Green synthesis of Equisetum arvense-mediated magnesium oxide nanoparticles and its

biomedical applications
Sulochana Govindharaj, S Rajeshkumar*

Nanobiomedicine Lab, Centre for Global Health Research, Saveetha Medical College and
Hospitals, Saveetha Institute of Medical and Technical Sciences, Chennai - 602105,
TamilNadu, India

*Corresponds to
Dr S Rajeshkumar
Professor
Nanobiomedicine Lab
Centre for Global Health Research
Saveetha Medical College and Hospital
Saveetha Institute of Medical and Technical Sciences
Chennai - 602 105, TN, India
[Link]@[Link]

Abstract
Background: Preparing and stabilizing various types of nanoparticles using herbal extract
has proven to be an intriguing prospective environmentally beneficial technology.
Magnesium oxide nanoparticles (MgONPs) are finding a broad range of applications in the
environmental and medical sciences due to their impressive antioxidant, anticancer, and
antibacterial activity. The traditional uses of Equisetum arvense included wound and ulcer
healing, renal issues, tuberculosis treatment, and bleeding control.
Aim: This work intended to synthesize magnesium oxide nanoparticles in an environmentally
friendly manner utilizing E. arvense, with potential uses in biomedicine.
Materials and method: Using an aquatic biological technique, E. arvense extract was used
to create magnesium oxide nanoparticles (MgONPs). The antibacterial activity of the
resulting MgO NPs against wound infections was evaluated. The ABTS, Nitric Oxide, DPPH,
FRAP, and H2O2 assays were used to measure antioxidant activity. Utilizing zebrafish
survivability during MgONPs treatment, cytotoxicity was evaluated.
Results: The green-produced MgONPs showed good antibacterial activities against clinical
infectious microbes, compared to the E. arvense control. It also demonstrated outstanding
biocompatibility and antioxidant activity.
Conclusion: The potential application of plants mediated NPs as antibacterial and an
antioxidant agent is the primary conclusion of the research.
Keywords: Green synthesis, antioxidant, anti-inflammatory, antimicrobial, eco-friendly,
biocompatibility.

Introduction
In recent days, the nanoparticles have been synthesized by using the green synthesis process.
It is also known as the biosynthesis process, the biosynthesis process was done by using the
plant-mediated nanoparticles. The green synthesis process efficacy of applications of natural
reducing agents, without using chemicals and less toxicity, perfect and stabilizing agent 1.
Magnesium oxide nanoparticles are one of the metal ion-based nanoparticles, a lot of research
is going into the metal-based nanoparticles by using plant-based. The metal-based
nanoparticles are used to synthesize the green synthesized method and it has maintained the
bioefficacy2. The Equisetum arvense is a perennial herb and native to the plant northern
Himalayas. The local name of the plant is Field horsetail or Horsetail, genus Equisetum, and
the species arvense3. Horsetail is one of the best treatments for skin disease, to treat pimples
and dermatitis, it has present the excellent wound-healing activity of the skin conditions 4. It
has been used for cosmetics like moisturizing agents, body shampoos, soaps, cosmetic
cleaners, and skin conditioning agents 5. E. arvense contains sterols, a rich source of vitamin
C, phenolic acid, and flavonoids6. It has also shown efficacy against fungi species 7. In
pharmaceutical studies, E. arvense has an additional medicinal therapy, and excellent
antioxidant and anti-inflammatory properties8.
The plant-based nanoparticles are used and they reduce toxicity and are ecofriendly. The
magnesium oxide nanoparticles have both metal and metal oxide based, they have different
biomedical and biochemical applications catalysis, tissue regeneration, and antimicrobial and
anticancer agents9. Magnesium oxide has excellent high stability, bioavailability, antiviral,
and antipathogenic activity10. The green method is used to develop the magnesium oxide
nanoparticle synthesis using cost-reduction, energy efficiency, removal of environmental
pollution, and biocompatibility11. The green synthesized magnesium oxide nanoparticles are
used in biomedical therapy such as cancer, inflammation, oxidation, and diabetics 12. The
concept of the research work has been to evaluate the preparation of nanoparticles,
antimicrobial activity against wound pathogens, time-kill curve assay, antioxidant, anti-
inflammatory activity, thrombolytic activity, cytotoxic effect against brine shrimp lethality
assay, and the embryonic toxicology using zebrafish embryos.

Materials methods
Preparation of plant extract:
At an Ayurvedic store in Poonamalli, Chennai, the dried E. arvense was bought. 150
milliliters of distilled water were combined with three grams of E. arvense. 150 milliliters of
E. arvense extract were thoroughly agitated and then heated to 60 degrees Celsius for 15 to
20 minutes in the heating mantle. After it was boiled and filtered through muslin material,
the extract was prepared and used for the manufacture of nanoparticles and additional study 13.
Preparation of nanoparticles:
The magnesium oxide nanoparticles were prepared using 50 mM of magnesium chloride
(MgCl), which was precursor to the magnesium oxide nanoparticle. To prepare the
magnesium chloride solution, 50 mL of distilled water and 50 mM of MgCl were combined.
The 50 mL magnesium chloride solution was combined with the 50 mL E. arvense extract.
Color changes were noticed in the solution after it was stirred for 48 hours at 600 rpm using a
magnetic stirrer. Up to 250–650 nm, the first stage of UV–visible spectroscopic
characterization was seen. After centrifuging the nanoparticle solution for ten minutes at
8000 rpm, the pellets were separated and distributed in various sterile containers14.
Antimicrobial activity:
E. arvense was used to perform the antibacterial activity for the green process of magnesium
oxide nanoparticles utilizing the agar well technique. Mueller Hinton Agar medium was
added to the sterile Petri plate. Sterile micro tips (9mm) are used to help prepare the agar well
diffusion technique in sterile petric plates. Fresh microbiological cultures, including E. coli,
S. aureus, Pseudomonas sp., and Candida albicans, were used to generate the various
microbial stains using Muller-Hinson broth. In the sterile swab in the agar plates, the
microbial broth culture was distributed uniformly. Following the addition of magnesium
oxide nanoparticles at three distinct concentrations (25, 50, and 100 µg/mL), the agar plates
were allowed to incubate for a full day at room temperature. The inhibitory zones were
measured in diameter on the plates after an incubation period. A pure aqueous extract of E.
arvense was utilized as the control in each microbiological culture15.
Time kill curve assay
To create a standardized vaccine, the isolated microbial broth was cultured. Nanoparticle
solutions were put into 96-well plates at different concentrations (25, 50, and 100 µg/mL).
Additionally, pure aqueous extract of E. arvense was used as the positive control, antibiotics
as the standard, and simply microbial culture was added as the negative control. At room
temperature, the plate was incubated, at regular intervals of 0, 2, 4, 6, 8, and 24 hours,
observations for microbial cultures have been collected. It was able to determine the killing
rate by plotting the total number of living cells as log10 (CFU/mL) against time16.
Anti-inflammatory activity
Bovin serum albumin denaturation assay
Anti-inflammatory efficacy of green production of magnesium oxide nanoparticles using E.
arvense, the prevention of albumin denaturation approach—which was previously
investigated by Mizushima et al.—has been somewhat modified. Five different
concentrations (10 – 50 µg/mL) were added to the nanoparticles. Adding a 1% aqueous
solution of the fractional representation of bovine albumin together with test extracts to the
reaction mixture. To modify the pH of the reaction mixture, a very small amount of 1N HCl
was added. The sample extracts were chilled and the turbidity was measured at 660 nm after
20 minutes of incubation at 37 °C and 20 minutes of heating at 51 °C.

Egg albumin denaturation assay

0.2 mL of egg albumin, PBS (2.8 mL of Phosphate Buffer Saline, keeping a pH range of 6.4),
and the five different concentrations of nanoparticle solutions (10 - 50 µg/mL) were added to
the 5 mL reaction mixture. Double-distilled water in an equivalent volume is used as a
control. In addition to determining the absorbance of standard values, standard medications
like diclofenac sodium were employed. After 15 minutes of incubation at room temperature,
the mixed solutions were heated to 50°C for five minutes to bring the water to a boil.
Measure the absorbance at 660 nm after the liquids have cooled. The formula was used to
determine the percentage of inhibition of protein denaturation.
Membrane stabilization assay
Preparation of Red Blood cell (RBC) suspension.
A euthanized human volunteer was selected based on health, and blood was drawn into
centrifuge tubes only after the donor had abstained from NSAID use for two weeks before the
experiment. The tubes underwent three rounds of sterilization using the same volume of
regular saline after being centrifuged for ten minutes at 3000 rpm. Following that, using a
standard saline solution, the volume of the blood was measured and reconstituted at a 10%
v/v concentration.
Heat-induced method
One milliliter of 10 % RBC suspension and one milliliter of magnesium oxide nanoparticles
made with E. arvense at different concentrations (10–50 µg/mL) made up the reaction
mixture (2 ml). Ordinary saline was added to the control test tube in place of magnesium
oxide nanoparticles made with E. arvense. A popular standard was vitamin C. Each
centrifuge tube holding the reaction mixture was incubated for 30 minutes at 56°C in a water
bath. Following the incubation time, the tubes were cooled using running tap water. The
absorbance of the supernatants was measured at 560 nm after the reaction mixture was
centrifuged for five minutes at 2500 rpm. The experiment was performed in duplicate for
each test sample17.

Antioxidant activity
DPPH assay
A solution of 10 mL methanol and 0.1 mM DPPH was prepared. Five varying amounts of
nanoparticle solutions (10 - 50 µg/ml) were combined with 1 mL of DPPH methanol
solutions. Following that, the mixed solutions were allowed to sit at room temperature for 30
minutes in a dark incubator. Ascorbic acid served as the standard, while DPPH solution
served as the control. A UV-visible spectrophotometer was used to detect the absorbance at
517 nm following the completion of the incubation. The greater activity of scavenging free
radicals is indicated by the lower absorbance. Using the DPPH assay, the percentage of
inhibition was calculated for each concentration of nanoparticles based on the percentage of
antioxidant activity.
H2O2 assay
The Halliwell technique [Halliwell et al., 1987] has brought about a few changes to the
assay's completion. All of the solutions were made from the beginning. In 1.0 mL, the
following ingredients make up the reaction's solution: A mixture of 100 µL of 28 mM 2-
deoxy-2-ribose (dissolved in phosphate buffer, pH 7.4), 500 µL of various nanoparticle
concentration solutions (10 to 50 µg/mL), 200µL of 1.04 mM EDTA and 200 mM Fecl 3,
100µL of H2O2 (1.0 mM), and 100µL of ascorbic acid (1.0 mM) were used. A 60-minute
incubation period at 37°C allowed for the determination of the degree of deoxyribose
breakdown using the TBA reaction. The absorbance is measured by comparing it to the blank
solution at 532 nm. The vitamin E was used as a positive control.
FRAP (Ferric Reducing Antioxidant Power) assay:
This technique works by decreasing the ability of Fe 3+ to Fe2+ ions in the sample. Ferric-
tripyridyltriazine complex to decrease the ferrous form in the presence of low pH, TPTZ
(2,4,6-Tri(2-pyridyl)-s-triazine), and the formulations indicated the blue color has been
absorbed in the UV-Visible spectrophotometer at 593 nm. Aqueous extract (0.7 mL) and 2.3
mL of the FRAP formulation reagents were combined with five different doses of
nanoparticle solutions (10 - 50 µg/mL). The mixed solutions were then left to incubate for 30
minutes at room temperature in the dark. The ascorbic acid used in the standard was
substituted with double-distillation water for the control. A UV-visible spectrophotometer
was used to detect the absorbance at 593 nm following the incubation period. To demonstrate
the increase in the reduction capabilities, the absorbance value was improved.
ABTS assay
The chemical compound ABTS (2, 2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid)). To
create the stock solution, 7 mM of ABTS was combined with distilled water, and 2.45 mM of
potassium per sulphate was combined with an equivalent volume of aqueous solution. After
the mixed solutions were incubated for 12 to 16 hours at room temperature in the dark, and
used. The working solution and the measuring absorbance at 734 nm were made by diluting
the stock solution in methanol. A working ABTS solution of 2 mL was combined with five
different concentrations of nanoparticle solutions (10 - 50 µg/mL), and the mixture was left to
incubate for 10 minutes at room temperature in the dark. Following that, a volume of ABTS
working solution equal to one milliliter was added as the control. The standard was butylated
hydroxytoluene or BHT. At 734 nm, the absorbance was measured with a spectrophotometer.
The percent of inhibition was determined as the percentage of antioxidant activity in each
concentration of the nanoparticles on the DPPH assay.
Nitric acid assay
Griess reagent (0.5 ml; 1% sulfanilamide, 2% H3PO4, and 0.1% N-(1-naphthyl)
ethylenediamine dihydrochloride) was added) is used to measure nitrite ions, which are
produced when sodium nitroprusside spontaneously releases nitric oxide in physiological pH
aqueous solution. The creation of nitrite ions is inhibited by nitrite scavengers, which
compete with oxygen. Sodium nitroprusside (10 mM) was mixed with different
concentrations of dissolved nanoparticle solutions (10 - 50 µg/mL) in phosphate-buffered
saline, and the combination was then allowed to sit at room temperature for 2.30 hours. After
that, a control experiment with an identical volume of double-distilled water was conducted.
It was determined that the absorbance was 546 nm. Instead of quercetin, a positive control
was employed18.

Thrombolytic activity
Using a sterile glass slide, one drop of blood was taken and incubated at the ideal temperature
for 45 minutes. Add the 20 µg/mL solution of magnesium oxide nanoparticles after the blood
has clotted. Afterward, without adding any solution, compare with the control. The ideal
temperature for the glass slide was maintained for 90 minutes, and the incubation hours were
recorded to track the lysis of the clot. Blood was removed and then re-infused ten times
following the incubations19.
Cytotoxic effect
Brine shrimp lethality assay
The test organism for the cytotoxic test was Artemia salina (nauplii), and the lethality assay
of brine shrimp was employed in its execution. Thirty milligrams of Artemia salina (brine
shrimp egg) were introduced to the saltwater in a sealed black container. The black container
with aeration was used to aid in the hatching of the nauplii eggs. Artemia salina eggs hatch
and transform into larvae after 24 hours. Following that, five distinct doses of nanoparticles
were injected into each of the six-well plates that held ten individual Artemia salina hatchings
(5, 10, 20, 40, and 80 µg/mL). The control was added solely to the saltwater and the six well
plates were incubated for a full day at room temperature. The number of nauplii in the 6-well
plate was counted when the incubation concluded20.
Embryonic toxicology
The fish tank had been preserved under excellent environmental conditions, the pH was
preserved in the range of 6.8 to 8.4, and the zebrafish were bought from neighbourhood
merchants in Tamilnadu. Fish foods like blood worms or ideal meals, which are both
commercially accessible, were fed to the fish twice a day. Where the breading tanks were
filled with three male and one female zebrafish. The eggs were carefully removed without
causing any damage to the viable embryos that were formed. Three times, the eggs were
cleaned in an E3 medium that was devoid of methylene blue. With 10 embryos in 2 mL of
solution per well, the eggs were put into three different well plates, measuring 6, 12, and 24.
In this experiment, the E3 medium that was diluted from the stock using sterilized water was
utilized. Once that was established, the pH was kept between 7.2 and 7. 3. In a study
conducted 24 to 96 hours after conception, embryos in good health were exposed to different
concentrations of nanoparticle solutions (5, 10, 20, 40, and 80 µg/mL). Nanoparticles and the
E3 medium solution were combined before the embryos were used. The embryos were
counted and the dead ones were taken out after the incubation period. All of the experimental
plates were kept at 28°C and covered with foil sheets to avoid light interference.
Evaluation of Zebrafish Embryos
Using a stereo microscope, the developmental stages of the fertilized zebrafish embryos were
examined. Following a 24-78-hour post-fertilization period, the embryos were introduced to
different concentrations of nanoparticle solutions (5, 10, 20, 40, and 80 µg/mL). The study's
endpoint is the proportion of embryos that die and hatch within the time frame. Under the
microscope, any deformity or anomaly was detected for both the treatment group and the
control group21.
Results:
Preparation of Magnesium oxide nanoparticles
The colors of the nanoparticles' solution at the beginning and end stages of the green
synthesis of E. arvense-mediated magnesium oxide nanoparticles were pale brown and dark
brown, respectively. This demonstrates magnesium oxide nanoparticles are present and
formed.

Figure 1: UV-visible absorption spectra of magnesium oxide nanoparticles after

bioreduction kinetics of the reaction of E. arvense aqueous extract in the concentration
range of 250 - 650 nm with different time intervals.
The most popular method for examining the optical characteristics of nanoparticles is UV-
visible spectroscopy. Bio reduction kinetics of the reaction between an aqueous extract of E.
arvense at varied time intervals and concentrations between 250 and 650 nm. Because surface
plasmon vibrations were excited, a maximum absorbance peak was seen at 380 nm for the
synthesizing of magnesium oxide nanoparticles (Figure 1).
Antimicrobial activity

Figure 2: Image representing Inhibition zone and time-kill curve assay of magnesium
oxide nanoparticles against treated Clinical pathogens especially A) C. albicans B)
Pseudomonas sp. C) S. aureus and D) E. coli
Antimicrobial activity against wound infections was demonstrated by the magnesium oxide
nanoparticles. Figure 2 shows the zone of inhibition for magnesium oxide nanoparticles
against wound pathogens at varying doses (25, 50, and 100 µg/mL). In 16 millimeters,
Pseudomonas sp exhibited the largest zone of inhibition at the higher level (100 µg/mL) and
the lowest level (25 µg/mL) in 12 millimeters. Similarly, C. albicans and S. aureus observed
the inhibitory zone in 14 millimeters at the higher level (100 µg/mL) and 11 millimeters at
the lower level (25 µg/mL). The E. coli was then detected in Figure 2 at the lowest
concentration (25 µg/mL) in 9 millimeters and at the highest level (100 µg/mL) in 14
millimeters, respectively. The control was observed the same level of zone of inhibition (9
mm) in clinical pathogens.

Time kill curve assay

When compared to the control group, the naturally developed MgONPs' time kill kinetic
experiment against clinical pathogens showed concentration-based antimicrobial
effectiveness. Throughout the course of the study, a growth minimized the Pseudomonas sp.
count was detected in the MgONPs concentrations of 25, 50, and 100 µg/mL when compared
to the control group. Within the first two hours, there was a notable decrease in the number of
Pseudomonas sp., indicating rapid microbial action, at least at the higher dose of 100 µg/mL.
MgONPs shown concentration-dependent microbiological efficiency against C. albicans in
the figure, akin to that of Pseudomonas sp. Within the first hour, MgONPs at all
concentrations significantly reduced the number of Candida albicans compared to the control,
demonstrating strong antibacterial activity. MgONPs had concentration-dependent
antibacterial activity against S. aureus in the figure, however the microbial count diminished
more gradually. A reduction in S. aureus count was less noticeable even at such a high
concentration (100 µg/mL), suggesting a lower susceptibility to MgONPs than other
pathogens. When compared to validated clinical pathogens, these results clearly highlighted
the concentration-dependent antibacterial capabilities of MgONPs. Pseudomonas sp.,
Candida albicans, and S. aureus showed initial susceptibility to MgONPs, but E. coli showed
a more gradual reaction. By comparing the results with the control group, it is possible to see
how much MgONPs have helped lower bacterial counts and how promising they are as an
antibacterial agent for clinical management.

Pseudomonas sp.
The assay for the time-kill curve revealed that magnesium oxide nanoparticles exhibited
resistance against microbial growth over 1-4 hours. Figure 2 displays the time-kill curve
assay conducted against Pseudomonas sp. The addition of magnesium oxide nanoparticles
inhibited the growth of Pseudomonas sp. A decrease in metal ions is the mechanism of
magnesium oxide nanoparticles, or magnesium nanoparticles mediated by E. arvense. It can
pierce the cell wall of microorganisms, and metal ions harm the wall. The microbiological
caused growth to slow down and death to occur. It was measured at 600 nm using the ELISA
reader. Because the control sample included entirely bacteria, it proliferated at a greater rate.
Similar to the standard values, a greater growth reduction was observed in the fourth hour at
the higher dose (100 µg/mL). The bacterial growth in the fourth hour was also lowered by the
lower concentration (25 µg/mL), respectively.

Candida albicans
The magnesium oxide nanoparticles' resistance to microbial growth was observed using the
time-kill curve assay, which required one to four hours. The time-kill curve experiment
against Candida albicans is shown in Figure 2, where the addition of magnesium oxide
nanoparticles inhibited the growth of the Candida albicans. A reduction of metal ions is the
method by which magnesium oxide nanoparticles or magnesium nanoparticles mediated by
E. arvense, work. Metal ions harm the microbial cell wall and can infiltrate it. The
microorganism caused mortality and reduced growth. At 600 nm, it was quantified using an
ELISA reader. Because the control sample entirely contained bacteria, it proliferated at a
greater rate. Similar to the standard values, a greater growth reduction was observed in the
fourth hour at the higher dose (100 µg/mL). The bacterial growth in the fourth hour was also
lowered by the lower concentration (25 µg/mL), respectively.

S. aureus
The assay for the time-kill curve revealed that magnesium oxide nanoparticles exhibited
resistance against microbial growth over 1-4 hours. Figure 2 depicts the time-kill curve assay
conducted against S. aureus. The addition of magnesium oxide nanoparticles inhibited S.
aureus growth. A decrease in metal ions is the mechanism of magnesium oxide nanoparticles,
or magnesium nanoparticles mediated by E. arvense. It can pierce the cell wall of
microorganisms, and metal ions harm the wall. The microbiological caused growth to slow
down and death to occur. It was measured at 600 nm using the ELISA reader. Because the
control sample included primarily bacteria, it expanded at a greater rate. Similar to the
standard values, a greater growth reduction was observed in the fourth hour at the higher dose
(100 µg/mL). The bacterial growth in the fourth hour was also lowered by the lower
concentration (25 µg/mL), respectively.

Escherichia coli
The assay for the time-kill curve revealed that magnesium oxide nanoparticles exhibited
resistance against microbial growth over 1-4 hours. The time-kill curve experiment against
Escherichia coli is depicted in Figure 2, where the addition of magnesium oxide
nanoparticles inhibited bacterial growth. A decrease in metal ions is the mechanism of
magnesium oxide nanoparticles, or magnesium nanoparticles mediated by E. arvense. It can
pierce the cell wall of microorganisms, and metal ions harm the wall. The microbiological
caused growth to slow down and death to occur. It was measured at 600 nm using the ELISA
reader. Because the control sample included solely bacteria, it grew at a greater rate. Similar
to the standard values, a greater growth reduction was observed in the fourth hour at the
higher dose (100 µg/mL). The bacterial growth in the fourth hour was also lowered by the
lower concentration (25 µg/mL), respectively.

Thrombolytic activity
The clotted drop of blood was added to the 20µg/mL of magnesium oxide nanoparticles,
which have a thrombolytic property. After mixing the clotted blood with the nanoparticle
solution for 20 minutes, the clotted blood liquefied once more, as represented in Figure 3.

Anti-inflammatory activity:

Figure 3: Image representing the anti-inflammatory activity of Magnesium oxide

nanoparticles using BSA assay, MSA assay, and EA assay and the thrombolytic activity.
Bovine serum albumin denaturation assay
The magnesium oxide nanoparticles were found to be effective at all concentrations (10 -50
µg/mL) in the anti-inflammatory activity assessments conducted using the bovine serum
albumin denaturation assay. The 50 µg/mL showed the highest percentage of inhibition
(78.4%), which was followed by the 40 µg/mL at 72.9 %, 30 µg/mL at 67.7 %, 20 µg/mL at
54.3 %, and 10 µg/mL at 44.6 %. In comparison to typical values, Figure 3 shows that
magnesium oxide nanoparticles may have an anti-inflammatory effect.

EA assay
Using the egg albumin denaturation assay, magnesium oxide nanoparticles were evaluated for
their anti-inflammatory action and shown to be effective at all concentrations (10 -50
µg/mL). The 50 µg/mL showed the highest percentage of inhibition (78.6 %), which was
followed by the 40 µg/mL (68.4 %), 30 µg/mL (64.1 %), 20 µg/mL (59.3 %), and 10 µg/mL
(53.7 %). When compared to conventional values, Figure 3 shows that magnesium oxide
nanoparticles may have an anti-inflammatory effect.
MSA assay:
Magnesium oxide nanoparticles were found to be effective at all concentrations (10 -50
µg/mL) in the anti-inflammatory activity assessments conducted using the membrane
stabilization assay. The samples with the highest percentage of inhibition were those
containing 50 µg/mL (84.8 %), 40 µg/mL (78.7 %), 30 µg/mL (73.3 %), 20 µg/mL (65.4 %),
and 10 µg/mL (54.5 %). Figure 3 shows that when compared to typical values, magnesium
oxide nanoparticles may have an anti-inflammatory effect.
Antioxidant activity
Figure 4: Image representing the antioxidant activity of Magnesium oxide nanoparticles
using DPPH assay, H2O2 assay, ABTS assay, FRAP assay, and EA assay.

DPPH assay
Magnesium oxide nanoparticles were found to be effective at all concentrations (10 -50
µg/mL) when their antioxidant activity was evaluated using the DPPH test. The 50 µg/mL
showed the largest percentage of inhibition, 89.57 %; the 40 µg/mL showed 84.12 %; the 30
µg/mL showed 81.09 %; the 20 µg/mL showed 74.76 %; and the 10 µg/mL showed 62.44 %.
In comparison to conventional values, the Figure 4 shows that magnesium oxide
nanoparticles may have an antioxidant effect.

H2O2 assay
The H2O2 assay, which measures antioxidant activity, demonstrated the effectiveness of
magnesium oxide nanoparticles at all concentrations (10 -50 µg/mL). The samples with the
highest percentage of inhibition were those with 50 µg/mL (86.9 %), 40 µg/mL (73.16 %), 30
µg/mL (63.5 %), 20 µg/mL (53.7 %), and 10 µg/mL (48.6 %). When compared to
conventional values, Figure 4 shows the possibility antioxidant effect of magnesium oxide
nanoparticles.

FRAP assay.
The efficacy of magnesium oxide nanoparticles was demonstrated in all concentrations (10 -
50 µg/mL) by the FRAP assay, which was used to assess antioxidant activity. Following the
50 µg/mL at 86.32 %, 40 µg/mL at 80.94 %, 30 µg/mL at 77.51 %, 20 µg/mL at 73.14 %,
and 10 µg/mL at 67.86 %, the highest percentage of inhibition was documented in this
sample. When magnesium oxide nanoparticles are compared to conventional values, Figure 4
shows that they may have an antioxidant effect.

ABTS assay
The assessment of antioxidant activity using ABTS assay showed the efficacy of magnesium
oxide nanoparticles in all various concentrations where (10 -50 µg/mL). The highest
percentage of inhibition was noted in the 50 µg/mL at 87.58 %, followed by the 40 µg/mL at
80.95 %, 30 µg/mL at 78.72 %, 20 µg/mL at 70.64 %, and 10 µg/mL at 65.81 %. Figure 4,
illustrates that magnesium oxide nanoparticles have an antioxidant potential effect compared
with standard values.

Nitric oxide assay

Magnesium oxide nanoparticles were found to be effective at all concentrations (10 -50
µg/mL) when their antioxidant activity was evaluated utilizing a nitric oxide analysis. Eighty-
eight per cent inhibitions were seen at 50 µg/mL, the maximum percentage of inhibition was
found at forty µg/mL (79.15 %), thirty µg/mL (78.01 %), twenty µg/mL (72.83 %), and ten
µg/mL (67.45 %). When magnesium oxide nanoparticles are compared to conventional
values, Figure 4 shows was represented the antioxidant effect against Nitric oxide assay.

Toxicology studies
Figure 5: The graph represents the cytotoxic effect of E. arvense-mediated magnesium
oxide nanoparticles using brine shrimp lethality assay and Embryonic toxicology using
Zebrafish embryos.

Brine shrimp lethality assay

Magnesium oxide nanoparticles' cytotoxic effect is depicted in Figure 5, where it is noted that
the particles' toxicity was reduced. 80 % of live naupliis was found to have the highest
concentration (40 & 80 µg/mL), while 100 % of live nauplii was found to have the lowest
concentration (5 & 10 µg/mL). A reduced percentage of living nauplii has been seen with
increasing nanoparticle concentration. It was found that at increasing concentrations,
magnesium oxide nanoparticles were less harmful.

Embryonic toxicology
The hatching rate of zebrafish embryos after treated the magnesium oxide
nanoparticles.
A range of quantities, including 5, 10, 20, 40, and 80 µg/mL, were introduced to the
magnesium oxide nanoparticles mediated by E. arvense. The different levels were
administered to the zebrafish embryos, which were then examined under a microscope for a
duration of 0 to 24 hours. The hatching rate of zebrafish embryos treated with magnesium
oxide nanoparticles is depicted in Figure 5. 100 % of the zebrafish eggs hatched out at the
lowest concentrations (5, 10 & 20 µg/mL). The zebrafish embryos that hatched at a rate of
80% had the highest concentration of nanoparticles. The zebrafish embryos did not hatch
later than expected, and the magnesium oxide nanoparticles exhibited reduced toxicity.

The viability rate of zebrafish embryos after treated the magnesium oxide nanoparticles
A range of quantities, including 5, 10, 20, 40, and 80 µg/mL, has been applied to the
magnesium oxide nanoparticles mediated by E. arvense. After applying the different levels to
the zebrafish embryos, the embryos were examined under a microscope for a duration of 0 to
76 hours. Following treatment with magnesium oxide nanoparticles, Figure 5 displays the
viability rate of the zebrafish embryos. The zebrafish embryos exhibit a 100 % survivability
rate when seen through the lens of the lowest concentrations (5 & 10 µg/mL). Eighty percent
of the zebrafish eggs hatched at nanoparticle doses of 20 and 40 µg/mL. A 60 % viability rate
has been observed at the highest concentration of magnesium oxide nanoparticles. The
zebrafish larvae's delayed growth and the treated embryos' deformity were not seen, despite
the toxicity of the magnesium oxide nanoparticles.

Discussion:
The green synthesis of magnesium oxide nanoparticles using E. arvense to produce the
biological applications and the synthesis process. The E. arvense aqueous extract with the
magnesium chloride solution was treated. It was reduced to magnesium ions to produce the
magnesium oxide nanoparticles. Previous research work was done by using the A.
precatorius extract mixed with magnesium chloride solutions and it formed magnesium oxide
nanoparticles22. The synthesis of magnesium oxide nanoparticles observed the changing of
color from pale brown to dark brownish. Similarly, the A. precatorius-mediated magnesium
oxide nanoparticles showed a color change from a brownish color to a dark brownish color.
The magnesium oxide nanoparticles were observed at the peak of 272 nm using UV
characterization23. In similar work, the absorbance peak of the magnesium oxide
nanoparticles showed at 385 nm24.
In present research work has shown very good antimicrobial activity against wound
pathogens. In similarly, the magnesium oxide nanoparticles showed antibacterial efficacy
against E. coli25. The previous work, the magnesium oxide nanoparticles of antimicrobial
activity against C. albicans, E. coli, Pseudomonas sp, S. aureus, and B. subtilis, respectively.
The measurement of the inhibitory zone is 14 mm, 14 mm, 13 mm, 10 mm, and 11mm 26. In
the anti-inflammatory activity, naturally, the E. arvense-mediated magnesium oxide
nanoparticles have excellent anti-inflammatory properties. In other research work, the % of
inhibition of hemolysis was observed in the highest concentration, there is a dose-dependent
manner27. Naturally, the E. arvense-mediated magnesium oxide nanoparticles have excellent
antioxidant properties. In another work, E. arvense methanolic extract demonstrated a higher
% of inhibition was shown in ABTS and FRAP, and the DPPH assay showed the % of
inhibition. The methanolic extract resulted from higher antioxidant activity28.
The magnesium oxide nanoparticles have a thrombolytic property. Previous research work
was an investigation of cardioprotective medications from plant sources including
determining the thrombolytic activity of extracts from A. bilimbi, C. viscosum, and D.
quercifolia29. In which the cytotoxic effect, brine shrimp lethality assay was done. It showed
less toxicity of the magnesium oxide nanoparticles. Similarly, magnesium oxide
nanoparticles using Phyllanthus emblica had the least cytotoxic effects observed in the higher
concentrations30. The investigation showed that magnesium nanoparticles mediated by Cassia
oleoresin had maximal cytotoxicity in cases where a maximum of 10µL is utilized 31.
Normally, the brine shrimp cytotoxicity bioassay test can be utilized as an initial assessment
for research into the potential for pesticide and anticancer effects 32. In the present research
work was done by embryonic toxicology using zebrafish embryos, the magnesium oxide
nanoparticles showed less toxicity, without any malformation and abnormalities. In another
research study, in treatments of zebrafish eggs, the formed MgO NPs demonstrated no
toxicological characteristics33. In comparison to the control group, the results exhibited an
increasing viability rate at all tested concentrations (100–1000 µg/mL), suggesting minimal
liability. At all the concentrations, no abnormalities in development were identified, and the
mortality concentration of silver nanoparticles was found to be 750 µg/mL34.
Limitation:
An In-vitro study was assessed on the E. arvense-mediated magnesium oxide nanoparticles.
In future studies develop characterizations such as SEM, TEM, FTIR, XRD, and EDX. The
magnesium oxide nanoparticles will develop the gel form and the gel will be used for the
wound healing activity.
Conclusion:
The current research work was done by using the green synthesis of E. arvense-mediated
magnesium oxide nanoparticles. The analysis of in-vitro study UV spectroscopy, antioxidant,
anti-inflammatory, antimicrobial, time-kill curve assay, and the toxicology study whereas
embryonic toxicology using zebrafish, and brine shrimp lethality assay was also estimated by
the E. arvense mediated magnesium oxide nanoparticles. In which future studies to develop
the characterization and the biomedical applications in in-vivo studies.
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