BIOFORMULATION OF SILVER NANOPARTICLES FROM BETEL LEAF (Piper betel) EXTRACT AND ITS ANTIOXIDANT, CYTOTOXIC AND ANTIBACTERIAL PROPERTIES David Gayadang
1 1 Graduate
Student, Saint Mary’s University School of Graduate Studies, Bayombong, Nueva
Vizcaya, Philippines 2 Teacher I, DepEd-Ifugao, Philippines 3 Director, Center for Natural Sciences, Saint Mary’s University,
Bayombong, Nueva Vizcaya, Philippines
1. INTRODUCTION Nanotechnology has progressed significantly in research
over the last century. Nanoparticles (NPs) are categorized based on their
shape, size, and chemical properties. Different methods have been used to study
other physical and chemical characteristics of NPs. These include X-ray
diffraction (XRD), X-ray photoelectron spectroscopy (XPS), infrared (IR),
scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer–Emmett–Teller (BET), and particle size analysis Parray et al. (2021). The field of nanotechnology has
grown rapidly in this century.
Zhang et al. (2016) noted that new progress in
nanoscience and nanotechnology has completely transformed how we detect, treat,
and stop different illnesses across all aspects of human existence. Silver
nanoparticles (AgNPs) are particularly important and
captivating among various types of tiny metal particles used in medical
applications. Silver nanoparticles account for more than 23% of all nano
products. Silver nanoparticles possess a wide variety of properties that makes
them valuable for different purposes. In a world threatened by the rise of
antimicrobial resistance, the use of silver nanoparticles in drug delivery
systems may help solve this emerging problem. Therefore, their large-scale
synthesis has the potential of being extremely useful, especially in
healthcare. Metals
like gold (Au), silver (Ag), palladium (Pd), zinc (Zn), copper (Cu), and iron
(Fe) are chosen due to their specific structures and shapes. These metals
exhibit distinct d–d transition features and strong localized surface plasmon
resonance (LSPR) effects. This has been discussed in studies by Khan et al. (2017), Mohan
et al. (2014), and Dreaden et al. (2012).
Silver nanoparticles find significant applications in medicine and healthcare.
Unlike regular medicines, nanoparticles appear to work well with the body. This
compatibility allows for precise treatment, making therapies more effective and
less harmful. As a result, silver nanoparticles hold great promise as
treatments. They possess various properties like fighting microbes, viruses,
and cancer, aiding in clot dissolution, preventing blood clotting, and assisting
in diagnosis and imaging. In the
world of green chemistry, an emerging field that aims to make chemical
processes more eco-friendly, silver nanoparticles are considered safe for
humans and animals when used in small amounts. They also don’t cause
considerable damage to the environment which sets them apart from other metal
nanoparticles. Several studies have suggested different ways for the
eco-friendly synthesis of metal nanoparticles, like using microorganisms and
plant extracts, that are safer such that of Makarov
et al. (2014) and Prasad et al. (2018).
Currently, there's an increasing demand to create nanoparticles in ways that
are kinder to the environment, avoiding harmful chemicals during the production
process, as discussed by Whitesides (2003).
From the perspective of the replacement of hazardous and non-renewable
chemicals, the utilization of plant extract as a reductor in the synthesis of
metal nanoparticles is an emerging method that is widely developed Ahmed et al. (2016) . Hence exploring sources of
biosynthesized silver nanoparticles from indigenous plants would be a great way
to discover and utilize the potential of ethnographic plants in the
Philippines. One of
these ethnographic plants is the Betel leaf (Piper betel) which is renowned for
its extensive healing properties. Phytochemical studies on piper betel leaves
showed the presence of alkaloids, tannins, carbohydrates, amino acids, and
steroidal components. The biomolecules present create its effects like being a
laxative and anti-parasitic substance. Piper betel freshens breath, aids the
heart, and has many roles like fighting fungi, protecting cells, aiding
digestion, and more. It also affects the brain, cools fever, fights cancer,
reduces inflammation, and influences the immune system and blood clotting.
Parts of the piper betel used are the stem, roots, leaves, stalks, and fruits.
The numerous curative properties of the betel leaf extracts include
anti-diabetic, anti-mutagenic, anti-inflammatory, antibacterial, antioxidative,
and anti-hemolytic. Staphylococcus aureus and Escherichia coli have stopped
their activity against betel leaf extracted in water Chakraborty and Shah (2011), and ethanol Mahfuzul Hoque et al. (2011). Manigauha et al. (2009) noted that the methanolic extracts
from betel leaves exhibit abilities to reduce, and scavenge DPPH radicals,
counteract superoxide anions, and degrade deoxyribose. The Betel leaves are
also reported to possess the ability to fight cancer, especially against
tobacco carcinogens Chang et al. (2002) , Wu et al. (2004). Research by Rai et al. (2011) also revealed that betel leaves can
hinder the development of oral cancer, stomach cancer, and breast cancer. Piper
betel L. or betel
vine also called “Ikmo” in Pangasinan and “Gawed” in Cordilleran provinces has about 100 varieties in
the world. It is a creeping plant that is common among East African and
Southeast Asian countries. Use of Betel leaf in the Philippines is common to
tribes in the Cordillera Administrative Region in their practice of “Moma”/” Mama”/” Nganga” etc. or chewing of betel nut. This
research aimed to explore the potential of the betel leaf in the bioformulation
of silver nanoparticles as an antibacterial, antioxidant, and cytotoxic agent.
This will be relevant in the development of new drugs formulated from endemic
plants in the Philippines. 2. MATERIALS AND METHODS Research Environment The research focused on the bioformulation of silver
nanoparticles (AgNPs) from betel leaf extract. The
antibacterial, cytotoxicity, and antibacterial properties were tested. Samples
were collected at Hapid, Lamut, Ifugao and were brought
to the SMU Center for Natural Sciences for
experimentation. Methods
and Procedures 1)
Plant extract preparation · The collected betel leaves from Hapid, Lamut, Ifugao were brought to the SMU Center for Natural Sciences and oven-dried at 70 degrees Celsius for 3 days. · The dried sample was blended to pulverize the leaves and was soaked in 500ml of 95% ethyl alcohol. · After 72 hours, the mixture was filtered using a glass funnel and cotton to remove the undissolved leaf particles. The mixture was then put into a water bath for the extract to be evaporated at 40 degrees Celsius until it had a syrup consistency. 2)
Preparation of Silver Nitrate A 95:5 ratio was used, and 3.4g of silver
nitrate was combined with 100 mL of distilled water. The solution was stirred
continuously. Another 100 mL of distilled water was combined with 100 mL of
silver nitrate solution. 3)
Bioformulation of Silver Nanoparticles
(AgNPs) from Silver Nitrate In
an Erlenmeyer flask, 38 mL of AgNO3 and 2 mL of leaf extract were
combined and stirred continuously using a glass stirring rod. This was stirred
until a chemical reaction occurred. This was observed once the silver nanoparticles
change color from yellow to dark brown. 4)
High-speed centrifuge The 95% AgNO3 and 5% leaf extract
were placed in a centrifuge tube measuring 10 mL each. For 25 minutes, the
solution was placed in a centrifuge at 6000 rpm. This was to separate the solid
and liquid parts of the solution in layers and then further separated them
through decantation. 5)
Percentage Yield The
solid precipitates of the solution were measured using an electronic weighing
scale to obtain the percentage yield in the 95% AgNO3 and 5% leaf
extract. 6)
UV-VIS Spectrophotometry In a solution (1:4 diluted water) of the
reaction mixture, the UV-Vis spectrum was used to measure the bioformulated AgNPs. It was
compared with 4.5mL of distilled water as a blank. 7)
Antibacterial activity To
test for the antibacterial activity of the sample, the disc diffusion method
was used. Nutrient Agar (NA) was used to screen the in vitro antibacterial
activity of the sample. ·
Preparation of Culture, Media, Positive Control,
Negative Control, and Extract Nutrient
agar and gulaman bar were cooked. This was
mixed with 1000mL tap water and prepared in three Erlenmeyer flasks. The
bacteria culture was incubated at 35 ℃ for 24 hours. After the
extraction, it was filtered using cotton balls and placed in a beaker. Filter
disc of 6mm diameter was immersed in the positive control: 0.25g/L
Streptomycin, negative control: Ethyl alcohol and Betel leaf extract for 24
hrs. ·
Dispensing media in a petri dish Nutrient
agar was allowed to cool down before it was dispensed into the bottom of the
petri dish. Then it was gently rotated
to evenly distribute the medium without any splash over the sides. The tissue
paper was used to wipe any moisture on the cover of the petri dish, as there
should be no moisture on the cover to avoid any water droplets on the colony
formed in the medium. ·
Swabbing of bacteria The
inoculum suspension was swabbed uniformly with the bacteria namely Staphylococcus
aureus (Gram-positive) and Escherichia coli (Gram-negative). It was
dried for 10 minutes. For 24 hours, the filter paper discs were soaked in the
positive control, negative control, and leaf extract and then placed on the
surface of the medium and the compound. This was allowed to diffuse for 5
minutes. ·
Incubation The
setups were kept for incubation at 37℃ for 24 hours. The plates
were kept for measuring the Zone of Inhibition. Inhibition zones were found
around the disc and were measured using a Vernier caliper.
This was done in three replicates. 8)
DPPH Radical Scavenging Assay The
concentrated betel leaf extract was used to make a stock solution. The aliquot
was taken to 500 ppm dilution and 500 Catechin as control (1mg/mL). One mL of
prepared stock solution was mixed with four mL of 0.1 Mm DPPH in a separate
plastic cuvette. This procedure was done in triplicate. Absorbance readings
were monitored using a UV-Vis spectrophotometer. 9)
Brine Shrimp Lethality Assay Artificial
seawater was used in hatching brine shrimps into their larval stage.
Forty-eight (48) hours were allowed to pass before confirming the petri dish
for the hatched eggs. Confirming the hatchlings, the extract was prepared by
getting an aliquot of the extract. 3 mL of the extract was mixed with 3 mL of
artificial seawater. The extract was then mixed and placed in replicate vials. Using
two-fold dilution, 15 vials were prepared with 1000ppm, 500ppm, 250ppm, 125ppm,
and 65 ppm extract and AgNPs of Betel leaf. The
lethality was measured every three hours for 24 hours. In each vial, 10 brine
shrimps were added per concentration. This was to measure the mortality of the
brine shrimps in each vial per concentration at a given time. The mortality
rate was manually counted using a magnifying glass and a flashlight. 3. RESULTS AND DISCUSSIONS 3.1. Visual Characterization on the
Formation of Silver Nanoparticles The utilization of plants to create silver
nanoparticles has gained interest lately. This approach is quick,
environmentally friendly, safe, and cost-effective. It offers a one-step method
for biosynthesis, as explained by Pal et al. (2019).
UV-VIS Spectrophotometry was used to measure the bioformulated
AgNPs. After combining the varying concentrations of
the crude extract and silver nitrate solution, the formulation of
biosynthesized silver nanoparticles is expected. The presence of biosynthesized
silver nanoparticles was verified by the color change in all five varying
concentrations of solution from greenish to dark brown. This is in accord with
the study done by Samuel et al. (2020) who reported
the same color changes due to the reduction of Ag+ ions. Change into dark brown
color was also observed by Lagashetty et al. (2019) in their
biosynthesis of silver and gold using the same sample. During the synthesis, the color change was observable
with constant stirring of the mixture. Solutions were continuously mixed until
there was the formation of cloudy particles suspended in the mixture. The color
changes were observed within 10 minutes of constant stirring. 3.2. UV-Vis To detect the visual characteristic and spectrum
absorbance of the biosynthesized AgNPs, a UV-vis
spectrophotometer was used. Due to surface plasmon resonance (SPR) the AgNPs are expected to give a peak at a particular
wavelength which will confirm its presence. The tables below show the
absorbance values of the silver AgNPs in the
different concentrations. Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Five different concentrations of the solution were prepared and triplicates were made from each concentration.
Distilled water as a blank was first placed in the 5ml cuvette for UV-Vis’s
spectroscopy. Then it was followed by the different concentrations of the
solution which were tested for initial characterization and were tested again
after 30 minutes. Initial confirmation of the presence of AgNPs
was done by the change in visual color. The summary table on the averages of
the different concentrations shows that AgNPs are
characterized based on their highest absorbance in the spectrophotometer. The
UV-Vis showed a mean absorbance of 3.80 at 400nm for the initial and 4.00 at
300-400nm at 30 minutes of characterization. This is similar
to that of Nguyen et. al (2021) with peaks in
the wavelength of 400-450 nm. The study of Lagashetty et al. (2019) has a close
result with the SPR at 430 nm indicating the presence of silver nanoparticles.
A higher absorbance peak was observed by Samuel et. al at 460 nm. 3.3. Antibacterial Activity The synthesized AgNPs were
screened in vitro for their antibacterial activity against E. Coli and S. aureus by
agar disc diffusion method. The results obtained are presented in the following table. Table 8
Betel leaf obtained a mean inhibition zone of
14.65 mm against S. aureus and 12.57 mm against E. coli. The
antibacterial activity was assayed by measuring the diameter of the inhibition
zone formed around the well. Table 9
Meropenem and clindamycin were used as a positive
control. The reduction of bacterial
growth rate on increasing the concentration of Ag nanoparticles is evident. AgNPs in Sample 5 (higher concentration) exhibited the
highest antibacterial activity for Staphylococcus
aureus (11.25 mm and Escherichia coli
(11.20 mm)—both partially active. The lowest activity was observed in
Sample 1(lowest concentration) for both S.
aureus and E. coli (10.16 mm). In
general, biosynthesized AgNPs ranges from partially
active to inactive against S. aureus and E. coli with increasing
antibacterial property with increasing level of concentration. It may appear in
the table that the raw extract has a higher antibacterial property than the
biosynthesized AgNPs which is in contrast with a
comparative study done by Nguyen et. al (2021) but it is also
important to take into consideration the concentration used by the researcher
in this study as compared with other studies which are relatively lower. The
results however can be compared to the study of Foronda & Cajucom (2023) wherein their
samples exhibited higher zones of inhibition than their extracting agent and
negative control. 3.4. Cytotoxic Properties (BSLA) A brine shrimp lethality assay was used to determine
the cytotoxic properties of the sample. The lethality was measured every three
hours for 24 hours. In each vial, 10 brine shrimps were added per
concentration. This is to measure the mortality of the brine shrimps in each
vial per concentration in a given time Foronda & Cajucom (2023). The results
of the test are shown in the following tables below. Table 10
Table 11
Table 12
Table 13
Table 14
Table 15
The
tables show that at 1000 ppm for the first three hours, no shrimps died. On the other hand, in
the succeeding hours, the mortality rate increases. According to
Meyer et al., if the crude plant extract has an LC50 value of less than 1000
µg/mL, it is toxic (active) while if it is greater than 1000 µg/mL, it is non-toxic
(inactive). The LC50 for the first three hours is greater than 1000 ppm thus
the silver nanoparticles from the crude extract of betel leaf are non-toxic but
as time increases, the LC50 decreases to less than 1000 ppm resulting in more
toxic (active) AgNPs. The 4:46 concentration of the
biosynthesized AgNPs showed toxicity at the 24th
hour being the lowest concentration achieving toxicity. At 4:45 concentration,
brine shrimps were completely eliminated at the 21st
hour being the fastest-acting sample. 3.5. Antioxidant Activity The antioxidant activity of the aqueous extract and
plant AgNPs was evaluated using DPPH scavenging. 15
vials with 1000ppm, 500ppm, 250ppm, 125ppm, and 65 ppm extract and AgNPs of Betel leaf were tested. Using the DPPH assay, the biosynthesized
AgNPs in plant extracts were compared to the raw
extract and the Cathechin as a positive control. The
result of the test is shown below. Table 16
Highest RSA achieved is 81.39% with the raw extract.
This reveals that biosynthesized AgNPs in the plant
samples yield lower RSA. All samples have lower RSA compared to the positive
control ranging from 68 – 80%. Nevertheless, the sample yields good results
with RSA higher than 50% and is very close to the result of the positive
control. The results showed that the extract and biosynthesized AgNPs are still effective as an antioxidant agent. 4. CONCLUSION AND RECOMMENDATION This study determined that silver nanoparticles can be synthesized using betel leaf extract. It is confirmed via visual characterization as observed by the changing color of the sample from green to dark brown. Further confirmation of the biosynthesized AgNPs is shown by the results of the UV-vis spectroscopy with maximum absorbance reading at around 300-400 nm. The raw extract of betel leaf has manifested higher effectivity in terms of antibacterial, antioxidant, and cytotoxic properties. Still, the biosynthesized silver nanoparticles showed antibacterial and cytotoxic properties, more so with their antioxidant property very close to the positive control. The biosynthesis of AgNPs in betel leaf still holds possibilities that can be explored more. This work may be further developed with a more in-depth characterization and applications of silver nanoparticles. The researchers recommend characterizing silver nanoparticles to a higher level of characterization and a wider range of concentrations should be used to yield significant results. Other properties of biosynthesized silver nanoparticles may even be explored.
CONFLICT OF INTERESTS None. ACKNOWLEDGMENTS The researchers would like to give special thanks to Dr. Regina D. Ramel, academic dean of the school of SMU School of Graduate Studies for the inspiration as well as to the SMU Center for Natural Sciences laboratory assistants Mr. Regidor Almendral and Mr. Michael Catacutan for their incomparable dedication and support throughout the experimentation process. REFERENCES Ahmed, S., Ahmad, M., Swami, B. L., & Ikram, S. (2016). A Review on Plant Extract Mediated Synthesis of Silver Nanoparticles for Antimicrobial Applications: A Green Expertise. Journal of Advanced Research, 7(1), 17–28. https://doi.org/10.1016/j.jare.2015.02.007. Chakraborty, D., Shah, B., (2011). Antimicrobial, Antioxidative and Antihemolytic Activity of Piper Betel Leaf Extracts, International Journal of Pharmacy and Pharmaceutical Sciences, 3(3),192-199. Chang, M. J.W., Ko, C.Y., Lin, R.F. and Hsiesh, L.L. (2002). Biological Monitoring of Environment Exposure to Safrole and the Taiwanese Betel Quid Chewing. Archives of Environmental Contamination and Toxicology, 43(4), 432-437. (2002). https://doi.org/10.1007/s00244-002-1241-0. Foronda, A. G. R., & Cajucom, D. E. L. (2023).
Anti-Bacterial, Cytotoxicity and Antioxidant Properties of the Isolated
Flavonoids Extract from White Dragon Fruit (Hylocereus Undatus) Peels and
Flesh. International Journal of Engineering Technologies and Management Research,
10(4), 1–13. https://doi.org/10.29121/ijetmr.v10.i4.2023.1305. Kolak, U., Osturk, M., Ozgokce, F., & Ulubelen, A. (2006).
Norditerpene Alkaloids from Delphenium Linearilobum and Antioxidant Activity.
Phytochemistry 67, 2170-2175. https://doi.org/10.1016/j.phytochem.2006.06.006. Lagashetty, A., Ganiger, S., Shashidhar (2019). Synthesis, Characterization
and Antibacterial Study of Ag-Au Bi-metallic Nanocomposite by Bioreduction
Using Piper Betel Leaf Extract, Heliyon, 5, 1-6 https://doi.org/10.1016/j.heliyon.2019.e02794.
Mahfuzul Hoque, M., Rattila, S., Asaduzzaman
Shishir, M., Bari, M.L., Inatsu, Y., Kawamoto, S., (2011). Antibacterial Activity of Ethanol Extract of
Betel Leaf (Piper betle L.) Against Some Food Borne Pathogens, Bangladesh
Journal of Microbiology, 28(2), 58-63. https://doi.org/10.3329/bjm.v28i2.11817.
Manigauha, A., Ali, H., Maheshwari, M.U. (2009). Antioxidant Activity of Ethanolic Extract of Piper Betel Leaves. Journal of Pharmaceutical Research, 2, 3, 2009, 491-494. Nguyen,
N. et. al (2021). Comparative Study of the Silver Nanoparticle Synthesis
Ability and Antibacterial Activity of the Piper Betle L. and Piper Sarmentosum
Roxb. Extracts, Journal of Nanomarerials, Vol. 2021, https://doi.org/10.1155/2021/5518389.
Olowa, L., and Nuneza, O. (2013). Brine Shrimp Lethality Assay of the Ethanolic Extracts of Three Selected Species of Medicinal Plants from Iligan City. Philippines, 2(11), 74-77. Parray, J., and Mir, M., and Shameem,
N. (2021). Nanotechnology and Nanoparticles.
https://doi.org/10.1002/9781119714897.ch1. Rai, M.P. et al. (2011). Piper Betel Linn (Betel Vine), the Maligned Southeast Asian Medicinal Plant Possesses Cancer Preventive Effects : Time to Reconsider the Wronged Opinion, Asian Pacific Journal of Cancer Prevention, 12, 2149-2156. Vishwanath, R., & Negi, B. (2021). Conventional and Green Methods of Synthesis of Silver Nanoparticles and their Antimicrobial Properties, Current Research in Green and Sustainable Chemistry, 4. 100205. ISSN 2666-0865, https://doi.org/10.1016/j.crgsc.2021.100205. Samuel,
H., Nachimuthu, S., Sadhasivam, B., & Ponnusamy, R. (2020).
Biological Synthesis of Silver Nanoparticles from Leaf Extract of Piper Betel and
its Antibacterial Properties. International Conference on Physics and Chemistry
of Materials in Novel Engineering Applications. https://doi.org/10.1063/5.0019754.
Valdes, CO. (2004). Betel Chewing in the Philippines. Arts of Asia, 34, 104-115. Wu, M. T., Wu, D. C., Hsu, H. K., Kao, E. L., & Lee, J. M. (2004).
Constituents of areca chewing related to esophageal cancer risk in Taiwanese
men. Diseases of the Esophagus, 17(3), 257–259, https://doi.org/10.1111/j.1442-2050.2004.00419.x.
Zhang, X. F., Liu, Z. G., Shen, W., & Gurunathan, S. (2016, September 13). Silver Nanoparticles : Synthesis, Characterization, Properties, Applications, and Therapeutic Approaches. International Journal of Molecular Sciences, 17(9), 1534. https://doi.org/10.3390/ijms1709153.
© IJETMR 2014-2023. All Rights Reserved. |