(1)PROJECT PROFILE

Project Title:
In vitro: Anti-bacterial Inhibitory Activity of Green Synthesized Silver
Nanoparticle Derived from Mango (Mangifera indica) Leaf Extract against
Streptococcus pneumoniae

Names of Project Proponent/s:

Dampog, Precious Dianne Flaga
Espancho, Ariane Donaire
So, Ghene Mae Cortes
Region: CARAGA Division: Surigao del Sur
School: Gamut National High School Grade level: 11&12
Project Duration (number of months): 2 months
Email: ghenemae080405@gmail.com Contact Number: 09700931204
(2) CATEGORY OF RESEARCH (4) THEME

(3) _____Physical Science _____Food safety

___/__Life Science _____Water conservation
_____Robotics and Intelligent Machines _____Renewable Energy
_____Mathematics and Computational _____Cyber security
_____Traffic/ road congestion
___/__Health
______Individual
_____Disaster mitigation
___/___Team
_____Agriculture and Environment
_____Others (please specify)
__________________________

(5) EXECUTIVE SUMMARY

With the proliferation of antibiotic resistance in bacteria due to the overuse of

antibiotics, some antibiotics are found to be ineffective. Antibiotic resistance would
hinder the treatment of diseases. On that note, thus study aims to explore the
utilization of Mangifera indica as an alternative source of organic silver nanoparticles
and to evaluate the antibacterial inhibitory activity.

The green synthesis approach creates a non-toxic nanomaterial that can be recycled
with minimal waste byproducts. Plant leaves containing phytochemicals are essential
for developing these nanoparticles, which have antioxidant and antibacterial
properties. (Singh, J. et. al, 2018). Leaf extracts show high antibacterial activity and
inhibition zone against Streptococcus Pneumoniae (Abiv et al.2020).

The plant material will be collected in Barangay Gamut, Tago, Surigao del Sur and
will be identified in the Department of Environment and Natural Resources (DENR).
Subsequently, M. indica leaves will be gathered for the preparation of leaf extract.
The leaves will be washed, air-dried, pounded, and blended into a powder. The
extract will then be mixed with AgNO3 and deionized water, and an antibacterial test
will be conducted. Lastly, antibacterial test will be performed using the Agar Diffusion
Assay with vancomycin as positive control and distilled water as negative control.

(6) INTRODUCTION

The troublesome multidrug-resistant organisms that are most frequently seen are
Acinetobacter baumannii and ESL-producing E. coli, methicillin-resistant S. aureus,
vancomycin-resistant Enterococcus, Klebsiella pneumonia, and penicillin-resistant
Streptococcus pneumonia. A highly drug-resistant strain of Mycobacterium
tuberculosis. (Faizan Abul Qais et al, 2019). Streptococcus pneumonia is bacteria
that are commonly found in the nose and throat of children and adults. S. pneumonia
can infect the lungs (pneumonia) or ears (otitis media), but it is considered invasive
when it is found in the blood, spinal fluid (e.g., meningitis), or other site that normally
does not have bacteria present. S. pneumoniae has developed resistance to multiple
antibiotics including penicillin, macrolides, fluoroquinolone and sulfamethoxazole-
trimethoprim. To treat this multidrug-resistant bacteria causing diseases drugs like
Levofloxacin, linezolid, clindamycin, or vancomycin may also be used in severe
cases where first- and second-line therapies are ineffective (Cools et al., 2021a,
Cools et al., 2021b, Scelfo et al., 2021).

The alarming effects of antibiotic resistance give rise to alternative medicine to find
solution to this life threatening microbial resistance causing disease. Nanotechnology
has been use in many industries including medicinal and therapeutic drug deliveries.
A common application of Nanomedicine is in therapeutic drug delivery, where
nanoparticles containing drugs for therapeutic treatment of disease are introduced
into the body and act as vessels that deliver the drugs to the targeted area. The
nanoparticle vessels, which can be made of organic or synthetic components, can
further be functionalized by adjusting their size, shape, surface charge, and surface
attachments (proteins, coatings, polymers, etc.(Tajabadi et.el, 2019).Green
approach of synthesizing silver nanoparticles is preferred because it is nontoxic and
eco-friendly. Nanobiotechnology which involves using living organisms (such as
animals, plants and microorganisms) in production of nanoparticles is a technology
currently been explored for drug or bioactive component delivery (Shahcheraghi et
al., 2022).
(6.1) RATIONALE/SIGNIFICANCE

This study aims to evaluate the potency of silver nanoparticles derived from mango
leaves against streptococcus pneumonia. The result of the study may give additional
information to the Department of Health (DOH) and Food and Drug Administration
(FDA) on the importance of silver nanoparticles derived Mangifera indica as natural
source of green synthesized nanoparticles against Streptococcus pneumonia. Also
this study will lead the said agencies to push using natural medicine particularly
silver nanoparticles derived from Mangifera indica as an antimicrobial agent. For
pharmaceutical companies, the results of the study will provide them baseline
information in discovering Mangifera indica to be used as raw material in the
production of antimicrobial agents against Streptococcus pneumonia.

This study will also benefit the community, as it may provide them knowledge that
Mangifera indica can be a natural source of silver nanoparticles and can be used as
safe, effective, and affordable alternative antibacterial medicine. Further; for the
future researchers, the results of the study will be a source of information relates to
antimicrobial properties of certain plants against Streptococcus pneumoniae.
(6.2)SCIENTIFIC BASIS/THEORETICAL FRAMEWORK/ MATHEMATICAL
THEORY INVOLVED

The antibacterial inhibitory activity of Green Synthesized Silver Nanoparticles

(AgNP’s) derived from Mango (Mangnifera Indica L.) leaf extract against
streptococcus pneumonia can be explained with a conceptual framework that is
presented below.

Figure 1. The Schematic paradigm showing the independent and dependent

variables of the present study.

The diagram shows the independent, mediatory, and dependent variables. Where in
the independent variables, it indicates the samples that will be going to use in the
study that includes silver nanoparticles derived from mango leaf extract, vancomycin
as a positive control, and distilled water as a negative control. Once the samples are
collected, the study will proceed with several processes, including preparation and
collection, ethanolic plant sample extraction, phytochemical testing, synthesis of
AgNPs from mango leaf samples, and evaluation of antibacterial inhibitory activity.
The output, or dependent variable, will be the antibacterial agent silver nanoparticles
derived from mango leaves.

Mango trees are tropical trees that are known for their sweet and juicy fruit. They
belong to the flowering plant genus Mangifera, which is part of the Anacardiaceae
family. Mango trees can grow up to 100 feet tall and can live for several decades
(Mahindra, 2022).

Streptococcus pneumoniae has a complex relationship with its obligate human host.
They can cause severe disease when bacterial and host factors allow them to invade
essentially sterile sites, such as the middle ear spaces, lungs, bloodstream and
meninges. (Weiser et.al, 2018).

The most common treatments for bacterial infections are antibiotic drugs. Bacteria
can develop antibiotic resistance that will no longer have reactivity to antibiotic
treatments. One of the crucial mechanisms of the bacteria is quorum sensing.
Bacteria communities can communicates in this way for adaptation and survival.
Quorum sensory regulates more than 10% of Streptococcus pneumoniae. The set of
gene are vital in virulence factor production, motility, motility-sessility switch, and
biofilm development. (Moradili et.al 2019)

The antibacterial activity of AgNPs was tested by a well-diffusion method on E. coli

and S. aureus, and the obtained results exhibited a promising antibacterial activity of
the as-synthesized nanoparticles.(Sundeep et.al,2017).
(6.3) OBJECTIVES

This study aims to determine the effectiveness of Mangifera indica derived from
Silver Nanoparticles against Streptococcus pneumoniae. Specifically, it aims to:

1. Determine the amount of bioactive compounds present in the Mangifera indica

extract using HPLC.

2. Determine the amount of Mangifera indica leaves that can produce substantial
amount of silver nanoparticles.

3. Determine the mean zone of inhibition of silver nanoparticles derived from

Mangifera indica extract, vancomycin, and distilled water.

4. Determine if there is a significant difference in the zone of inhibition among silver

nanoparticles, vancomycin, and distilled water against Streptococcus pneumoniae.

5. Determine the significant differences of the zone of inhibition among the various
amount of silver nanoparticles derived from young mango leaves.

(7) REVIEW OF LITERATURE

Streptococcus Pneumonia

Acute bacterial infections are brought on by Streptococcus pneumoniae. Louis

Pasteur initially discovered the bacteria, also known as pneumococcus, in the saliva
of a rabid patient in 1881. Lobar pneumonia and pneumococcus were originally
associated in 1883, but until the advent of the Gram stain in 1884, between 1915
and 1945, various kinds of pneumonia were mistaken for pneumococcal pneumonia.
It described the pneumococcal capsular polysaccharides' chemical makeup and
antigenicity in human illness. By 1940, more than 80 different pneumococci
serotypes had been identified. The search for effective pneumococcal vaccines
dates back to 1911; however, after the discovery of penicillin in the 1940s, interest in
pneumococcal vaccination waned. It wasn't until the late 1960s that efforts to create
a polyvalent pneumococcal vaccine were once more focused. In 1977, the United
States approved the use of the first pneumococcal vaccination. In 2000, the first
conjugate pneumococcal vaccination received US approval.

A gram-positive, lancet-shaped bacterium called Streptococcus pneumoniae is the

culprit behind community-acquired pneumonia. It is the most dangerous bacterial
pathogen in the world, causing over 190 million infections and over 1.2 million deaths
annually. This Gram-positive bacterium can colonize the human nasopharynx
asymptomatically in up to 95% of babies and 25% of adults. S. in a capsule. The
basis for the classification of the bacterium that causes pneumonia is the capsular
polysaccharide. Pneumonia is pathogenic for humans. There have been discovered
92 different serotypes as of 2011.

Mango (Mangifera indica) Leaf Extract

Aside from its highly well-known fruits, the mango tree is an evergreen that has a
variety of traditional medicinal uses. Mangoes are native to South and Southeast
Asia, and in 2018, there were 55.4 million tonnes of mangoes produced worldwide
(the figure also includes guavas and mangosteens). India, China, Thailand,
Indonesia, Pakistan, Mexico, Brazil, Bangladesh, Nigeria, and the Philippines are the
top nations for mango production. Pruning produces significant amounts of
agricultural leftovers, including leaves, flowers, stems, and bark, in addition to its
economically significant portion (fruit), which presents farmers with disposal
challenges. Mango leaves (MLs) are a possible source of minerals, namely
potassium. iron, sodium, calcium, magnesium, potassium, phosphorus, nitrogen, and
vitamins, i.e. B, E, C, and A. Protein is a significant biomacromolecule found in
mango leaves. In impoverished nations, MLs can be used as an alternate source of
cattle nutrition to help with the livestock food deficit. The probable mineral source for
mango leaves is calcium. potassium, phosphorus, nitrogen, calcium, iron, sodium,
magnesium, boron, zinc, manganese, and vitamins, i.e. The concentrations of
several minerals in mango leaves were reported by Ali et al. (2020) as 589 (K), 480
(P), 343 (Fe), 98 (Mg), 368 (Ca), 28 (Na), 14 (Zn), 3 (Mn), and 2 (N) mg/100 g dry
weight (DW). Another study discovered that mango leaves included Ca (4.41%), Mg
(1.58%), K (0.55%), Na (0.23%), N (2.60%), and P (0.40%).

Particularly for the use of plant extracts as food supplements or additives, extraction
with food-grade solvents has been considered as the preferred condition for
obtaining acceptable yields while safeguarding the valuable bioactive features of
plant components. Recent studies have largely debated on affordable and
environmentally sustainable extraction methods of bioactive compounds from plant
materials that minimize the use of solvents. There are still numerous plants that are
not appropriately utilized, despite the rising interest in using natural medicine. The
Mangifera indica leaves, one of the least-exploited plants, and mangiferin, its main
component, are the subject of the current study. Mango leaves are also employed for
religious and ceremonial purposes.

Silver Nanoparticles
Researchers and industry professionals from all over the world have recently
become interested in the developments in the field of silver nanoparticles. Due to
their effective catalytic powers, antimicrobial traits, and distinctive optical behavior,
the use of silver nanoparticles has significantly increased. Numerous industries,
including biomedical applications, water purification technology, electronics, and
renewable energy, stand to benefit greatly from the ongoing research and
discoveries around silver nanoparticle-based solutions. Silver nanoparticles are still
the subject of significant research due to their potential to transform a variety of
industries, opening the door for many exciting new advancements in the near future.

By interacting with proteins that contain sulfur, silver nanoparticles bind to the
surface bacterial cell membrane, preventing cell membrane penetration and
respiratory processes, which results in cell death. Therefore, the interaction of a
molecule containing a thiol from a respiratory enzyme cell with a silver nanoparticle
may stop bacteria from respiring. In addition to interacting with the membrane, the
anti-bacterial cell of silver nanoparticles also penetrates the bacteria. In order to stop
DNA and cell duplication, silver nanoparticles rush inside the bacterial cell and
compress the DNA. Due to their potential for high yields and protein production,
microbes have garnered a lot of interest for their use in the synthesis of AgNPs. This
strategy can be difficult to maintain in terms of culture and growth, though offers
multiple benefits such as being environmentally friendly, easy to handle, energy-
efficient, low in toxicity, and cost-effective. Neo-clerodane flavonol glycosides,
ergosterol, iridoid glycosides, phytoecdysones, and other polyphenols are among the
phytochemicals that play a role in the green production of nanoparticles as reducing,
capping, and stabilizing agents.

Antibacterial Inhibitory Activity

An antibacterial inhibitory activity refers to a substance’s capacity to stop bacterial

development or reproduction. In order to create novel antibiotics to fight antibiotic-
resistant bacteria, a variety of natural and synthetic substance have had their
antibacterial capabilities examined.

Antibiotics, which are made by microorganisms naturally, are a significance class of

antibacterial substances. The increase of germs of germs that are resistant to
antibiotics has, however, rekindled interest in finding and creating fresh antibacterial
medicines.
Numerous studies have focused on exploring the antibacterial potential of plant
extracts, essential oils, and their bioactive compounds. For example, studies on
plants such as garlic, turmeric, and oregano have shown promising antibacterial
inhibitory activities against various bacteria, including multi-drug resistant strains.

In addition to natural products, researchers have also investigated the antibacterial

properties of synthetic compounds, Nanoparticles, such as silver nanoparticles, have
gained attention due to their strong antibacterial activities against a wide range of
bacteria. Other synthetic compounds, such as quinolones and sulfonamides, have
also been extensively studied for their inhibitory effects on bacterial growth.

Furthermore, the combination of different antibacterial agents has been explored to

enhance their inhibitory activities. This approach aims to overcome bacterial
resistance and improve treatment efficacy. Synergistic interactions between different
antibiotics can lead to increased antibacterial activity, making them more effective in
treating infections.

In recent years, there has also been a focus on using advanced technologies, such
as high-throughout screening and computer- aided drug design, to discover new
antibacterial compounds. These approaches enable researchers to screen large
libraries of compounds and predict their inhibitory activities against specific bacterial
targets.

(8) METHODOLOGY

This chapter outlines the research design, sampling design, laboratory procedures
and statistical analysis to be employed in the study. The laboratory procedures
include the step-by-step protocol on testing for phenolic content of Mango leaf
extract, synthesis and characterization of AgNP’s, and antibacterial property.

RESEARCH DESIGN

This work will apply the post-test true experimental research strategy in analyzing
the antibacterial of AgNP’s generated from Mango leaf extract against Streptococcus
pneumonia. The zone of inhibition will be evaluated in millimeter following the
treatment of manufactured AgNPs. The posttest true experimental research design is
a sort of experimental study in which the group (the plate containing previously
grown microorganisms) gets treatment of experimental drug (AgNPs), negative
control (placebo drug) and positive control (vancomycin). After 24 to 48 hours of
incubation, the zones of inhibition of the therapy will be measured in millimeter (mm)
using the vernier caliper (Health Services Research techniques, 2010). (Health
Services Research methods, 2010).

Table 1: Research Design Showing Zone of Inhibition of Silver Nanoparticles derived

from Mangifera indica leaf extract, Vancomycin, and Distilled water against
S.pneumoniae
Set-ups Zone of Inhibition (mm) of Mango Leaf Extract
against S. pneumoniae
Experimental R1 R2 R3 Average
Silver Nanoparticles X X X X
(AgNPs) derived from
Mangifera indica leaf
extract
Positive Control: X X X X
Vancomycin
Negative Control: X X X X
Distilled water

Legend:
R = Replicate X = Zone of Inhibition

SAMPLING DESIGN

The researchers will use the non-probability purposive sampling design in the
collection of the plant materials (Mango leaf extract). The sample will be collected in
Gamut, Tago, Surigao del Sur and will be transported to the laboratory for the
synthesis of AgNPs and antibacterial screening.

DATA GATHERING

A. Preparation of Leaf Extract

Freshly collected leaves of M.indica will be washed with distilled water and will be air
dried for 4 days. After drying, the plant material will be pounded into smaller particles
and then blended to powder using an electric blender. A 200grams of the powdered
samples is to be stored in airtight containers and will be kept under normal room
temperature. The extracts will then be filtered using Whatman no. 1 filter paper and
the filtrates concentrated in vacuo at 40ºC using a rotary evaporator (Akerele et al.,
2008).

B. Synthesis of AgNPs

In order to optimize the process of preparation of AgNPs samples, about 10 ml of

0.10M AgNO3 will be dissolved in 80 ml of deionized water and mixed with 10 ml of
Mango leaf extract solution under vigorous stirring at room temperature for 5 min.
Optimization of silver nanoparticles formation will be made using heat mediated
synthesis by heating the mixture to 80 °C for 10 mins. After the process, the
synthesized silver nanoparticles will be cooled down at a room temperature before
subjecting to UV-Vis Spectrometry characterization at 420nm. The procedures will
follow the study of Prathna, T et al., 2013 entitled “Heat Mediated Synthesis of Silver
Nanoparticles Using Citrus lemon (Lemon) Extract”

C. Characterization of Silver Nanoparticles

The UV–vis spectra of Ag NPs and plant extract will be measured at a wavelength
range of 300-600nm. The extract color change from yellowish to reddish brown is
exhibited by Ag reduction in reaction with plant extract. The change in UV-vis spectra
for AgNPs will be manifested by the broadening of the peak, which indicates the
formation of nanoparticles due to slow reduction rates.
D. Test for antibacterial Property (Guevera et. al, 2004)

d.1. Preparation of Innoculum

Colonies of Streptocuccos pneumoniae will be inoculate onto 5 mL Mueller-Hinton

broth (MHB) and will be incubate at 350C for 2 hours to allow bacteria to grow. It will
also be adjust using 0.5 McFarland standard solutions with sterile NSS. This will
serve as the standardized inoculum.

d.2. Preparation of Seeded Agar Plate

About Twenty mL of Mueller-Hinton Agar, MHA, will be dispense into each of the
sterile petri dishes to a depth of 4 mm and will be cooled down and solidify. A sterile
cotton swab will be dipped onto the standardized organism (inoculum) suspension,
pressed and rotated firmly against the inside of the tube just above the fluid to
remove the excess liquid. The entire surface of the solidified agar will be swabbed
evenly to ensure even and complete distribution of the inoculum. The plates will be
allowed to dry for 15 minutes. This preparation must be done in the biosafety
cabinet.
d.3. Phytochemical Analysis using HPLC Technique
 Fig 2: Schematic lay-out of HPLC System
(International Journal of Chemical Studies 2020; 8(6): 2590-2596.
Retrieved from www.chemijournal.com )

The image depicts the process of performing phytochemical analysis using the
HPLC technique. The HPLC system is comprised of several components which are
detailed in the image. The Mobile Phase Reservoir stores the mobile phase needed
for the analysis. The eluent system is degassed in the Degasser, and the solvent
delivery system is managed by the Pump which facilitates the flow of the mobile
phase through the system. The Injector is responsible for introducing the sample to
the system. The Column Compartment controls the column temperature, while the
Detector detects each component in the separated mixture after elution from the
column. The recorded data is then converted into meaningful information by the Data
Processor (Recorder). Finally, any excess or impure liquid is collected in the Waste.

Statistical Analysis

In this study, the researchers will employ the single factors Analysis of Variance
(ANOVA) and Least Significant Difference (LSD) test to determine the significant
difference on the mean zones of inhibition exhibited by AgNPs derived from Mango
leaf extract (Experiment Drug), Vancomycin (positive control ) and distilled water
(NegativeControl). Calculations on mean zones of inhibition and standard deviation
of the replicated tests must also be conducted in this study.

(9) EXPECTED OUTPUTS AND POTENTIAL IMPACTS

After this research, the amount of bioactive compounds present in Mango leaf
extract will determine the alkaloids, flavonoids, benzophenones and phenolic
compounds will also be found, it is expected that silver nanoparticles derived from
Mango leaf extract is a potential antibacterial against streptococcus pneumoniae with
the results of this study, the society will be informed about Mangifera indica which
can be potential alternative antibacterial treatment against S. pneumoniae. Scientist
will also benefit in conducting future research as this will fill the gaps in knowledge
about the plant and its inhibitory effect against S. pnemoniae. Patients who primarily
suffer from the effect of S. pnemoniae will be treated using Mangifera Indica. The
agencies like Philippine Institute of Traditional and Alternative Health Care (PITACH)
and Department of Health (DOH) that are tasked to address health issues will learn
more about the effectiveness of Silver Nanoparticles derived from Mango Leaf
extract against S. pneumoniae and recommended Mangifera Indica as alternative
treatment to diseases caused by S. pnemoniae.

(10) WORK PLAN AND TARGET DELIVERABLES

WORK PLAN

Research Title:
In vitro: Anti-bacterial Inhibitory effect of Silver Nanoparticle Derived from Mango
(Mangifera indica Linn) leaf Extract against Streptococcus pneumoniae
Name of Proponent/s:
Dampog, Precious Dianne Flaga
Espancho, Ariane Donaire
So, Ghene Mae Cortes

Timeline/Duration:
Two (2) Months
Description:
The primary purpose of this Research Proposal is to test the inhibitory
activity of green synthesized silver nanoparticles derived from mango leaf extract
against streptococcus pneumonia.

Time Frame Activities/Tasks Persons Output

Responsible Expected
September 11, Attend orientation on the Research Research Work
2023 Mechanics and Guidelines of proposal Plan, Estimated
Likha (Research Proposal) Proponents, Cost and
Competition, which is based Coach and Materials
on the DepEd Order No. 480, School Head
s. 2023, “Observance of the
2023 National Science
Month”
September 12 Seeking approval and Research Relevant and
–15, 2023 validation of Research Proponents Timely
Problem, Statement of the and Research Research Title
 Problem, and Research Title Coach
September 16- Crafting of Chapter 1 to Research Crafted
18, 2023 Chapter 2 of Research Proponents Introduction and
Proposal (Introduction and and Research Review of
Review of Related Literature) Coach Related
Literature

September 19- Crafting of Chapter 3 Research Crafted

22, 2023 (Methodologies) Proponents Methodologies
and Research
Coach
September 23, Evaluating the Research Research Corrected
2023 Proposal from the Research Proponents, Research
Perspective Research Proposal
Coach,
Research
Critiques and
School Head
September 24- Modifying research proposal Research Revised
25, 2023 by utilizing the revisions Proponents Research
provided by the Research and Research Proposal of
Professionals Coach Chapter 1 to 3
September 26- Preparing the Research Research Finalized
30, 2023 Proposal for submission Proponents papers for
and Research Presentation
Coach
October 3, Submission of Soft Copy Research Submitted the
2023 version of the Research Proponents , final copy of
Proposal Research Research
Coach, Proposal
October 9, Presentation of Research Research Approval
2023 Proposal Proponents Research
Proposal
October 11, Submission of research Research Submitted
2023 specimens for testing, Proponents , Research
screening, and analysis in Research Proposal
the laboratory Coach, and
Laboratory
Expertise
November 11, Release of results from the Research Results of the
2023 laboratory testing, screening, Proponents , test from the
and analysis of the research Research laboratory
specimens Coach, and
Laboratory
Expertise
November 12, Analyzing and interpreting Research Analyzed and
2023 the data gathered from the Proponents interpreted
Research Study and Research Survey Results
Coach
November 15, Submission of the final Research Publication of
2023 Research Study and awaiting Proponents the Research
for publication and Research Study
Coach
(11) REFERENCES
A. Afreen, et al. Phytochemical-assisted biosynthesis of silver nanoparticles from
Ajuga bracteosa for biomedical applications. Materials Research Express, 7 (7)
(2020), p. 75404

Ali, B.A.; Alfa, A.A.; Tijani, K.B.; Idris, E.T.; Unoyiza, U.S.; Junaidu, Y. Nutritional
Health Benefits and Bioactive Compounds of Mangifera indica L (Mango) Leaves
Methanolic Extracts.

Kharchoufi, S.; Parafati, L.; Licciardello, F.; Muratore, G.; Hamdi, M.; Cirvilleri, G.;
Restuccia, C. Edible coatings incorporating pomegranate peel extract and biocontrol
yeast to reduce Penicillium digitatum postharvest decay of oranges. Food Microbiol.
2018, p. 74, 107–112.

Kumar, Manoj, Vivek Saurabh, Maharishi Tomar, Muzaffar Hasan, Sushil Changan,
Minnu Sasi, Chirag Maheshwari, Uma Prajapati, Surinder Singh, Rakesh Kumar
Prajapat, Sangram Dhumal, Sneh Punia, Ryszard Amarowicz, and Mohamed
Mekhemar. "Mango (Mangifera indica L.) Leaves: Nutritional Composition,
Phytochemical Profile, and Health-Promoting Bioactivities." Antioxidants (Basel) 10,
no. 2 (February 2021): 299

Lavelle, E. C., Ward, R. W. (2021). Mucosal Vaccines — Fortifying the Frontiers.

Nat. Rev. Immunol. doi: 10.1038/s41577-021-00583-2

M. Guilger-Casagrande, R. de Lima. Synthesis of silver nanoparticles mediated by

fungi: A review. Frontiers in Bioengineering and Biotechnology, 7 (2019), p. 287-
287.

Pariona. 2018. The Top Mango Producing Countries in the World.

P.N.V.K. Pallela, et al. Ultra Small, mono dispersed green synthesized silver
nanoparticles using aqueous extract of Sida cordifolia plant and investigation of
antibacterial activity. Microbial Pathogenesis, 124 (2018), pp. 63-69.

Princwill-Ogbonna, I.; Ogbonna, P.; Ogujiofor, I. Proximate Composition, Vitamin,

Mineral and biologically Active Compounds Levels in Leaves of Mangifera indica
(Mango), Persea americana (Avocado pea), and Annona muricata (Sour sop). J.
Appl. Sci. Environ. Manag. 2019,pp. 23, 65.

Ryan Gierke, MPH; A. Patricia Wodi, MD; and Miwako Kobayashi, MD, MPH.
Pneumococcal Disease. Center for Disease Control and Prevention. 2021.

Trimble, A., Connor, V., Robinson, R. E., Mclenaghan, D., Hancock, C. A., Wang, D.,
et al. (2020). Pneumococcal Colonisation is an Asymptomatic Event in Healthy
Adults Using an Experimental Human Colonisation Model. PloS One 15, 2020, pp.
1–12.