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Philippine Science High School

Main Campus

Cytotoxicity of Lagundi Extract on


Human Colorectal Carcinoma and
Lung
Non-Small Cell Carcinoma

Jose Sandino A. Bandonil


Marc Patrick C. Celon

Richard Dean Clod C. dela Cruz


September 2012

Cytotoxicity of Lagundi Extract on Human Colorectal


Carcinoma and Lung Non-Small Cell Carcinoma
by
Jose Sandino A. Bandonil
Marc Patrick C. Celon
Richard Dean Clod C. dela Cruz

Submitted to the Faculty of the


Philippine Science High School Main Campus
in partial fulfillment of the requirements for
Science and Technology Research 2
September 2012

ABSTRACT

Cancer is one of the most lethal conditions known to man. According to the latest statistics, it
caused 7.6 million deaths, or 13% of worldwide total deaths in 2008; among these, 70%
came from low and middle-income countries. To counter this, a cheap and widely accessible
drug must be produced. In order to start the research the aforementioned need, this study
aims to discover the cytotoxicity of the crude extract of lagundi (Vitex negundo), a common
shrub in Africa and South Asia, against human colorectal carcinoma (HCT-116) and lung
non-small cell carcinoma (A549) cells. The crude extract will be extracted from 10 mg of
powdered lagundi, through the Soxhlet extraction process. This extracts cytotoxicity will
then be determined through the MTT assay. The cytotoxicity can be measured through the
IC50. The extracts cytotoxicity will be compared with that of Adriamycin PFS a known
effective anticancer drug and dimethyl sulfoxide (DMSO). The respective IC 50 results taken
from the different treatments will be computed through linear regression, given the
absorbance of each treated unit. The MTT assay will also be applied, with the same
treatments, on Chinese hamster ovarian cells (AA8). This will determine the feasibility of
lagundi extract as an anticancer drug, for an extract cytotoxic to cancer cells but ineffective
against normal cells will make it possible for such a drug to be produced. Data analysis will
be done through one-tailed independent T-test. An value of 0.05 will be used to determine
the effectivity of the treatments.

APPROVAL SHEET
This research work entitled, Cytotoxicity of Lagundi Extract on Human
Colorectal Carcinoma and Lung Non-Small Cell Carcinoma by Jose Sandino A.
Bandonil, Marc Patrick C. Celon, and Richard Dean Clod C. dela Cruz, presented to the
Faculty of the Philippine Science High School Main Campus in partial fulfillment of the
requirements in Science and Technology 2, is hereby accepted.

Ana Victoria M. Lloren


Research Adviser

ACKNOWLEDGEMENTS

To our parents, who never stopped supporting us physically and mentally


To our Research adviser, Maam Bick Lloren, for pushing us to our limits
To Dr. Sonia D. Jacinto, for allowing us to use her facilities for this study and for giving us
the scientific know-how we needed
To our friends, for hearing out our concerns when we needed to blow off pressure
To our teachers, who helped us become more efficient workers
And finally, to Pisay, for arming us with the knowledge and wisdom we needed to
accomplish this project

Thank you very much

TABLE OF CONTENTS
Page
Approval Sheet...........................................................................................................................i
Acknowledgements...................................................................................................................ii
Table of Contents......................................................................................................................iii
List of Tables.............................................................................................................................v
List of Figures...........................................................................................................................vi
Introduction................................................................................................................................1
Background of the Study...............................................................................................1
Statement of the Problem...............................................................................................1
Significance of the Study ..............................................................................................2
Scope and Limitations ..................................................................................................3
Review of Related Literature.....................................................................................................5
Cancer............................................................................................................................5
Human Colon Carcinoma..............................................................................................6
Lung Non-Small Cell Carcinoma..................................................................................7
Lagundi (Vitex negundo)................................................................................................8
Cytotoxicity Assays.......................................................................................................9
Soxhlet Extraction........................................................................................................11
Materials and Methods............................................................................................................13
Process Flowchart........................................................................................................13
Procurement of Materials and Equipment...................................................................14
Extraction of the Crude Extract...................................................................................14
Cytotoxicity Assay for Lagundi Extract......................................................................15

Data Analysis...............................................................................................................17
Results and Discussion............................................................................................................18
Extraction of the Crude Extract...................................................................................18
Cytotoxicity Assay on HCT116 Cells.........................................................................18
Cytotoxicity Assay on A549 Cells..............................................................................19
Cytotoxicity Assay on AA8 Cells...............................................................................19
Data Analysis...............................................................................................................19
Summary and Conclusion........................................................................................................21
Recommendations....................................................................................................................22
Bibliography............................................................................................................................23
Appendix A: Complete Results with Absorbance Values....................................................26

List of Tables
Table
1
2

Title
HCT116 IC50 Values
Complete MTT Results for HCT-116 Cells

Page
18
26

List of Figures
Figure
Title
1
96-well plate after MTT assay on HCT116.
2
HCT116 IC50 Value Comparison

Page
19
20

INTRODUCTION
Background of the Study
Cancer is one of the deadliest conditions that man has ever encountered. Current
statistics compiled by the World Health Organizations International Agency for Research on
Cancer show that cancer is currently the world leading cause of mortality, causing 7.6 million
deaths or 13% of total 2008 deaths. 70% of these deaths occurred in low and middle-income
countries. Current projections predict 13.1 million deaths by 2030. The five main types of
cancer worldwide are of the lung, of the stomach, of the liver, of the colorectum, and of the
breast, in decreasing order for number of deaths (World Health Organization, 2012).
Meanwhile in the Philippines, cancer, or malignant neoplasms, ranks third in the ten leading
causes of mortality, and is superseded only by heart diseases and vascular diseases. Cancer
has a mortality rate of 49.5% in the Philippines and was the cause of 43,043 deaths in 2006.
It can also be noticed that the Philippine cancer incidence and mortality rate has been steadily
increasing, except for the years 2001 to 2006, when the mortality rate stabilized at 48.5%
0.3% (Department of Health, 2006). Top cancer sites in the Philippines include the breast, the
lungs, the liver, the colorectum, and the cervix uteri, in decreasing number of cases
(GLOBOCAN 2008, 2008).

Statement of the Problem


This project aims to determine the cytotoxicity of lagundi through the testing of its
crude extracts. The crude extract will be extracted through Soxhlet extraction, after it will be
subjected to rotary evaporation. The crude extract will undergo the MTT assay to determine
1

its cytotoxicity against human colorectal carcinoma (HCT-116) and lung non-small cell
carcinoma (A549) cells. While crude extracts from lagundi are already known to be cytotoxic
against HCT116 cells, this study will validate such claims. Crude extract treatments will
undergo different concentrations: 50 g/mL, 25 g/mL, 12.5 g/mL, and 6.25 g/mL. The
cytotoxicity will be measured through the half maximal inhibitory concentration (IC 50),
which measures the highest concentration of the tested chemical that can kill half the cells in
a well. During the assay, temperature and % CO 2 level will be kept constant in the incubator.
The crude extracts IC50 will be compared to those of Adriamycin PFS a well known
anticancer drug and dimethyl sulfoxide (DMSO), the solvent with which the extract was
dissolved for assay purposes. However, Adriamycin PFS will be tested with the
concentrations 25 g/mL, 12.5 g/mL, 6.25 g/mL, and 3.125 g/mL because of its
reputation as an effective drug.
The crude extract will also be tested against Chinese hamster ovary (AA8) cells. This
is so that the effect of the extract on normal cells can also be discovered. It is ideal that the
crude extract is not cytotoxic against AA8 cells. Such a result will mean that lagundi is a
possible natural anti-lung cancer drug. It will also be ideal if results have a low variance, so
that the effect of the crude extract on humans in vivo will be more predictable.

Significance of the Study


Current scientific research on anticancer cures has found several possible anticancer
cures. However, no cure has yet been developed that can be mass-produced. This is because
available treatments for cancer demand either expertise on cancer treatment or a chemical
produced only on small quantities. Such requirements drive up the cost of cancer treatment.
2

A cancer drug that is widely available in the Philippine archipelago and one that can be easily
mass produced will lower the price of cancer treatment and make it more accessible to the
general populace.
An anticancer drug derived from lagundi will do just that. Not only is lagundi a wellknown cough medicine, it is also found in almost all parts of the Philippines. Lagundi is
promoted by the Department of Health as a cheap cough medicine, and being used in local
health centers for the same function. A study that proves the possible anticancer capabilities
of the plant will give lagundi medicine a larger market and thus earn Philippine producers
millions or even billions of dollars in revenue. This can increase the governments tax
revenue, making possible several improvements in infrastructure and in other sectors. In
other words, this small project by Philippine Science High School students may serve as a
tipping point to usher in several changes in the Philippines.

Scope and Limitations


This study will be conducted in the Philippine Science High School Main Campus
(PSHS-MC) Research Unit and in the Cell Biology Laboratory of the University of the
Philippines Institute of Biology (UP-IB). The extraction process for the lagundi crude extract
will be done through Soxhlet extraction in the PSHS-MC Research Unit. The crude extract
will be taken from 10 mg of powdered lagundi leaves. From the crude extract, 4 mg will be
taken and dissolved in 1 mL dimethyl sulfoxide (DMSO). This solution will be used for the
MTT assay. 100 L of extract with different concentrations will be transferred to each well
during the assays treatment process. The same amount will be transferred to each well for
the control treatments Adriamycin PFS and DMSO.
3

The MTT cytotoxicity assay will test the crude extracts effect against HCT116, A549
and AA8 through the measurement of the crude extracts IC50. The lower the IC50, the more
cytotoxic the extract is. The ideal IC 50 is below 20; however, an IC 50 at the range between 20
and 25 is said to still be acceptable for crude extracts. It is because crude extracts might have
a compound that retards the effect of the cytotoxic compound. The MTT assay will be done
in the UP-IB Cell Biology Laboratory.

REVIEW OF RELATED LITERATURE


Cancer
Cancer is one of the most lethal conditions known to man. To counter this, the World
Health Organization (WHO) founded a research agency dedicated to cancer research called
the International Agency for Research on Cancer (IARC). Statistics compiled by IARCs
GLOBOCAN 2008 show that cancer caused 13%, or 7.6 million deaths, of all total deaths
around the world. Of these, 70% occurred in low and middle income countries. Current
projections predict 13.1 million deaths in 2030. The five main types of cancer are of the lung,
of the stomach, of the liver, of the colorectum and of the breast, in decreasing order in
number of deaths (WHO, 2012). In the Philippines, cancer ranks third in the ten leading
causes of mortality, counting 43,043 deaths and having a 49.5% mortality rate. The
Philippine cancer incidence rate and mortality rate have been increasing, except for the years
2001 to 2006, when the mortality rate stabilized (Department of Health, 2006). Leading
cancer sites in the Philippines are in the breast, the lung, the liver, the colorectum and the
cervix uteri (GLOBOCAN 2008, 2008).
Cancer is merely a disease in which abnormal cells divide uncontrollably instead of
undergoing the normal cell cycle. Instead of undergoing apoptosis when they age or are
damaged, they continue multiplying in a process called metastasis. The change from a normal
cell and tumor cell stems from several events, but the start of these changes occur a cells
DNA is either damaged or altered. The damage to the DNA causes mutations that alter the
normal cell division and growth process (National Cancer Institute, 2012). These changes
usually occur due to four important factors: the organisms genetic material, physical
carcinogens such as radiation, chemical carcinogens like asbestos, and biological
5

carcinogens, which include bacterial or viral infections like Hepatitis A. Aging also increases
the possibility of cancer development (WHO, 2012).
In an effort to remedy cancer, huge efforts are being done to look for possible cures.
Several possible natural cures have been found, according to an article by Hafidh, et. al
(2009). In the same article are examples of plants that tested positive to cytotoxicity. The list
includes garlic (Allium sativum), which has been tested against the colon cancer cell line, and
Mangor ginger (Curcuma mangga), assessed to be cytotoxic against the DU-145 (human
prostate cancer), NCI-H460 (non-small cell lung cancer), and MCF-7 (breast cancer) cell
lines. Hafidh et. al. (2009) also goes on to list possible anticancer chemicals. These chemicals
include flavonoids, flavonols, sterols, carotenoids, and phenols. The flavone flavopiridol has
in fact tested positive to cytotoxicity against lung non-small cell carcinoma (A549) cells.
Inhibition of RNA and protein synthesis in the A549 cells has been pinpointed to contribute
to the flavones cytotoxicity (Bible et. al., 1996). Another example would be camptothecin,
an extract taken from the tree Camptotheca acuminata. It is known to be cytotoxic against
HCT116 and MCF-7 cancer cells (Gupta et al., 1997).
Human Colon Carcinoma
Colon cancer is a cancer concentrated on the colon and rectal portions of the body. It
is one of the most common types of cancer in the world, accounting for 10% of total cancer
patients worldwide. Most of these patients are concentrated on developed countries, with
Australia and New Zealand having the highest incidence rates in the world, with Western
Europe and Southern Europe at second and third highest, respectively. However, while
developed countries have higher incidence rates, developing countries have higher mortality
rates than the developed countries (GLOBOCAN 2008, 2008). The cancer is characterized by
tumor penetration through the bowel wall. Prognosis is done by inspecting for the presence
6

of nodal involvement and distant metastases. Nodal involvement may signal metastases in
other regions of the body. Aside from these two, however, cancer stage and race has also
been observed to play a role in the survival of the patient (National Cancer Institute, 2012).
For in vitro testing of extracts on colon cancer, HCT116 is used. HCT116 is a cancer
cell line classified under Biosafety Level 1. The cell line is epithelial, and is adherent to
surfaces. Products of cells from this particular cell line include carcinoembryonic antigen and
keratin. The cell line has a mutation in codon 13 of the ras protooncogene, which might
account for its cancerous behavior (American Type Culture Collection, 2011). This cell line
is one of the possible cells that start human colon cancer, which ranks as the fourth most
common cause of death from cancer; it is outranked only by cancer of the liver, cancer of the
stomach, and cancer of the lung, in increasing order for number of deaths (World Health
Organization, 2012).
Lung Non-Small Cell Carcinoma
Lung non-small cell carcinoma is a type of epithelial lung cancer that arises from lung
epithelial cells between the central bronchi to the terminal alveoli. It has three main types:
squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. Squamous cell
carcinomas start near a central bronchus, while adenocarcinoma and large cell carcinoma
start in peripheral lung tissue. The main risk factor for this type of cancer is smoking;
however, exposure to some carcinogenic chemicals, exposure to second-hand smoke, and
radiation to the chest or the breast are also important risk factors. Symptoms of the condition
may include chest pain, breathing problems, weight and appetite loss, hoarseness, and facial
swelling. In the United States, the 5-year survival rate of lung cancer depends on the cancer
stage: 49% for local stages, 16% for the regional stage, and 2% for the distant stage. Current
7

treatments cannot cure most patients with non-small cell lung cancer (National Cancer
Institute, 2012).
For in vitro testing, the A549 cell line is used for the efficacy of certain extracts
against non-small cell carcinoma cells. This cell line was started from lung carcinoma cells
taken from a 58-year old Caucasian male. The A549 cell line was established by D.J. Giard.
Each cell of the cell line divides every 22 hours, and is adherent to the surface where they
were established. Studies by M. Lieber, et. al. show that A549 cells could synthesize lecithin
from desaturated fatty acids through the cytidine diphosphocholine pathway. The cell line is
classified under Biosafety Level 1 (American Type Culture Collection, 2009). According to a
recent study by Diaz et. al. (2003), vitexicarpin, a flavone constituent of Vitex negundo
leaves, is cytotoxic to some varieties of cell lines.
Lagundi (Vitex negundo)
Lagundi is a shrub commonly found in Southeast Asia and Africa. It can be
recognized due to its tri- or penta-foliate leaves, which soon gives rise to bluish-purple
flowers. It is recognized across Asia as a folk medicine that is able to cure headaches,
sinusitis, and venereal diseases. In the Philippines, it is considered as an anticancer in folk
medicine (Vishwanathan, et. al., 2010). The shrub has been proven to be an anti-cough
medicine, and is endorsed by the Philippine Department of Health. It is currently being
considered as a replacement for dextrothemorphan in the public health system.
Phytochemical screenings of the plants ethanolic leaf extract have shown that it contains
alkaloids, iridoids, phenolic acids, flavonols, and flavonoids (Lagundi/vitex negundo/fiveleaved chaste tree: herbal medicinal plants/alternative medicine in the Philippines, 2012).
Vitex negundo has been found to have anti-inflammatory and analgesic properties through
antihistamine and prostaglandin-inhibiting activities. The plant has also been found to be an
8

antioxidant, an enzyme inhibitor, a hepato-protector, a laxative, and an antivenin. It was also


discovered to have a cytotoxic effect on COLO-320 tumor cells. Also, vitexicarpin, one of
the flavones found in lagundi leaves, has been found to have cytotoxic potency against a
human cancer cell line panel in a study by Diaz et. al. (2003). Lagundi has been tested on
Swiss albino mice against Daltons Ascitic Lymphoma (DAL), and on Ehrlich Ascites
Carcinoma (EAC) by Dewade et. al. (2010) and Saluja et. al. (2010), respectively.
Cytotoxicity Assays
The studies on Vitex negundos cytotoxicity done by Dewade et. al and Saluja et. al.
was done in vivo. In other words, they were done on live animals. Another type of testing is
in vitro, or studies on organisms through the use of its isolated components. Cytotoxicity
assays are one of those processes used for in vitro studies. These assays measure how toxic a
certain object is to a certain cell. These assays observe cell damage through observable
indicators such as color. There are three types of cytotoxicity assays, all of which are
classified based on the parameters of the measurements: metabolic function/ATP, membrane
integrity, and cell population (NoAb BioDiscoveries, n.d.).
There are several cytotoxicity assays that can be used. One is the MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay,
which uses the cells ability to absorb a formazan product through the application of
tetrazolium salts. However, the medium used for the assay can distort the results, as well as
the presence of albumin (Chen et. al., 2004). The MTS assay is used in some cytotoxicity
studies, such as the cytotoxicity measurement of nanoparticles through the use of human skin
fibroblasts (Dechsakulthorn et. al, 2007). Another assay is the LDH (lactate dehydrogenase)
assay, which tests the viability of a cell through the measure of cell membrane strength. A
modification of the LDH assay adds lysis buffer, allowing the measurement of cell viability
9

in percentages (Vanderbilt, 2007). This assay was used on the assessment of the effect of
Ritonavir, an HIV protease inhibitor, on human endothelial cells (Zhong et. al, 2002). One
more example will be the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. This assay is the same as the MTS assay because they both use tetrazolium
salts and are both colorimetric in nature. However, the MTT assay is better in terms of
reproducible results. Addition of media, cell population of each well, and the amount of MTT
dye were discovered to interfere with results (Twentyman et. al., 1987). This assay is widely
used, and was used on a study on the effect of a cyclooxygenase-2 inhibitor on non-small cell
lung cancer cell lines and on the cytotoxicity of other anticancer agents (Hida et. al, 2000).
The half maximal inhibitory concentration (IC 50), which is the amount of concentration
needed for a solution to have an effect on 50% of the experimental unit, measures the
cytotoxicity of the possible anticancer compound. This is done through the interpretation of
the graph of the curve of the function of binding as the concentration logarithmically
increases (50% of what? How exactly are EC50 and IC50 defined?, n.d.). The above assays
can also be used for the testing of the cytotoxicity of extracts.
Soxhlet Extraction
The extracts that will be used for the above assays can be extracted through Soxhlet
extraction. Soxhlet extraction is a method of extraction developed by Franz von Soxhlet on
1879. It involves a still pot, a thimble, a condenser, and a distillation path. The solvent is
placed in the still pot, while the extract source wrapped in filter paper will be placed in the
thimble. The condenser facilitates the condensation of the solvent-extract mixture, and the
distillation path which the mixture passes on its way back to the still pot (Universiti Malaysia
Sabah, n.d.). Before the start of extraction, however, there are specific protocols for different
10

types of samples. For dry samples, the sample will be weighed into the thimble. Moist
samples must first be dried for 2-3 hours at 10312 C after being weighed before its
addition to the thimble. For liquid samples, it should first be mixed with sand and anhydrous
sodium sulfate before being added to the thimble (Behr Labor-Technik, n.d.).
A common solvent used for Soxhlet extraction is ethyl alcohol. Ethyl alcohol is a
colorless liquid with a mild odor. Its boiling point is at 78 C, while the freezing point is -114
C. It is a miscible compound with the chemical formula C 2H5OH. Ethyl alcohol is highly
flammable and can cause severe eye irritation and moderate skin irritation. When ingested, it
may cause gastrointestinal irritation along with nausea, vomiting, and diarrhea. Inhalation
may cause nausea, headache, dizziness, and under extreme conditions, unconsciousness or
even coma. Mutagenic and reproductive effects can also be detected when in chronic
amounts (North American Fire Arts Association, 2001).

11

MATERIALS AND METHODS


Process Flowchart
Procurement of AA8
and A549 cells

Procurement of
equipment

Procurement of
powdered lagundi

Extraction of crude
extract

Cytotoxicity assay on
HCT116 cells

Cytotoxicity assay on
A549 cells

Cytotoxicity assay on
AA8 cells

Data analysis

12

Procurement of Materials and Equipment


The powdered lagundi that will be used for the experiment will be sourced from
HerbSeeds, an online seller that was found in Sulit.com. The powdered lagundi will come
from Quezon province, and will be shipped to the Metro Manila through LBC. The
equipment that will be used for the extraction of the crude extract will be borrowed from the
Philippine Science High School Main Campus (PSHS-MC) Research Unit. The A549 and
AA8 cells that will be used for testing will be bought from the Institute of Biology of the
University of the Philippines. Likewise, the Institute of Biology will provide for the
chemicals that will be used as control for the cytotoxicity assay: Adriamycin PFS, also
known as Doxorubicin, and dimethyl sulfoxide (DMSO). The cytotoxicity assay will be done
in the Cell Biology Laboratory of the Institute of Biology.

Extraction of the Crude Extract


After procurement, the powdered lagundi will be extracted through Soxhlet
extraction. The Soxhlet extractor is a device developed by Franz von Soxhlet on 1879. It
involves a still pot, a thimble, a condenser, and a distillation path. The setup is as follows: the
thimble and the distillation path are both in one glass part. The thimble-distillation path part
is placed at the top of the still pot, while the condenser is placed atop the thimble-distillation
path tube. The still pot contains the solvent, while the thimble contains the extract source
wrapped in filter paper. The extraction works by heating the solvent in the still path. As it
evaporates and moves up the tube it encounters the thimble. The evaporated solvent brings
along with it the chemicals of the crude extract. It soon encounters the condenser, where the
solvent mixed with the chemical is condensed in the presence of relatively lower
13

temperatures caused by cold water. The condensed mixture goes down the distillation path
and into the still pot, and the process is repeated again (Universiti Malaysia Sabah, n.d.).
Powdered lagundi, the extract source, was a dry sample, thus it was weighed before being
added to the thimble as said in Behr Labor-Techniks guidelines for Soxhlet extraction.
The extraction will use ethyl alcohol as its solvent. The solvent will initially be heated
at 78 C; however, the temperature will be lowered later so that the crude extracts chemicals
will not be disturbed or destroyed by the boiling action of the solvent. The extraction will go
for 18 hours, after which the crude extract will be separated from the mixture by undergoing
rotary evaporation until it has been reduced to a paste.

Cytotoxicity Assay for Lagundi Extract


After the extraction process for the lagundi extract, it will be subjected to a
cytotoxicity assay. The extract will be tested on human colorectal carcinoma (HCT116) and
human non-small cell lung carcinoma (A549) cells. The extract will be tested at different
concentrations: 25 L/mL, 12.5 L/mL, 6.25 L/mL, and 3.125 L/mL. Adriamycin PFS
will be used as positive control due to its proven efficacy against cancer cells, while DMSO
will be used as negative control.
The start of the assay will be marked by the seeding of the cell line to a 96-well plate.
The extract will be mixed with DMSO with a ratio of 4 mg extract per mL of DMSO. The
96-well plate will then be placed in an incubator and left for 24 hours. The incubator will be
set at 37 C temperature and at 5% CO2 content for the rest of the assay. While the cells in
the well plate are allowed to grow in the incubator, a master dilution plate will be used to set
the different concentrations that will be used for treatment. The process is done by adding
14

150 L of cell culture media to the first well, and 100 L of the culture media to the three
remaining wells. 50 L of the extract solution will be mixed with the first wells media. They
will then be mixed, and 100 L will be taken and then added to the next well; this will be
repeated until the fourth well is done. After the 24-hour period, the well plate is withdrawn
from the incubator and treated with the necessary chemicals. There will be three replicates
for the extract treatments and DMSO. However, Adriamycin PFS will only have two
replicates, because the drug is already known to have a very low IC 50. The treatment process
is done such that concentrations of the extracts decrease starting from the wells at the edge to
the wells at the center. The treatments will be taken from the prepared master dilution plate.
After the treatment process, the treated well plate will again be deposited in the incubator for
72 hours.
After 72 hours, the well plate will be taken from the incubator and treated with MTT
solution. The MTT solution has a concentration of 5 mg MTT for every mL of phosphate
buffer saline (PBS) (Minnesota State University, 2007). 20 L of the solution will be added
to each well. The 96-well plate will then again be left in the incubator for 3 hours. The
absorbance of the each well will then be taken after 3 hours through a plate reader. The basic
premise of the assay, however, is that if the yellow MTT turned to purple formazan after the
three-hour waiting period, cells in the well are alive. As the color gets closer to purple, the
higher the amount of living cells in the well. A program will be used for the computation of
IC50 through the input of each wells absorbance. The assay will also be done on Chinese
hamster ovarian cells (AA8) to measure the effect of the extract on normal cells.

Data Analysis
15

After obtaining the value of IC50 of each of the samples, we then test their
cytotoxicity by comparing it to Doxorubicin, a known anti-cancer chemical. For this, we
need to do an independent t-test to compare the extracts effectiveness with that of
Doxorubicin.
The independent t-test is a statistical test that determines the statistical significance
between the difference of results from two groups. The test judges the variability of results
relative to the mean difference. The result t is the difference berween means divided by the
variability of both groups. To determine the significance of the results, a p-value is derived
from the degrees of freedom, and compared to the t-value. If the p-value is less than or equal
to the t, then the difference is significant. However, if the results is not large enough, it is not
significant enough (The t-test, n.d.).

16

RESULTS AND DISCUSSION


Extraction of the Crude Extract
Ten grams of powdered lagundi was the source of the crude extract. The liquid
resulting from the Soxhlet extraction process was dark green and fluid. It also had the distinct
smell of ethanol, most likely arising from the ethanol used as a solvent during the extraction
process. A vial of this liquid underwent rotary evaporation. After rotary evaporation, the
liquid gave rise to a dark, sticky substance: the pure crude extract. The crude extract had no
smell.
Cytotoxicity Assay on HCT116 Cells
Four mg of the crude extract was used for the MTT assay on HCT116. Table 1 shows
the IC50 for HCT116 when treated with the lagundi crude extract and the positive control,
Doxorubicin, also known as Adriamycin PFS.
Table 1: HCT116 IC50 Values
Treatments
Doxorubicin
Lagundi Crude Extract

1
1.9151
22.5692

Replicates (IC50)
2
1.8450
34.3580

3
2.4492
22.6471

A picture of the 96-well plate used for the assays different replicates is shown in
Figure 1. The different concentrations are arranged in the following manner: the highest
concentration (50 g/mL) is treated to the cells in the edge wells, with the treated
concentration going down as the well gets nearer to the middle along the column. The lowest
concentration (25 g/mL) is treated in the fourth well from the edge. Each replicate occupies

17

three columns of a well plate, in effect forming a 3x4 rectangle where only one chemical is
treated to the cells.

Figure 1: 96-well plate after MTT assay for HCT116.


The 3x4 rectangle on the lower right
portion of the plate is the part where the
test for lagundi cytotoxicity was done.

Cytotoxicity Assay on A549 Cells


The cytotoxicity assay for this cell line is still ongoing.
Cytotoxicity Assay on AA8 Cells
The cytotoxicity assay for this cell line is still ongoing.
Data Analysis
The IC50 of lagundi crude extract and Doxorubicin will be compared through the onetailed T-test. Figure 2 shows a comparison between the IC 50 values for HCT116 between the
two treatments.

18

Figure 2: HCT116 IC50 Value Comparison


40
35
30
25
Doxorubicin

20

Lagundi Crude Extract


15
10
5
0
Replicate 1

Replicate 2

Replicate 3

The t-value between the two was 7.74. Thus, it can be said that there is a significant
difference between the two values. However, lagundi crude extract can still be said to be
cytotoxic on HCT116 cells since the IC50 value is near the ideal value of 20 g/mL.

19

SUMMARY AND CONCLUSIONS


With respect to the above partial results, it has been decided that lagundi crude extract
is a feasible anticancer drug for human colon carcinoma. The average IC50 taken from the
MTT assay done in the Institute of Biologys Cell Biology Laboratory is a value enough to
give the lagundi crude extract a status as a possible anticancer drug. The study has validated
the apparent cytotoxicity of lagundi crude extract against human colon carcinoma cells
(HCT116).
Cytotoxicity assays for A549 and AA8 cells are still ongoing.

20

RECOMMENDATIONS
It is recommended that the cytotoxic chemical responsible for the relatively low IC 50
in the assay be isolated. The timeframe for the study is too short for the said process, which is
said to take months of repeated cytotoxicity testing after every round of separation. The crude
extract should also be tested for compounds that may possibly retard the cytotoxicity of the
crude extract. Such testing will explain the IC50 results higher than 20 g/mL.
Subsequent studies on the same topic may also test compounds from lagundi against
other cancer cell lines. The lagundi compounds may also be tested against cancer cells in
vivo. This will determine the compounds effect on cancer cells when other body factors are
included in the experiment.
Crude extracts from other plants may also be tested against the cancer cells used in
the study. A combination of plants may help in the creation of an anticancer drug that is a
mixture of cytotoxic compounds taken from such plants. Such studies as the ones said above
may help in the creation of a cheap and widely-accessible anticancer drug.

21

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24

APPENDIX A
Complete Results with Absorbance Values
Table 2: Complete MTT Assay Results for HCT116
c

g/ml

Trial I (absorbance)

DMSO

1.752

Doxo

25

0.207

12.5

0.18

6.25

0.176

3.125

0.19

IC50

1.98
6

g/ml

DMSO

1.752

0.39
3
0.52
7
0.47
3

Lagund
i

50

0.235

25

1.469

12.5

1.845

6.25

1.091

1.82
7
0.20
1
0.44
2
1.88
8
0.36
9
22.5692

1.612

1.98
6
0.16
7
0.20
5
0.15
5
0.16
6

1.90
6

Trial III (absorbance)

0.184

1.88
9
0.20
2
0.32

0.175

0.37

0.203

0.206

0.34
6

0.215

0.213

1.9151
Trial I (absorbance)

IC50

1.82
7
0.2

Trial II (absorbance)

0.226
0.206

1.8450
Trial II (absorbance)

1.612
0.216
1.353
1.781
1.099

1.88
9
0.18
7
1.2
1.49
9
1.27
3
34.3580

0.842

1.90
6
0.22
7
1.39
1
1.24
1
1.01
9

0.57
1
0.20
4
0.20
5
0.18
9
0.21
1

0.35
2

2.4492
Trial III (absorbance)

0.842
0.195
0.168
0.402
0.432

0.57
1
0.20
2
0.39
0.57
5
0.36
3

0.35
2
0.44
9
0.15
3
0.35
4
0.65

22.6471

25