Green Synthesis of Strontium Nanoparticles Using Clove and Lemongrass and Its

Anticariogenic Activity and Antioxidant Activity

Rajeshkumar Shanmugam*, Sulochana Govindaraj, Vedha R Nair, Prenetha R,
Tharani Munusamy
Department of Pharmacology, Saveetha Dental College and Hospitals, Saveetha Institute of
Medical and Technical Sciences, Saveetha University, Chennai – 600077, Tamilnadu, India

*Corresponding Author
Dr S Rajeshkumar
Professor
Nanobiomedicine Lab, Centre for Global Health Research, Saveetha Medical College and
Hospital, Saveetha Institute of Medical and Technical Sciences, Saveetha University,
Chennai – 600077, Tamilnadu, India.
[email protected]
Sulochana Govindaraj, Tharani Munusamy
Nanobiomedicine Lab, Centre for Global Health Research, Saveetha Medical College and
Hospital, Saveetha Institute of Medical and Technical Sciences, Saveetha University,
Chennai – 600077, Tamilnadu, India.

Vedha R Nair, Prenetha R

Department of Pharmacology, Saveetha Dental College and Hospitals, Saveetha Institute of
Medical and Technical Sciences, Saveetha University, Chennai – 600077, Tamilnadu, India.

ABSTRACT:
Background: Dental issues have a significant impact on people's health globally.
Applications for strontium include biomedicine, cancer therapy, sensors, supercapacitors, and
catalysts. It can also be used in the treatment of bone tissue in dentistry. As a result, interest
in using Strontium-based nanoparticles in dentistry and medical applications has increased. It
has anti-diabetic, antibacterial, anti-toxin, anti-hepatotoxin, and antioxidant properties in
addition to its capacity to heal wounds.
Aim: The objective of the research is to determine the anticariogenic activity and antioxidant
activity of the green synthesis of strontium nanoparticles (SrNPs) using lemongrass and
clove.
Materials and methods: The research work was done by anticariogenic activity and the Free
radical scavenging activity using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay and H2O2
assay.
Result: Strontium nanoparticles have a dose-dependent increase zone of inhibition in
anticariogenic activity against Staphylococcus aureus and Streptococcus mutans, and
Enterococcus faecalis was showed in equal zone of inhibition in three different
concentrations. Similarly free radical scavenging activity of lemon grass and clove
formulation mediated Strontium nanoparticles, maximum inhibitory activity was observed at
maximum concentration.
Conclusion: SrNPs was showed a very good antioxidant and anticariogenic activity. The
higher inhibitory zone was observed by higher concentration (100 μg/mL) against the
cariogenic pathogens. Similarly to the free radical scavenging assay.

KEYWORDS: Green synthesis, Strontium Nanoparticles, Anticariogenic activity,

Antioxidant activity, biomedical application, Product Development, Sustainable.
INTRODUCTION:

Dental caries, a frequent type of dental disease, is attributed to microorganisms found in

dental plaque on the tooth surface. Streptococcus mutans, Streptococcus rattus,
Streptococcus cricetus, Streptococcus ferus, and Streptococcus sobrinus are among the
Streptococcus mutans that have a role in the production of dental biofilm and the formation
of dental caries. Streptococcus sobrinus and Streptococcus mutans pathogens are highly
infected in dental caries, and the pathogens growth decreased by using some herbs 1. Herbs
and herbal extracts contain a variety of phytochemicals that have biological qualities that
provide benefits to human health and reduce the risk of chronic disease 2. Streptococcus
mutans is challenging other clinical pathogens because the clinical microbes have habituating
properties in the oral cavity. In humans, a high quantity of sucrose was consumed; sucrose
was attached to teeth. Microorganisms convert sucrose into lactic acid, especially S. mutans.
S. mutans was forming the plague in the oral cavity 3. Today, oral problems are treated with
herbal-based products.

Humans have been exploring natural remedies for diseases since ancient times; more
recently, herbal medicines have gained popularity in dietary supplements, energy drinks,
multivitamins, mouthwashes, toothpastes, etc. These applications have widened the scope of
herbal treatment while also boosting its credibility 4. Lemongrass (Cymbopogon citratus) has
been shown to have antioxidant properties and is used for treating digestive problems,
diabetes, and inflammation. Citral epoxide, a component of lemon grass oil, has a higher
antibacterial activity than fungi, and has also been shown to successfully inhibit methicillin-
resistant Staphylococcus aureus (MRSA) strains5. Various studies had reported about the
antibacterial activity of lemongrass oil against the different pathogens including gram
negative and positive bacteria, fungi and yeast 6. Similarly, clove essential oil exhibits
antifungal, anticariogenic, and antimicrobial activities. Numerous antimicrobial compounds
against oral bacteria attributed with dental caries have been isolated from clove 7.
In another research work, nanoparticles are commonly used for the targeted bacteria.
Nanoparticles were used to treated the wound healing, detection of bacterial system, to avoid
the infectious disease and the treatment of ailment effectively in the antibiotic delivery
system. They are used to laminate the antibacterial agents and to boost up the pharmaceutical
materials8,9. In vitro, antibiotics, antimicrobial peptides and metal based ions such as
strontium (Sr), zinc (Zn), silver (Ag), etc,.. have demonstrated bactericidal effects against
several periodontal infections. Sr is particularly intriguing because it has been studied to
impede P. gingivalis, E. coli, A. actinomycetemcomitans and S. aureus growth. SrNPs was
used highly in bone regeneration method; they have a unique property to easily connect to the
particles8. SrNPs have a great antioxidant properties, some pharmaceutical drugs was coated
with Sr based antioxidant drug.

Oxidative stress is a condition that occurs when the balance between a cell's antioxidative
defense and oxidants is upset by the existence of too many oxidants, like ROS 10. Damaged
tissues produce reactive oxygen species (ROS), which is linked to oxidative stress. An
imbalance between excessive ROS generation and insufficient antioxidant defense systems is
classified as this mechanism11. The objective of the research is to determine the
anticariogenic activity and antioxidant activity of the green synthesised of strontium
nanoparticles (SrNPs) using lemongrass and clove.
MATERIALS AND METHODS:
Preparation of plant extract
1 g of clove and 1 g of dried leaves of Lemongrass were mixed with 100 mL of distilled H2O
after being placed at a heating mantle at 50 °C for 15-20 mins, the filtered solution using
Whatman No.1 filter paper, the solution was stored in the refrigerator.
Preparation of Strontium nanoparticles:

30 mM of strontium chloride was added in 50 mL of distilled water. The 50 mL of filtered

plant extract was added with the strontium chloride solution. Lemongrass and clove extract
with strontium chloride solution is acquired as shown in figure 1. This solution is placed in
the orbital shaker for 24 hours. After incubation, the solution was centrifuged for 10 minutes
at 8000 rpm (Rotation per minute). Then, SrNPs pellet was collected and supernatant was
discarded.

Antioxidant Activity:

(DPPH) 2,2-Diphenyl-1-picrylhydrazyl assay:

0.1 mM of 2,2-Diphenyl-1-picrylhydrazyl was mixed methanol solvent for the preparation

stock standard solution. The stock standard solution was dissolved in the methanol solvent to
make a working standard solution. In each assay, working standard solutions are freshly
prepared. Five various concentrations (10-50µg/mL) of herbal formulation mediated SrNPs
was added to the 200 microlitre of 2,2-Diphenyl-1-picrylhydrazyl working standard solution
in the 96 well ELISA plate. The plate was kept in the dark incubation at 37 degree celsius for
30 mins. After the incubation the plate was measured at 517 nm using ELISA reader.
Methanol servos as a blank in this study. The ascorbic acid was used as a standard. To
determine the % of inhibition was calculated by the following formula:

% DPPH Scavenging Activity = [(Ac – As) / Ac] × 100

Where, Ac - absorbance of the control (without sample), As - absorbance of the sample (with
sample)

Hydroxyl radical scavenging assay (H2O2 assay)

Halliwell et al method was used for performing H2O2 assay by the free radical scavenging
assay. 1 mL of reaction mixture with 100 µL of 28 mM of 2- deoxy-2-ribose was prepared.
Five different concentrations (10-50µg/mL) of herbal formulation mediated SrNPs was
added. After that, 100 µL of ascorbic acid, 200µL of ferric chloride, 200 µL of EDTA was
added that mixture solution. Finally, the tubes were kept in dark incubation for 1 hour at
room temperature. The completion of the incubation period, the tubes was measured at 532
nm against a blank solution. The positive control was used as vitamin E.

% of inhibition = [(Ab − As)/Ab] × 100

Where, Ab - absorbance of the control (without sample), and As - absorbance of the sample.

Anticariogenic Activity:
Rajeshkumar et al method was used for performing agar well diffusion technique by the
anticariogenic activity13. The lemongrass and clove mediated strontium nanoparticles
determined the anticariogenic activity using agar well diffusion method against
Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis. Muller Hinton agar
were sterilized using the autoclave for 15 mins at 121 degree celsius. The cariogenic bacteria
(Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis) was freshly prepared
overnight. The polystyrene tip (9mm) was used to cut the wells in the muller hinton agar
filled Petri plates. The fresh cariogenic culture was evenly spread in the media plates using
sterile cotton swabs. Three different concentrations (25, 50 and 100 μg/ml) of lemongrass and
clove mediated strontium nanoparticles were added into the wells. Amoxyrite was used as a
standard, it a antibiotics for the bacterial culture. The plates were incubated at 37 degree
celsius for 24 hrs. After the incubation period was completed, to estimate the zone of
inhibition in the wells were measured in diameter. The inhibitory zone was calculated in
millimeters (mm).
RESULT:

UV-visible Spectroscopy
2.500

2.000
Absorbance

1.500
24 h
1.000

0.500

0.000
370.0 420.0 470.0 520.0 570.0 620.0

Wavelength (nm)

Figure 1: The UV-visible spectroscopy of the green synthesis of Strontium

nanoparticles.
Figure 1 shows the outcomes of the UV-visible spectroscopy study for the green synthesis of
SrNPs, which demonstrated an elevated absorption peak at 435 nm after a 24-hour incubation
period. The various absorption peaks indicate the wavelengths at which the nanoparticles
exhibited the strongest absorbance, so signifying their unique optical features and verifying
effective synthesis at the given time intervals.
As shown in figure 2a, the lemongrass and clove formulation mediated Sr NPs was noted the
% of inhibition in the dose dependent on DPPH free radical assay. The minimum
concentration of 10 μg/ml, the % of free radical inhibition was observed to the 73% whereas
at maximum concentration 50 μg/ml the inhibition ranged from 73% to 82%. The % of free
radical inhibition by lemongrass and clove mediated SrNPs occurred in concentration
dependent manner. When the concentration increased the % of free radical inhibition also
increase or higher.
Figure 2: Antioxidant activity of green synthesis of SrNPs using A) DPPH assay and B)
H2O2 assay.
As shown in figure 2b, the lemongrass and clove formulation mediated Sr NPs was noted the
% of inhibition in the dose dependent on H2O2 free radical assay. The minimum concentration
of 10 μg/ml, the % of free radical inhibition was observed to the 50% whereas at maximum
concentration 50 μg/ml the inhibition ranged from 50% to 83%. The % of free radical
inhibition by lemongrass and clove mediated SrNPs occurred in concentration dependent
manner. When the concentration increased the % of free radical inhibition also increase or
higher.

Figure 3: Anticariogenic activity green synthesised SrNPs using agar well diffusion
technique against cariogenic pathogens ((a) S. aureus, (b) S. mutans & (c) E. faecalis).
Figure 3 (d): Graphical representation of anticariogenic activity of green synthesized
SrNPs against cariogenic pathogens treated with various concentration.
SrNPs was increase the three different concentrations and the inhibitory zone also raised in
the anticariogenic against Staphylococcus aureus and Streptococcus mutans. Similarly the
SrNPs was loaded into 3 wells for 3 concentrations to observe the same inhibitory zone in
Enterococcus faecalis. At least concentration (25 μg/mL) in 12 mm for inhibitory zone and
higher concentration (100 μg/mL) in 17 mm for inhibitory zone showed in Staphylococcus
aureus (Figure 3: (a)). At least concentration (25 μg/mL) in 13 mm for inhibitory zone and
higher concentration (100 μg/mL) in 15 mm for inhibitory zone showed in Streptococcus
mutans (Figure 3: (b)). At least concentration (25 μg/mL) in 9 mm for inhibitory zone at
higher concentration (100 μg/mL) in 9 mm for inhibitory zone showed in Enterococcus
faecalis (Figure 3: (b)). The SrNPs compared with amoxyrite (antibiotic) was loaded in 4 th
well respectively.
DISCUSSION:
In today's world, classic methods are recommended to the alternative therapies in novel
research. According to these green synthesis methods, they have a minimal side effect for the
drug delivery system. A multifaceted mechanism is involved in the exhibition of
antimicrobial potential of nanoparticles. Microbial cell adhesion of nanoparticles, and
reactive oxygen species (ROS), and their penetration inside the cells, have been
acknowledged as the most important and primary modes of antimicrobial action14.

In previous research work, Apsana et al was reported, antibacterial properties of O. sanctum

(tulsi) leaves extract it was reported that strontium nanoparticles exhibit the peak was
observed by the range in between 250 -350 nm, the peak was obtained by 274 nm in UV-
spectrum and showed antimicrobial activity against many bacterias S. aureus, E. coli etc…15.
Traditionally, stimulation of growth factors by using Strontium, to stimulate the ability of
calcium and regeneration of bone. Hence dentistry and medicinal field was used in the
strontium based nanoparticles because of its exhibited properties being similar to that of
calcium. In previous research work Mutreja et al, it was reported that strontium NPs
demonstrated antimicrobial activity against methicillin resistant Staphylococcus aureus16.
Similarly, In a study by Maghsoudi et al, on observed the anticariogenic activity of curcumin-
loaded polysaccharide nanoparticles against Streptococcus mutans, it was noted that the
inhibitory zone of curcumin-loaded chitosan nanoparticle held promising anti cariogenic
activity. They also reported the SrNPs showed to have more inhibitory effect when compared
to free curcumin17.

In previous research work, they had noted the antioxidant property of oolong tea mediated
strontium nanoparticles inhibited the % of inhibition. The different concentrations of the
SrNPs were (10-50) μg/mL with their % of inhibition was observed in 517 nm 55 %, 60 %,
74%, 76% and 80% respectively. The direct proposition to the concentration of the SrNPs
and the % of inhibition in the DPPH assay. In the H 2O2 assay, the different concentrations of
the SrNPs were (10-50) μg/mL with their % of inhibition was observed in 532 nm 52%, 60%,
68%, 72%, and 77% respectively. The green synthesis of SrNPs was less effective at 30
μg/mL. Commercial antibiotics were more effective than that of nanoparticles in the
antioxidant activity. In common the concentration level was increased in the H 2O2 assay, the
% of inhibition also rises18.

By the results we came to know that there was a dose-dependent increase in the both H 2O2
and DPPH activity of lemon grass and clove formulation mediated SrNPs. The antioxidant
activity based on concentration dependent, maximum inhibitory activity was observed at
maximum concentration.
Limitation
Herbs play a major role in dental treatment, specifically in tooth pain. The green synthesis of
nanoparticles has a less toxicity and safety with the combination of clinical eco-friendly.
Anticariogenic assay against the cariogenic pathogens was done by the application of drug
delivery in the dental problems. In future studies were to develop the green synthesised
SrNPs mediated dental varnish, mouthwash and toothpaste. These will make a product and
preliminary to check the animal model and clinical trial.
CONCLUSION:
A sensible method was investigated to create a green, environmentally acceptable way to
synthesize strontium nanoparticles using the extract of lemon grass and clove. The
synthesized lemongrass and clove formulation mediated SrNPs shows very good antioxidant
activity and similar to the anticariogenic activity against cariogenic pathogens. The results of
this work suggest that lemon grass and clove formulation mediated strontium nanoparticles
may eventually be used as anticariogenic agent in future dentistry and their biomedical
applications.
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