20nov2018 Paper 2

Gilo, Lorico, Terre | 1
Republic of the Philippines

Department of Education
National Capital Region
Division of Quezon City Schools
Quezon City
Cytotoxicity of Phytosynthesized Silver Nanoparticles (AgNPs) using Aqueous Extract

of Alugbati (Basella alba L.) on Selected Cancer Cell Lines
Life Science – Team Category

(Senior High School)
A Science Investigatory Project

Presented to DepEd NCR
Regional Science Fair 2018
S.Y. 2018 – 2019
*Arniel Joseph G. Gilo

Ernztin Andrea A. Terre
Hans Daniel Q. Lorico
Project Proponents
Quezon City Science High School

Regional, Quezon City, NCR
Mrs. Jackieline A. Gagaza

Research Adviser
Mr. Denis Dyvee R. Errabo

Research Associate I
De La Salle University
I. Title page
TABLE OF CONTENTS
I. Title page ............................................................................................................................................1

List of Figures ........................................................................................................................................... 3
List of Tables ............................................................................................................................................. 4
List of Equations ...................................................................................................................................... 4
I. ABSTRACT........................................................................................................................................5
II. INTRODUCTION ............................................................................................................................6
Background of the Study ......................................................................................................................... 7
Research Questions: .............................................................................................................................. 10
Research Hypotheses ............................................................................................................................. 10
Statistical Hypotheses ............................................................................................................................ 10
Objectives ................................................................................................................................................. 11
General ............................................................................................................................................................................11
Specific ............................................................................................................................................................................11
Significance of the Study ...................................................................................................................... 12
III. MATERIALS AND METHODOLOGY ..................................................................................... 14
Location and Duration of the Study .................................................................................................. 14
Materials ................................................................................................................................................... 14
General Procedure ................................................................................................................................. 15
IV. RESULTS........................................................................................................................................ 22
Ultraviolet-Visible Spectroscopy ........................................................................................................ 22
Fourier-Transform Infrared Spectroscopy...................................................................................... 23
SEM and EDAX Analyses .................................................................................................................... 25
TEM Analysis .......................................................................................................................................... 27
MTT Assay ............................................................................................................................................... 28
I. A549 Human Lung Adenocarcinoma ........................................................................................................28
II. MCF7 Human Breast Carcinoma ..............................................................................................................32
V. DISCUSSION................................................................................................................................. 35
VI. CONCLUSION AND RECOMMENDATIONS ...................................................................... 40
VII. BIBLIOGRAPHY .......................................................................................................................... 42
VIII. APPENDIX....................................................................................................................................... 46
Appendix A. List of Figures ................................................................................................................................46
Appendix B. List of Tables ..................................................................................................................................52
Appendix C. List of Equations ...........................................................................................................................52
Appendix D. MTT Assay/Statistical Analysis.............................................................................................53
Appendix E. Certificate of Authentication ...................................................................................................59
Appendix F. Certificate(s) of Appearance ...................................................................................................60
List of Figures
Figure 1: Alugbati (Basella alba L.) Leaves (Photo taken by Hans Lorico, 2018) ...................... 14
Figure 2: Silver Nitrate (AgNO3) (Photo taken by Hans Lorico, 2018) ....................................... 14
Figure 3: Deionized Water (dH2O) (Photo taken by Hans Lorico, 2018) .................................... 14
Figure 4: Flowchart of the General Procedure of the Study ......................................................... 15
Figure 5: Collection of Alugbati Leaves (Photo taken by Arniel Gilo, 2018) .............................. 16
Figure 6: Boiling of Alugbati Aqueous Extract (Photo taken by Hans Lorico, 2018) ................. 17
Figure 7: Phytosynthesis of AgNPs (Photo taken by Ernztin Terre, 2018) .................................. 18
Figure 8: U-2900 UV-Vis Spectrophotometer (Photo taken by Hans Lorico, 2018) ................... 19
Figure 9: FTIR Spectroscopy (Photo taken by Hans Lorico, 2018) ............................................. 20
Figure 10: SEM-EDAX (Photo taken by Arniel Gilo, 2018) ....................................................... 20
Figure 11: UV-Visible Spectra of the aqueous extract of silver nanoparticles (AgNPs) UV-
Visible Spectrophotometry ........................................................................................ 22
Figure 12: Infrared spectra of the aqueous extract of Alugbati .................................................... 23
Figure 13a and 13b: from top to bottom Infrared spectra of Silver nanoparticles, Infrared spectra
of the silver nanoparticles (blue) and aqueous extract (red) (Blue arrows indicate
peak transfer) ............................................................................................................. 24
Figure 14a and 14b: SEM images of AgNPs ................................................................................ 26
Figure 15a, 15b and 15c: SEM images of gold-coated AgNPs .................................................... 27
Figure 16: EDAX of phytosynthesized AgNPs ............................................................................ 27
Figure 17a, 17b and 17c: TEM Analysis taken from Niraimathi et al. ......................................... 28
Figure 18: Average Absorbance Values Obtained from the MTT Assay ..................................... 28
Figure 19: Micrograph of A549 cells before treatment (Photo taken by MCCL, 2018) .............. 30
Figure 20: Micrograph of A549 cells 72 hours after treatment at 100 g/mL (Photo taken by
MCCL, 2018) ............................................................................................................ 30
Figure 21: Percent Inhibition of Doxorubicin and the Silver Nanoparticles ................................ 31
Figure 22: Average Absorbance Values Obtained from MTT Assay (MCF7) ............................ 32
Figure 23: MCF-7 cells before treatment...................................................................................... 33
Figure 24: MCF-7 cells 72 hours after treatment with silver nanoparticles at lowest concentration
................................................................................................................................... 33
Figure 25: Percent Inhibition of the two setups on MCF-7 Breast Cancer Cells ......................... 34
Figure 26: Obtained UV-Vis spectra ............................................................................................ 36

Figure 27: Niraimathi et al.’s obtained UV-Vis spectra .............................................................. 36
Figure 28: Schematic proposed by Marslin et al. on the phytosynthesis of nanoparticles .......... 37
List of Tables
Table 1. Experimental design setup for the MTT Assay .............................................................. 21
Table 2. Mean absorbance values of various setups at varying concentrations............................ 29
Table 3. ANOVA table of absorbance values of various setups at varying concentrations ......... 29
Table 4. Mean Absorbance Values at 570 nm .............................................................................. 32
Table 5. ANOVA Table of Mean Absorbance Values at 570 nm ................................................ 33
List of Equations
Equation 1. Percentage of Cell Viability ...................................................................................... 21
Quezon City Science High School

Golden Acres Rd., cor. Misamis St., Bago Bantay, Quezon City
National Capital Region Science High School
Cytotoxicity of Phytosynthesized Silver Nanoparticles

(AgNPs) using Aqueous Extract of Alugbati (Basella
alba L.) on Selected Cancer Cell Lines
Arniel Joseph G. Gilo1, Hans Daniel Q. Lorico1, and Ernztin Andrea A. Terre1
1
Grade 11 STEM, Quezon City Science High School
I. ABSTRACT
There has been an increasing interest in targeting natural compounds found in plants as
potential anticancer agents because cancer poses now as the second leading cause of death world-
wide. Nanoparticles are gaining interest in the field of medicine due to their vast bioactivity po-
tential particularly on its cytotoxic properties on cancer cell lines. This study was conducted to
establish the cytotoxic activity of the phytosynthesized silver nanoparticles from the leaves of Ba-
sella alba L. (Indian spinach or Alugbati) on human breast (MCF7) and human lung (A549) cancer
cell lines. The phytosynthesized silver nanoparticles using alugbati aqueous extract were charac-
terized through UV-Vis, FTIR, and SEM-EDAX analyses. The UV-Vis spectra surface plasmon
resonance showed a peak of 442 nm. The FTIR showed the respective stretch of bonds and func-
tional groups of the synthesized nanoparticles, while SEM images show that nanoparticles were
spherical in shape and have a size ranging from 49 nm-71 nm. Elemental analysis by EDAX also
confirms the presence of silver nanoparticles. The cytotoxicity of the phytosynthesized silver na-
noparticles was tested via MTT assay. The results showed that the cytotoxic activity of phytosyn-
thesized silver nanoparticles against cancer cell lines, A549 and MCF7, was statistically signifi-
cant. More profound toxic morphological changes induced by AgNPs was observed in cancer cell
line A549 than with MCF7. The study concludes that it is possible to phytosynthesize silver nano-
particles using the aqueous extract of Alugbati with a potential of use as an anti-cancer agent.
KEYWORDS: Cancer, silver nanoparticles, Alugbati (Basella alba L.), Lung Cancer, Breast Cancer
II. INTRODUCTION
The field of nanotechnology ranks as one of the most vigorously explored areas in modern
research. As a result, recent studies have proven the proficiency and advantages of utilizing metal
nanoparticles (NPs) in various fields such as electronics, engineering, cosmetic, and biomedical
applications. Metal NPs have been widely used as an antibacterial agent in food storage, textile
coating and in a number of environmental applications. While NPs could be synthesized through
various methods, the synthesis of NPs through biological technique provides an advancement over
physical and chemical methods as it is less toxic, cost-effective and eco-friendly. A number of
published literatures such as those of Miura and Shinohara (2009)1, Mousavi (2018)2, report on
the green synthesis of silver nanoparticles (AgNPs) using several plants such as Artemesia tur-
comanica and etc.3
In this research work, the green synthesis of AgNPs using the aqueous extract of Basella
alba (Linn.) will be conducted . Basella alba (family: Basellaceae), locally known as alugbati, is
a perennial vine found in the tropics where it is commonly used as a green leafy vegetable. It serves
as a good source of vitamin A, vitamin C, and vitamin B9, iron, magnesium, and calcium, and has
been used in Indian traditional medicines as an aspirant, rubefacient, and for catarrh infections.
Leaves of Basella alba are helpful for the treatment of hypertension, malaria, and anemia, and they
also exhibit antifungal, anti-inflammatory and analgesic activities. Related literatures show that
1Nobuhiko Miura and Yasushi Shinohara, "Cytotoxic Effect and Apoptosis Induction by Silver Nanoparti-
cles in HeLa Cells," Biochemical and Biophysical Research Communications 390, no. 3 (2009): , accessed Novem-
ber 20, 2018, doi:10.1016/j.bbrc.2009.10.039.
2
Bita Mousavi, Farzaneh Tafvizi, and Saied Zaker Bostanabad, "Green Synthesis of Silver Nanoparticles Using Arte-
misia Turcomanica Leaf Extract and the Study of Anti-cancer Effect and Apoptosis Induction on Gastric Cancer Cell Line
(AGS)," Artificial Cells, Nanomedicine, and Biotechnology, 2018, doi:10.1080/21691401.2018.1430697.
3
Ibid
the Basella alba species are rich in ascorbic acid, carbohydrates, proteins, flavonoids, and phenols.
Furthermore, green synthesized AgNPs possess good antioxidant activity.4
Cancer, a band of disease compromising abnormal cell growth with the capacity to invade
or spread to the other parts of the body through metastasis, is the second leading cause of death in
the global scale based on the World Health Organization and has been held responsible for 8.8
million deaths in 2015. Around 70% of the mortality happens in countries with low to middle
incomes.5 The Philippine Statistics Authority also described cancer as the third primary cause of
death in the country, which kills 1 out of 10 Filipinos since 2004. Among the multiple types of
malignant tumor, lung and breast cancer are two of the most common causes of cancer-related
deaths worldwide.6 Lung cancer continues to be one of the leading causes of mortalities in the
Philippines. Concurrently, about 27.7 percent of female cancer-related deaths in the Philippines
are mainly attributed to breast cancer.7
This study aims to assert the anti-cancer properties of phytosynthesized silver nanoparticles
from Alugbati (Basella alba L.) as a substitute treatment to conventional cancer treatment methods
alongside decreasing certain adverse effects.
Background of the Study
According to the World Health Organization, cancer is the second leading cause of death
worldwide, accounting for 8.8 million deaths in 2015. Around 70% of deaths from cancer occur
4 K. L. Niraimathi, R. Lavanya, V. Sudha, and P. Brindha. "Green synthesis and characterization of silver nanoparticles
from aqueous extract of Basella alba and their in-vitro antioxidant potentials." International journal of pharmacy and pharmaceu-
tical sciences 6, no. 10 (2014): 393-396.
5 "Cancer." World Health Organization. Accessed October 12, 2018. http://www.who.int/news-room/fact-sheets/de-
tail/cancer.
6 "Common Cancer Types," National Cancer Institute, accessed September 25, 2018, https://www.can-
cer.gov/types/common-cancers.
7 "Cancer Country Profile: Philippines," Cancer Country Profile, 2014, , accessed October 24, 2018,
http://www.who.int/cancer/country-profiles/phl_en.pdf.
in countries with low and middle-incomes.8 This said, the Philippine Statistics Authority reveals
that after cardiovascular diseases, cancer has been the third primary cause of mortality among
Filipinos with a rate of 1 out of 10 deaths since 2004.
Lung and breast cancers are two of the most common occurring cancers in the world, with
lung cancer being the most lethal for the past few decades. On a global scale, an approximate of
1.76 million and 627,000 deaths were caused by lung and breast cancer, respectively. In the Phil-
ippines, lung cancer is the leading cause of cancer-related deaths among men, and the third among
women.9 It accounts for 34.2 percent of deaths induced by cancer.10 Meanwhile, breast cancer
affects 1 out of 13 women locally, making Philippines the most vulnerable to breast cancer among
the Asian countries.11 According to the World Health Organization, about 27.7 percent of female
cancer-related deaths in the Philippines are attributed to breast cancer.12
Nanotechnology is widely used in different fields of science such as aerospace engineering,
cosmetic products, environmental remediation, nanoelectronics, and most importantly in medical
healthcare due to its immense application.13 Nanoparticles are particles in free-state that range
from 1-100 nm in dimension. One example is silver nanoparticle (AgNP), a metal nanoparticle
that is commonly utilized because of its exceptional properties of chemical stability, good conduc-
tivity, catalytic, antibacterial, antiviral, antifungal, and anti-inflammatory activities. Currently,
AgNPs are well-known in cancer diagnosis and treatment.14 A biological alternative method in the
8 "Cancer." World Health Organization. Accessed October 12, 2018.

9 Ibid., "Cancer," World Health Organization.
10 "Cancer Country Profile: Philippines," Cancer Country Profile, 2014, accessed October 24, 2018.
11 Philippine Daily Inquirer, "Incidence of Breast Cancer Rising in PH, Say Experts," Inquirer News TRANSCRIPT
Dutertes 2nd State of the Nation Address Comments, , accessed November 09, 2018, https://newsinfo.inquirer.net/942804/philip-
pine-news-updates-breast-cancer-dr-christina-galvez-philippine-breast-cancer-society-bibeth-orteza.
12 "Cancer Country Profile: Philippines," 2014, accessed October 24, 2018.
13 Maqusood Ahamed, Mohamad S. Alsalhi, and M.k.j. Siddiqui. 2010. “Silver Nanoparticle Applications and Human
Health.” Clinica Chimica Acta411 (23-24): 1841–48. https://doi.org/10.1016/j.cca.2010.08.016.

14 Shakeel Ahmed et al., Journal of Advanced Research, January 2016, accessed October 11, 2018,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703479/.
form of plant-mediated synthesis of nanoparticles is used since synthesis of nanoparticles by chem-
ical and physical approach is perilous and toxic to the environment. This procedure is a green
chemistry approach that links nanotechnology to plants since plants are cheap and require low
maintenance.15 Even though much interest from the scientific community are directed towards
AgNPs, only a limited number of studies on AgNP synthesis using abundant materials are locally
found in the Philippines.
Plant extracts are subjects of intensive and timely research due to their medicinal properties
that have the potential to serve as natural remedies for different illnesses. Currently, there are in-
creasing number of studies on the feasibility of different bioactive extracts as anticancer agents.
These studies were able to expand the efficacy of commonly utilized chemotherapy drugs, includ-
ing Paclitaxel and Docetaxel, which show pre-immune functions when applied to the cancer pa-
tient.16 Agents that can elicit the activity of apoptosis in cancer cells are therefore considered po-
tentially significant for the development of anticancer chemotherapeutics.17 Alugbati (Basella alba
L.) is a common plant in the Philippines that has been a subject to various research studies and is
known for being rich in vitamins, minerals, and phytochemicals.
Basella alba L., locally named as alugbati, is an edible perennial herb that contains vitamins
A, C, and B9, iron, magnesium, and calcium. As herbal plants are globally known for their medic-
inal value and safety, Basella alba L. is usually cooked, turned into a juice or paste to treat a wide
15 Khadeeja Parveen, Viktoria Banse, and Lalita Ledwani. 2016. “Green Synthesis of Nanoparticles: Their Advantages
and Disadvantages.” https://doi.org/10.1063/1.4945168.
16 Lemos et al., "Anticancer Properties of Essential Oils and Other Natural Products," International Scholarly Research
Notices, March 25, 2018, accessed September 8, 2018, https://www.hindawi.com/journals/ecam/2018/3149362/.

17 Ajay Kumar et al., "An Essential Oil and Its Major Constituent Isointermedeol Induce Apoptosis by Increased Ex-
pression of Mitochondrial Cytochrome C and Apical Death Receptors in Human Leukaemia HL-60 Cells," Chemico-Biological
Interactions 171, no. 3 (2008): doi:10.1016/j.cbi.2007.10.003.
variety of illnesses. It is mostly utilized for therapeutic purposes, but it is currently gaining more
attention in the field of more serious diseases.18
This study aims to prove the anti-cancer properties of phytosynthesized silver nanoparticles
from alugbati (Basella alba L.) as substitute treatment to conventional cancer treatment methods
alongside decreasing certain adverse effects.
Research Questions:
1. Will the phytosynthesized silver nanoparticles exhibit cytotoxic activity on human lung
adenocarcinoma (A549) cancer cell line?
2. Will the phytosynthesized silver nanoparticles exhibit cytotoxic activity on human breast
(MCF7) cancer cell line?
Research Hypotheses
a. The phytosynthesized silver nanoparticles will exhibit cytotoxic activity on human lung
adenocarcinoma (A549) cancer cell line.
b. The phytosynthesized silver nanoparticles will exhibit cytotoxic activity on human breast
(MCF7) cancer cell line.
Statistical Hypotheses
Null:
1. The phytosynthesized silver nanoparticles will have no effect on the cellular viability of
human lung adenocarcinoma (A549) cancer cell line.
18 S. A. Deshmukh and D. K. Gaikwad, A review of the taxonomy, ethnobotany, phytochemistry and pharmacology of
Basella alba (Basellaceae). J App Pharm Sci, 2014; 4 (01): 153-165.

2. The phytosynthesized silver nanoparticles will have no effect on the cellular viability of
breast (MCF7) cancer cell line.
Alternative:
1. The phytosynthesized silver nanoparticles will have an effect on the cellular viability of
human lung adenocarcinoma (A549) cancer cell line.
2. The phytosynthesized silver nanoparticles will have an effect on the cellular viability of
breast (MCF7) cancer cell line.
Objectives
General
a. To determine the cytotoxic activity of the phytosynthesized silver nanoparticles (AgNPs)
through the MTT assay
Specific
a. Extract an aqueous alugbati (Basella alba L.) from the leaves to be used for the phytosyn-
thesis of AgNPs
b. Phytosynthesize the silver nanoparticles using alugbati (Basella alba L.) extract
c. Characterize phytosynthesized silver nanoparticles through FTIR, UV-VIS and
SEM/EDAX
d. Perform MTT assay to test the cytotoxicity of the phytosynthesized silver nanoparticles on
selected cancer cell lines
e. Analyze the significance of the data using one-way ANOVA
f. Perform post-hoc analysis of the sample’s calculated percentage cell viability through stu-
dent’s t-test
Significance of the Study
Cancer, a class of diseases characterized by the abnormal cell growth, is the second preemi-
nent cause of mortality in the world, being accountable to nearly 1 out of 6 deaths worldwide. An
Approximately 8.8 million deaths were recorded in the year 2015 alone. Furthermore, its impact
on the world’s economy is alarming and steadily increasing, amounting to a total annual economic
cost of approximately US $ 1.16 trillion in 2010.19 In the Philippine context, about 13 out of 100
males and 14 out of 100 females would have had suffered from a certain form of cancer if they
would have reached the age of 75. Meanwhile, out of 100 males and 100 females, 11 males and 7
females, respectively, would have died from cancer before reaching the age of 75.20
Early detection is key to curing cancer, but not all cancers can be detected at an early stage
enough for it to be alleviated. More than a third of all cancer cases can be cured when detected and
treated initially. Currently, surgery is the most efficient and universal form of medication against
cancer. Cancers of the mouth and larynx are small abnormal masses of tissue, thus they could be
treated with radiotherapy. Furthermore, there are specific types of cancer that can be remedied
through chemotherapy alone such as acute lymphocytic leukemia, which is common for children,
testicular cancer, and choriocarcinoma of the uterus. In some cases, several methods are used as
an auxiliary for surgery which further increase the probability of survival such as radiotherapy,
hormone therapy, and chemotherapy.
19 Ibid., “Cancer," World Health Organization.

20 Adriano Laudico et al., "2015 PHILIPPINE CANCER FACTS and ESTIMATES," Philcancer, 2015, accessed Sep-
tember 8, 2018, http://www.philcancer.org.ph/wp-content/uploads/2017/07/2015-PCS-Ca-Facts-Estimates_CAN090516.pdf.
Conventionally, metallic nanoparticles are produced from chemicals as reducing agents
and tend to pose serious biological threats due to their toxicity; thus, plant based biological mole-
cules derived from extracts is used as a safer alternative. Through extensively controlled assem-
blies, these plant-based biological molecules can be a perfect fit for the synthesis of metallic na-
noparticles.21 Silver nanoparticles (AgNPs) are a particular type of metallic nanoparticles that are
known for their various medical purposes due to their advantageous size and shape-depending
properties. Commonly amalgamated through green synthesis, these nanoparticles have been
proven in both in vivo and in vitro tests their antiproliferative and apoptosis-inducing properties,
resulting to a positivistic effect on the abnormal mass of tissues and the healthy normal tissues
surrounding the tumor.22
Basella alba L., locally named as alugbati, freely grows and adapts to even minuscule
patches of land, making it a viable herbal medicine. Herbal plants are naturally gifted at the syn-
thesis of medicinal compounds without possessing harmful synthetic additives/drugs as such in
modern treatments through technology. Related analyses prove the many medicinal values it con-
tains. As a good source of vitamins A, C, and B9, iron, magnesium, and calcium, Basella alba L.,
can be cooked, turned into a juice or paste to treat a wide range of illnesses. 23 Other studies point
out its flavonoid, saponin, carotenoid, amino acid and organic acid content, which richly qualifies
the vine under herbal medicine.
This study aims to prove the cytotoxic properties of Basella alba L. as a substitute medi-
cation to conventional cancer treatment methods alongside decreasing certain adverse effects.
21 Ibid., Ahmed et al.

22 "George Mihail Vlăsceanu et al., "Silver Nanoparticles in Cancer Therapy," Silver Nanoparticles in Cancer Therapy -
ScienceDirect, March 25, 2016, accessed September 1, 2018, https://www.sciencedirect.com/science/arti-
cle/pii/B9780323428637000025.
23 S. A. Deshmukh and D. K., 153-165
III. MATERIALS AND METHODOLOGY
Location and Duration of the Study
The synthesis, characterization, and testing for the cytotoxicity of silver nanoparticles
(AgNPs) on selected cancer cell lines lasted from October 9, 2018 to October 29, 2018. Alugbati
leaves were obtained from the school grounds of Quezon City Science High School (QCSHS) then
verified and authenticated at the University of the Philippine’s Institute of Biology’s Jose Vera
Santos Memorial Herbarium (PUH). The preparation of phytosynthesized AgNPs using alugbati
aqueous extract was done at the QCSHS Chemistry Laboratory and De La Salle University
(DLSU) Chemistry Instruments Laboratory. The characterization of AgNPs through FTIR and
UV-Vis spectroscopy were done at the DLSU Chemistry Instruments Laboratory while the SEM
and EDAX analyses were performed at the DLSU Solid State Physics Laboratory. The results of
the characterization of AgNPs were collected on the same day they were analyzed. The AgNPs
were sent to the UP-IB Mammalian Cell Culture Laboratory to test their cytotoxic activities (MTT
Assay) against human lung adenocarcinoma (A549) and human breast (MCF7) cancer cell lines.
Materials
Figure 1: Alugbati Figure 2: Silver Nitrate Figure 3: Deionized Water

(Basella alba L.) Leaves (AgNO3) (dH2O)
(Photo taken by Hans Lorico, 2018) (Photo taken by Hans Lorico, 2018) (Photo taken by Hans Lorico, 2018)
General Procedure
Figure 4: Flowchart of the General Procedure of the Study

Collection of Alugbati Leaves
Fresh alugbati leaves were obtained from the school grounds of Quezon City Science
High School. The plant samples were then verified and authenticated at the University of the
Philippine’s Institute of Biology’s Jose Vera Santos Memorial Herbarium (PUH).
Figure 5: Collection of Alugbati Leaves

(Photo taken by Arniel Gilo, 2018)
Preparation of Alugbati Aqueous Extract
One hundred and fifty (150) grams of leaves were weighed and washed with deionized
water before use. Following the 1:4 ratio, the leaves were crushed and mixed with 600 mL of
deionized water. The resulting mixture was covered with aluminum foil and boiled for 1 hour. It
was allowed to infuse overnight. After which, the aqueous solution was filtered using Whatman
Filter Paper No.1, and the filtrate was then kept at 4°C until further use.
Figure 6: Boiling of Alugbati Aqueous Extract

(Photo taken by Hans Lorico, 2018)
Phytosynthesis of Silver Nanoparticles (AgNPs)
The phytosynthesis of AgNPs was done according to the methods of Niraimathi et al. with
slight modifications.24 One hundred and fifty milliliters (150 mL) of the extract was added drop-
wise to three (3) mM of silver nitrate (AgNO3) solution. The resulting solution was covered with
aluminum foil to prevent photoactivation of the silver nitrate before stirring it at 500 rpm for thirty
(30) minutes. The color of the solution gradually changed from light yellow to golden brown. This
indicates that the silver nitrate was reduced and the AgNPs were formed. The reduced solution
was centrifuged at 10,000 rpm for fifteen (15) minutes, and the resulting pellet was then washed
and centrifuged repeatedly for four (4) times to remove any impurities adsorbed on the surface of
the AgNPs. The resulting powder was then lyophilized and stored at 4°C prior to use in experi-
mental work.
24 K. L. Niraimathi, R. Lavanya, V. Sudha, and P. Brindha. "Green synthesis and characterization of silver nanoparti-
cles from aqueous extract of Basella alba and their in-vitro antioxidant potentials." International journal of pharmacy and phar-
maceutical sciences 6, no. 10 (2014): 393-396.
Figure 7: Phytosynthesis of AgNPs

(Photo taken by Ernztin Terre, 2018)
Characterization of the Silver Nanoparticles (AgNPs)
a. Ultraviolet-Visible Spectroscopy
The bioreduced AgNP solution was subjected to UV-Visible spectroscopy to confirm the
formation and stability of phytosynthesized AgNPs. In addition to the 100% AgNP solution, a
dilute solution of 50% was also analyzed via UV-Vis spectroscopy. The spectra were recorded
with a U-2900 Spectrophotometer in the wavelength interval of 800.0 nm to 300.0 nm. The sam-
pling interval was set at 1.0 nm and the slit width at 1.50 nm.
Figure 8: U-2900 UV-Vis Spectrophotometer

b. Fourier-Transform Infrared Spectroscopy
The aqueous alugbati extract and bioreduced AgNP solution were analyzed through FTIR
spectroscopy. The samples were prepared and stabilized using potassium bromide (KBr) pellet
technique, and the spectra was recorded from the wavelength interval of 4000 to 500 cm . The -1
resulting spectra will be interpreted and analyzed in identifying the metabolites and phytochemi-
cals involved in the capping of the silver nanoparticles, as well as to explain the synthesis of the
AgNPs.
Figure 9: FTIR Spectroscopy

c. SEM-EDAX (Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy)
A thin film of the sample was obtained from a pinch of AgNP prepared on a carbon-coated
paper. The film of AgNPs was coated with gold through gold sputtering to further discern its mor-
phology. The film was allowed to dry and the resulting images were observed under SEM. Simul-
taneously, elemental analysis of the AgNPs was conducted through EDAX.
Figure 10: SEM-EDAX

(Photo taken by Arniel Gilo, 2018)
MTT Assay
The MTT cytotoxicity assay adapted from Mosmann (1983) was performed by the Mam-
malian Cell Culture Laboratory of the UP Institute of Biology. In detail, 4 x 10 -4cells/mL cells
were seeded (depending on the cell culture used) in sterile 96-well microtiter plates. The plates
were incubated overnight at 37°C and 5% CO2.
Treatments using the samples were obtained by doing eight two-fold dilutions from 100
µg/mL down to 0.78125 µg/mL. The negative control as well as the blank will be the setup using
DMSO, Doxorubicin as the positive control, while varying concentrations of silver nanoparticles
were used as the test setup. The silver nanoparticles were allowed to completely disperse prior to
administration. After the incubation of the cells, they were treated with the setups’ corresponding
substance. The setups containing the treated cells were incubated for 72 hours at 37°C and 5%
CO2
The media were removed afterwards. 3-(4,5-dimethylethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) dye at 5mg/mL PBS was added. For 4 hours, the cells was incubated at
37°C and 5% CO2. DMSO was used to dissolve the formazan crystals formed by the reduction of
the dye by the live cells. Absorbance was read at 570 nm.
Research Design
The table below shows the various variables and setups used in the conduct of the MTT
assay.
Table 1. Experimental design setup for the MTT Assay
Setup Samples used*

Test Phytosynthesized silver nanoparticles (AgNPs)
Positive Control Doxorubicin
Negative Control Dimethyl sulfoxide (DMSO)
*Varying concentrations: from 100 µg/mL down to 0.78125 µg/mL.
𝑂𝐷570 𝑜𝑓 𝑇𝑒𝑠𝑡
% 𝐶𝑒𝑙𝑙 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = ( ) × 100
𝑂𝐷570 𝑜𝑓𝑐𝑜𝑛𝑡𝑟𝑜𝑙
Equation 1. Percentage of Cell Viability

Statistical analysis
Data represented was the mean ± standard error of the mean (SEM). Three trials were per-
formed with two replicates each. Statistical significance was tested by one-way ANOVA and post-
hoc through student’s t-test, and p-values inferior to 0.05 was considered significant.
IV. RESULTS
Ultraviolet-Visible Spectroscopy
Figure 11: UV-Visible Spectra of the aqueous extract of silver nanoparticles

(AgNPs) UV-Visible Spectrophotometry
The spectra obtained from the bioreduced solution of silver nitrate indicates an apex at
442.0 nm at an absorbance of 3.364. The valley is found at 388.0 nm at an absorbance of 2.681.

Fourier-Transform Infrared Spectroscopy
Alcohol or Phenol
O-H Stretch
Amide Carbonyl
C=O Stretch
Figure 12: Infrared spectra of the aqueous extract of Alugbati

The spectra were obtained by Fourier-Transform Infrared Spectroscopy (FTIR) from the
aqueous extract of alugbati. The medium peak at 815.62 cm-1 indicates the presence of a C=C bond
from an alkene. The peaks observed at 1114.37 cm-1, 1327.02 cm-1, and at 1381.43 cm-1, indicates
the presence of a C-O stretching from a secondary alcohol, C-N from an aromatic amine, and a C-
H bending from an aldehyde respectively. The strongest peak observed at 1636.51 cm-1 indicates
a carbonyl C=O stretching from an amide. Lastly, the peak at 3442.29 cm-1 indicates the presence
of an alcohol or phenol O-H stretch. The functional groups are a characteristic of secondary me-
tabolites from plants such as flavonoids, tannins, phenolic acids and etc.
Figure 13a and 13b: from top to bottom Infrared spectra of Silver nanoparticles, Infrared spectra of
the silver nanoparticles (blue) and aqueous extract (red) (Blue arrows indicate peak transfer)
Comparing to FTIR spectra of the aqueous plant extract, there was a shift in the O-H peak
from 3404 cm-1 to 3402 cm-1 indicating the bonding of the O-H group to the silver nanoparticles.25
Moreover, the amide C=O absorption peak transfer from 1628.33 cm-1 towards 1657.47 cm-1 in-
dicates the bonding of the amide C=O group to the silver nanoparticles. Lastly, comparing the
1382 cm-1 peak from Mousavi et al.’s study, there was a similar peak observed at 1387 cm-1. This
is related to the stretching vibration of N=O group, which is formed by NH2 amine oxidation and
reduction of the silver cation into metallic silver. 26
SEM and EDAX Analyses

Images of the silver nanoparticles (AgNPs) were captured through the Scanning Electron
Microscope (SEM). The AgNPs were coated with gold to increase visibility under SEM. As shown
in the images, the size of the spherical-shaped AgNPs ranges from 49 nm to 71 nm. Energy-dis-
persive X-ray spectroscopy (EDAX) analysis was done to identify the elemental chemical compo-
sition of AgNPs. In the spectrum, it is shown that there are peaks indicating of silver (Ag) and
chlorine (Cl) content in 3Kev. The results of the said analysis had shown that the AgNPs consist
almost entirely of silver (Ag) with 59.77% and of chlorine with 40.23% in atomic percentage (see
Figure 15 and Table 1).
25 Niraimathi, K. L., Lavanya, R. V., Sudha, and Brindha, P., "Green synthesis and characterization of silver nanoparti-
26 Bita Mousavi, Farzaneh Tafvizi, and Saied Zaker Bostanabad, "Green Synthesis of Silver Nanoparticles Using Arte-
misia Turcomanica Leaf Extract and the Study of Anti-cancer Effect and Apoptosis Induction on Gastric Cancer Cell Line
(AGS)," Artificial Cells, Nanomedicine, and Biotechnology, 2018, doi:10.1080/21691401.2018.1430697.
Figure 14a and 14b: SEM images of AgNPs

Figure 15a, 15b and 15c: SEM images of gold-coated AgNPs
Figure 16: EDAX of phytosynthesized AgNPs
Table 2: EDAX of synthesized elements during the formation of silver nanoparticles

(AgNPs) through the leaves of Basella alba L.
Element Weight % Atomic % Error %
ClK 18.11 40.23 4.29
AgL 81.89 59.77 2.69
TEM Analysis
According to the study by Niraimathi et al., the resulting structure of the agglomerated
nanoparticles have a size which ranges from 22.6 to 25 nm. Moreover, the morphology of the silver
nanoparticles was characterized in the same study through TEM and were said to have a spherical
shape with a mean diameter of 24.79 nm. 27
27 Ibid., Niraimathi.
Figure 17a, 17b and 17c: TEM Analysis taken from Niraimathi et al.28
MTT Assay
I. A549 Human Lung Adenocarcinoma
1.600
1.400
1.200
Absorbance Values
1.000
0.800
0.600
0.400
0.200
0.000
100 50 25 12.5 6.25 3.125 1.5625 0.78125
Concentration of Sample (Doxorubicin and AgNPs) µg/mL
DMSO Doxorubicin AgNPS
Figure 18: Average Absorbance Values Obtained from the MTT Assay
The MTT Assay is an assay used in determining cellular viability through colorimetric
means. The mitochondria of functioning cells have the ability to reduce the MTT dye into dark
violet, insoluble formazan crystals, which can then be quantified by measuring the absorbance at
570 nanometers. The higher absorbance value obtained, more cells are viable and alive.
28
Ibid., Niraimathi.
Table 2. Mean absorbance values of various setups at varying concentrations
Concentration DMSO Doxorubicin AgNPs

100 1.238 0.486 1.146
50 1.275 0.288 1.149
25 1.289 0.645 1.181
12.5 1.307 0.891 1.201
6.25 1.297 0.963 1.234
3.125 1.307 1.030 1.212
1.5625 1.309 1.152 1.262
0.76125 1.397 1.311 1.309
Figure 18 and Table 2 above shows the mean absorbance values obtained from varying
concentrations of the samples, with DMSO, dimethyl sulfoxide, acting both as the negative control
and blank. While Doxorubicin, an anticancer drug, acting as the positive control. Statistical anal-
ysis through ANOVA shows that there are significant differences between the three setups and
their corresponding obtained absorbance values.
Table 3. ANOVA table of absorbance values of various setups at varying concentrations

Source of
SS df MS F P-value F critical
Variation
Between
0.86540092 2 0.43270046 10.9642864 0.00054886 3.46680011
Groups
Within
0.82875523 21 0.03946453
Groups
Total 1.69415616 23
Table 3 further illustrates the significant differences between the setups. A p-value of
0.00054886 was obtained, which satisfies the parameter p>0.01. Moreover, the obtained F value
of 10.96 is also greater than the F critical value, further stipulating that there are significant differ-
ences among the set-ups. Post-hoc analysis through student’s t-test shows that doxorubicin exhib-
ited a significantly higher inhibitory effect on the A549 cells. Nevertheless, the phytosynthesized
silver nanoparticles were able to significantly impact the growth of the A549 lung cancer cells,
expressing a t-test statistical value of 10.17 > 1.89 (critical value  = 0.05).
Figure 19: Micrograph of A549 cells before Figure 20: Micrograph of A549 cells 72
treatment (Photo taken by MCCL, 2018) hours after treatment at 100 g/mL (Photo
taken by MCCL, 2018)
The micrographs above show the morphological changes in the cellular structure of the
lung cancer cells 72 hours after treatment with the phytosynthesized silver nanoparticles. This
shows that the nanoparticles were able to induce necrosis and apoptosis into the A549 cancer cell
line. The graph below shows the respective percentage inhibition  SEM induced by the positive
control and the test setup on the cell line.

60
50
40
Percent Inhibition
30
20
10
0
100 50 25 12.5 6.25 3.125 1.5625 0.78125
-10
Doxorubicin AgNPS
Figure 21: Percent Inhibition of Doxorubicin and the Silver Nanoparticles
Although the obtained value of percentage of inhibition for the silver nanoparticles was
significantly lower compared to the positive control, doxorubicin, (See Figure 21) it is essential to
note that doxorubicin is already an established cancer drug, and the purest analytical grade of the
drug was used in the assay. Meanwhile, in the phytosynthesis of silver nanoparticles which relied
on the methods of Niraimathi et al., the aqueous extract obtained was only 25% aqueous extract,
which may explain the low cytotoxicity exhibited by the nanoparticles. However, the assays were
able to prove that even at dilute concentrations, the silver nanoparticles were already toxic and
able to inhibit the proliferation of A549 cells and induce apoptosis.

II. MCF7 Human Breast Carcinoma
1.400
1.200
Absorbance Values
1.000
0.800
0.600
0.400
0.200
0.000
100 50 25 12.5 6.25 3.125 1.5625 0.78125
DMSO Doxorubicin AgNPS
Figure 22: Average Absorbance Values Obtained from MTT Assay (MCF7)
The same assay was conducted to test the cytotoxic properties of the silver nanoparti-
cles on the breast cancer cell line, MCF-7.
Table 4. Mean Absorbance Values at 570 nm

DMSO Doxorubicin AgNPs
0.984 0.247 1.068
0.938 0.221 1.010
1.052 0.216 1.011
1.079 0.261 0.963
1.069 0.282 1.032
1.135 0.350 1.177
0.960 0.427 1.085
0.955 0.578 0.876
In the testing the cytotoxic activity of silver nanoparticles, DMSO was again used as the
negative control and the blank, while doxorubicin was used as the positive control. Statistical test,
through ANOVA, showed that there exist significant differences between the groups and their
corresponding obtained absorbance values.
Table 5. ANOVA Table of Mean Absorbance Values at 570 nm

Source of
SS df MS F P-value F critical
Variation
Between
2.62673062 2 1.31336531 137.432575 8.646E-13 3.46680011
Groups
Within
0.20068511 21 0.00955643
Groups
Total 2.82741573 23
Table 5 illustrates the ANOVA table of the values which shows the resulting F value of
137.43 which is immensely greater than the critical value of 3.47. However, further post-hoc anal-
ysis through student’s t- test show that only doxorubicin was able to significantly impact the cells’
viability, with the silver nanoparticles showing insignificant and inconsistent results on inhibiting
the viability of the cells.
Figure 23: MCF-7 cells before treatment Figure 24: MCF-7 cells 72 hours after treat-
ment with silver nanoparticles at lowest con-
centration
100
80
60
Percent Inhibition
40
20
0
100 50 25 12.5 6.25 3.125 1.5625 0.78125
-20
-40
Doxorubicin AgNPS
Figure 25: Percent Inhibition of the two setups on MCF-7 Breast Cancer Cells
The micrographs (see figure 24) show that there existed a zone of inhibition in the MCF-7
cells after being treated with the lowest concentration of silver nanoparticles (0.786125 g/mL)
exhibiting a 54.9112% inhibition, showing that the nanoparticles have the potential to be a potent
anticancer agent. Even at the lowest concentration the nanoparticles were able to induce apoptosis
and necrosis to the cells. The inconsistencies in the data may be attributed to the insolubility of the
nanoparticles in deionized water. The exact concentration was not able to be administered to the
cells, causing a vast number of outliers in the data. Nevertheless, the assay proved that the silver
nanoparticles were able inhibit the activity of the cells to some extent.
V. DISCUSSION
The search for treatment to cancer is one of the most important research concerns in the
field of medicine. Treatments ranging from chemotherapy, surgery, radiation therapy, and now
29
even immunotherapy, are some of the ways that cancer is currently being treated. Even so, re-
search is still ongoing towards the field of the use of natural products as novel methods in treating
cancer.
The use of nanomaterials in medicine, nanomedicine, is one of the research interests in the
present with regards to novel methods in cancer treatment. Nanomaterials are defined as materials
whose at least one dimension is under 100 nm. Due to their decreased size and higher surface area,
they perform considerably better than their macro- counterparts. 30
Recently, plant-mediated synthesis or phytosynthesis of silver nanoparticles are gaining
attention due to the protocol being safer and less toxic compared to the conventional method of
metallic nanoparticle synthesis using toxic and inorganic compounds.31 Moreover, compared to
the biosynthesis of nanoparticles through microorganism-mediated synthesis, such as by bacteria,
fungi, and yeast, the process of phytosynthesis is more reliable. Due to these reasons, the potential
of phytosynthesized nanoparticles are gaining attention in various scientific disciplines.
In this study, silver nanoparticles were phytosynthesized using the aqueous extract from
Alugbati (Basella alba L.). The initial confirmation that the silver ions were reduced to silver
nanoparticles was the formation of a yellowish-brown color. The UV-Vis spectra obtained (see
Figure 3) shows that the peak of the sample was at 442 nm. This was similar to obtained UV-Vis
29 "Types of Cancer Treatment," National Cancer Institute, accessed October 14, 2018, https://www.cancer.gov/about-
cancer/treatment/types.
30 Elham Abbasi et al., "Silver Nanoparticles: Synthesis Methods, Bio-applications and Properties," Critical Reviews in
Microbiology, 2014, doi:10.3109/1040841x.2014.912200.

31 Ibid., Ahmed et al.
spectra by Niraimathi which confirmed the formation of silver nanoparticles at 435 nm (See Fig-
ures 13 and 14). 32
Figure 26: Obtained UV-Vis spectra Figure 27: Niraimathi et al.’s 33 obtained UV-Vis spec-
tra
The FTIR spectra revealed the presence of secondary metabolites in the plant extract such
as flavonoids, phenolic acids, and etc. (See Figure 6). Moreover, the shifting of the peaks in the
transmittance spectra as shown in Figure 7, show the transfer and bonding of the molecules from
the phytochemicals in the extract to the nanoparticles. Marslin et al. further elaborates on the pro-
cess involving the in vitro synthesis of nanoparticles, which allows reduction of metal ions into
stable nanoparticles with the aid of phyto-reductants from the plant extract. 34
32 K. L. Niraimathi, R. Lavanya, V. Sudha, and P. Brindha. "Green synthesis and characterization of silver nanoparti-
33 ibid
34 Gregory Marslin et al., "Secondary Metabolites in the Green Synthesis of Metallic Nanoparticles," Materials 11, no.
6 (2018): , accessed October 22, 2018, doi:10.3390/ma11060940.

Figure 28: Schematic proposed by Marslin et al. 35 on the phytosynthesis of nanoparticles
The potential of phytosynthesized silver nanoparticles in treating malignancies has been
explored in several papers. In 2017, Razumov et al. explored the selective cytotoxicity of manga-
nese nanoparticles against glioblastoma cells. The nanoparticles exhibited a selectivity index for
glioblastoma cells, U-251; therefore, concluding that manganese nanoparticles have the potential
to be an oncolytic agent. Meanwhile in 2018, Mousavi et al. examined the cytotoxic potential of
silver nanoparticles while also investigating its ability to induce apoptosis on cancer cell lines.
However, the bioactive properties of which are mainly dependent on the extract used as the cap-
ping agent.
The cytotoxicity of the phytosynthesized silver nanoparticles using the aqueous extract of
alugbati (Basella alba L.) may be attributed to two things (1) the presence of a bioactive compound
in alugbati and (2) the ability of silver nanoparticles to induce mitochondrial and DNA damage
from the generation of reactive oxygen species (ROS). In 2018, Sheng- Xiang Lv and Xiao Quiao
35 Gregory Marslin et al., "Secondary Metabolites in the Green Synthesis of Metallic Nanoparticles," Materials 11, no.
6 (2018): , accessed October 22, 2018, doi:10.3390/ma11060940.

discussed the properties of isovitexin, a bioactive compound found in alugbati, and its ability to
induce autophagy and apoptosis in liver cancer cells36. On the other hand, reactive oxygen species
are essential in the activation of various pathways such as in the expressions of ho-1 and mt-2A
37
genes, which are established genes inducing oxidative stress. The generation of ROS mainly
comes from the mitochondria, through the mitochondrial electron transport system.
In 2017, Dayem et al. further establishes the role of reactive oxygen species in the bioac-
tivity of metallic nanoparticles. Nanoparticles are mainly incorporated into the cell by endocytosis,
with clathrin or caveolin proteins playing an essential role in the cellular uptake of nanoparticles38.
Once inside the cell, there are four main methods in the generation of intracellular ROS, (1) catal-
ysis of free-radical reactions, (2) growth factor activation, (3) interaction with the mitochondria,
and (4) activation of NOX (NADPH oxidases). In the mitochondria, the disruption of the electron-
transport chain through the activation of NOX and other NADPH-related enzymes as well as the
39
depolarization of the mitochondrial membrane and allows the NP-related generation of ROS.
Thus, the cytotoxicity of nanoparticles may be linked to DNA damage and chromosomal aberra-
tions induced by the reactive oxygen species (ROS). Showing that silver nanoparticles could be a
potent anticancer agent.
In this study, the nanoparticles which were phytosynthesized using the aqueous extract of
alugbati yielded, although inconclusive, positive results. The inconclusive results may be at-
tributed to the insolubility of the silver nanoparticles in an aqueous solution, which caused the real
36 Lv, Sheng-Xiang, and Xiao Qiao. "Isovitexin (IV) induces apoptosis and autophagy in liver cancer cells through en-
doplasmic reticulum stress." Biochemical and biophysical research communications 496, no. 4 (2018): 1047-1054.
37 Nobuhiko Miura and Yasushi Shinohara, "Cytotoxic Effect and Apoptosis Induction by Silver Nanoparticles in HeLa
Cells," Biochemical and Biophysical Research Communications 390, no. 3 (2009): , accessed October 22, 2018,
doi:10.1016/j.bbrc.2009.10.039.
38 Ahmed et al. "The role of reactive oxygen species (ROS) in the biological activities of metallic nanoparticles." Inter-
national journal of molecular sciences 18, no. 1 (2017): 120.

39 Xia, Tian, Michael Kovochich, Jonathan Brant, Matt Hotze, Joan Sempf, Terry Oberley, Constantinos Sioutas, Jo-
anne I. Yeh, Mark R. Wiesner, and Andre E. Nel. "Comparison of the abilities of ambient and manufactured nanoparticles to in-
duce cellular toxicity according to an oxidative stress paradigm." Nano letters 6, no. 8 (2006): 1794-1807.
concentration of the nanoparticles be variable. Nevertheless, it could be inferred from the data,
that even at dilute concentrations the nanoparticles were able to exhibit potent anti-cancer proper-
ties.
The study no longer tested the silver nanoparticles on normal cells since multiple literature
have already cited the selectivity of silver nanoparticles on normal cells. In the study of Ortega et
al. which focuses on the properties of nanoparticles synthesized by yeast, they stated that AgNPs
have a higher inhibition efficacy on tumor cells than they do on normal cells. 40 Mousavi et al.,
compared the cytotoxic effects of Artemesia turcomanica synthesized silver nanoparticles on gli-
oblastoma cells and L-929 cells, stating that after 24 hours of incubation, higher inhibitory con-
centration was obtained on the activity against the glioblastoma cells than on the normal cells. 41
This is essential, because it is important that the silver nanoparticles express selective cytotoxicity
towards the cancer cell line, without harming the normal cell line.
40 Ortega, Francisco G., Martín A. Fernández-Baldo, Jorge G. Fernández, María J. Serrano, María I. Sanz, Juan J. Díaz-
Mochón, José A. Lorente, and Julio Raba. "Study of antitumor activity in breast cell lines using silver nanoparticles produced by
yeast." International journal of nanomedicine 10 (2015): 2021.
41 Mousavi, Bita, Farzaneh Tafvizi, and Saied Zaker Bostanabad. "Green synthesis of silver nanoparticles using Arte-
misia turcomanica leaf extract and the study of anti-cancer effect and apoptosis induction on gastric cancer cell line (AGS)." Arti-
ficial cells, nanomedicine, and biotechnology (2018): 1-1
VI. CONCLUSION AND RECOMMENDATIONS
The results of the study established that the synthesis of silver nanoparticles using the aque-
ous extract of alugbati (Basella alba L.) is feasible and can be an alternative way to other conven-
tional means of synthesizing silver nanoparticles. The formation of the silver nanoparticles was
due to the reduction of silver ions by the phytochemicals in the aqueous extract, confirmed by the
UV-Vis, FT-IR, and SEM-EDAX analyses.
This study established that the phytosynthesized silver nanoparticles using alugbati aque-
ous extract may have a future potential for possible treatment of cancer.
The researchers recommend the following:
1. Assessment of the molecular mechanisms involved in the cytotoxic activity of the
phytosynthesized silver nanoparticles through other assays such as:
a. TUNEL Assay
b. DAPI staining assay
c. Annexin V/PI Assay
d. MTT Assay on AA8, L-929, and HuVEC cells
2. Improvement of the methodology involved in the synthesis of nanoparticles and
incorporation of other factors.
3. Further characterization of the phytochemical profile of the plant by GC-MS
4. Usage of other parts of plants for extraction, investigation and comparison to exist-
ing studies.
5. Testing of cytotoxic properties against other cell lines

Overall, the research proved that the synthesis of silver nanoparticles through environmen-
tally viable and safe methods are possible. Lastly, the selective cytotoxic properties of the phyto-
synthesized silver nanoparticles using the aqueous extract of alugbati allow it to be a potential
agent in treating cancer.

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VIII. APPENDIX
Appendix A. List of Figures
Figure 1: Alugbati Leaves Figure 2: Silver Nitrate Figure 3: Deionized Water
Figure 5: Boiling of Alugbati

Figure 4: Collection of Alugbati Leaves
Aqueous Extract
Figure 6: Phytosynthesis of Silver Nanoparti-

Figure 7: UV-Vis Spectroscopy
cles
Figure 8: FTIR Spectroscopy Figure 9: SEM-EDAX
Figure 10: UV-Visible Spectra of the aqueous extract of

silver nanoparticles (AgNPs) UV-Visible Spectrophotometry
Figure 11: Infrared spectra of the aqueous extract of Alugbati

Figure 12a and 12b: from top to bottom

Infrared spectra of Silver nanoparticles, Infrared spectra of the silver nanoparticles (blue) and
aqueous extract (red)
Figure 13a and 13b: SEM images of AgNPs

Figure 14a, 14b and 14c: SEM images of gold-coated AgNPs
Figure 15: EDAX of phytosynthesized AgNPs

Figure 16a, 16b and 16c: TEM Analysis taken from Niraimathi et al.
Figure 18: Niraimathi et al.’s

Figure 17: Obtained UV-Vis spectra
obtained UV-Vis spectra
Figure 19: Schematic proposed by Marslin et al. on the phytosynthesis of nanoparticles

Appendix B. List of Tables

Table 1. Experimental design setup for the MTT Assay
SETUP Sample used:
Phytosynthesized silver nano-
Test Varying concentrations:
particles (AgNPs)
from 100 µg/mL down to
Positive Control Doxorubicin
0.78125 µg/mL.
Negative Control Dimethyl sulfoxide (DMSO)
Table 2. EDAX of synthesized elements during the formation of

silver nanoparticles (AgNPs) through the leaves of Basella alba L.
Appendix C. List of Equations

% Cell Viability = OD570 of Test/OD570 of control × 100
Equation 1. Percentage of Cell Viability
Appendix D. MTT Assay/Statistical Analysis
Table 6. Percent inhibition computed from the absorbance readings of each concentration
of the positive control, doxorubicin, against A549 cells
Doxorubicin
Conc (µg/mL)
Trial 1 Trial 2
5 52.6111 56.3939 62.9167 64.9560
1.6666667 75.3883 75.2346 73.5732 77.0910
0.5555556 47.9983 45.1478 40.9589 46.6539
0.1851852 24.7330 25.9763 25.1732 31.5291
0.0617284 19.9210 24.6162 22.1628 24.1134
0.0205761 16.1693 18.8498 17.9872 21.1975
0.0068587 8.9478 12.7187 6.1813 8.0967
0.0022862 -0.4511 3.5074 1.9272 2.1895
IC50 2.050 µg/mL 2.333 µg/mL
of AgNPs against A549 cells
AgNps
Conc (µg/mL)
Trial 1 Trial 2
100 7.7213 11.2358 4.8774 4.5864
50 10.9863 11.8216 6.1240 11.6094
25 7.8157 7.8919 11.7234 11.1565
12.5 7.9384 6.8882 9.5810 2.1461
6.25 4.9808 7.8959 -0.0962 0.4017
3.125 15.2034 6.4729 4.1588 3.1201
1.5625 2.9380 4.8682 2.4830 0.8591
0.78125 6.4620 2.6172 1.7557 1.0643
IC50 >100 µg/mL >100 µg/mL
of the positive control, doxorubicin, against MCF7 cells
Doxorubicin
Conc (µg/mL)
Trial 1 Trial 2
6.25 72.5799 73.5436 61.3412 64.6943
3.125 77.9461 77.3569 63.0905 67.9470
1.5625 78.9556 79.5791 70.1967 69.2758
0.78125 76.7496 72.0062 49.5845 63.3558
0.390625 73.0562 72.9022 66.8776 68.2173
0.1953125 65.6273 70.6309 64.4817 60.9756
0.0976563 52.1477 60.9795 49.3925 53.7495
0.0488281 37.9699 28.2895 39.7548 38.8792
IC50 0.0558 µg/mL 0.0961 µg/mL
of Ag NPS against MCF7 cells
AgNPs
Conc (µg/mL)
Trial 1 Trial 2
100 19.4043 -11.0819 -22.9558 -4.3004
50 -2.2727 15.2357 -17.5018 -22.0044
25 8.8854 20.8106 -3.2887 -2.5579
12.5 9.6423 19.8289 20.0688 -2.5229
6.25 12.7791 7.8522 8.7657 -6.3804
3.125 -3.5533 16.0261 -5.8890 -8.2445
1.5625 1.2445 4.1349 -23.2938 -33.9763
0.78125 -4.8872 -6.2030 54.9112 -3.5503
IC50 >100 µg/mL >100 µg/mL
Table 10. Mean Inhibition Values (A549) Table 11. Mean Inhibition Values (MCF7)
DMSO Doxorubicin AgNPs DMSO Doxorubicin AGNPs
0 39.4796 4.73682 0 68.0398 -4.7335
0 50.2145 6.75688 0 71.5851 -6.6358
0 30.1265 6.43125 0 74.5018 5.9624
0 17.9019 4.42562 0 65.4240 11.7543
0 15.1356 2.19703 0 70.2633 5.7542
0 12.3673 4.82587 0 65.4289 -0.4152
0 5.99075 1.85805 0 54.0673 -12.9727
0 1.1955 1.9832 0 36.2234 10.0677
Table 12. Absorbance readings of treated A549 cells

Trial 1
DMSO Dox AgNPs
1.274 1.240 0.596 0.548 1.160 1.116
1.236 1.250 0.306 0.308 1.106 1.096
1.260 1.235 0.649 0.684 1.150 1.149
1.265 1.287 0.960 0.945 1.175 1.188
1.323 1.260 1.034 0.974 1.227 1.190
1.320 1.250 1.077 1.043 1.089 1.202
1.279 1.292 1.171 1.122 1.248 1.223
1.354 1.337 1.352 1.299 1.259 1.310
Trial 2
DMSO Dox AgNPs
1.223 1.216 0.376 0.423 1.170 1.136
1.329 1.285 0.286 0.253 1.183 1.211
1.364 1.296 0.651 0.596 1.203 1.221
1.353 1.323 0.836 0.823 1.210 1.230
1.390 1.215 0.887 0.956 1.267 1.251
1.355 1.302 1.015 0.984 1.276 1.279
1.312 1.352 1.188 1.128 1.301 1.274
1.487 1.409 1.323 1.269 1.347 1.321
Table 13. Absorbance readings of treated MCF7 cells

Trial 1
DMSO Dox AgNPs
1.081 1.202 0.313 0.302 0.920 1.268
1.189 1.187 0.262 0.269 1.215 1.007
1.235 1.331 0.270 0.262 1.169 1.016
1.251 1.321 0.299 0.360 1.162 1.031
1.222 1.376 0.350 0.352 1.133 1.197
1.314 1.444 0.474 0.405 1.428 1.158
1.094 1.397 0.596 0.486 1.230 1.194
1.021 1.107 0.660 0.763 1.116 1.130
Trial 2
DMSO Dox AgNPs
0.801 0.850 0.196 0.178 1.015 0.861
0.717 0.660 0.179 0.174 0.809 0.840
0.763 0.879 0.164 0.168 0.848 0.842
0.776 0.968 0.193 0.191 0.697 0.894
0.802 0.875 0.232 0.193 0.765 0.892
0.728 1.055 0.271 0.251 0.944 0.965
0.579 0.769 0.319 0.308 0.831 0.903
0.851 0.839 0.483 0.405 0.381 0.875
Table 15. Mean Absorbance Values

Table 14. Mean Absorbance Values (A549)
(MCF7)
DMSO Doxorubicin AgNPs DMSO Doxorubicin AgNPs
1.238 0.486 1.146 0.984 0.247 1.068
1.275 0.288 1.149 0.938 0.221 1.010
1.289 0.645 1.181 1.052 0.216 1.011
1.307 0.891 1.201 1.079 0.261 0.963
1.297 0.963 1.234 1.069 0.282 1.032
1.307 1.030 1.212 1.135 0.350 1.177
1.309 1.152 1.262 0.960 0.427 1.085
1.397 1.311 1.309 0.955 0.578 0.876
Table 16. t-Test: Paired Two Sample for Means

Blank Test
Mean 1.30228125 1.2115
Variance 0.0020091 0.00312479
Observations 8 8
Pearson Correlation 0.89723545
Hypothesized Mean Difference 0
df 7
t Stat 10.1681902
P(T<=t) one-tail 9.5823E-06
t Critical one-tail 1.89457861
P(T<=t) two-tail 1.9165E-05
t Critical two-tail 2.36462425

Positive Control v test Test
Mean 0.8456875 1.2115
Variance 0.12006666 0.00312479
Observations 8 8
df 7
t Stat 3.51366114
P(T<=t) one-tail 0.00490547
P(T<=t) two-tail 0.00981093

Positive Control Blank
Mean 1.30228125 0.8456875
Variance 0.0020091 0.12006666
Observations 8 8
df 7
t Stat 4.14376645
P(T<=t) one-tail 0.00216436
P(T<=t) two-tail 0.00432871

Blank Test
Mean 1.021375 1.02766667
Variance 0.00515495 0.00784644
Observations 8 8
df 7
t Stat -0.206439
P(T<=t) one-tail 0.42116209
P(T<=t) two-tail 0.84232417

Doxorubicin Test
Mean 0.32275 1.02766667
Variance 0.01566791 0.00784644
Observations 8 8
Pearson Correlation -0.3133928
df 7
t Stat -11.423245

Doxorubicin Negative Control
Mean 0.32275 1.021375
Variance 0.01566791 0.00515495
Observations 8 8
Pearson Correlation -0.2833946
df 7
t Stat -12.274389
Appendix E. Certificate of Authentication

Appendix F. Certificate(s) of Appearance


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Gilo, Lorico, Terre | 1

Republic of the Philippines

Cytotoxicity of Phytosynthesized Silver Nanoparticles (AgNPs) using Aqueous Extract

Life Science – Team Category

A Science Investigatory Project

*Arniel Joseph G. Gilo

Quezon City Science High School

Mrs. Jackieline A. Gagaza

Mr. Denis Dyvee R. Errabo

I. Title page ............................................................................................................................................1

Figure 26: Obtained UV-Vis spectra ............................................................................................ 36

Quezon City Science High School

Cytotoxicity of Phytosynthesized Silver Nanoparticles

comanica and etc.3

Furthermore, green synthesized AgNPs possess good antioxidant activity.4

are mainly attributed to breast cancer.7

alongside decreasing certain adverse effects.

Background of the Study

Filipinos with a rate of 1 out of 10 deaths since 2004.

cancer-related deaths in the Philippines are attributed to breast cancer.12

Nanotechnology is widely used in different fields of science such as aerospace engineering,

cosmetic products, environmental remediation, nanoelectronics, and most importantly in medical

tivity, catalytic, antibacterial, antiviral, antifungal, and anti-inflammatory activities. Currently,

8 "Cancer." World Health Organization. Accessed October 12, 2018.

Health.” Clinica Chimica Acta411 (23-24): 1841–48. https://doi.org/10.1016/j.cca.2010.08.016.

form of plant-mediated synthesis of nanoparticles is used since synthesis of nanoparticles by chem-

found in the Philippines.

known for being rich in vitamins, minerals, and phytochemicals.

Notices, March 25, 2018, accessed September 8, 2018, https://www.hindawi.com/journals/ecam/2018/3149362/.

attention in the field of more serious diseases.18

alongside decreasing certain adverse effects.

adenocarcinoma (A549) cancer cell line?

(MCF7) cancer cell line?

adenocarcinoma (A549) cancer cell line.

(MCF7) cancer cell line.

human lung adenocarcinoma (A549) cancer cell line.

Basella alba (Basellaceae). J App Pharm Sci, 2014; 4 (01): 153-165.

breast (MCF7) cancer cell line.

human lung adenocarcinoma (A549) cancer cell line.

breast (MCF7) cancer cell line.

a. To determine the cytotoxic activity of the phytosynthesized silver nanoparticles (AgNPs)

through the MTT assay

c. Characterize phytosynthesized silver nanoparticles through FTIR, UV-VIS and

selected cancer cell lines

e. Analyze the significance of the data using one-way ANOVA

Significance of the Study

hormone therapy, and chemotherapy.

19 Ibid., “Cancer," World Health Organization.

Conventionally, metallic nanoparticles are produced from chemicals as reducing agents

surrounding the tumor.22

thesis of medicinal compounds without possessing harmful synthetic additives/drugs as such in

the vine under herbal medicine.

21 Ibid., Ahmed et al.

III. MATERIALS AND METHODOLOGY

Location and Duration of the Study

Figure 1: Alugbati Figure 2: Silver Nitrate Figure 3: Deionized Water

Figure 4: Flowchart of the General Procedure of the Study

Collection of Alugbati Leaves

Philippine’s Institute of Biology’s Jose Vera Santos Memorial Herbarium (PUH).

Figure 5: Collection of Alugbati Leaves

Preparation of Alugbati Aqueous Extract

Figure 6: Boiling of Alugbati Aqueous Extract

Phytosynthesis of Silver Nanoparticles (AgNPs)

Figure 7: Phytosynthesis of AgNPs