bowers and Wong41 presented the technique used the particles was revealed to be entirely spherical,
in this paper to estimate creatinine levels. The use as demonstrated by the SEM image in Figure 1A.
of commercial kits (Sensa core electrolyte, Ben- The amorphous and smooth nature of natural poly-
galuru, Karnataka, India) enabled the estimation mers was implicated by the SEM scans.
of the levels of serum electrolytes, such as chlo-
ride, sodium, potassium, and calcium ions42.

Detection of Total Genomic

DNA Degradation
As demonstrated in another academic work, the
salting out extraction technique was used to detect
DNA degradation via agarose gel electrophoresis
after Labarca and Paigen43 The ImageJ software,
as an average optical density value, enabled the as-
sessment of the intensity of DNA fragmentation.

Histopathological Evaluation
Processing for paraffin sectioning followed
the immediate incorporation of 10% buffer-neu-
tral formalin solution for one to two days to the
kidney and liver tissues. In line with Tousson44,
the histopathological assessment was enabled by
staining the sections with hematoxylin and eosin
(H&E).

Immunohistochemical Detection
of P53 Immunoreactivities
The use of the Avidin Biotin Complex (ABC)
enabled detection in the kidney and liver sections
of the expression of apoptotic P53 immunoreac-
tivities (P53-ir)2.

Immunohistochemical Detection
of Bcl2 Immunoreactivities
Similarly, the use of ABC enabled detection in
the kidney and liver sections of the expression of
anti-apoptotic Bcl2 immunoreactivities (Bcl2-ir)2.

Statistical Analysis
An unpaired t-test to evaluate the salient vari-
ances among treatment groups enabled the under-
taking of statistical analysis where data were ex-
pressed as average values ± SE. The threshold for
the biochemical data representing the criterion of
statistically significant data was 0.05. The SPSS Figure 1. A-B, Scanning Electron Microscopy (SEM) and
Transmission electron microscopy (TEM) images of the Ephe-
statistical version 21 software package (IBM dra-coated nanoparticles. C, Fourier transform infrared spec-
Corp., Armonk, NY, USA) was used to conduct troscopy (FTIR) spectra at wavenumber of 4,000 to 400 cm-1 of
all statistical analyses. Ephedra and Ephedra nanoparticles coated with flax seeds extract.

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 Alanda (Ephedra foeminea) nanoparticles as a modulator of genomic DNA degradation in mice

Transmission Electron Microscopy (TEM) Kidney Functions and Electrolytes

Figure 1B demonstrates that TEM image where Biomarker
very monodisperse nanoparticles are generated Upon comparing the control mice, a sub-
without defects, validating the synthesis of high stantial reduction of Ca++ and Na+ (p<0.05) in
quality. Similarly, the amorphous and spherical EST was observed, while a substantial rise in
nature of the nanoparticles was revealed. An av- Cl- and K+ (p<0.05) was witnessed in the cre-
erage size of 40 nm was observed in the particles. atinine and serum levels, as portrayed in Table
The generation of an amorphous polymer-drug I. Nevertheless, upon comparison with the EST
structure is indicated, as per prior evidence. group, EST treated with Alanda demonstrated a
substantial rise in Ca++ and Na+ levels (p<0.05)
Fourier Transform Infrared Spectroscopy while a substantial reduction in Cl- and K+ levels
(FTIR) (p<0.05) of creatinine and urea were also wit-
The potential alterations in the structure follow- nessed.
ing the addition of flax seed extract are assessed af-
ter the chemical bonding and functional groups of Liver Functions Biomarker
the prepared Ephedra nanoparticles are determined Upon comparing the control mice, a substan-
via the FTIR. The stretching of the CH- groups of tial reduction of the albumin and total protein
alkanes and the hydroxyl (-OH) groups of aliphatic (p<0.05) was observed in EST, while a substantial
alcohol demonstrated sharp peaks at 2,929 cm-1 and rise (p<0.05) in the serum alkaline phosphatase
3,430 cm-1, respectively, as per Figure 1C that shows (ALP), Glutamic-oxaloacetic transaminase (GOT),
the formulation of Ephedra-coated particles and and Glutamic-pyruvic transaminase (GPT) con-
the observed characteristic peaks of the Ephedra. currently was witnessed (Table II). Nevertheless,
The stretching of the carbonyl group C=O and PO2 upon comparison with the EST group, EST treated
caused the observed peaks of 1,037 cm-1 and 1,621 with Alanda exhibited a substantial rise in albumin
cm-1. Consequently, when the nanoparticles had flax (p<0.05) while also demonstrating a substantial re-
seed extract added to them, no alteration occurred in duction (p<0.05) in the total protein, ALP, GOT,
the positioning of such peaks. Thus, as demonstrat- and GPT (Table II).
ed in Figure 1C, the absence of interactions between
flax seed extracts and Ephedra is proven by the Total Genomic Degraded DNA in
nearly equivalent spectra displayed by the Ephedra the Kidney and Liver
nanoparticles and Ephedra materials. The total genomic DNA degradation in the kid-
ney and liver is portrayed in Figure 2. Compared
Zeta Potential with FSG (KG1, KG2, LG1, and LG2) and the neg-
An anionic nature was observed in the Ephe- ative control, the sparse fragmentation witnessed in
dra nanoparticles, as suggested by their negative the Alanda NPs group (KG4 and LG4) can be ig-
zeta potential of -25 mV. Neeraj et al34 note that nored. Conversely, serious necrosis (smear shape)
increased bio-adhesive strength is demonstrated was linked to a late stage of apoptosis witnessed
by polyanions, in comparison to polycations. The in the small amount of DNA damage provoked by
stability of the Flax seed gum loaded with Ephedra the EST and Alanda groups (KG3, KG5, LG3, and
was guaranteed by the high zeta potential value. LG5). Nevertheless, EST-induced DNA damage to

Table I. Changes in serum electrolytes and kidney function levels.

Urea Creatinine Na+ K+ Ca++ Cl-
(gm/dl) (gm/dl) (mmol/l) (mmol/l) (mmol/l) (mmol/l)

Control 26.75#±1.38 0.52#±0.18 136.6#±8.8 4.19#±0.24 1.12#±0.01 101.5#±8.9

FSG 26.15#±1.62 0.55#±0.13 137.0#±7.4 4.22#±0.31 1.09#±0.09 107.0#±7.5
Alanda 33.0#*±1.15 0.59#±0.22 131.0#*±6.6 4.59#*±0.30 0.91#*±0.03 109.5#*±8.5
Alanda Nps 25.25#±1.55 0.40#±0.03 135.5#±9.6 4.74#±0.22 1.06#±0.03 100.8#±6.6
EST 54.5*±2.96 0.98*±0.06 124.5*±7.1 6.06±0.54 0.89*±0.01 116.4*±8.2
EST+ Alanda Nps 27.0#±0.91 0.45#±0.02 134.5#±9.7 4.50#*±0.29 1.01#±0.01 107.8#*±6.7

Data are expressed as mean±S.E.M. (*) and (#) significant difference compared to control and Ehrlich solid tumor (EST) at
p<0.05 respectively.

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Table II. Changes in serum liver enzymes (GPT, GOT, ALP, total protein, albumin) levels.
GPT GOT Alb ALP T. protein
(U/I) (U/l) (gm/dl) (U/I) (gm/dl)

Control 44.5#±1.44 141.0#±3.81 4.38#±0.03 188.3#±4.6 5.92#±0.12

FSG 45.1#±1.60 147.0#±6.44 4.21#±0.18 192.5#±9.07 5.66#±0.38
Alanda 60.26#*± 4.25 171.5#*±9.39 3.86#*±0.35 229.0#*±12.5 5.12#*±0.33
Alanda Nps 42.0#±1.581 139.3#±4.07 4.12#±0.06 204.8#±5.27 5.77#±0.04
EST 82.3*±3.09 295.0*±4.42 3.43*±0.09 483.3*±16.5 4.70*±0.08
EST+ Alanda Nps 59.3#*± 3.64 168.0#*±6.28 4.23#±0.45 208.5#±9.5 5.23#*±0.08

Ehrlich solid tumor (EST); Flax Seed Gum (FSG); alkaline phosphatase (ALP).
Data are expressed as mean ± S.E.M. (*) and (#) significant differences compared to control and EST, respectively, at p<0.05.

Figure 2. Stained agarose gel of genomic degraded DNA of liver (L) and kidney (K) demonstrating apoptotic and necrotic
cell deaths induced by EST. G1, control; G2, FSG; G3, Alanda; G4, Alanda NPs; G5, EST; G6, treated EST with Alanda NPs.

the tissues of the kidney and liver (KG6 and LG6) Histological Alterations in the Liver
was mitigated by the protective impact of Alanda As shown in Figures 3A-B and 3D focusing on
NPs within the EST group treated with Alanda NPs. liver sections, a network extending from a central
vein to the edges of the hepatic lobules where the
Kidney Histopathology Findings portal tracts appeared form due to the radiating
As demonstrated in Figures 3A-D, standard cords of hepatocytes configured in the form of
histological structures were observed in the med- strands or anastomosing cords that surround the
ullary and cortical areas of the renal and glomeruli normal central vein, thereby demonstrating a stan-
tubules within the Alanda NPs group (G4), FSG dard histological pattern in the Alanda NPs group
(G2), and control group (G1). Conversely, Figure (G4), FSG (G2), and control group (G1). Contrast-
4C portrays atrophy in the tubular and glomeru- ingly, Figure 3C portrays severe inflammatory cell
li cells and moderate degeneration in the Alanda infiltration and tissue degeneration within the liv-
(G3) kidney sections. Figure 3E demonstrates at- er sections of the mice treated with Alanda (G3).
rophy in tubular cells, moderate degeneration, and As demonstrated by Figure 3E, congestion in the
marked inflammatory cellular infiltration in the portal and central veins or marked dilation, cel-
kidney sections of the EST group (G3), also dis- lular infiltrations, and marked atrophied vacuolar
playing variable histological alterations in certain degeneration of hepatocytes were observed in the
portions of the urinary tubules and glomeruli. Fig- EST group (G5). In addition, Figure 3F displays the
ure 3F illustrates mild degeneration of the urinary marked diffuse necrosis of hepatic tissue, moder-
and moderate atrophy of the tubular cells within ate inflammatory cells, and moderate cytoplasmic
the kidney sections of the EST treated with Alanda vacuolization of hepatocytes within the liver sec-
NPs group (G6). tions of the EST treated with Alanda NPs (G6).

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 Alanda (Ephedra foeminea) nanoparticles as a modulator of genomic DNA degradation in mice

Figure 3. Photomicrographs of mice kidney and liver sections in the different experimental groups stained with Haematoxy-
lin & Eosin. A-B, D, Liver sections in control (G1), FSG (G2), and treated mice Alanda NPs (G4) groups revealed normal histo-
logical pattern with normal central vein is surrounded by radiating cords of hepatocytes (Hp) and normal central vein (Cv). C,
Liver sections in Alanda (G3) revealed extensive degeneration of tissue and inflammatory cells infiltrations (arrows). E, Liver
section in EST (G5) revealed degeneration of hepatocytes (arrow heads) and marked inflammatory cells infiltrations (arrows).
F, Liver sections in treated EST with Alanda NPs (G6) revealed a moderate diffuse necrosis of hepatic tissue (arrow heads) and
moderate inflammatory cells infiltrations (arrows) (Magnification power is 25). A-B, D, Kidney sections in control (G1), FSG
(G2) and treated mice Alanda NPs (G4) groups revealed normal histological structures of the glomruli (G) and renal tubules
(Tb). E: Kidney sections in EST (G5) revealed marked inflammatory cellular infiltration (arrows) and moderate degenerative
and, atrophy in tubular cells tublar cells marked degenerated (arrow heads). F: Kidney sections in treated EST with Alanda
NPs (G6) group revealed a moderate atrophy in tubular cells, with mild degenerated urinary (Magnification power is 50 um).

Bcl2 Immunohistochemical liver sections of the distinct groups. A significant-

Alterations in the Liver ly positive reaction for Bcl2-ir (grade 5) in he-
Figures 4 and 5 portray the distribution and patocytes was observed within the liver sections
detection of Bcl2 immunoreactivity (Bcl2-ir) in of the Alanda NPs, FSG, and control groups. The

Figure 4. Immunohistochemical localization of anti-apoptotic Bcl2 and apoptotic P53- dependent pathway in liver and kid-
ney sections in different groups, indicated a marked downregulation of Bcl2 expression in EST and elevation in P53 on liver
and kidney, while and elevation of Bcl2 expression and depletion in P53 in EST+Alanda NPs on liver and kidney (Magnifica-
tion power is 25 and 50 um respectively).

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 R.M. Elgharabawy, E. Tousson, I.E. El Sayed, A.H. Abd El-Aleim, M. Elabd, et al

Figure 5. Data represent the expression level of Bcl2 and P53 in liver and kidney tissues in different experimental groups.
Columns with different signs are significantly different (p<.05) Where (*) and (#) significant difference compared to control
and EST respectively.

liver sections in the Alanda group displayed a cortical areas was observed within the liver sec-
moderately positive reaction for Bcl2-ir (grade 3). tions of the Alanda NPs, FSG, and control groups.
In addition, the liver sections in the EST group The kidney sections in the EST group displayed
displayed mildly positive reactions for Bcl2-ir a faintly positive reaction for Bcl2-ir (grade 1),
(grade 2). Upon comparison with the control while the Alanda group displayed a mildly posi-
group, a substantial reduction in the intensity of tive reaction for Bcl2-ir (grade 2). Upon compar-
Bcl2-ir was observed in the liver sections of the ison with the control group, a substantial rise in
EST group. Contrastingly, moderately positive re- the intensity of Bcl2-ir was observed in the liver
actions for Bcl2-ir (grade 4) were witnessed in the sections of the EST group. Nonetheless, mildly
liver sections of the EST treated with the Alanda positive reactions for Bcl2-ir (grade 3) were wit-
NPs group. nessed in the kidney sections of the EST treated
with the Alanda NPs group.
Bcl2 Immunohistochemical Alterations
in the Kidney P53 Immunohistochemical
Figures 4 and 5 show the distribution and de- Alterations in the Liver
tection of Bcl2 immunoreactivity (Bcl2-ir) in kid- Figures 4 and 5 illustrate the distribution and
ney sections of the distinct groups. A significant- detection of P53 immunoreactivity (P53-ir) in liver
ly positive reaction for Bcl2-ir (grade 5) in renal sections of the distinct groups. A faintly positive
and glomeruli tubules within the medullary and reaction for P53-ir (grade 2) in the hepatocytes was

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 Alanda (Ephedra foeminea) nanoparticles as a modulator of genomic DNA degradation in mice

observed within the liver sections of the Alanda of such substances in safeguarding the hepato-
NPs, FSG, and control groups. The liver sections cyte against membrane fragility. The change of
in the Alanda group displayed a moderately posi- hepatic enzymes and tissue architectures charac-
tive reaction for P53-ir (grade 4), whereas the liver terize the EST-induced hepatoxicity witnessed in
sections of the EST group displayed a strongly pos- separate academic works, thereby corroborating
itive reaction for P53-ir (grade 5). Upon compari- the findings of this study10,14. Additionally, the ex-
son with the control group, a substantial rise in the haustion of albumin and total protein levels in-
intensity of P53-ir was observed in the liver sec- duced by EAC is also reported in another research
tions of the EST group. Nonetheless, mild to mod- paper46. Khali et al27 note that the antioxidant and
erate positive reactions for P53-ir (grade 3) were scavenging properties, in tandem with the stim-
witnessed in the liver sections of the EST treated ulating impact on antioxidative defense systems
with the Alanda NPs group. of Alanda NPs, aid the hepatic protective effect
of the substance, which decreases the activities of
P53 Immunohistochemical ALP, ALT, and AST upon its incorporation.
Alterations in the Kidney A renal tissue injury induced by EST could be
Figures 4 and 5 display the distribution and the cause of the diminished level of sodium and
detection of P53 immunoreactivity (P53-ir) in calcium ions and the heightened levels of chloride,
kidney sections of the distinct groups. A faintly urea, creatinine, and potassium ions, thereby re-
positive reaction for P53-ir (grade 1) in the renal flecting the changes to kidney functions induced
and glomeruli tubules within the medullary and by EST. Conversely, these changes in electro-
cortical areas was observed within the kidney lytes and kidney functions are mitigated when
sections of the Alanda NPs, FSG, and control EST is treated with Alanda NPs. Waste products
groups. The kidney sections in the Alanda group are excreted as the kidney loses efficiency upon
displayed a moderately positive reaction for P53- increased levels of creatinine and urea, causing
ir (grade 2), whereas the kidney sections of the reduced glomerular filtration rates. As Salama et
EST group displayed a strongly positive reaction al47 observe, a rupture in the integrity of the cell
for P53-ir (grade 3). Upon comparison with the membrane, loss of function, and cellular leak-
control group, a substantial rise in the intensity age in the kidney are indicated by the mentioned
of P53-ir was observed in the kidney sections of biomarkers. Moreover, increased levels of creati-
the EST group. Nevertheless, mildly positive re- nine and urea were found to be the outlet through
actions for P53-ir (grade 2) were witnessed in the which EST provokes renal dysfunction in anoth-
liver sections of the EST treated with the Alanda er study13, thus corroborating the findings of this
NPs group. study. Similarly, reductions in sodium ions and
heightened levels of chloride, creatinine, urea,
and potassium ions were found to cause substan-
Discussion tial alterations in renal functionality and kidney
injury within the Ehrlich ascites tumor, as per
As Aldubayan et al10 and Kabel et al45 observe, another academic work that reflects the results of
the increased sensitivity to chemotherapy and this paper4.
the rapid advancement rate of Ehrlich carcinoma Chromosomal aberrations, mutations, and
make it almost an exact equivalent to human can- DNA damage occurred due to the aggregation
cers. The development of tumors in animals sig- of reactive oxygen species (ROS) within wom-
nificantly impacts organ function. The exhaustion en’s bodies in tandem with breast cancer devel-
of total albumin and protein levels, alongside the opment, as per another research paper reflecting
rise in ALP, AST, and ALT activities, characterize the results of this academic work8. As Abd El-
the liver dysfunction provoked by the Ehrlich sol- daim et al13,14 observe, ROS plays a significant
id tumor, as this paper shows. Nevertheless, such role in provoking tissue damage after applying
alterations to the liver functions were positively oxidative stress that demolishes cellular homeo-
mitigated when the Est was treated with Alanda stasis once EST is induced, resulting in changes
NPs. It is possible that a reduction in the leakage to the kidney and liver structure, per the results
of enzymes into blood circulation occurs due to of this paper. Given that the kidney section of the
the stabilization of the hepatic cellular membrane EST group displayed renal toxicity in the form
damage as a result of rises in ALP, ALT, and of atrophy of glomeruli and tubular cells as well
AST levels in sera, which validate the capability as marked degeneration, the findings of the im-

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 R.M. Elgharabawy, E. Tousson, I.E. El Sayed, A.H. Abd El-Aleim, M. Elabd, et al

munohistochemical and histopathological evalu- tinct academic work50. Lastly, heightened Bcl2
ation of kidney tissues were corroborated by the expression and reduced P53 expression in renal
biochemical findings of this paper. and hepatic tissues were found in EST-carrying
By decreasing the severe effects of EST on mice treated with Alanda NPs, per the results of
renal and hepatic tissue architecture, DNA dam- this study.
age, and serum levels of kidney and liver func-
tion, the results of this paper were presented.
Furthermore, the Bcl2 expressions in renal and Conclusions
hepatic tissues were reduced, while the P53 ex-
pressions in these tissues were elevated. Khalil Highly safe and efficacious treatments are now
et al27 note that antioxidant activities may be the embodied by alternative medicine, such as herb-
source of the beneficial effects of Alanda NPs. al medicine, in the fight against cancer. A nov-
The mitochondrial destruction and accumulation el synthesized nanoparticle design for treatment
of inflammatory cells caused by the migration is presented in this study. A new potential drug
and proliferation of tumor cells are attributed to combat cancer could be Alanda NPs extracts,
to the effect of the infiltration of Ehrlich tumor per the promising results discussed. Significantly
cells in internal organs in an academic work5. fewer side effects could result from implementing
While leaving cell replication multiplication un- such new medication, particularly in contrast to
touched, apoptosis is a significant procedure that chemotherapy.
maintains cell numbers at optimal levels. Al-Ra-
sheed et al48 and Alapati et al49 state that the sup-
pression of cancer, ageing, regulation of the cell
cycle, apoptosis, and the expression of genes are Data Availability
The data used to support the findings of this study are avail-
involved in the transcription factor of the protein able from the corresponding author upon request.
P53. The activation of DNA-repairing proteins
is enabled by P53 and how it stops the cell cycle
at G1 and G2. Upon comparison with the control
group that detects apoptosis induced by EST, the Ethics Approval
Our study was approved by the Institutional Animal Care
Bcl2 expressions were substantially elevated in and Use Committee (IACUC – SCI – TU – 0311) and had
the kidney and liver tissues in the EST group the capability to underwrite this study, which was guided by
while the apoptotic P53 expression was similarly the rules provided by the Moral Committee of the Faculty of
elevated. Anti-apoptotic effects are implicated Science at Tanta University.
within Alanda NPs as the alterations in Bcl2 and
P53 expressions are altered by the treatment of
Alanda NPs to the EST. Apoptosis can arise after Conflicts of Interest
oxidative stress and serious cellular genotoxicity The authors declare that they have no conflicts of interest.
occur due to such cellular and molecular actions.
Free radical-induced damage in endothelial cells
is safeguarded by natural antioxidants stemming Authors’ Contributions
from E. foeminea extracts, as per another study27 Ehab Tousson, Rehab M. Elgharabawy, Ibrahim E. El Sayed:
that corroborates the results of this work. The conception and design of the study; Rehab M. Elgharabawy,
findings of this study found that the expressions Amira S. Ahmed, Doaa T. Gebreel: analysis and interpretation
of P53 and Bcl2 proteins displayed an inverse of data; All authors: drafting the article and making critical re-
visions related to the relevant intellectual content of the man-
correlation. Aldubayan et al10 note bidirectional uscript; Rehab M. Elgharabawy, Ehab Tousson: final approval
alterations of protein expression patterns of P53 of the version of the article to be published.
and Bcl2. Thus, there is potential for apoptosis
to take place in the kidney and liver within EST,
given the reduction in Bcl2 antiapoptotic cells
and the rise in P53 apoptotic cells. A substantial ORCID ID
rise in P53 immunoreactivity as a result of the Rehab M. Elgharabawy: 0000-0002-4399-8195
Ehab Tousson: 0000-0001-9722-3603
Ehrlich tumor was also observed in another pa- Ibrahim E. El Sayed: 0000-0002-6418-987
per10. Similarly, heightened P53 expression in the Abd El-Aleim H.: 0000-0002-4625-8651
hepatic tissue of mice and diminished liver anti- Mervat Elabd: 0000-0001-5943-8347
oxidant systems are provoked by EAC, per a dis- Doaa T. Gebreel: 0000-0002-3526-7683

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 Alanda (Ephedra foeminea) nanoparticles as a modulator of genomic DNA degradation in mice

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