ArticlePDF Available

Cytotoxicity, Anti-diabetic, and Hepato-protective Potential of Ajuga bracteosa-conjugated Silver Nanoparticles in Balb/c Mice

Authors:
Article

Cytotoxicity, Anti-diabetic, and Hepato-protective Potential of Ajuga bracteosa-conjugated Silver Nanoparticles in Balb/c Mice

Abstract and Figures

Background: Ajuga bracteosa is a traditional herb used against various diseases. Objective: Current research aimed to investigate the anti-diabetic and hepato-protective effect of green synthesized silver nanoparticles (ABAgNPs) using Ajuga bracteosa aqueous extract (ABaqu). Methods: In vitro anti-diabetic and cytotoxic effects were carried out via α- glucosidase inhibition, brine shrimp lethality, and protein kinase inhibition assays. For in vivo screening of 200 mg/kg and 400 mg/kg of both ABAgNPs and ABaqu in alloxan-induced and CCl4-induced Swiss albino mice were used. Liver and kidney functional markers, hematology, and histopathological studies were carried out after 14 days of administration. Results: In vivo antidiabetic and anti-cancerous effects showed valuable anti-hyperglycemic and hepato-protective potential when mice were treated with ABaqu and ABAgNPs. A significant reduction in the blood glucose level was recorded when ABaqu and ABAgNPs were administrated orally compared to Glibenclamide treated group. Significant reduction in ALT, AST, ALP, urea, uric acid, and creatinine was recorded in ABaqu and ABAgNPs treated diabetic mice. The hepato-protective findings indicated that ALT, ALP, AST were elevated in CCl4-induced mice while declined in both ABAgNPs and ABaqu treated CCl4-induced mice. Histopathological examination revealed that ABAgNPs have hepato-protective activity. Conclusion: It was concluded that ABAgNPs and ABaqu possessed strong anti-diabetic and hepato-protective phytoconstituents which could be used in the prevention of diseases.
Content may be subject to copyright.
Send Orders for Reprints to reprints@benthamscience.net
Current Pharmaceutical Biotechnology, 2021, 22, 1-19 1
LETTER ARTICLE
1389-2010/21 $65.00+.00 © 2021 Bentham Science Publishers
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential of Ajuga
bracteosa-conjugated Silver Nanoparticles in Balb/c Mice
Sadia Nazer1, Saiqa Andleeb2,*, Shaukat Ali2, Nazia Gulzar1, Abida Raza3, Habib Khan1, Kulsoom Akhter4
and Muhammad Naeem Ahmed4
1Microbial Biotechnology laboratory, Department of Zoology, University of Azad Jammu and Kashmir, Muzaffarabad,
13100, Pakistan; 2Department of Zoology, Government College University, Lahore, Pakistan; 3National Institute for
Lasers and Optronics (NILOP), Pakistan Atomic Energy Commission, Islamabad, Pakistan; 4Department of Chemistry,
University of Azad Jammu and Kashmir, Muzaffarabad, 13100, Pakistan
Abstract: Background: Ajuga bracteosa is a traditional herb used against various diseases.
Objective: Current research aimed to investigate the anti-diabetic and hepato-protective effect of green
synthesized silver nanoparticles (ABAgNPs) using Ajuga bracteosa aqueous extract (ABaqu).
Methods: In vitro anti-diabetic and cytotoxic effects were carried out via α- glucosidase inhibition,
brine
shrimp lethality, and protein kinase inhibition assays. For in vivo screening of
200 mg/kg and 400
mg/kg
of both ABAgNPs and ABaqu in alloxan-induced and
CCl4-induced Swiss albino mice
were
used. Liver and kidney functional markers, hematology, and histopathological studies were carried
out after 14 days of administration.
Results: In vivo antidiabetic and anti-cancerous effects showed valuable anti-hyperglycemic and hepato-
protective potential when mice were treated with ABaqu and ABAgNPs. A significant reduction in the blood
glucose level was recorded when ABaqu and ABAgNPs were administrated orally compared to Glibenclamide
treated group. Significant reduction in ALT, AST, ALP, urea, uric acid, and creatinine was recorded in
ABaqu and ABAgNPs treated diabetic mice. The hepato-protective findings indicated that ALT, ALP, AST
were elevated in CCl4-induced mice while declined in both ABAgNPs and ABaqu treated CCl4-induced
mice. Histopathological examination revealed that ABAgNPs have hepato-protective activity.
Conclusion: It was concluded that ABAgNPs and ABaqu possessed strong anti-diabetic and hepato-
protective phytoconstituents, which could be used in the prevention of diseases.
A R T I C L E H I S T O R Y
Received: November 27, 2020
Revised: January 19, 2021
Accepted: January 21, 2021
DOI:
10.2174/1389201022666210421101837
Keywords: Ajuga bracteosa, anti-diabetic activity, anti-cancerous activity, mice, CCl4, silver nanoparticles, alloxan
1. INTRODUCTION
Diabetes mellitus is a chronic metabolic disorder, pres-
ently affecting more than 100 million people worldwide [1-
3]. About 3.3% of deaths were recorded in the whole world
due to diabetes, now diabetes is expected to be the 7th lead-
ing reason for death [4]. Complications related to diabetes
generate social and economic burdens [5]. Cardiovascular,
retinopathy, kidney disorder, foot ulcers, amputations, chron-
ic wounds, and infertility diseases are diabetic related [6, 7].
Now, it becomes a global problem to meet the requirement
of the anti-diabetic agents to treat the disease [8]. Cancer is
also characterized as a multifactorial disease, involves the
invasion of abnormal and uncontrolled growth cells, and
leads to the formation of tumors [9]. Hepatocellular carci-
noma (HCC) is a highly malignant disease, accounts for 80%
*Address correspondence to this author at the Microbial Biotechnology
laboratory, Department of Zoology, University of Azad Jammu and Kash-
mir, Muzaffarabad, 13100, Pakistan; E-mail: drsaiqa@gmail.com
to 90% of primary liver cancer. Several studies also reported
the association between diabetes mellitus and HCC [10-13].
The major risk factors for HCC worldwide are chronic infec-
tion with hepatitis B and C, alcoholic and metabolic liver
diseases [14], obesity, environmental pollutants, aflatoxin
exposure [15], and nitrosamine consumption [16]. Because
of the high death rate associated with cancer and the side
effects of chemo and radiotherapy, many cancer patients
seek alternative methods of treatment [17].
There are different techniques nowadays that have been
used for the production of drugs as anti-diabetic and anti-
cancerous agents. In recent times, the idea of merging smart
drug delivery is a highly efficient way to synthesize the na-
noparticles with enhanced delivery, bioavailability, and safe-
ty profiles [18]. Various nanomaterials and nano-devices
using AgNPs have been developed for the early diagnosis
and treatment of cancer and diabetes with the least adverse
effects [19, 20]. Since the last decades’ metallic nanoparti-
cles via the green synthesis approach have received consid-
2 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
erable interest because of biocompatibility, biological activi-
ty, and environment-friendly process compared to the con-
ventional chemical process [21, 22]. Moreover, the devel-
opment and improvement of biological activities of medici-
nal plants combining with metal nanoparticles are highly
appealing. These bio-conjugated nanoparticles often found to
display excellent anticancer, antibiotic, anti-diabetic activity,
and act as drug delivery [23]. Ajuga bracteosa is used
against different diseases such as skin disease (Dermatitis),
gastrointestinal disease, (Diarrhea), parasitic disease, (Malar-
ia), water and foodborne diseases (Typhoid fever and Chol-
era) [24]. Nazer et al. [25] demonstrated the antibacterial
potential of green synthesized silver nanoparticles using A.
brateosa and showed that plants could be useful against the
emerging multidrug-resistant bacterial strains. Therefore, the
current research aimed to evaluate the anti-diabetic and anti-
cancerous effect of Ajuga bracteosa-conjugated silver nano-
particles because these diseases are closely associated with
each other as discussed above and could be prevented via a
green synthesized therapeutic agent. The findings of current
research could be potential for the development of biogenic
based anti-diabetic and anti-cancerous nano-therapeutic
drugs.
2. MATERIALS AND METHOD
2.1 Ethical Statement
All experiments have been designed to avoid distress,
unnecessary pain, and suffering to the experimental animals.
All procedures were conducted following international regu-
lations referred to as Wet op de dierproeven (Article 9) of
Dutch Law.
2.2 Chemicals Used
All chemicals and reagents were obtained from Sigma
Aldrich (Germany), Merck (Germany), and Sigma Al-
drich (Switzerland). Silver nitrate (AgNO3), dimethyl
sulphoxide (DMSO), sulphuric acid (H2SO4), DPPH,
nin-
hydrin, ethanol, gallic acid, FolinCiocalteu reagent,
rutin,
aluminum chloride (AlCl3), sodium hydroxide (NaOH), sodi-
um nitrate (NaNO2), chloroform, ethyl acetate: acetic acid,
acetone, butanol, p-anisaldehyde,
hydrochloric acid, potassi-
um persulfate, methanol, glacial acetic acid, gelatin solution,
iron chloride (FeCl3), Millon’s reagent, Benedict’s solution,
Wagner’s reagent/ Mayor’s reagents, ethyl acetate, ammonia,
sodium carbonate,
alloxan,
diethyl ether, Glibenclamide,
sodium chloride (NaCl), Diclofenac sodium,
tetra carbon
chloride (
CCl4), olive oil, silymarin,
crystal violet, doxoru-
bicin, Alpha-glucosidase, sodium buffer, p-nitophenyl α-
D-carbonate, Heparin,
sodium carbonate
, ascorbase,
2.3. Extract preparation, Silver Nanoparticles Synthesis,
and Characterization
The roots and aerial parts of A. bracteosa were cleaned
with running tap water to remove dust and air-dried at room
temperature (20 ºC±2) under shade. The dried material was
crushed into a fine powder and used for the aqueous extract
preparation via maceration [25, 26]. Silver nanoparticles
using ABaqu were synthesized by a simple ratio method
[21]. The preliminary characterization of green synthesized
nanoparticles (ABAgNPs) was done through a UV-viz spec-
trophotometer (between 200 nm to 800 nm). The morpholo-
gy (shape and size) of ABAgNPs and ABaqu were con-
firmed through a scanning electron microscope. The dried
pellets of ABaqu and ABAgNPs were also used for FTIR
spectrometric analysis.
2.4. In Vitro Activities
2.4.1. P
hyto-constituent’s Analysis
Both ABAgNPs and ABaqu were analyzed for amino
acids, tannins, alkaloids, terpenoids, quinones, saponins,
steroids, flavonoids, carbohydrates, proteins, glycosides, and
phenolics. [27-29]. The total phenols were estimated using
the FolinCiocalteu reagent method [30] and it was ex-
pressed as mg/g gallic acid equivalent using the following
equation based on the calibration curve: y = 0.476x + 0.8, R2
= 0.996, where y was the absorbance and x was the gallic
acid equivalent (mg/g). Total flavonoid contents were quan-
tified via the method illustrated by Zou et al. [31] and it was
expressed as mg/g rutin equivalent using the following equa-
tion based on the calibration curve: y = 0.333x + 0.069, R2 =
0.999, where y was the absorbance and x was the rutin
equivalent (mg/g). Rutin was used as a standard for the cali-
bration curve.
2.4.2. Protein Kinase Inhibition Assay
In this assay, Streptomyces strain was used with slight
modification [32]. This procedure was performed under
sterile conditions. Streptomyces strain (cultivated) was
inoculated into tryptone soy broth (TS broth) and poured
into the Petri dishes. Filter paper discs impregnated with
ABaqu and ABAgNPs and also standard were set aside on
the surface of formed loan media incubated for 24 to 36 h
to target growth of the strain. The formation of a
clear/bald zone around the disc showed the inhibition of
mycelia and spore formation. The positive control taken
was surfactin, whereas the negative control was DMSO.
2.4.3. Brine Shrimp Toxicity Assay
Artemia salina lethality assay was carried out with
small modifications [33]. The larvae of Brine shrimp were
hatched in the bi-partitioned tank with filled artificial
seawater at 37°C. The incubation temperature was 21°C
to 30°C. After two days of hatching, nauplii (mature)
were collected and dispersed on a 96-well plate. The dilu-
tion was made of stock solutions with seawater and test
samples (ABaqu and ABAgNPs; 100 mg/ml). The final
concentrations (25 µg/ml, 200 µg/ml, 100 µg/ml, and 50
µg/ml) were used to test the lethality of brine shrimp.
Furthermore, the diluted solutions were then transferred
into each well containing dried yeast, 20 nauplii, then the
death rate of these larvae was calculated. The negative
control group contained seawater, and nauplii, whereas
the positive controls contained seawater, doxorubicin (4
mg/ml), and the nauplii. Plates were placed at room tem-
perature under fluorescence light for 24 h. This test was
performed in triplets and the total number of dead nauplii
was calculated in each well. The lethality was calculated
as a percentage of positive control, and LD50 was also
determined.
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 3
2.4.4. Alpha-Glucosidase Inhibition Activity
It was evaluated using the previously illustrated meth-
od with small modifications [34]. Alpha-glucosidase (1
mg) was dissolved in phosphate buffer (100 ml; pH 6.8),
having 200 mg of bovine serum albumin. The mixture
contained 250 µl of ABaqu and ABAgNPs each at a con-
centration of 1 mg/ml in phosphate glucopyranoside (250
µl: 5 mM) was then incubated for 5 min at 37 ºC. After
incubation, 250 µl of the alpha-glucosidase (0.15 unit/ml)
was poured and further incubated at 37 ºC for 15 min.
Additionally, 2000 ml of sodium buffer (490 µl: pH 6.8)
and p-nitrophenyl α-D-carbonate (200 mM) were added to
the reaction mixture, and the absorbance was noted at 400
nm using UV-spectrophotometer. The reaction mixtures
without ABaqu and ABAgNPs were taken as the blanks
and ascorbase were taken as a positive control. The
ABaqu and ABAgNPs that were required to scavenge
50% of radical (IC50) were determined by using five dif-
ferent concentrations of ABaqu as well as ABAgNPs. The
calculation of percentage inhibition (IAG%) was carried
out as: %=[(Ac-As)/Ac]x100.
Where Ac and As denoted
the absorbance of the control and test samples (ABaqu and
ABAgNPs), respectively
.
2.5. In Vivo Activities
2.5.1. Experimental Animal
The male Swiss albino mice (23-30 g) were purchased
from the animal facility center of the National Institute of
Health (NIH), Islamabad, Pakistan. The animals were accli-
matized for 2 weeks before the beginning of the current re-
search. The surrounding environment (laboratory conditions)
was maintained as 5 % humidity, for 12 h day/night cycles,
and the temperature was 25±2 °C. The handling and special
care of the Swiss albino mice were according to the estab-
lished public health guidelines in Guide for Care and Use of
Laboratory Animals (2011). The feed (commercial chow)
and water consumption patterns were observed throughout
the study period.
2.5.2. Acute Oral Toxicity
Acute oral toxicity was performed with a limit test at
1000 mg/kg as a single dose for both ABaqu and ABAgNPs
according to the adopted procedures described previously
with slight modifications [35]. The Swiss albino mice were
selected randomly after acclimatization to the laboratory
conditions and kept in their cages for at least 5 days before
dosing. The animals were kept without food for one day be-
fore dosing but had access to water. Three groups having
five healthy Swiss male mice weighing 2330 g were accli-
matized, such as the control group treated with vehicle (give
vehicle name and dose quantity); animals treated with
ABaqu: and animals treated with ABAgNPs. On the first
day, after fasting, the first group vehicle control was given
distilled water orally, 2nd and 3rd groups were administrated
with 1000 mg/kg of ABaqu and ABAgNPs separately. Food
was given after dosing of 1- 2 h. All the groups were closely
observed for 5 h and then at regular intervals for 14 days.
The bodyweight of the animals was monitored on 1st day, 7th
day, and 14th day. At the end of the experiment, liver and
kidney were excised and weighed. The serum was separated
for the hematological and biochemical evaluation. The be-
havioral signs of toxicity (salivation, changes in eyes, skin,
mucous membranes, hair, circulatory, respiratory, autonomic
and central nervous systems, motor activity, lethargy, trem-
ors, diarrhea, convulsion, or sleep) were recorded according
to the specifications of the OECD.: ‘Guidelines for the Test-
ing of Chemicals” [36].
2.5.3. In Vivo Anti-Diabetic Activity
Two doses (low and high doses; 200 mg/kg and 400
mg/kg) of both ABaqu and ABAgNPs were used FOR in
vivo anti-diabetic screening. Swiss albino mice were divided
into three groups; each group contained 6 mice: Group I
(Control), Group II (vehicle: Saline solution), except group
III (Alloxan (Al) treated: diabetic mice; n=30). Diabetes was
induced by a single intraperitoneal injection of Alloxan (150
mg/kg body weight) to overnight fasted mice. Alloxan dis-
solved in 0.9 % of normal saline. After some days diabetes
was confirmed from the tail clip sampling. A blood glucose
level greater than 200 mg/dl was considered as diabetes [37].
After diabetes confirmation, Alloxan treated mice were fur-
ther divided into 5 groups (n=6) for antidiabetic prevention
such as Group IV (Glibenclamide treated), Group V (ABaqu
200 mg/kg treated), Group VI (ABaqu 400 mg/kg treated)
Group VII (ABAgNPs 200 mg/kg treated), and Group VIII
(ABAgNPs 400 mg/kg treated). All treatments were admin-
istered orally in diabetic mice through drinking water for 15
days. The bodyweight of all experimental mice was meas-
ured via weighing balance at the beginning and the end of
the experimental study. Blood sugar level was estimated
after 1st day, 4th day, 7th day, and 14th day by collecting blood
via tail clip sampling. All mice were sacrificed at the end of
the experiment, and blood samples were collected by cardiac
puncture. Biochemical assays such as sugar level, liver func-
tional markers (ALAT, AST, and ALP), renal functional markers
(serum urea, uric acid, creatinine), and hematological parameters
were analyzed.
2.5.4. In Vivo Anti-Cancerous Activity
The anti-cancerous activity was performed by a modified
method [38]. In in vivo anti-cancerous screening, two doses
(low and high doses) of both ABaqu (200 mg/kg and 400
mg/kg) and ABAgNPs (200 mg/kg and 400 mg/kg) were
used. Swiss albino mice were divided into three groups; each
group contained 6 mice: Group I (Control; healthy mice),
Group II (vehicle: olive oil), except group III (CCl4 treated
mice; n=30). Group III mice were treated with an intraperi-
toneal injection of 50 % CCl4 in olive oil for six consecutive
weeks to induce liver cancer. After six weeks, CCl4-induced
mice were further divided into 5 groups (n=6) for anti-cancer
prevention such as Group IV (silymarin drug-treated), Group
V (ABaqu 200 mg/kg treated), Group VI (ABaqu 400 mg/kg
treated) Group VII (ABAgNPs 200 mg/kg treated), and
Group VIII (ABAgNPs 400 mg/kg treated). All groups were
treated intra-peritoneal for 30 days. CCl4-induced mice were
regularly observed for4 h after initial administration and then
once a day for consecutive two weeks. During the experi-
ment, behavioral and morphological changes were also ob-
served. The bodyweight of all experimental mice was meas-
ured at the beginning and the end of the experimental study.
After the treatment, mice were fastened overnight, sacrificed,
and blood was collected for biochemical analysis such as
4 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
liver function markers and hematological parameters. Mac-
roscopic characteristics of the heart, liver, and kidney were
also recorded.
2.6. Statistical Analysis
Each experiment was conducted thrice the time. Statisti-
cal analyses were performed using GraphPad Prism for Win-
dows (version 5.03) and also used to plot graphs with error
bars of standard errors of the means (SEM). Statistical icons:
single letter such a =p0.05; double letter such as aa
=p0.01; triple letter such as aaa, =p0.001.
3. RESULTS
3.1. Synthesis and Characterization of ABAgNPs
When an aqueous extract of Ajuga bractoesa (ABaqu)
was mixed with the silver ions, the reduction process oc-
curred. Before the AgNO addition, ABaqu extract had yel-
lowish-brown color, while after boiling for 15-20 min, the
color changed to dark brown, which indicated the formation
of silver nanoparticles ABAgNPs (Fig. 1) using ABaqu ex-
tract. Color change from yellowish-brown to dark brown
indicates the biosynthesis of ABAgNPs. Brown color solu-
tion is due to SPRP. Further, the synthesis of ABAgNPs was
confirmed through the UV-Viz spectrum at a range between
200-800 nm wavelength. The absorbance was recorded at
400 nm confirmed the ABAgNPs synthesis (Fig. 1). SEM
image showed relatively tube-like, individual as well as sev-
eral aggregates of nanoparticles with a diameter ranging
from 5µm up to 50 µm (Fig. 1). The IR spectrum had shown
bands at 1021 cm-1, 1108 cm-1, 1319 cm-1, 1543 cm-1, 2011
cm-1, and 3361 cm-1. The 3361 cm-1 correspond to O-H and
H stretching and confirmed the presence of alcohol and phe-
nol’s (Fig. 1). The band at 1543 cm-1 corresponds to Carbon-
yl stretching, 1319 cm-1 corresponds to N-H and C-N
stretching indicating the presence of aromatic amino groups
and proteins. The absorption bands at 1108 cm-1 and 1021
cm-1 might have contributed by the CO group of the poly-
saccharides (Fig. 1). All figures and descriptions of synthesis
and characterization of ABAgNPs have been published by
Nazer et al. [25].
3.2. Phyto-constituent Screening
In the current research, qualitative phytochemical screen-
ing of ABAgNPs and ABaqu was done. Various phytochem-
ical constituents, such as tannins, saponins, alkaloids, free
amino acids, proteins, quinones, carbohydrates, phenols,
terpenoids, glycosides, steroids, and flavonoids were detect-
ed in both ABAgNPs and ABaqu. The flavonoid contents in
ABAgNPs and ABaqu were found to be 18.41±0.005 mg
RHE/g, and 15.61±0.01 mg RHE/g of extract. The standard
curve for rutin hydrate was plotted and found to be linear in
Fig. (1). Synthesis and Characterization of green nanoparticles using A. bracteosa aqueous extract. A) UV-Viz spectra analysis, B) Scanning
electron microscopy of synthesized silver nanoparticles and A. bracteosa aqueous extract; C) FTIR of Green synthesized nanoparticles
(Nazer et al., 2020). (A higher resolution / colour version of this figure is available in the electronic copy of the article).
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 5
the range of 0.066-0.166 mg/ml. The total phenolic contents
in ABAgNPs and ABaqu extract were found to be
272.0±0.01 µg GAE/g, and 268±0.0495 µg GAE/g of ex-
tract, respectively.
3.3. Acute Oral Toxicity Studies
ABAgNPs and ABaqu did not show any toxicity signs
and symptoms at 1000 mg/kg throughout the observation
period. No significant difference was recorded in the body
and organ weights of all treated and control animals (vehicle,
ABAgNPs, and ABaqu). At the end of acute toxicity evalua-
tion tests, no lesions or any other abnormal changes in color,
size, shape, and texture were found on the vital organs (liver
and kidney) of treated animals with ABAqu and ABAgNPs.
The behavioral parameters such as skin, hair fall, eyes color,
urination, diarrhea, salivation, and sleep were normal during
the study. No toxic symptom, death/mortality, and coma
were observed. All treated mice lived up to 14 days after the
administration of ABaqu, ABAgNPs, and control (vehicle).
Hematological results revealed that no significant difference
was observed among all treated groups. No significant dif-
ference was recorded in biochemical markers (ALT, AST,
and ALP) of liver function tests. The microscopic outcomes
did not show any unrefined pathological and any other
changes in the treatment groups when compared to the con-
trol mice. It was observed that both the kidney and liver re-
served their normal textures without any anomalous altera-
tions in color and appearance. Significantly, no structural
modifications were detected in different tissues using a spe-
cific resolution of 400 µm. The normal texture of the liver,
such as a central vein, endothelial cells, hepatocytes, Kupfer
cells, and sinusoids was seen intact in all treated groups (Fig.
2). Therefore, we can say that ABaqu and ABAgNPs might
be nontoxic at 1000 mg/kg and the oral LD50 is bigger than
the tested dose in the Swiss albino mice.
3.4. In Vivo Anti-Diabetic Activity
3.4.1. Morphological and Behavioral Changes
During the experiment, certain behaviors (sluggish body
movements, shivering, and tired eyes) and morphological
changes (weakening of body hairs and kyphosis) were ob-
served in the Al-induced diabetic group. It was observed that
most of the mice became diabetic on first administration.
The bodyweight of control and vehicle (saline) treated mice
were increased (30.4±0.5 g to 35±0.8 g) and (31±1.0 g to
36.8±1.0 g) while the bodyweight of the Al-induced diabetic
mice was significantly declined to 23.4±0.3 g. ABaqu (200
mg/kg and 400 mg/kg) treated Al-induced diabetic mice
group showed a slight reduction from 38.8±1.7 g to 35.6±1.2
g, and 36.0±0.5 g to 31.0±1.4 g. However, there was no sig-
nificant difference recorded in Al-induced diabetic mice
treated with ABAgNPs at 200 mg/kg and ABAgNPs at 400
mg/kg, respectively.
3.4.2. Blood glucose Level
The blood glucose level was estimated during the exper-
imental study on 1st day, 4th day, 7th day, and 14th day to
evaluate the anti-diabetic effect of ABAgNPs and ABaqu.
Oral administration of the ABaqu and ABAgNPs for 14 days
produced a significant reduction in the blood glucose level
compared to Glibenclamide treated groups. Results revealed
that control (normal control group) and vehicle (saline)
groups exhibited normal blood glucose levels throughout the
experimental study. However, the Al-induced diabetic mice
showed significantly increased sugar levels from 161.2±5.8
mg/dl (1st day) to 294.0±1.9 mg/dl (14th day). It was noted
that Al-induced diabetic mice treated with ABaqu reduced
the blood sugar level on the 14th day at both doses
(202.2±2.0 mg/dl at 200 mg/kg and 205.2±5.6 mg/dl at 400
mg/kg) compared to Al-induced diabetic mice (294.0±1.9
mg/dl). Similarly, Al-induced diabetic mice treated with
ABAgNPs significantly reduced the blood sugar level on the
Fig. (2). Histological features of Balb/c mice skin treated with DH2O, ABAgNPs, and ABaqu during acute oral toxicity test. EC= endothelial
cells, CV= central vein, KC= Kupfer cells, H= hepatocytes, and S= sinusoids. (A higher resolution / colour version of this figure is available
in the electronic copy of the article).
6 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
14th day at both doses (191.2±3.5 mg/dl at 200 mg/kg and
188.4±3.5 mg/dl at 400 mg/kg) compared to Al-induced dia-
betic mice. We can say that both ABAgNPs and ABaqu had
anti-hyperglycemic potential compared to a drug used (Table
1).
3.4.3. Liver Functional Markers
Results showed that Al-induced diabetic mice had in-
creased levels of ALT (152.8±1.7 U/L), AST (215.0±2.5
U/L), and ALP (383.8±19.3 U/L) compared to control
(65.4±2.3 U/L, 54.2±4.1 U/L, and 190.2±5.5 U/L) and vehi-
cle (69.6±2.3 U/L, 59.8±1.9 U/L, and 196.4±4.6 U/L). On
the other hand, a significant decrease in ALT, AST, and ALP
(101.4±2.7 U/L, 123.6±1.7 U/L, 283.6±11.6 U/L) were ob-
served when Al-induced diabetic mice treated with
Glibenclamide. A significant decrease found in marker levels
were recorded when Al-induced diabetic mice treated with
ABaqu at 200 mg/kg (121±4.3 U/L, 100.2±3.5 U/L, and
259.4±11.5 U/L) and 400 mg/kg (110±3.5 U/L, 96.4±3.6
U/L, and 245±15 U/L). Tremendous results were recorded
when Al-induced diabetic mice were treated with ABAgNPs
(Table 2). It was observed that liver markers were signifi-
cantly reduced at both concentrations of ABAgNPs such as
at 200 mg/kg (106.6±4.1 U/L, 98.4±2.9 U/L, and 219.8±13.5
U/L) and 400 mg/kg (95.0±4.2 U/L, 87.8±2.4 U/L, and
194.4±14.8 U/L).
3.4.4. Renal Functional Markers
Results indicated that there was significant increase of
urea, uric acid, and creatinine found in Al-induced diabetic
mice (47.8±2.1 mg/dl, 32.4±1.0 mg/dl, and 2.0±0.2 mg/dl)
compared to control group (27.4±1.8 mg/dl, 19.8±0.9 mg/dl
and 0.6±0.1 mg/dl) and vehicle group (26.0±1.4 mg/dl,
20.2±0.8 mg/dl and 0.7±0.0 mg/dl). Glibenclamide showed a
significant decrease in renal functional markers compared to
Al-induced diabetic mice (Table 2). Urea, uric acid, and cre-
atinine levels were decreased in ABaqu treated Al-induced
diabetic mice at 200 mg/kg (39.6±0.8 mg/dl, 22.0±1.1 mg/dl
and 1.0±0.0 mg/dl), and at 400mg/kg (39.4±0.7 mg/dl,
22.2±0.9 mg/dl and 1.0±0.0 mg/dl). Similarly, ABAgNPs
treated Al-induced diabetic mice showed significant reduc-
tion in urea, uric acid and creatinine level at 200 mg/kg
(35.8±1.4 mg/dl, 20.4±0.5 mg/dl, and 1.0±0.0 mg/dl), and at
400 mg/kg (36.0±1.8 mg/dl, 19.8±0.7 mg/dl, and 0.9±0.0
mg/dl) (Table 2).
3.4.5. Hematology
The level of RBC was recorded as significantly low in
Al-induced diabetic group (2.7±0.1 106/µl) matched to con-
trol, vehicle and Glibenclamide treated groups (5.5±0.1
106/µl, 5.2±0.2 106/µl, and 3.2±0.2 106/µl). ABaqu (200
mg/kg) treated Al-induced diabetic mice showed a low level
of RBC (2.5±0.2 106/µl) and ABaqu (400 mg/kg) treated Al-
induced diabetic mice showed 2.9±0.1 106/µl level of RBC.
On the other hand, there was the level of RBC in ABAgNPs
(200 mg/kg) and ABAgNPs (400 mg/kg) treated Al-induced
diabetic mice was recorded as 3.1±0.1 106/µl and 3.5±0.1
106/µl. A significant reduction in hemoglobin level was rec-
orded in Al-induced diabetic mice (7.0±0.7 g/dl) compared
to all other treated groups. The hemoglobin level in control
and vehicle groups was recorded as 14.4±0.5 g/dl and
14.2±0.4 g/dl while in ABaqu (200 mg/kg and 400 mg/kg)
treated Al-induced diabetic mice was recorded as 12.4±0.5
g/dl and 12.1±0.2 g/dl (Table 3). Similarly, the hemoglobin
level in ABAgNPs (200 mg/kg) and ABAgNPs (400 mg/kg)
treated Al-induced diabetic mice was recorded as 12.8±0.5
g/dl and 12.4±0.5 g/dl. Hematocrit level was increased in Al-
induced diabetic mice (48.4±1.3%) and in Glibenclamide
treated Al-induced diabetic mice (45.0±1.4%) compared to
normal and vehicle-treated groups (36.4±1.4% and
36.8±1.5%). Hematocrit levels were found normal in all oth-
er treated groups. MCV level decreased in ABaqu (200
mg/kg and 400mg/kg) treated Al-induced diabetic mice
Duration
Treatments
Days (mg/dl)
1st
4th
7th
14th
Control
155.6±5.4
154.6±3.7
155.6±5.4
157.6±5.5
Vehicle (Saline)
158.8±5.5
156.0±4.7
158.8±5.5
157.2±5.1
Diabetic (Alloxan; Al)
161.2±5.8
232.6±7.0
281.2±5.8
294.0±1.9aaa,bbb
AL+Glibenclamide
159.2±6.4
186.0±2.9
159.2±6.4
187.2±3.5ccc
AL+ABaqu (200 mg/kg)
165.0±3.7
224.6±7.4
165.0± 3.7
202.2±2.0ddd
AL+ABaqu (400 mg/kg)
159.0±8.6
217.2±6.6
159.0±8.6
205.2±5.6e
AL+ABAgNPs (200 mg/kg)
168.4±4.4
188.6±4.1
168.4±4.4
191.2±3.5fff
AL+ABAgNPs (400 mg/kg)
151.2±4.8
187.0±8.2
151.2±4.8
188.4±3.5ggg
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 7
Table 2. A significant difference in liver and kidney function markers among Al-induced diabetic mice with saline, Glibenclamide,
ABAgNPs, and ABaqu extract.
Parameters
Treatments
Level (U/L)
Level (mg/dl)
ALT
AST
ALP
Urea
Uric acid
Creatinine
Control
65.4±2.3
54.2±4.1
190.2±5.5
27.4±1.8
19.8±0.9
0.6±0.1
Vehicle (Saline)
69.6±2.3
59.8±1.9
196.4±4.6
26.0±1.4
20.2±0.8
0.7±0.0
Diabetic (Alloxan; Al)
152.8±1.7aaa
215.0±2.5aaa
383.8±19.3aaa
47.8±2.1aaa
32.4±1.0aaa
2.0±0.2aaa
AL+Glibenclamide
101.4±2.7bbb,ccc
123.6±1.7bbb,ccc
283.6±11.6bbb,ccc
36.8±0.8ccc,bb
20.2±2.4ccc
0.9±0.1ccc
AL+ABaqu (200 mg/kg)
121.0±4.3ddd
100.2±3.5ddd
259.4±25.8ddd
39.6±0.8d
22.0±1.1ddd
1.0±0.0ddd
AL+ABaqu (400 mg/kg)
110.0±3.5eee
96.4±3.6eee
245.0±33.6eee
39.4±0.7ee
22.2±0.9eee
1.0±0.0eee
AL+ABAgNPs (200 mg/kg)
106.6±4.1ff
98.4±2.9ff
219.8±13.5ff
35.8±1.4fff
20.4±0.5ff
1.0±0.0ff
AL+ABAgNPs (400 mg/kg)
95.0±4.2ggg
87.8±2.4ggg
194.4±14.8ggg
36.0±1.8ggg
19.8±0.7ggg
0.9±0.0ggg
‘a’ indicates the significant difference between control and Diabetic (Alloxan (AL), ‘b’ indicates the significant difference between control and AL+Treated Glibenclamide, ‘c’ indi-
cates the significant difference between Diabetic (Alloxan (AL), and AL+Treated Glibenclamide, ‘d’ indicates the significant difference between Diabetic (Alloxan (AL), and
AL+ABaqu (200 mg/kg), ‘e’ indicates the significant difference between Diabetic (Alloxan (AL), and AL+ABaqu (400 mg/kg), ‘f’ indicates the significant difference between Dia-
betic (Alloxan (AL), and AL+ABAgNPs (200 mg/kg), ‘g’ indicates the significant difference between Diabetic (Alloxan (AL), and AL+ABAgNPs (400 mg/kg). Statistical icons:
single letter such a =p0.05; double letter such as aa =p0.01; triple letter such as aaa, =p0.001.
Table 3. A significant difference in hematological parameters among Al-induced diabetic mice with saline, Glibenclamide,
ABAgNPs, and ABaqu extract.
Parameters
Treatments
RBC
(106/µl)
Hb (g/dl)
Hemato-
crit (%)
MCV (fL)
Platelets
(103/µl)
Neutro-
phils
(%)
Eosino-
phils
(%)
Mono-
cytes
(%)
Lympho-
cyte
(%)
WBC
(103/µl)
Control
5.5±0.1
14.4±0.5
36.4± 1.4
80.2± 1.5
256.6±17.
6
31.2±0.9
3.8±0.4
3.8±0.4
77.8±1.1
3.7±0.2
Vehicle (Saline)
5.2±0.2
14.2±0.4
36.8±1.5
78.4±1.9
271.4±18.
7
30.6±1.1
2.4±0.2
4.0±0.3
75.2±1.9
3.8±0.2
Diabetic (Allox-
an; Al)
2.7±0.1aa
a
7.0±0.7aaa
48.4±1.3aa
a
51.4±1.0aa
a
627.4±4.4
35.6±0.5
3.6±0.2
3.0±0.3
56.4±1.8
7.9±0.3aa
a
AL+Glibenclami
de
3.2±0.2bb
b
13.0±0.7cc
c
45.0±1.4bb
66.2±1.8bb
370.0±5.7
31.4±0.5
2.6±0.2
3.2±0.4
70.8±2.1
4.9±0.3b
bb,ccc
AL+ABaqu (200
mg/kg)
2.5±0.2
12.4±0.5dd
d
35.8±1.6dd
d
62.6±2.1dd
d
380.2±4.1
30.6±1.1
2.8±0.4
3.0±0.3
62.6±0.9
5.0±0.1d
dd
AL+ABaqu (400
mg/kg)
2.9±0.1
12.1±0.2ee
e
39.0±1.0ee
61.6±1.2ee
378.2±6.6
32.4±1.2
3.2±0.4
3.2±0.4
65.6±1.5
4.8±0.2ee
e
AL+ABAgNPs
(200 mg/kg)
3.1±0.1
12.8±0.5fff
36.8±1.0fff
64.2±2.6fff
375.4±4.6
32.2±0.7
2.8±0.4
3.2±0.4
67.4±1.5
7.0±0.6ff
f
AL+ABAgNPs
(400 mg/kg)
3.5±0.1
12.4±0.5gg
g
33.4±1.2gg
g
68.6±0.7gg
g
323.8±10.
0
31.0±0.7
3.0±0.3
3.4±0.4
70.0±0.6
4.5±0.1g
gg
‘a’ indicates the significant difference between control and Diabetic (Alloxan (AL), ‘b’ indicates the significant difference between control and AL+Treated Glibenclamide, ‘c’ indi-
cates the significant difference between Diabetic (Alloxan (AL), and AL+Treated Glibenclamide, ‘d’ indicates the significant difference between Diabetic (Alloxan (AL), and
AL+ABaqu (200 mg/kg), ‘e’ indicates the significant difference between Diabetic (Alloxan (AL), and AL+ABaqu (400 mg/kg), ‘f’ indicates the significant difference between Dia-
betic (Alloxan (AL), and AL+ABAgNPs (200 mg/kg), ‘g’ indicates the significant difference between Diabetic (Alloxan (AL), and AL+ABAgNPs (400 mg/kg). Statistical icons:
single letter such as a =p0.05; double letter such as aa =p0.01; triple letter such as aaa, =p0.001.
(62.6±2.1% and 61.6±1.2%), and in ABAgNPs (200 mg/kg
and 400mg/kg) treated Al-induced diabetic mice (64.2±2.6%
and 68.6±0.7%). The MCV level in Glibenclamide treated
Al-induced diabetic mice was recorded as 66.2±1.8%. Re-
sults revealed that Al-induced diabetic mice showed a signif-
icant decrease in MCV level (51.4±1.0%) compared to all
treated groups. The level of MCV in the control and the ve-
hicle-treated group was recorded as 80.2±1.5% and
8 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
78.4±1.9%. There was a significant increase in the platelets
level recorded in Al-induced diabetic mice (627.4±4.4 103/
µl) compared to control and vehicle-treated groups
(256.6±17.6 103/µl and 271.4±18.7 103/µl). Platelets level
significantly decreased in ABaqu (200 mg/kg and 400
mg/kg) and ABAgNPs (200 mg/kg and 400 mg/kg) treated
Al-induced diabetic mice (380.2±4.1103/µl, 378.2±6.6,
375.4±4.6 103/µl, and 323.8±10.0 103/µl). Similarly, the re-
duced level was recorded in Glibenclamide-treated Al-
induced diabetic mice (370.0±5.7103/µl). The neutrophil
level remained normal in all groups except Al-induced dia-
betic mice (35.6±0.5%), where a slight increase was found.
Eosinophils and monocytes were found normal in all groups.
Lymphocytes level decreased in Al-induced diabetic mice
(56.4±1.8 %) compared to the control and vehicle-treated
group (77.8±1.1% and 75.2±1.9%). Lymphocytes level was
increased in ABaqu (200 mg/kg and 400 mg/kg) treated Al-
induced diabetic mice (62.6±0.9% and 65.6±1.5%), whereas
a significant increase was also found in ABAgNPs (200
mg/kg and 400 mg/kg) treated Al-induced diabetic mice
(67.4±1.5 % and 70.0±0.6 %) and Glibenclamide treated Al-
induced diabetic mice (70.8±2.1 %). There was a significant
increase in the WBC level recorded in Al-induced diabetic
mice (7.9±0.3 103/ µl) compared to control and vehicle-
treated groups (3.7±0.2 103/µl and 3.8±0.2 103/µl). WBC
level was decreased in ABaqu (200 mg/kg and 400 mg/kg)
and ABAgNPs (400 mg/kg) treated Al-induced diabetic mice
(5.0±0.1 103/µl, 4.8±0.2 103/µl, and 4.5±0.1 103/µl). While
increased level was recorded in ABAgNPs (400 mg/kg)
treated Al-induced diabetic mice (7.0±0.6 103/ µl). The re-
duced level was recorded in Glibenclamide-treated Al-
induced diabetic mice (4.9±0.3 103/µl) (Table 3).
3.5. In Vivo Anti-Cancerous Activity
3.5.1. Morphological Analysis
During the experiment, certain behaviors (slow body
movements, dark color urine, and eye color change) and
morphological changes (loss of body hairs and loss of appe-
tite) were observed in CCl4-induced mice. No mortality was
recorded during the experimental study. The body weights of
all treated and control animals were recorded on the 1st day
of the experiment and the 30th day (after the experiment). It
was observed that CCl4-induced mice exhibited lower
body weight after treatment (24.2±2.6 g) compare to
normal control (35.4±2.7 g) and vehicle-treated mice
(32.8±1.9 g). The body weights of the vehicle and the control
group remained the same throughout the study, while the
bodyweight of CCl4-induced treated mice with ABAgNPs at
200 mg/kg and 400 mg/kg was reduced as 32.4±1.4 g to
23.8±1.8 g and 37.8±1.3 g to 30.6±1.5 g. Similar results
were shown by ABaqu at both 200 mg/kg and 400 mg/kg
concentrations used. ABaqu showed reduced body weight at
both 200 mg/kg and 400 mg/kg (35.2±3.4 g to 20.4±0.7 g)
and (33.4±2.4 g to 27.0±3.4 g).
3.5.2. Macroscopic Studies of Organs
The organs (liver, heart, and kidney) of all treated groups
and control were excised after 30 days and macroscopic
characteristics were recorded. The livers extracted from con-
trol and vehicle groups were red-brown in color, bright look,
moist, and damp surface. On the other hand, yellowish-
brown spots like steatosis (infiltration of liver cells with fats;
fatty liver), lesions, blunt edges, less moist surface, and a
less sharp look were recorded in CCl4-induced mice. While
red-brown color, bright look, the moist and damp surface of
the liver was also observed in CCl4-induced mice treated
with ABaqu at 400 mg/kg and CCl4-induced mice treated
with silymarin. CCl4-induced mice treated with ABAgNPs
and ABaqu showed a light brownish color with mild steato-
sis compared with the CCl4-induced mice. Kidneys of CCl4-
induced mice were small in size, squeezed, hard, light
brown, and fatty deposition while soft, brownish in color,
healthy kidneys were observed in other CCl4-induced mice
treated with silymarin, ABAgNPs, and ABaqu. Macroscopic
characteristics of the heart in CCl4-induced mice and other
treated groups were also observed. Results indicated that the
heart was in small size, shrink, and hard in structure com-
pared to all other treated and control groups. Hearts of CCl4-
induced treated groups were bright, sharp, and reddish-
brown.
3.5.3. Body Organs Weight
CCl4-induced mice showed a reduction in liver weight
(1.836±0.0 g) compared to control and vehicle (2.204±0.41 g
and 1.99± 0.18 g). In contrast, CCl4-induced mice treated
with silymarin, ABaqu (400 mg/kg), and ABAgNPs (200
mg/kg and 400 mg/kg) showed increased liver weight
(2.032±0.0 g, 2.266±0.0 g, 2.046±0.0g and 2.45±0.10 g).
The kidney weight of CCl4-induced mice was reduced
(0.182±0.02 g) compared to control and vehicle (0.218±0.05
g and 0.216±0.05 g). On the other hand, CCl4-induced mice
treated with silymarin, ABaqu (200 mg/kg and 400 mg/kg),
and ABAgNPs (200 mg/kg and 400 mg/kg) showed in-
creased kidney weight (0.214±0.05 g, 0.196±0.01 g,
0.232±0.06 g, 0.198±0.0 g, and 0.24±0.05 g) compared to
CCl4-induced mice. The heart weight of CCl4-induced mice
was decreased (0.190±0.02 g) compared to control and vehi-
cle (0.202±0.01 g and 0.208±0.02 g). On the other hand,
CCl4-induced mice treated with silymarin, ABaqu (200
mg/kg and 400 mg/kg), and ABAgNPs (200 mg/kg and 400
mg/kg) showed increased heart weight (0.208±0.03 g,
0.202±0.0 g, 0.20±0.01 g, 0.206±0.01 g, and 0.198±0.0 g)
compared to CCl4-induced mice.
3.5..4 Biochemical Parameters
It was revealed that the ALT level was significantly ele-
vated (134.8±7.9 U/L) in intraperitoneal administrated CCl4-
induced mice paralleled to control and vehicle-treated mice
(63.8±2.4 U/L and 67.8±2.7 U/L), respectively (Table 4).
Treated CCl4-induced mice with ABaqu (200 mg/kg and 400
mg/kg) and ABAgNPs (200 mg/kg and 400 mg/kg) showed
significant declined in ALT level (95.2±5.1 U/Land 94.9±5.1
U/L) and (84±6.9 U/L and 68±3.9 U/L). On the other hand,
CCl4-induced mice treated with ABaqu (200 mg/kg and 400
mg/ kg) showed a significant reduction in AST level (86±5.6
U/L and 84.2±3.5 U/L) while CCl4-induced mice treated
with ABAgNP (200 mg/kg and 400 mg/ kg) showed a highly
significant reduction in AST level (72.8±3.7 U/L and
69.2±5.7 U/L) (Table 4). CCl4-induced mice treated with
ABaqu showed a reduction in ALP level (291.0±22.3 U/L) at
200 mg/kg, whereas CCl4-induced mice treated with ABaqu
at 400 mg/kg showed an increased level of ALP 355.8±17.5
U/L compared to CCl4-induced mice. There was a significant
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 9
decrease in the ALP level when CCl4-induced mice were
treated with ABAgNPs at both doses (258.8± 14.3 U/L at
200 mg/kg and 246.6 ±16.5 U/L at 400 mg/kg). A significant
reduction in ALT, AST, ALP levels were recorded in CCl4-
induced mice when treated with silymarin (62.2±2.9 U/L,
50.4± 3.2 U/L, and 179.2±7.0 U/L) (Table 4).
3.5.4. Hematology
White blood cell levels were noted as high (6.5±0.5 103
µl) in CCl4-induced mice compared to control (3.5±0.2 103
µl) and vehicle-treated mice (3.7±0.2 µl). CCl4-induced mice
treated with ABAqu (200 mg/kg and 400 mg/kg) showed a
slight decline in WBC (5.2±0.5±103 µl and 5.5±0.3 103 µl),
whereas CCl4-induced mice treated with ABAgNPs (200
mg/kg and 400 mg/kg) showed a significant reduction in
WBC (4.2±0.4 103 µl and 4.4±0.3 103 µl) matched to CCl4-
induced mice. The current results showed that there was a
reduction in RBC in CCl4-induced mice (3.0±0.2 106/µl).
ABaqu (200 mg/kg and 400 mg/kg) treated CCl4-induced
mice displayed an increased in RBC (3.1±0.2 and 3.7±0.2
106/µl). On the other hand, RBCs concentration was signifi-
cantly increased in ABAgNPs treated CCl4-induced mice
(4.4±0.3 106/µl and 4.3±0.3 106/µl) at both doses (200 mg
/kg and 400 mg/kg) (Table 5). Hematocrit level was almost
the same in control as well as all treated groups. Blood plate-
let count (PLT) showed a decreased level in CCl4-induced
mice (133±17.9 103/µl) while silymarin treated CCl4-induced
mice showed a significant increase (234.2±13.4 103/µl). The
CCl4-induced mice treated with ABaqu (200 mg/kg and 400
mg/kg) and ABAgNPs (200 mg/kg and 400 mg/kg) demon-
strated a significant increase in dose-dependent manner.
Lymphocyte level was reduced in CCl4-induced mice
(61.0±2.6%) compared to control (73.2±1.9%) and vehicle-
treated mice (73.4±2.4%). On the other hand, CCl4-induced
mice treated with ABaqu (200 mg/kg and 400 mg/kg) and
ABAgNPs (200 mg/kg and 400 mg/kg) showed a significant
rise in lymphocyte level (65.6±3.7%, 64.2±2.5%,
66.8±2.6%, and 74.6±3.7%). Silymarin treated CCl4-induced
mice also showed an increased level of lymphocytes. Neu-
trophils were increased in CCl4-induced mice, while a signif-
icant reduction was recorded in CCl4-induced mice treated
with ABAgNPs at both concentrations (27.0±2.7% at 200
mg/kg and 32.4±2.5% at 400 mg/kg). Silymarin treated
CCl4-induced mice also showed a significant reduction.
Monocytes were improved in CCl4-induced mice compared
to control and vehicle groups. It was observed that ABaqu
(200 mg/kg) and ABAgNPs (400 mg/kg) showed a reduction
in monocytes while ABaqu (400 mg/kg) and ABAgNPs (200
mg/kg) showed increased level compared to CCl4-induced
mice (Table 5). Eosinophils were increased in CCl4-induced
mice (3.2±0.2%) while significant reduction was recorded
when CCl4-induced mice treated with ABaqu (2.6±0.4% at
400 mg/kg) and ABAgNPs (2.2±0.2% at 200 mg/kg and
2.4±0.2% at 400 mg/kg).
3.5.5. Histopathological Studies
Histopathological sections of liver, heart, and kidney of
CCl4-induced Swiss albino mice and CCl4-induced mice
treated with silymarin, ABaqu, and ABAgNPs were ob-
served (Figs. 3-5). Heart histology shows the presence of a
normal heart with integrated tissue patterns in control and
vehicle groups. On the other hand, the hearts of CCl4-
induced mice had lost tissue integrity. However, no differ-
ence was observed in CCl4-induced mice treated with si-
lymarin, ABaqu, and ABAgNPs (Fig. 3). Control animals
showed regular hepatic architecture with a central vein, dis-
tinct hepatocytes, endothelial cells, Kupfer cells, and sinus-
oidal spaces (Fig. 4). In the CCl4-induced mice, the hepatic
architecture was disrupted and upset, indicating an abnor-
mality among hepatic cells. Inflammation, tissue necrosis,
congested Kupfer cells, and lack of central vein were also
recorded. The cell membranes and central veins were disin-
tegrated. The sinusoidal spaces were dilated (Fig. 4). This
phenomenon was brought back to near normal hepatic
Parameter
Treatments
Unit (U/L)
ALT
AST
ALP
Control
63.4±2.8
51.2±2.7
167.4±12.2
Vehicle (Olive oil)
67.8±2.7
56.6±3.4
175.6±11.2
Diseased (CCl4)
134.8±7.9 aaa,bbb
109.0±3.6 aaa,bbb
345.6±16.6 aaa,bbb
CCl4+ silymarin)
62.2±2.9 ccc
50.4±3.2 ccc
179.2±7.0 ccc
CCl4+ABaqu (200 mg/kg)
95.2±5.1 ddd
86.8±5.6 d
355.8±17.5
CCl4+ABaqu (400 mg/kg)
94.9±5.0 eee
84.2±3.5 ee
291.0±22.3
CCl4+ABAgNPs (200 mg/kg)
84.0±6.9 fff
72.8±3.7 fff
258.8±14.3 ff
CCl4+ABAgNPs (400 mg/kg)
68.8±3.9 ggg
69.2±5.7 ggg
246.6±16.5 gg
10 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
Table 5. A significant difference in hematological parameters among CCl4-induced treated mice with olive oil, silymarin,
ABAgNPs, and ABaqu extract.
Parameter
Treatments
WBC
(103/µl)
RBC
(106/µl)
Hemoglobin
(g/dl)
Hematocrit
(%)
Platelets
(103/µl)
Lymphocyte
(%)
Neutrophils
(%)
Monocytes
(%)
Eosinophils
(%)
Control
3.5±0.2
5.0±0.1
12.7±0.4
33.8±1.8
264.6±20.4
73.2±1.9
24.4±2.3
2.2±0.2
2.2±0.2
Vehicle (Olive oil)
3.7±0.2
4.0±0.3
12.5±0.2
35.2±2.4
271.4±18.7
73.4±2.4
20.0±2.6
2.3±0.2
2.4±0.2
Diseased (CCl4)
6.5±0.4
aaa
3.0±0.2
aaa
10.8±0.4 a,b
33.4±2.0
133.0±17.9 aaa
61.0±2.6
45.6±2.8aaa
3.0±0.4
3.2±0.2
aaa
CCl4+ silymarin)
3.7±0.1
ccc
4.5±0.2
cc
12.4±0.5
30.4±1.4
234.2±13.4
71.4±2.8
34.4±3.1
2.2±0.2
2.2±0.2
CCl4+ABaqu (200
mg/kg)
5.2±0.5
3.1±0.2
11.0±0.3
30.6±1.8
195.2±26.5
65.6±3.7
40.2±3.5
2.8±0.2
3.0±0.3
CCl4+ABaqu (400
mg/kg)
5.5±0.3
3.7±0.2
11.8±0.4
32.4±1.5
201.8±37.6
64.2±2.5
36.8±4.5
3.4±0.4
2.6±0.4
CCl4+ABAgNPs (200
mg/kg)
4.4±0.3 ff
4.4±0.3 f
12.6±0.4
35.8±2.0
210.8±13.4
66.8±2.6
27.0±2.7
3.2±0.4
2.2±0.2
CCl4+ABAgNPs (400
mg/kg)
4.2±0.4
ggg
4.3±0.3 g
12.0±0.3
37.0±2.0
258.4±34.9 g
74.6±3.7
32.4±2.5 gg
2.2±0.2
2.4±0.2
‘a’ indicates the significant difference between control and diseased (CCl4), ‘b’ indicates the significant difference between vehicle and CCl4+Treated silymarin, ‘c’ indicates the
significant difference between cancer (CCl4), and CCl4+Treated silymarin, ‘d’ indicates the significant difference between cancer (CCl4), and CCl4+ABaqu (200 mg/kg), ‘e’ indicates
the significant difference between cancer (CCl4), and CCl4+ABaqu (400 mg/kg), ‘f’ indicates the significant difference between cancer (CCl4), and CCl4+ABAgNPs (200 mg/kg), ‘g’
indicates the significant difference between cancer (CCl4), and CCl4+ABAgNPs (400 mg/kg). Statistical icons: single letter such as a =p0.05; double letter such as aa =p0.01; triple
letter such as aaa, =p0.001.
Fig. (3). Histology of heart tissues in control, vehicle, and CCl4-induced mice treated with silymarin, ABaqu, and ABAgNPs.
(A higher
resolution / colour version of this figure is available in the electronic copy of the article).
architecture in the CCl4-induced mice treated with ABaqu
and ABAgNPs, demonstrating significant protection of the
liver. ABAqu showed slight prevention of CCl4 liver toxici-
ty. ABAgNp and Silymarin preparation treatment appeared
to significantly prevent CCl4 toxicity. Histology of kidneys
from control, vehicle, diseased and CCl4-induced mice treat-
ed with silymarin, ABaqu, and ABAgNPs was observed. Fig.
(5) reveals the congestion of glomerular capillaries and in-
flammation in CCl4-induced mice. Patchy necrosis and hem-
orrhage were also seen in CCl4-induced mice. CCl4-induced
mice treated with ABAgNPs showed normal kidney structure
with mild inflammation, clear glomerular capillaries, proxi-
mal convoluted tubule, distal convoluted tubule, and promi-
nent squamous cells (Fig. 5).
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 11
3.6. In Vitro Activities
3.6.1. Alpha-glucosidase Inhibition Effect
The result suggests that A. bractoesa under in vitro con-
ditions exhibit good α- glucosidase inhibition activity. The
percentage inhibition of ABaqu and ABAgNPs was maxi-
mum of 91.74% and 85.14 % at 50 µg/ml concentration,
while for the positive control, acarbose maximum inhibition
was 90.1 % at 50 µg/ml concentration.
3.6.2. Cytotoxic Effects
In the current study, the toxic effect of green synthe-
sized nanoparticles (ABAgNPs) was analyzed compared
to Ajiga bractoesa extract (ABaqu). It was recorded that
at a minimum concentration of 25 µg/ml of ABaqu and
ABAgNPs the mortality was 73% and 55%, respectively,
whereas when the concentration of ABaqu increased from
50 µg/ml to 200 µg/ml the mortality was about 76%, 73%
to 93%. The mortality percentage of ABAgNPs also in-
creased when concentration increased from 50 µg/ml to
200 µg/ml (83% to 96%). The nanoparticles (ABAgNPs)
were shown to have an LD50 value of 27.57 µg//ml. Pro-
tein kinase inhibition assay demonstrated ABAgNPs have
anticancerous potential as it showed maximum inhibited
zone (25.5± 0.5 mm) compared to both ABaqu (14.5± 0.5
mm) and positive control surfactin (22.0±0.0 mm).
4. DISCUSSION
4.1. Synthesis and Characterization
In the current research, ABAgNPs synthesis from Ajuga
bractoesa was primarily identified by color change. This
color change is due to the presence of phytoconstituents of
Fig. (4). Histology of kidney tissues in control, vehicle, and CCl4-induced mice treated with silymarin, ABaqu, and ABAgNPs.
[DCT= distal convoluted tubule, PCT= proximal convoluted tubule, H=hemorrhage SC= squamous cell, G= glomerulus, PN=patchy
necrosis].
(A higher resolution / colour version of this figure is available in the electronic copy of the article).
Fig. (5). Histology of liver tissues in control, vehicle, and CCl4-induced mice treated with silymarin, ABaqu, and ABAgNPs. [E= en-
dothelial cells, CV= central vein, KC= Kupfer cells, H= hepatocytes, S= sinusoids].
(A higher resolution / colour version of this figure is
available in the electronic copy of the article).
12 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
ABaqu extract, such as flavonoids, alkaloids, steroids, sapo-
nins, and they act as a reducing agent that reduced silver ions
(Ag+) to a silver atom (Ag0). The findings are consistent with
previous literature [39, 40]. They also reported similar
changes in color during nanoparticle formation. The brown
color appears in the solution indicating the formation of
AgNPs in the mixture as a result of the reduction of Ag+
ions to Ag metal through reducing agents present in the me-
dicinal plants, such as phenolics, terpenoids, proteins, en-
zymes, flavones, polysaccharides, and amino acid, etc [41,
42]. The UVViz spectra at 425460 nm are an indication of
the surface plasmon resonance bands, which play a vital role
in the size, and morphology of nanoparticles [43, 44]. Previ-
ous studies suggested that a peak located between 400 and
450 nm has been observed for AgNPs and might be attribut-
ed to spherical and cylindrical nanoparticles [42]. In the cur-
rent study, the absorbance peak was recorded at 400 nm that
confirmed the ABAgNPs synthesis. Our findings are in
agreement with Almalah et al. [45], who used C. zylinicum
bark extract as a reducing agent and the formation of silver
nanoparticles occurred rapidly. The ABAgNPs spectrum via
FTIR displayed that functional groups found in it play an
important role in the capping and stability of AgNPs [46].
The bioactive molecules that exist in plant extract are im-
portant for the bio-reduction of Ag+ as well as act as capping
and stabilizing agents [47, 48]. In this study FTIR result of
ABAgNPs showed N-H and C-N stretching, indicating the
presence of aromatic amino groups and proteins, CO
stretching group confirmed the presence of polysaccharides,
and O-H and H stretching indicated the alcohols and phe-
nol’s presence. Femi-Adepoju et al. [49] studied the silver
nanoparticles synthesized from Gleichenia Pectinata indicate
C-H stretching of aromatic compounds, C=O stretching
for carbonyl compounds, corresponding to C-C and C-N
stretching present in amide links serving as the stabilizing
and capping agents. FTIR result indicates that the interaction
of alcohol, carbonyl, and terpenoid groups with silver was
responsible for silver nanoparticles synthesis and serving as
strong binding sites. These results were supported by various
researchers [50]. Spherical AgNPs of an average diameter of
8-10 nm were synthesized by using fruit extract of cucumber
(Cucumis sativus) [51]. Qais et al. [52] evaluated the leaves
extract of Murraya koenigii and used it to synthesize AgNPs,
which were found to be spheroidal in shape with 520 nm
particle size. Ali et al. [53] studied that Ajuga bracteosa con-
tains antioxidant and antimicrobial phytoconstituents. In the
current research, the qualitative analysis of both ABaqu and
Ajuga bractoesa synthesized silver nanoparticles
(ABAgNPs) agree with the FTIR results, which revealed the
presence of various phytochemicals such as terpenoids, tan-
nins, flavonoids, saponins, steroids, alkaloids, free amino
acids, phenols, quinone, and glycosides. Both flavonoids and
phenolic compounds are involved in free radicals scaveng-
ing, inhibition of lipid peroxidation, and act as bio-reductants
during metal nanoparticle synthesis [54].
4.2. Oral Toxicity Effect
Globally world’s population (80%) used medicinal plants
(homeopathic remedies) for primary health care [55] because
herbal products are not dangerous, safe, and have a low toxic
effect [56]. But some traditionally used medicine showed
adverse effects on human health, so it is necessary to conduct
in vivo toxicity studies on them. Therefore, to evaluate the
toxic effect of medicinal plants, the animal model is an im-
portant tool [57]. In Pakistan rural population depends on
traditional medicines to cure health problems [58, 59]. In the
current research, A. bracteosa was used because it is being
used for the cure of urinary and gastrointestinal disorders,
fungal, bacterial, worm inflammation, diabetes, and cancer
[60, 61]. It exhibited an anti-cancerous effect in rats and car-
dio-stimulant action in animals [62].
The acute toxicity study is necessary to determine the
safer dose range to manage the clinical signs and symptoms
of the designed drugs [63]. The current results revealed that
the dose of 1000 mg/kg is not toxic. Therefore, a 400 mg/ml
dose was selected for further studies. The behavior of the
treated groups was recorded as normal due to the proper in-
take of food and water. We can say that the metabolic pro-
cesses of proteins, carbohydrates, and lipids are normal in-
side the animal bodies when treated with the ABaqu and
ABAgNPs. Our results are in agreement with the findings of
Iversen et al. [64]. They showed that these phytochemicals
play an important physiological function in the body. Current
findings revealed that no significant difference was recorded
between the body weights of control and treated groups,
which may lead to the positive effect of silver nanoparticles
and ABaqu. The findings of oral acute toxicity were agreed
with Sah et al. [65]. On the other hand, our results are incon-
sistent with Abba et al. [66], who reported the increase in
liver and kidney weight at 200 mg/kg and 600 mg/kg doses.
Many researchers examined the liver and kidneys of mice to
evaluate the toxic and safety of herbal drugs [67].
Hematological parameters screening can be used to de-
termine the harmful effect of any foreign particle including
plant extracts on blood [68]. In the current research, the he-
matological parameters (WBC, RBC, Hb, Hematocrit, Plate-
lets, Eosinophils, Neutrophils, Lymphocytes, Monocytes,
MCV) were analyzed and no statistically significant differ-
ence was recorded among all treated groups. Results re-
vealed that ABaqu and ABAgNPs may not possess any toxic
substances that can cause abnormalities or anemia. Our re-
sults are consistent with the findings of Loha et al. [69]. In
the current research, all biochemical parameters didn’t show
significant changes and our findings agreed with the out-
comes of Loha et al. [69]. Any change in the biochemical
parameters revealed liver damage, RBC damage, and muscle
damage [69]. The toxic effect of any metabolite or drug
damaged the organs [70]. Our results indicated that
ABAgNPs and ABaqu had no toxic substances involved in
the tissue-damaging. Our findings disagreed with the work of
Abba et al. [66], who showed the kidney tissue morphology
differences, changes in hematological parameters, and bio-
chemical parameters when treated with 400 mg/kg and 600
mg/kg. The acute oral toxicity results showed that green syn-
thesized silver nanoparticles using A. bracteosa (ABAgNPs)
and ABaqu extract had no toxicity effect in Swiss albino
mice and safe.
4.3. In Vitro Cytotoxic Studies
Brine shrimp lethality assay is a frequently used
method for the toxicological screening of natural materi-
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 13
als in the process of new drug development [71]. The pre-
vious literature reveals that the toxicity of silver nanopar-
ticles depends on the concentration [72]. Zia et al. [72]
revealed that silver nanoparticles have great potential for
protein kinase assay. ABAgNPs were a very effective
kinase inhibitor as a result of protein kinase inhibition.
The allosteric binding potential of an extract to the kinase
(active or inactive sites) might lead to the development of a
new tool for chemo-preventive measures [73].
The previous
studies reported that protein kinases participated in cell
differentiation and proliferation, apoptosis, and various
metabolic processes. Thus, it would be possible that the
inhibitory activities of silver nanoparticles on protein
kinases might be clinically beneficial for the treatment
of cancer [74].
4.4. Anti-diabetic Effect
Diabetes mellitus is the most common and widespread
disease, characterized by hyperglycemia and metabolic
changes of proteins, carbohydrates, and lipids [75]. This dis-
ease is associated with various organs (brain, heart, kidney,
pancreas, intestine) and disorders (renal, cardiovascular, eye,
and neurological) in the long term and with a variety of other
symptoms like weight loss, blurred vision, increase in blood
and urine sugar delayed wound healing, fatigue, and polyu-
ria, etc. [76, 77]. It was observed that hyperglycemia activity
elevated several mechanisms in different organs such as in
intestine/gut (glucose absorption, α-glucosidase, and amyl-
ase activity), adipocyte (lipolysis , and lipid peroxidation
products ), liver (glycogen breakdown , triacylglycerols ,
gluconeogenesis ), kidney (generation of free radicals ,
oxidative stress ), and pancreatic cells (early insulin secre-
tion ), respectively (Fig. 6) while some mechanisms in skel-
etal muscle and kidney were demoted (glucose uptake ,
glucose utilization , glycogenesis , and antioxidant en-
zymes ). In the current research, all hyperglycemic activi-
ties were shown by Al-induced diabetic mice, affected the
body weight, glucose level, liver functional markers, renal
functional markers, and hematological parameters in Al-
induced diabetic mice. It was observed that body weight,
RBC, Hb, MCV, and lymphocyte were reduced while ALT,
AST, ALP, urea, creatinine, uric acid, WBC, hematocrit,
platelets, neutrophils, eosinophils were increased in Al-
induced diabetic mice (Fig. 6). These parameters impaired
the kidney, brain, pancreas, intestine, heart, and liver, and
causes various disorders (Fig. 6). Our results are agreed with
the previous literature [76, 77].
The treatments include exercise, medication, and proper
diet but effective diabetes treatment is the use of hypergly-
cemic drugs and the use of insulin [78]. Nowadays. medici-
nal plants are being used for the treatment of various chronic
diseases [79, 80]. Previous research illustrated the use of
medicinal plants for the prevention and treatment of diabetes,
which may be considered as safe therapy compared to con-
ventional therapy [81, 82]. Several parts of medicinal plants
such as seed, roots, flower, and aerial parts have been used
for diabetic treatment [83]. Many herbal plants have been
used in anti-diabetic remedies i.e. Acv acia arabica, Bam-
busa arundinasia, Boswellia carterii, Coriandrum sativum,
Glycyrrhiza glabra, Myrtus communis, Rosa damascene,
Oxalis corniculata, Portulaca oleracea, Punica granatum,
Vitis vinifera, and Rosa canina [83-88]. Researchers used
Al-induced and streptozotocin-induced diabetic mice to evaluate
the anti-diabetic effect of medicinal plants [86, 88-90]. In the cur-
rent research, the whole plant (aerial parts and roots) of Aju-
ga bracteosa was used to screen the anti-diabetic activity in
Al-induced diabetic mice.
Now a days, silver nanoparticles are being used in the
field of medicine and exploring as anti-diabetic, anti-
oxidative, anti-inflammatory, anti-coagulant, cytotoxic, and
antimicrobial activities [91-93]. Current research agreed with
the outcomes of Das et al. [94], who reported that silver na-
noparticles synthesized using Ananas comosus showed high
activity against α-glucosidase activity and indicated a posi-
tive role in diabetes treatment. Similar findings were shown
by Rajaram et al. [95], Abideen and VijayaSankar, [96], and
Sengottaiyan et al. [97]. They showed the anti-diabetic effect
of green synthesized AgNO3 using Tephrosia tinctoria, sea-
weeds, and Solanum nigrum. It was observed that medicinal
plants play an important role in the treatment of diabetes
(hypoglycemic activity) through several mechanisms, such
as regeneration of pancreatic tissue, improvement of serum
Fig. (6). Hyperglycemia actions in alloxan-induced diabetic mice, impact on liver and kidney functional markers, hematology, and various
organs. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
14 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
insulin, and Langerhans islets, improvement of antioxidant
function, hepatocyte cells, pancreas tissue, and kidney struc-
tures, suppression of apoptosis of peri-insular cells, inhibit
degenerative changes in the beta cells of the pancreas, pene-
tration of lymphocytes into pancreatic islets, an inflammato-
ry cytokine, elevating serum insulin secretion, glucose oxida-
tion, enhancement of the glucose uptake level, glycogenesis,
with reduction of total cholesterol, triglyceride, and low-
density lipoprotein cholesterol, improving cardio-protective
atherosclerotic and indices, improvement of antioxidant en-
zyme (superoxide dismutase), and total antioxidant capacity
as well as suppression of oxidative stress markers [83-88].
In the current research, ABAgNPs using A. bracteosa
and its ABaqu extract showed a positive hypoglycemic effect
in Al-induced diabetic mice (Fig. 6) due to the presence of
phytochemical constituents. As we know that ABAgNPs and
ABaqu have alkaloids, flavonoids, glycosides, carbohy-
drates, tannins, phenols, amino acids, and saponins. These
phytochemicals possess a significant hypoglycemic effect.
Our findings agreed with Hsu et al. [98]. A. bracteosa pos-
sessed antioxidant activities that can reduce the diabetic ef-
fect. Our results agreed with the previous literature [99, 100].
In the present study, we observed that the phytochemicals
found in ABAgNPs and ABaqu have a great impact on bod-
yweight, liver functional markers, renal functional markers,
and hematological parameters. It was observed that
ABAgNPs and ABaqu increased the body weight, RBC, Hb,
MCV, and lymphocytes in Al-induced diabetic mice while
reduced the level of ALT, AST, ALP, urea, creatinine, uric
acid, WBC, platelets, hematocrits, neutrophils, eosinophils,
and blood glucose level in Al-induced diabetic mice (Fig. 6).
Treatment with AgNPs extract produced a highly significant
increase in the blood hemoglobin of diabetic animals [101].
All these parameters involved in the improvement and en-
hancement of various mechanisms such as glucose oxidation
, glucose uptake , β cell protection , glycolysis , α-
glucosidase inhibition activity , α-amylase inhibition activi-
ty , adipogenesis , cholesterol , glucose absorption ,
gluconeogenesis , and blood glucose , respectively (Fig. 6)
in Al-induced diabetic mice. These processes reduced the
level of organ damage, symptoms, and reduce the occurrence
of various disorders associated with diabetes (Fig. 6).
4.6. Anti-cancerous Effect
The biological activities of the liver including bacteria
destruction in blood, bile acid secretion, detoxification, and
blood clotting factor generation are very complex. Various
factors such as drugs, microbes, xenobiotic compounds, and
metabolites in the liver) triggered liver injury [102]. Carbon
tetrachloride (CCl4) is one of the most common hepato-toxic
agents and is regularly used in the hepato-protective assay in
which toxic free radicals are produced [103]. CCl4 induced
hepatoxicity is caused by oxidative stress. Two mechanisms
are involved in the hepatotoxicity caused by CCl4 (Fig. 7).
When CCl4 enters the body, the biotransformation process
starts with the help of the CYP2E1 oxidase system (cyto-
chrome P450), involved in the metabolism of synthetic com-
pounds to generate CCl3*, CHCl2*, and CCl3OO- *under a low
partial pressure of oxygen and high partial pressure of oxy-
gen, as a result, causing apoptosis and steatosis or fatty liver
[104, 105] (Fig. 5). The findings of current research agreed
with Kiezcka and Kappus [105] that CCl4-induced liver inju-
ry in an animal model and caused oxidative stress, promoting
lipid peroxidation and damaging hepatocellular membrane.
Various researchers reported that both increased enzyme
activity and cell membrane permeability contribute to hepat-
ic structural injury [106]. In the first mechanism, we can see
that CCl3OO- * accepts the protons from unsaturated fatty
acids found in the membrane and deactivates the anti-
oxidative enzymes, and caused lipid peroxidation. Due to the
inhibition of anti-oxidative enzymes, the accumulation of
O2.-and H2O2 occurred causing hepatic injury and formation
of free radicals. On the other hand, various parts of the liver,
such as Kupfer cells, endothelial cells, and sinusoidal cells
were affected by the exposure of CCl4. Kupfer cells secrete a
number of cytokines (TNF-α, IL-1, IL-6, IL-8), chemokines
(KC/GRO, IP-109, MIP-2, MCP-1), and pro-inflammatory
mediators like NO, which initiates hepatic inflammation and
toxicity (Fig. 7). Similar characteristics were observed in the
current research. Histopathology of the liver indicated the
aggregations of Kupfer cells, which indicates the inflamma-
tion of the liver, loss of endothelial cells, and sinusoidal cells
due to activation of cytokines, chemokines, and NO because
they are involved in the pathogenesis. These mediators cause
Lipid peroxidation, Inflammation, Apoptosis, Steatosis, and
Fatty liver, lead to cause hepatic injury (Fig. 7). Overall, we
can say that CCl4 induced hepatotoxicity due to lipid peroxi-
dation, deactivation of anti-oxidative enzymes, and genera-
tion of free radicals [107, 108]. The liver injury had a great
impact on the liver functional markers, and hematological
parameters. We observed that RBC, Hb, platelets, lympho-
cytes are declined in CCl4 induced mice while the levels of
neutrophils, monocytes, eosinophils, and WBC were elevat-
ed (Fig. 7).
In the current research, CCl4 induced mice showed a re-
duction in body and organ weight compared to control. Our
results are agreed with Meena and Paulraj [109] and Dutta et
al. [110]. On the other hand, CCl4 induced mice treated with
ABAgNPs and ABaqu extract showed significantly in-
creased body weight compared to CCl4 induced mice and
agreed with the findings of Shah et al. [111]. They showed
that C. nudiflora and Croton bonplandianus Baill. increased
the body and organ weight when administered in CCl4 in-
duced mice. The liver biomarkers ALT, ALP, AST were
elevated in CCl4 induced mice, while the declined level was
found in CCl4 induced mice when treated with ABAgNPs
and ABaqu extract, which indicating the maintenance of cell
integrity and stabilization of structures and membranes. His-
topathology of the liver supported the current findings. Cur-
rent research indicated the hepato-protective effect of
ABAgNPs and ABaqu extract. Silver nanoparticles and
ABaqu possessed phytochemical constituents that blocked
the production of cytokines, chemokines, and inflammatory
mediators, and ultimately prevented CCl4-induced hepatic
injury, lipid peroxidation, deactivation of anti-oxidative en-
zymes, and generation of free radicals. Similar results were
made by Mandal et al. [112] and Jeyachandran et al. [113].
They showed the anticancer activity of Anisomeles mala-
barica and Solidago microglossa, respectively.
The results of the current research revealed the potential
hepato-protective activity of green synthesized silver nano-
particles ABAgNPs using A. bracteosa and ABaqu extract in
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 15
CCl4-induced Swiss albino mice. This protective effect was
due to the presence of potent bioactive compounds (flavo-
noids, phenols, saponins, tannins, alkaloids, glycosides, etc)
in the A. bracteosa. These phytochemical constituents, most-
ly antioxidants have a positive impact on the reduction of
chronic diseases. Our findings agreed with Dai and Mum-
mer, [114]. In the current research, the hepato-protective
potential of ABAgNPs and ABaqu extract was first investi-
gated by suppressing the CCl4-induced oxidative stress and
inflammation in the livers of CCl4-induced Swiss albino
mice and morphological changes attenuated by CCl4. Similar
results were shown by various researchers [115-118]. They
showed the presence of various bioactive materials in medic-
inal plants with remarkable biological activities such as anti-
oxidant, anti-inflammatory, antibacterial, antiseptic, and anti-
cancerous. Based on the previous literature, we can say that
natural sources, especially medicinal plants possess anti-
cancerous compounds that play an important role in liver
protection via free radical’s neutralization.
CONCLUSION
From the whole research work, it was concluded that
biogenic synthesized ABAgNPs could play an important role
in the field of bio-nanomedicine due to the presence of phy-
toconstituents such as flavonoids, phenols, carbohydrates,
and amino acids. ABAgNPs and ABaqu could be used as a
potential therapeutic agent for drug formulation against can-
cer, and diabetic diseases. In future studies, the impact of
various physicochemical parameters such as pH, tempera-
ture, polymer modification using different poly-
mers/chemicals on the stability of ABAgNPs should be ob-
served The imaging and bio-distribution of ABAgNPs in
animal models should be screened for the development of a
potential therapeutic drug that could be effective against both
infectious and non-infectious diseases.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
The manuscript has been read and approved by all the
authors and that the criteria for authorship have been met.
HUMAN AND ANIMAL RIGHTS
All experiments have been designed to avoid distress,
unnecessary pain, and suffering to the experimental animals.
All procedures were conducted following international regu-
lations referred to as Wet op de dierproeven (Article 9) of
Dutch Law.
CONSENT FOR PUBLICATION
The approval for publication of this article has been taken
from all the authors.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings
of this research are available within the article.
FUNDING
No
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
The authors are grateful to the Department of Physics,
University of Azad Jammu and Kashmir, Muzaffarabad and
National Institute for Lasers and Optronics (NILOP), Paki-
stan Atomic Energy Commission, Islamabad, Pakistan, for
providing research facilities
Fig. (7). Schematic demonstration showing the mechanisms involved in CCl4-induced liver injury and hepato-protective effect of green syn-
thesized silver nanoparticles ABAgNPs and ABaqu extract. (A higher resolution / colour version of this figure is available in the electronic
copy of the article).
16 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
REFERENCES
[1] Bhuvaneshwari, J.; Khanam, S.; Devi, K. In-vitro enzyme inhibi-
tion studies for antidiabetic activity of mature and tender leaves of
Mangifera indica var. Totapuri. Res. Rev. J. Microbiol. Biotech-
nol, 2014, 3, 36-41.
[2] Guariguata, L.; Whiting, D.R.; Hambleton, I.; Beagley, J.; Lin-
nenkamp, U.; Shaw, J.E. Global estimates of diabetes prevalence
for 2013 and projections for 2035. Diabetes Res. Clin. Pract.,
2014, 103(2), 137-149.
http://dx.doi.org/10.1016/j.diabres.2013.11.002 PMID: 24630390
[3] Gondi, M.; Prasada Rao, U.J. Ethanol extract of mango (Man-
gifera indica L.) peel inhibits α-amylase and α-glucosidase activi-
ties, and ameliorates diabetes related biochemical parameters
in streptozotocin (STZ)-induced diabetic rats. J. Food Sci. Tech-
nol., 2015, 52(12), 7883-7893.
http://dx.doi.org/10.1007/s13197-015-1963-4 PMID: 26604360
[4] Singh, D.; Jain, M.; Upadhyay, K.; Khandelwal, N.; Verma, H.N.
Green synthesis of silver nanoparticle using Argemone Mexicana
leaf extract and evaluation of their antimicrobial activities. Dig. J.
Nanomater. Biostruct., 2011, 2(5), 483-489.
[5] Zhao, Y.; Jiang, Z.; Guo, C. New hope for type 2 diabetics: target-
ing insulin resistance through the immune modulation of stem
cells. Autoimmun. Rev., 2011, 11(2), 137-142.
http://dx.doi.org/10.1016/j.autrev.2011.09.003 PMID: 21964164
[6] Sanghera, D.K.; Blackett, P.R. Type 2 diabetes genetics: beyond
GWAS. J. Diabetes. Metabol, 2012, 3(198), 6948.
http://dx.doi.org/10.4172/2155-6156.1000198
[7] Zatalia, S.R.; Sanusi, H. The role of antioxidants in the pathophys-
iology, complications, and management of diabetes mellitus. Acta
Med. Indones., 2013, 45(2), 141-147.
PMID: 23770795
[8] El-Amrani, F.; Rhallab, A.; Alaoui, T.; El-Badaoui, K.; Chaki, S.
Hypoglycaemic effect of Thymelaea hirsuta in normal and strep-
tozotocin-induced diabetic rats. J. Med. Plants Res., 2009, 3(9),
625-629.
[9] Lee, S.J.; Yook, S.; Yhee, J.Y.; Yoon, H.Y.; Kim, M.G.; Ku, S.H.;
Kim, S.H.; Park, J.H.; Jeong, J.H.; Kwon, I.C.; Lee, S.; Lee, H.;
Kim, K. Co-delivery of VEGF and Bcl-2 dual-targeted siRNA
polymer using a single nanoparticle for synergistic anti-cancer ef-
fects in vivo. J. Control. Release, 2015, 220(Pt B), 631-641.
http://dx.doi.org/10.1016/j.jconrel.2015.08.032 PMID: 26307351
[10] Fujita, K.; Iwama, H.; Miyoshi, H.; Tani, J.; Oura, K.; Tadokoro,
T.; Sakamoto, T.; Nomura, T.; Morishita, A.; Yoneyama, H.; Ma-
saki, T. Diabetes mellitus and metformin in hepatocellular carci-
noma. World J. Gastroenterol., 2016, 22(27), 6100-6113.
http://dx.doi.org/10.3748/wjg.v22.i27.6100 PMID: 27468203
[11] Kralj, D.; Virović Jukić, L.; Stojsavljević, S.; Duvnjak, M.;
Smolić, M.; Čurčić, I.B. Hepatitis C virus, insulin resistance, and
steatosis. J. Clin. Transl. Hepatol., 2016, 4(1), 66-75.
http://dx.doi.org/10.14218/JCTH.2015.00051 PMID: 27047774
[12] Klil-Drori, A.J.; Azoulay, L.; Pollak, M.N. Cancer, obesity, diabe-
tes, and antidiabetic drugs: is the fog clearing? Nat. Rev. Clin. On-
col., 2017, 14(2), 85-99.
http://dx.doi.org/10.1038/nrclinonc.2016.120 PMID: 27502359
[13] Mantovani, A.; Targher, G. Type 2 diabetes mellitus and risk of
hepatocellular carcinoma: spotlight on nonalcoholic fatty liver
disease. Ann. Transl. Med., 2017, 5(13), 270.
http://dx.doi.org/10.21037/atm.2017.04.41 PMID: 28758096
[14] Bosch, F.X.; Ribes, J.; Diaz, M.; Cleries, R. Primary liver cancer.:
‘Worldwide incidence and trends. Gastroenterol., 2004, 127, 5-
16.
http://dx.doi.org/10.1053/j.gastro.2004.09.011
[15] Hiotis, S.P.; Rahbari, N.N.; Villanueva, G.A.; Klegar, E.; Luan,
W.; Wang, Q.; Yee, H.T. Hepatitis B vs. hepatitis C infection on
viral hepatitis-associated hepatocellular carcinoma. BMC Gastro-
enterol., 2012, 12(10), 64.
http://dx.doi.org/10.1186/1471-230X-12-64 PMID: 22681852
[16] Farazi, P.A.; DePinho, R.A. Hepatocellular carcinoma pathogene-
sis: from genes to environment. Nat. Rev. Cancer, 2006, 6(9),
674-687.
http://dx.doi.org/10.1038/nrc1934 PMID: 16929323
[17] Nazeema, T.H.; Suganya, P.K. Synthesis and characterization of
silver nanoparticle form two medicinal plants and its anticancer
property. Int. J. Res Eng. Tech., 2014, 2, 49-56.
[18] Kalaydina, R.V.; Bajwa, K.; Qorri, B.; Decarlo, A.; Szewczuk,
M.R. Recent advances in “smart” delivery systems for extended
drug release in cancer therapy. Int. J. Nanomedicine, 2018, 13,
4727-4745.
http://dx.doi.org/10.2147/IJN.S168053 PMID: 30154657
[19] Jeyaraj, M.; Rajesh, M.; Arun, R.; MubarakAli, D.; Sathishkumar,
G.; Sivanandhan, G.; Dev, G.K.; Manickavasagam, M.; Premku-
mar, K.; Thajuddin, N.; Ganapathi, A. An investigation on the cy-
totoxicity and caspase-mediated apoptotic effect of biologically
synthesized silver nanoparticles using Podophyllum hexandrum
on human cervical carcinoma cells. Colloids Sur, B, 2013, 102,
708-717.
[20] Saratale, G.D.; Saratale, R.G.; Benelli Kumar, G.A.; Pugazhendhi,
D.S.; Kim, H.S. Anti-diabetic potential of silver nanoparticles
synthesized with Argyreia nervosa leaf extract high synergistic
antibacterial activity with standard antibiotics against foodborne
bacteria. J. Cluster Sci., 2017, 28(3), 1709-1727.
http://dx.doi.org/10.1007/s10876-017-1179-z
[21] Salem, S.S.; Fouda, A. Green Synthesis of Metallic Nanoparticles
and Their Prospective Biotechnological Applications: an Over-
view. Biol. Trace Elem. Res., 2021, 199(1), 344-370.
http://dx.doi.org/10.1007/s12011-020-02138-3 PMID: 32377944
[22] Silva, S.; Costa, E.M.; Costa, M.R.; Pereira, M.F.; Pereira, J.O.;
Soares, J.C.; Pintado, M.M. Aqueous extracts of Vaccinium co-
rymbosum as inhibitors of Staphylococcus aureus. Food Control,
2015, 51, 314-320.
http://dx.doi.org/10.1016/j.foodcont.2014.11.040
[23] Patra, J.K.; Baek, K.H. Antibacterial activity and synergistic anti-
bacterial potential of biosynthesized silver nanoparticles against
foodborne pathogenic bacteria along with its anticandidal and an-
tioxidant effects’. Front. Microbiol., 2017, 8, 167.
http://dx.doi.org/10.3389/fmicb.2017.00167 PMID: 28261161
[24] Castro, A.; Coll, J.; Arfan, M. neo-Clerodane diterpenoids from
Ajuga bracteosa. J. Nat. Prod., 2011, 74(5), 1036-1041.
http://dx.doi.org/10.1021/np100929u PMID: 21539300
[25] Nazer, S.; Andleeb, S.; Ali, S.; Gulzar, N.; Iqbal, T.; Khan,
M.A.R.; Raza, A. Synergistic Antibacterial Efficacy of Biogenic
Synthesized Silver Nanoparticles using Ajuga bractosa with
Standard Antibiotics: A Study Against Bacterial Pathogens. Curr.
Pharm. Biotechnol., 2020, 21(3), 206-218.
http://dx.doi.org/10.2174/1389201020666191001123219 PMID:
31573882
[26] Hafeez, K.; Andleeb, S.; Ghousa, T.; Mustafa, R.G.; Naseer, A.;
Shafique, I.; Akhter, K. Phytochemical screening, alpha-
glucosidase inhibition, antibacterial and antioxidant potential of
Ajuga bracteosa Extracts’. Curr. Pharm. Biotechnol., 2017, 18(4),
336-342.
http://dx.doi.org/10.2174/1389201018666170313095033 PMID:
28294059
[27] Paudel, A. Phytochemical and biological screening of Rhododen-
dron campanulatum.. Dissertation, Nepal Tribhuvan University,
2005.
[28] Trease, G.E.; Evans, W.C. Pharmacognosy, 15th Edn.; Saunders,
2002, pp. 214-393.
[29] Parekh, J.; Chands, S. Phytochemical screening of some plants
from Western regions of India. Plant Arch., 2008, 8, 662.
[30] Zhou, K.; Yu, L. Total phenolic contents and anti-oxidant proper-
ties of commonly consumed vegetables grown in Colorado’. Food
Sci. Technol., 2006, 39, 1155-1162.
[31] Zou, Y.; Lu, Y.; Wei, D.; San Francisco, F. Antioxidant activity of
a flavonoid-rich extract of Hypericum perforatum L. in vitro. J.
Agric. Food Chem., 2004, 52(16), 5032-5039.
http://dx.doi.org/10.1021/jf049571r PMID: 15291471
[32] Fatima, H.; Khan, K.; Zia, M.; Ur-Rehman, T.; Mirza, B.; Haq,
I.U. Extraction optimization of medicinally important metabolites
from Datura innoxia Mill.: an in vitro biological and phytochemi-
cal investigation. BMC Complement. Altern. Med., 2015, 15(1),
376.
http://dx.doi.org/10.1186/s12906-015-0891-1 PMID: 26481652
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 17
[33] Bibi, G.; Ullah, N.; Mannan, A.; Mirza, B. Antitumor, cytotoxic
and antioxidant potential of Aster thomsonii extracts. Afr. J.
Pharm. Pharmacol., 2011, 5(2), 252-258.
[34] Dewi, R.T.; Iskandar, Y.M.; Hanafi, M.; Kardono, L.B.; Angelina,
M.; Dewijanti, I.D.; Banjarnahor, S.D. Inhibitory effect of koji
Aspergillus terreus on α-glucosidase activity and postprandial hy-
perglycemia. Pak. J. Biol. Sci., 2007, 10(18), 3131-3135.
http://dx.doi.org/10.3923/pjbs.2007.3131.3135 PMID: 19090111
[35] Mulisa, E.; Asres, K.; Engidawork, E. Evaluation of wound heal-
ing and anti-inflammatory activity of the rhizomes of Rumex ab-
yssinicus J. (Polygonaceae) in mice. BMC Complement. Altern.
Med., 2015, 15, 341.
http://dx.doi.org/10.1186/s12906-015-0878-y PMID: 26423525
[36] OECD. Guidelines for the Testing of Chemicals. OECD 423.
Acute Oral Toxicity Acute Toxic Class Method; Organization for
Economic Cooperation and Development: Paris, 2001.
[37] Tanquilut, N.C.; Tanquilut, M.R.C.; Estacio, M.A.C.; Torres,
E.B.; Rasario, J.C.; Reyes, B.A.S. Hypoglycemic effect of Lager-
stroemia speciosa (L.) Pers. on alloxan-induced diabetic mice N.
C. J. Med. Plants Res., 2009, 3(12), 1066-1071.
[38] Arockia, J.; Paul, J. KarunaiSelvi, B.; Karmegam, N. Biosynthesis
of silver nanoparticles from Premna serratifolia L. leaf and its an-
ticancer activity in CCl4-induced hepato-cancerous Swiss albino
mice’. Appl. Nanosci., 2015, 5, 937-944.
http://dx.doi.org/10.1007/s13204-014-0397-z
[39] Ahmed, S.; Mudasir, S.U.; Babu, A.; Sawami, L.; Ikram, S. Green
synthesis of silver nanoparticles using Azadiranchta indica aque-
ous leaf extract. J. Radiat. Res. Appl. Sci., 2016, 9(1), 1-7.
http://dx.doi.org/10.1016/j.jrras.2015.06.006
[40] Nahar, K.; Aziz, S.; Bashar, M.S.; Haque, M.A.; Al-Reza, S.M.
Synthesis and characterization of Silver nanoparticles from Cin-
namomum tamala leaf extract and its antibacterial potential. Int. J.
Nanodimens., 2020, 11(1), 88-98.
[41] Ibrahim, H.M. Green synthesis and characterization of silver
nanoparticles using banana peel extract and their antimicrobial ac-
tivity against representative microorganisms. J. Radiat. Res. Appl.
Sci., 2015, 8(3), 265-275.
http://dx.doi.org/10.1016/j.jrras.2015.01.007
[42] Zaheer, Z.; Rafiuddin, Silver nanoparticles to self-assembled
films: green synthesis and characterization. Colloids Surf. B Bio-
interfaces, 2012, 90, 48-52.
http://dx.doi.org/10.1016/j.colsurfb.2011.09.037 PMID: 22055624
[43] Bonde, S.R.; Rathod, D.P.; Ingle, A.P.; Ade, R.B.; Gade, A.K.;
Rai, M.K. Murraya koenigiimediated synthesis of silver nano-
particles and its activity against three human pathogenic bacteria.
Nanosci. Methods, 2012, 1, 25-36.
http://dx.doi.org/10.1080/17458080.2010.529172
[44] Mallikarjun, K.; Narsimha, G.; Dillip, G.; Praveen, B.; Shreedhar,
B.; Lakshmi, S. Green synthesis of silver nanoparticles using
Ocimum leaf extract and their characterization. Dig. J. Nano-
mater. Biostruct., 2011, 6, 181-186.
[45] Almalah, H.I.; Alzahrani, H.A.; Abdelkader, H. Green Synthesis
of Silver Nanoparticles using Cinnamomum zylinicum and their
synergistic effect against Multi-DrugResistance Bacteria. J. Nano-
technol. Res., 2019, 1(3), 095-107.
[46] Ashok, K.D. Rapid and green synthesis of silver nanoparticles
using the leaf extracts of Parthenium hysterophorus: a novel bio-
logical approach. Int. Res. J. Pharmacol., 2012, 3(2), 169-171.
[47] Dada, A.O.; Adekola, F.A.; Odebunmi, E.O. Kinetics and equilib-
rium models for sorption of Cu(II) onto a novel manganese nano-
adsorbent. J. Dispers. Sci. Technol., 2016, 37(1), 119-133.
http://dx.doi.org/10.1080/01932691.2015.1034361
[48] Dada, A.O.; Ojediran, O.J.; Dada, F.E.; Olalekan, A.P.; Awakan,
O.J. Green synthesis and characterization of silver nanoparticles
using Calotropis Procera extract. J. Appl. Chem. Sci. Interface.,
2017, 8(4), 137-143.
[49] Femi-Adepoju, A.G.; Dada, A.O.; Otun, K.O.; Adepoju, A.O.;
Fatoba, O.P. Green synthesis of silver nanoparticles using terres-
trial fern (Gleichenia Pectinata (Willd.) C. Presl.): characteriza-
tion and antimicrobial studies. Heliyon, 2019, 5(4), e01543.
http://dx.doi.org/10.1016/j.heliyon.2019.e01543 PMID: 31049445
[50] Jyoti, M.; Baunthiyal, M.; Singh, A. Characterization of silver
nanoparticles synthesized using Urtica dioica Linn.leaves and
their synergistic effects with antibiotics. J. Radiat. Res. Appl. Sci.,
2016, 9, 217-227.
http://dx.doi.org/10.1016/j.jrras.2015.10.002
[51] Roy, N.; Gaur, A.; Jain, A.; Bhattacharya, S.; Rani, V. Green
synthesis of silver nanoparticles: an approach to overcome toxici-
ty. Environ. Toxicol. Pharmacol., 2013, 36(3), 807-812.
http://dx.doi.org/10.1016/j.etap.2013.07.005 PMID: 23958974
[52] Qais, F.A.; Shafiq, A.; Khan, H.M.; Husain, F.M.; Khan, R.A.;
Alenazi, B.; Alsalme, A.; Ahmad, I. Antibacterial Effect of Silver
Nanoparticles Synthesized Using Murraya koenigii (L.) against
Multidrug-Resistant Pathogens.Bioorganic Chem. Appl; , 2019, p.
4649506.
[53] Ali, T.; Naqash, A.; Wadoo, R.; Rashid, R.; Bader, G.N. Antimi-
crobial potential and determination of total phenolic and flavonoid
content of aerial part extracts of Ajuga bracteosa Wall ex. Benth.
Pharm. Communi., 2018, 8(3), 114-118.
http://dx.doi.org/10.5530/pc.2018.3.24
[54] Makarov, V.V.; Love, A.J.; Sinitsyna, O.V.; Makarova, S.S.;
Yaminsky, I.V.; Taliansky, M.E.; Kalinina, N.O. “Green” nano-
technologies: synthesis of metal nanoparticles using plants. Acta
Naturae, 2014, 6(1), 35-44.
http://dx.doi.org/10.32607/20758251-2014-6-1-35-44 PMID:
24772325
[55] Ekor, M. The growing use of herbal medicines: issues relating to
adverse reactions and challenges in monitoring safety. Front.
Pharmacol., 2014, 4, 177.
http://dx.doi.org/10.3389/fphar.2013.00177 PMID: 24454289
[56] Ibrahim, M.B.; Sowemimo, A.A.; Sofidiya, M.O.; Badmos, K.B.;
Fageyinbo, M.S.; Abdulkareem, F.B.; Odukoya, O.A. Sub-acute
and chronic toxicity profiles of Markhamia tomentosa ethanolic
leaf extract in rats. J. Ethnopharmacol., 2016, 193, 68-75.
http://dx.doi.org/10.1016/j.jep.2016.07.036 PMID: 27426507
[57] Ugwah-Oguejiofor, C.J.; Abubakar, K.; Ugwah, M.O.; Njan, A.A.
Evaluation of the antinociceptive and anti-inflammatory effect of
Caralluma dalzielii. J. Ethnopharmacol., 2013, 150(3), 967-972.
http://dx.doi.org/10.1016/j.jep.2013.09.049 PMID: 24140204
[58] Hussain, S.; Malik, F.; Mahmood, S. Review: an exposition of
medicinal preponderance of Moringa oleifera (Lank.). Pak. J.
Pharm. Sci., 2014, 27(2), 397-403.
PMID: 24577932
[59] Jan, S.A.; Khan, Z.; Zeb, S.A.; Khalil, A.T. Shah. S.H. Ethnobot-
any and Research Trends in Trachyspermum ammi L. (Ajowan);
A Popular Folklore Remedy. Am.-Eurasian J. Agric. Environ.
Sci., 2015, 15(1), 68-73.
[60] Akriti, P.; Jadona, M.; Katarea, Y.K.; Singoura, P.K.; Rajakb, H.;
Chaurasiyaa, P.K.; Patila, U.K. Pawara. R.S. Ajuga bracteosa
wall: A review on its ethnopharmacological and phytochemical
studies. Pelagia Research Library. Pharm. Sin., 2011, 2(2), 1-10.
[61] Abhishek, V.; Kaur, S.P. Chemical investigation of medicinal
plant Ajuga bracteosa. J. Nat. Prod. Plant Res., 2011, 1(1), 37-45.
[62] Ghufran, M.S.; Qureshi, R.A.; Batool, A.; Kondratyuk, T.P.;
Guilford, J.M.; Marler, L.E. Evaluation of selected indigenous
medicinal plants from the western Himalayas for cytotoxicity and
as potential cancer chemopreventive agents. Pharm. Biol., 2009,
47, 533-538.
http://dx.doi.org/10.1080/13880200902873847
[63] Saleem, U.; Ahmad, B.; Ahmad, M.; Erum, A.; Hussain, K.; Irfan
Bukhari, N. Is folklore use of Euphorbia helioscopia devoid of
toxic effects? Drug Chem. Toxicol., 2016, 39(2), 233-237.
http://dx.doi.org/10.3109/01480545.2015.1092040 PMID:
26453021
[64] Iversen, P.O.; Nicolaysen, G. Waterfor life, Tidsskrift for Den
Norske Laegeforening: tidsskrift for praktisk medicin. Ny Raekke,
2003, 123(23), 3402-3405.
[65] Sah, S.; Bala, N.; Basu, R.; Das, S. Acute Toxicity Study of Silver
Nanoparticle Coupled with Euphorbia thymifolia’. J. Nanosci.
Technol., 2018, 4, 412-414.
http://dx.doi.org/10.30799/jnst.125.18040402
[66] Abba, S.; Omotoso, O.D.; Joseph, M.I. Hemorrhagic centrolobar
necrosis and cytoplasmic vacuolation of the hepatocytes in syzyg-
ium guineense chronic treated mice. Int. J. Anat. Appl. Physiol.,
2018, 4(4), 99-102.
18 Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 Nazer et al.
[67] Satyapal, U.S.; Kadam, V.J.; Ghosh, R. Hepatoprotective activity
of livobond a polyherbal formulation against CCl4 induced hepa-
totoxicity in rats. Int. J. Pharmacol., 2008, 4(6), 472-476.
http://dx.doi.org/10.3923/ijp.2008.472.476
[68] Yakubu, A.; Adua, M.M.; Adamude, H. Welfare and hematologi-
cal indices of weaner rabbits as affected by stocking density. Pro-
ceedings of the 9th World rabbit congress, Verona, Italy2008.
[69] Loha, M.; Mulu, A.; Abay, S.M.; Ergete, W.; Geleta, B. Acute
and subacute toxicity of methanol extract of Syzygium guineense
leaves on the histology of the liver and kidney and biochemical
compositions of blood in rats. Evid. Based Complement. Alternat.
Med., 2019, 2019, 5702159.
http://dx.doi.org/10.1155/2019/5702159 PMID: 30956682
[70] Debelo, N.; Afework, M.; Debella, A.; Debella, A.; Makonnen,
E.; Ergete, W.; Geleta, B. Assessment of hematological, biochem-
ical and histopathological effects of acute and sub-chronic admin-
istration of the aqueous leaves extract of Thymus schimperi in rats.
J. Clin. Toxicol., 2016, 6(286), 2161-0495.
http://dx.doi.org/10.4172/2161-0495.1000286
[71] Sangian, H.; Faramarzi, H.; Yazdinezhad, A.; Mousavi, S.J.; Za-
mani, Z.; Noubarani, M.; Ramazani, A. Antiplasmodial activity of
ethanolic extracts of some selected medicinal plants from the
northwest of Iran. Parasitol. Res., 2013, 112(11), 3697-3701.
http://dx.doi.org/10.1007/s00436-013-3555-4 PMID: 23922204
[72] Zia, G.; Sadia, H.; Nazir, S.; Ejaz, K.; Ali, S.; Ihsan-Ul-Haq, ;
Iqbal, T.; Khan, M.A.R.; Raza, A.; Andleeb, S. Ihsan-ul-Haq, Iq-
bal, T.; Khan M.A.R.; Raza A.; Andleeb, S. In vitro Studies on
cytotoxic, DNA protecting, antibiofilm and antibacterial effects of
biogenic silver nanoparticles prepared with Bergenia ciliata rhi-
zome extract’. Curr. Pharm. Biotechnol., 2018, 19(1), 68-78.
http://dx.doi.org/10.2174/1389201019666180417160049 PMID:
29667550
[73] Smyth, L.A.; Collins, I. Measuring and interpreting the selectivity
of protein kinase inhibitors. J. Chem. Biol., 2009, 2(3), 131-151.
http://dx.doi.org/10.1007/s12154-009-0023-9 PMID: 19568781
[74] Yao, J.C.; Shah, M.H.; Ito, T.; Bohas, C.L.; Wolin, E.M.; Van
Cutsem, E.; Hobday, T.J.; Okusaka, T.; Capdevila, J.; de Vries,
E.G.; Tomassetti, P.; Pavel, M.E.; Hoosen, S.; Haas, T.; Lincy, J.;
Lebwohl, D.; Öberg, K. RAD001 in Advanced Neuroendocrine
Tumors, Third Trial (RADIANT-3) Study Group. Everolimus for
advanced pancreatic neuroendocrine tumors. N. Engl. J. Med.,
2011, 364(6), 514-523.
http://dx.doi.org/10.1056/NEJMoa1009290 PMID: 21306238
[75] Alvin, C.P.; David Allesio, D.D. Endocrine pancreas and pharma-
cotherapy of diabetes mellitus and hypoglycaemia.Goodman and
Gilman’s the Pharmacological Basis of Therapeutics, 12th ed;
Brunton, L.; Chabner, B.; Knollman, B., Eds.; McGraw-Hill: New
York, 2011, p. 1237.
[76] Islam, D.; Huque, A. Sheuly, Mohanta, L.C.; Das, S.K.; Sultana,
A.; Lipy, E.P.; Prodhan, U.K. Hypoglycemic and hypolipidemic
effects of Nelumbo nucifera flower in Long-Evans rats. J. Herb-
med Pharmacol., 2018, 7, 148-154.
http://dx.doi.org/10.15171/jhp.2018.25
[77] Maideen, N.M.P.; Balasubramaniam, R. Pharmacologically rele-
vant drug interactions of sulfonylurea antidiabetics with common
herbs. J. Herb med. Pharmacol., 2018, 7, 200-210.
[78] Azizi, F.; Hatami, H.; Janghorbani, M. Epidemiology and control
of common disease in Iranian Tehran; Eshtiagh Pres, 2007.
[79] Bahramsoltani, R.; Sodagari, H.R.; Farzaei, M.H.; Abdolghaffari,
A.H.; Gooshe, M.; Rezaei, N. The preventive and therapeutic po-
tential of natural polyphenols on influenza. Expert Rev. Anti In-
fect. Ther., 2016, 14(1), 57-80.
http://dx.doi.org/10.1586/14787210.2016.1120670 PMID:
26567957
[80] Asadi-Samani, M.; Bagheri, N.; Rafieian-Kopaei, M.; Shirzad, H.
Inhibition of Th1 and Th17 cells by medicinal plants and their de-
rivatives: a systematic review. Phytother. Res., 2017, 31(8), 1128-
1139.
http://dx.doi.org/10.1002/ptr.5837 PMID: 28568565
[81] Mina, C.N.; Farzaei, M.H.; Gholamreza, A. Medicinal properties
of Peganum harmala L. in traditional Iranian medicine and mod-
ern phytotherapy: a review. J. Tradit. Chin. Med., 2015, 35(1),
104-109.
http://dx.doi.org/10.1016/S0254-6272(15)30016-9 PMID:
25842736
[82] Farzaei, M.H.; Bahramsoltani, R.; Abbasabadi, Z.; Rahimi, R. A
comprehensive review on phytochemical and pharmacological as-
pects of Elaeagnus angustifolia L. J. Pharm. Pharmacol., 2015,
67(11), 1467-1480.
http://dx.doi.org/10.1111/jphp.12442 PMID: 26076872
[83] Farzaei, F.; Morovati, M.R.; Farjadmand, F.; Farzaei, M.H. A
Mechanistic Review on Medicinal plants used for diabetes melli-
tus in traditional Persian medicine. J. Evid. Based Complementary
Altern. Med., 2017, 22(4), 944-955.
http://dx.doi.org/10.1177/2156587216686461 PMID: 29228789
[84] El-Sayed, M.I. Effects of Portulaca oleracea L. seeds in treatment
of type-2 diabetes mellitus patients as adjunctive and alternative
therapy. J. Ethnopharmacol., 2011, 137(1), 643-651.
http://dx.doi.org/10.1016/j.jep.2011.06.020 PMID: 21718775
[85] Pourghassem-Gargari, B.; Abedini, S.; Babaei, H.; Aliasgarzadeh,
A.; Pourabdollahi, P. Effect of supplementation with grape seed
(Vitis vinifera) extract on antioxidant status and lipid peroxidation
in patient with type II diabetes. J. Med. Plants Res., 2011, 5,
2029-2034.
[86] Agila, K.N.; Kavitha, R. Antidiabetic, antihyperlipidaemic and
antioxidant activity of Oxalis corniculata in alloxan induced dia-
betic mice. J. Nat. Sci. Res., 2012, 2, 9-17.
[87] Lee, A.S.; Lee, Y.J.; Lee, S.M.; Yoon, J.J.; Kim, J.S.; Kang, D.J.;
Lee, H.S. Portulaca oleracea ameliorates diabetic vascular in-
flammation and endothelial dysfunction in db/ dbmice; Evid.
Based Complementary Altern. Med, 2012, p. 741824.
[88] Al-Qalhati, I.R.; Waly, M.; Al-Attabi, Z. AL-Subhi, L.K. Protec-
tive effect of Pteropyrum scoparium and Oxalis corniculata
against streptozotocin-induced diabetes in rats. FASEB, 2016, 30,
1176-1184.
[89] Sen, S.; Roy, M.; Chakraborti, A.S. Ameliorative effects of
glycyrrhizin on streptozotocin-induced diabetes in rats. J. Pharm.
Pharmacol., 2011, 63(2), 287-296.
http://dx.doi.org/10.1111/j.2042-7158.2010.01217.x PMID:
21235594
[90] Ozcan, F.; Ozmen, A.; Akkaya, B.; Aliciguzel, Y.; Aslan, M.
Beneficial effect of myricetin on renal functions in streptozotocin-
induced diabetes. Clin. Exp. Med., 2012, 12(4), 265-272.
http://dx.doi.org/10.1007/s10238-011-0167-0 PMID: 22083509
[91] P, P.S.; T, K.S. Antioxidant, antibacterial and cytotoxic potential
of silver nanoparticles synthesized using terpenes rich extract of
Lantana camara L. leaves. Biochem. Biophys. Rep., 2017, 10, 76-
81.
http://dx.doi.org/10.1016/j.bbrep.2017.03.002 PMID: 29114571
[92] Annu, A.S.; Kaur, G.; Sharma, P.; Singh, S.; Ikram, S. Fruit waste
(peel) as bio-reductant to synthesize silver nanoparticles with an-
timicrobial, antioxidant and cytotoxic activities. J. Appl. Biomed.,
2018, 16(3), 221-231.
http://dx.doi.org/10.1016/j.jab.2018.02.002
[93] Patra, J.K.; Das, G.; Kumar, A.; Ansari, A.; Kim, H.; Shin, H-S.
Photo-mediated Biosynthesis of Silver Nanoparticles Using the
Non-edible Accrescent Fruiting Calyx of Physalis peruviana L.
fruits and investigation of its radical scavenging potential and cy-
totoxicity activities. J. Photochem. Photobiol. B, 2018, 188, 116-
125.
http://dx.doi.org/10.1016/j.jphotobiol.2018.08.004 PMID:
30266015
[94] Das, G.; Patra, J.K.; Debnath, T.; Ansari, A.; Shin, H.S. investiga-
tion of antioxidants, antibacterial, antidiabetic, and cytotoxicity
potential of silver nanoparticles synthesized using the outer peel
extract of Ananas comosus (L.). PLoS One, 2019, 14(8), 0220950.
http://dx.doi.org/10.1371/journal.pone.0220950
[95] Rajaram, K.; Aiswarya, D.C.; Sureshkumar, P. Green synthesis of
silver nanoparticle using Tephrosia tinctoria and its anti-diabetic
activity. Mater. Lett., 2015, 138, 251-254.
http://dx.doi.org/10.1016/j.matlet.2014.10.017
[96] Abideen, S. VijayaSankar, M. In-vitro Screening of Antidiabetic
and Antimicrobial Activity against Green Synthesized AgNO3 us-
ing Seaweeds. J. Nanomed. Nanotechnol., 2015, •••, 6.
[97] Sengottaiyan, A.; Aravinthan, A.; Sudhakar, C.; Selvam, K.;
Srinivasan, P.; Govarthanan, M.; Manoharan, K.; Selvankumar, T.
Synthesis and characterization of Solanum nigrum-mediated silver
Cytotoxicity, Anti-Diabetic, and Hepato-Protective Potential Current Pharmaceutical Biotechnology, 2021, Vol. 22, No. 0 19
nanoparticles and its protective effect on alloxan-induced diabetic
rats. J. Nanostruct. Chem., 2016, 41-48.
[98] Hsu, C.H.; Liao, Y.L.; Lin, S.C.; Hwang, K.C.; Chou, P. The
mushroom Agaricus Blazei Murill in combination with metformin
and gliclazide improves insulin resistance in type 2 diabetes: a
randomized, double-blinded, and placebo-controlled clinical trial.
J. Altern. Complement. Med., 2007, 13(1), 97-102.
http://dx.doi.org/10.1089/acm.2006.6054 PMID: 17309383
[99] Shayganni, E.; Bahmani, M.; Asgary, S.; Rafieian-Kopaei, M.
Inflammaging and cardiovascular disease: Management by medic-
inal plants. Phytomedicine, 2016, 23(11), 1119-1126.
http://dx.doi.org/10.1016/j.phymed.2015.11.004 PMID: 26776956
[100] Rouhi-Boroujeni, H.; Heidarian, E.; Rouhi-Boroujeni, H.; Deris,
F. RafieianKopaei, M. Medicinal plants with multiple effects on
cardiovascular diseases. Curr. Pharm. Des., 2017, 23, 999-1015.
http://dx.doi.org/10.2174/1381612822666161021160524 PMID:
27774898
[101] Qasim, AL-Daami; Lamia, Al-Mashhedy Hypoglycemic effect by
assay some glucoregµlatory enzymes and hematological parame-
ters using silver nanoparticles of peel Raphanus sativus L aqueous
extract in male Rats. J. Phys. Conf. Ser., 2019, 1294, 062047.
http://dx.doi.org/10.1088/1742-6596/1294/6/062047
[102] Wang, Y.; Jiang, Y.; Fan, X.; Tan, H.; Zeng, H.; Wang, Y.; Chen,
P.; Huang, M.; Bi, H. Hepato-protective effect of resveratrol
against acetaminophen-induced liver injury is associated with in-
hibition of CYP-mediated bioactivation and regulation of SIRT1-
p53 signaling pathways. Toxicol. Lett., 2015, 236(2), 82-89.
http://dx.doi.org/10.1016/j.toxlet.2015.05.001 PMID: 25956474
[103] Kemelo, M.K.; Pierzynová, A.; Kutinová Canová, N.; Kučera, T.;
Farghali, H. The involvement of sirtuin 1 and heme oxygenase 1
in the hepatoprotective effects of quercetin against carbon tetra-
chloride-induced sub-chronic liver toxicity in rats. Chem. Biol. In-
teract., 2017, 269, 1-8.
http://dx.doi.org/10.1016/j.cbi.2017.03.014 PMID: 28347707
[104] De Groot, H.; Littauer, A.; Hugo-Wissemann, D.; Wissemann, P.;
Noll, T. Lipid peroxidation and cell viability in isolated hepato-
cytes in a redesigned oxystat system: evaluation of the hypothesis
that lipid peroxidation, preferentially induced at low oxygen par-
tial pressures, is decisive for CCl4 liver cell injury. Arch. Bio-
chem. Biophys., 1988, 264(2), 591-599.
http://dx.doi.org/10.1016/0003-9861(88)90325-6 PMID: 3401014
[105] Kieczka, H.; Kappus, H. Oxygen dependence of CCl4-induced
lipid peroxidation in vitro and in vivo. Toxicol. Lett., 1980, 5(3-4),
191-196.
http://dx.doi.org/10.1016/0378-4274(80)90058-2 PMID: 7466845
[106] Contreras-Zentella, M.L.; Hernández-Muñoz, R. Is liver enzyme
release really associated with cell necrosis induced by oxidant
stress? Oxid. Med. Cell. Longev., 2016, 2016, 3529149.
http://dx.doi.org/10.1155/2016/3529149 PMID: 26798419
[107] Ighodaro, O.M.; Akinloye, O.A. Sapium ellipticum (Hochst) Pax
leaf extract: antioxidant potential in CCl4-induced oxidative stress
model. Bull. Fac. Pharm. Cairo Univ., 2018, 56(1), 54-59.
http://dx.doi.org/10.1016/j.bfopcu.2017.11.001
[108] Lu, Y.; Chen, J.; Ren, D.; Yang, X.; Zhao, Y. Hepatoprotective
effects of phloretin against CCl4 induced liver injury in mice.
Food Agric. Immunol., 2017, 28(2), 211-222.
http://dx.doi.org/10.1080/09540105.2016.1258546
[109] Meena, R.; Paulraj, R. Oxidative stress mediated cytotoxicity of
TiO2 nano anatase in liver and kidney of Wistar rat. Toxicol. Envi-
ron. Chem., 2012, 94(1), 146-163.
http://dx.doi.org/10.1080/02772248.2011.638441
[110] Dutta, S.; Chakraborty, A.K.; Dey, P.; Kar, P.; Guha, P.; Sen, S.;
Kumar, A.; Sen, A.; Chaudhuri, T.K. Amelioration of CCl4 in-
duced liver injury in swiss albino mice by antioxidant rich leaf ex-
tract of Croton bonplandianus Baill. PLoS One, 2018, 13(4),
e0196411.
http://dx.doi.org/10.1371/journal.pone.0196411 PMID: 29709010
[111] Shah, M.D.; D’Souza, U.J.A.; Iqbal, M. The potential protective
effect of Commelina nudiflora L. against carbon tetrachloride
(CCl4)-induced hepatotoxicity in rats, mediated by suppression of
oxidative stress and inflammation. Environ. Health Prev. Med.,
2017, 22(1), 66.
http://dx.doi.org/10.1186/s12199-017-0673-0 PMID: 29165163
[112] Mandal, S.; Phadtare, S.; Sastry, M. Interfacing biology with
nanoparticles. Curr. Appl. Phys., 2005, 5, 118-127.
http://dx.doi.org/10.1016/j.cap.2004.06.006
[113] Jeyachandran, R.; Mahesh, A.; Cindrella, L. DEN-induced cancer
and its alleviation by Anisomeles malabarica (L.) R.Br. ethanolic
leaf extract in male albino mice. Int. J. Cancer, 2007, 3, 174-179.
http://dx.doi.org/10.3923/ijcr.2007.174.179
[114] Dai, J.; Mumper, R.J. Plant phenolics: extraction, analysis and
their antioxidant and anticancer properties. Molecules, 2010,
15(10), 7313-7352.
http://dx.doi.org/10.3390/molecules15107313 PMID: 20966876
[115] Ajay, G.O.; Olagunju, J.A.; Ademuyiwa, O.; Martins, O.C. Gas
chromatography mass spectrometry analysis and phytochemical
screening of ethanol root extract of Plumbago zeylanica, Linn. J.
Med. Plants Res., 2011, 5(9), 1756-1761.
[116] Eidi, A.; Mortazavi, P.; Bazargan, M.; Zaringhalam, J. Hepatopro-
tective activity of cinnamon ethanolic extract against CCI4-
induced liver injury in rats. EXCLI J., 2012, 11, 495-507.
PMID: 27547174
[117] Satish, S.S.; Janakiraman, N.; Johnson, M. Phytochemical analy-
sis of Vitex altissima L. using UV-VIS, FTIR and GC-MS. IJPSR,
2012, 4(1), 56-62.
[118] Rajaram, K.; Moushmi, M.; Velayutham Dass Prakash, M.;
Kumpati, P.; Ganasaraswathi, M.; Sureshkumar, P. Bioactive
studies between wild plant and callus culture of Tephrosia tincto-
ria Pers. Aplp. Biochem. Biotechnol, 2013, 1-16.
DISCLAIMER: The above article has been published in Epub (ahead of print) on the basis of the materials provided by the author. The Edito-
rial Department reserves the right to make minor modifications for further improvement of the manuscript.
... ; Silver nanoparticles have acquired prominent place in disease management because of their unique properties owing to small dimension and large surface area, mechanical and thermal stability, anti-inflammatory and antimicrobial activity [38]. In the current study, the descriptions of synthesis and characterization of ABAgNPs have been published by Nazer et al. [39]. The results of this study, following the guidelines of the OECD, showed that ABaqu and ABAgNPs showed non-toxic effect on mice skin at a dosage of 1000 mg/kg body weight. ...
Preprint
Full-text available
Background: Wound therapy is complicated, uncomfortable for the patient, and costly for the health-care system. Silver nanoparticles (AgNP) have antibacterial characteristics that can prevent bacterial infection in wounds and speed up wound healing. Objective: The aim of current research was to investigate the wound healing and anti-inflammatory potential of biogenic synthesized silver nanoparticles (ABAgNP) using Ajuga bracteosa (ABaqu) in Swiss albino mice. Methods: In vivo wound healing and anti-inflammatory activities were carried out using Bala/c mice. For in vivo screening of 200 mg/kg and 400 mg/kg of both ABAgNPs and ABaqu were used. Liver and kidney functional markers, hematology, and histopathological studies were carried out after 14 days of administration. Results: The obtained biogenic nanoparticles were characterized, dermal toxicity, wound excision repairing, and formalin-induced paw edema assays were performed in Swiss albino mice. Dermal toxicity showed that tested concentrations of ABaqu and ABAgNPs were safe. No adverse effects, changes, and alteration in the skin of treatment groups as well as the control vehicle group (petroleum jelly) were recorded. Results revealed that the enhanced wound contraction was observed in ABaqu, ABAgNP, and the Nitrofuranose treated groups from 7th to 11th days. The anti-inflammatory activity in formalin-induced paw edema model illustrated the potential use of silver nanoparticles ABAgNPs and ABaqu as a reducing or inflammation inhibiting agents due to the release of acute inflammatory mediators. Conclusion: Therefore, it was concluded that both silver nanoparticles (ABAgNP) and Ajuga bracteosa (ABaqu) extracts could be used as a wound healing and anti-inflammatory agents.
Article
Full-text available
The green synthesis of nanoparticles (NPs) using living cells is a promising and novelty tool in bionanotechnology. Chemical and physical methods are used to synthesize NPs; however, biological methods are preferred due to its eco-friendly, clean, safe, cost-effective, easy, and effective sources for high productivity and purity. High pressure or temperature is not required for the green synthesis of NPs, and the use of toxic and hazardous substances and the addition of external reducing, stabilizing, or capping agents are avoided. Intra- or extracellular biosynthesis of NPs can be achieved by numerous biological entities including bacteria, fungi, yeast, algae, actinomycetes, and plant extracts. Recently, numerous methods are used to increase the productivity of nanoparticles with variable size, shape, and stability. The different mechanical, optical, magnetic, and chemical properties of NPs have been related to their shape, size, surface charge, and surface area. Detection and characterization of biosynthesized NPs are conducted using different techniques such as UV–vis spectroscopy, FT-IR, TEM, SEM, AFM, DLS, XRD, zeta potential analyses, etc. NPs synthesized by the green approach can be incorporated into different biotechnological fields as antimicrobial, antitumor, and antioxidant agents; as a control for phytopathogens; and as bioremediative factors, and they are also used in the food and textile industries, in smart agriculture, and in wastewater treatment. This review will address biological entities that can be used for the green synthesis of NPs and their prospects for biotechnological applications.
Article
Full-text available
Background Multi-drug resistance in bacterial pathogens is a rising concern today. Green synthesis technology has been utilized to cure infectious diseases. Objectives The aim of the current research was to highlight the antibacterial, antioxidant, and phytochemical screening of green synthesized silver nanoparticles using Ajuga bracteosa. Methods Extract of A. bracteosa was prepared by maceration technique. Silver nanoparticles were synthesized using A. bracteosa extract and were confirmed by UV-vis spectrophotometer, Scanning electron microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The antibacterial, anti-biofilm, cell proliferation inhibition, TLC-Bioautography, TLC-Spot screening, antioxidant, and phytochemical screening were also investigated. Results UV-viz spectrum and Scanning electron microscopy indicated the synthesis of green nanoparticles at 400 nm with tube like structures. FTIR spectrum showed the functional groups have a role in capping and stability of AgNP. Agar well diffusion assay represents the maximum antibacterial effect of ABAgNPs against Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, and Pseudomonas aeruginosa at 0.10 g/ml concentration compared to ABaqu. Two types of interactions among nanoparticles, aqueous extract, and antibiotics (Synergistic and additive) were recorded against tested pathogens. Crystal violet, MTT, TLC-bio-autography, and spot screening supported the findings of antibacterial assay. Highest antioxidant potential effect in ABaqu 14.62% (DPPH) and 13.64% (ABTS) while 4.85% (DPPH) and 4.86% (ABTS) was recorded in ABAgNPs. Presence of phytochemical constituents showed pharmacological importance. Conclusion It has been concluded that green synthesis is an innovative technology in which natural products are used against infectious pathogens. Current research shows the significant antibacterial effect against infectious agents.
Article
Full-text available
Currently, green nanotechnology-based approaches using waste materials from food have been accepted as an environmentally friendly and cost-effective approach with various biomedical applications. In the current study, AgNPs were synthesized using the outer peel extract of the fruit Ananas comosus (AC), which is a food waste material. Characterization was done using UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, scanning electronic microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) analyses. The formation of AgNPs has confirmed through UV-visible spectroscopy (at 485 nm) by the change of color owing to surface Plasmon resonance. Based on the XRD pattern, the crystalline property of AgNPs was established. The functional group existing in AC outer peel extract accountable for the reduction of Ag+ ion and the stabilization of AC-AgNPs was investigated through FT-IR. The morphological structures and elemental composition was determined by SEM and EDX analysis. With the growing application of AgNPs in biomedical perspectives, the biosynthesized AC-AgNPs were evaluated for their antioxidative, antidiabetic, and cytotoxic potential against HepG2 cells along with their antibacterial potential. The results showed that AC-AgNPs are extremely effective with high antidiabetic potential at a very low concentration as well as it exhibited higher cytotoxic activity against the HepG2 cancer cells in a dose-dependent manner. It also exhibited potential antioxidant activity and moderate antibacterial activity against the four tested foodborne pathogenic bacteria. Overall, the results highlight the effectiveness and potential applications of AC-AgNPs in biomedical fields such as in the treatment of acute illnesses as well as in drug formulation for treating various diseases such as cancer and diabetes. Further, it has applications in wound dressing or in treating bacterial related diseases.
Article
Full-text available
Development of multidrug resistance among pathogens has become a global problem for chemotherapy of bacterial infections. Extended-spectrum β -lactamase- (ES β L-) producing enteric bacteria and methicillin-resistant Staphylococcus aureus (MRSA) are the two major groups of problematic MDR bacteria that have evolved rapidly in the recent past. In this study, the aqueous extract of Murraya koenigii leaves was used for synthesis of silver nanoparticles. The synthesized MK-AgNPs were characterized using UV-vis spectroscopy, FTIR, XRD, SEM, and TEM, and their antibacterial potential was evaluated on multiple ES β L-producing enteric bacteria and MRSA. The nanoparticles were predominantly found to be spheroidal with particle size distribution in the range of 5–20 nm. There was 60.86% silver content in MK-AgNPs. Evaluation of antibacterial activity by the disc-diffusion assay revealed that MK-AgNPs effectively inhibited the growth of test pathogens with varying sized zones of inhibition. The MICs of MK-AgNPs against both MRSA and methicillin-sensitive S. aureus (MSSA) strains were 32 μ g/ml, while for ES β L-producing E. coli , it ranged from 32 to 64 μ g/ml. The control strain of E. coli (ECS) was relatively more sensitive with an MIC of 16 μ g/ml. The MBCs were in accordance with the respective MICs. Analysis of growth kinetics revealed that the growth of all tested S. aureus strains was inhibited (∼90%) in presence of 32 μ g/ml of MK-AgNPs. The sensitive strain of E. coli (ECS) showed least resistance to MK-AgNPs with >81% inhibition at 16 μ g/ml. The present investigation revealed an encouraging result on in vitro efficacy of green synthesized MK-AgNPs and needed further in vivo assessment for its therapeutic efficacy against MDR bacteria.
Article
Full-text available
Plant medicine is the oldest form of health care known to mankind. Syzygium guineense is one of the many species of Ethiopian medicinal plants which has a long history of use as remedies for various ailments such as dysentery, diarrhea, and hypertension. In many countries, herbal medicines and related products are introduced into the market without safety or toxicological evaluation. The aim of this study was to investigate the histopathological effect of 80% methanol extract of S. guineense on liver and kidney and blood parameters of rats. For acute toxicity study, rats were randomly divided into three groups (n=4). The control group received distilled water, while the experimental groups received a single dose of 2000 mg/kg and 5000 mg/kg 80% methanolic extract of S. guineense leaves per oral. For subacute toxicity study, the rats were randomly divided into three groups (n=6). The control group received distilled water, while the experimental groups received 500 mg/kg and 1500 mg/kg 80% methanol extract of S. guineense leaves orally for 28 days. At the end of the experiment, blood samples were collected for hematology and clinical chemistry evaluations. Gross pathology and histopathology of liver and kidneys were assessed. In the acute toxicity study, rats treated with 2000 mg/kg and 5000 mg/kg showed no toxicological signs observed on behavior, gross pathology, and body weight of rats. In the subacute toxicity study rats have showed no significant changes on behavior, gross pathology, body weight, and hematological and biochemical parameters, whereas both experimental groups had a lower blood glucose level compared with the control group (p < 0.05). There were no significant differences in the gross and histopathology of the liver and kidneys of experimental animals in extract exposed groups and their counterpart control. The 80% methanol extract of S. guineense does not produce adverse effects in rats after acute and subacute treatment. Before marketing a S. guineense leaf based remedy, subchronic and chronic toxicity evaluations need to be done.
Article
Full-text available
A World Health Organization survey indicated that about 70-80% of the world population rely on non-conventional medicine mainly of plant sources in their primary healthcare. In Ethiopia over 85% of the population relies on traditional medicine for the fight against various diseases. Syzygium guineense is one of such medicinal plants in use. It is traditionally used as a remedy for diarrhea, stomach pain and intestinal cramps. The toxic effects of these plants are often not put into considerations when they are being used. In this study we attempted to investigate the toxic effect of Syzygium guineense. On the histopathology of the liver of mice we choose the Liver because it is the major metabolic organ. Chronic administration of the aqueous of the extract of Syzygium guineense was found to be toxic to the hepatocytes as the micrograph showed increase vaculations, and shrinkage of the hepatocytes. There was also a general decrease in body weight and enlarged liver. Keywords: Hepatocyte; Toxic; Syzygium; Guineense; Acqeous. Abbreviations: EHNRI: Ethiopian Health and Nutrition Research Institute; ANOVA: Analysis of Variance.
Article
Full-text available
Advances in nanomedicine have become indispensable for targeted drug delivery, early detection, and increasingly personalized approaches to cancer treatment. Nanoparticle-based drug-delivery systems have overcome some of the limitations associated with traditional cancer-therapy administration, such as reduced drug solubility, chemoresistance, systemic toxicity, narrow therapeutic indices, and poor oral bioavailability. Advances in the field of nanomedicine include “smart” drug delivery, or multiple levels of targeting, and extended-release drug-delivery systems that provide additional methods of overcoming these limitations. More recently, the idea of combining smart drug delivery with extended-release has emerged in hopes of developing highly efficient nanoparticles with improved delivery, bioavailability, and safety profiles. Although functionalized and extended-release drug-delivery systems have been studied extensively, there remain gaps in the literature concerning their application in cancer treatment. We aim to provide an overview of smart and extended-release drug-delivery systems for the delivery of cancer therapies, as well as to introduce innovative advancements in nanoparticle design incorporating these principles. With the growing need for increasingly personalized medicine in cancer treatment, smart extended-release nanoparticles have the potential to enhance chemotherapy delivery, patient adherence, and treatment outcomes in cancer patients.