Manuscript Final
Manuscript Final
Manuscript Final
A Research Presented to
In Partial Fulfillment
Title Page i
Contents page ii
Research Abstract iii
1.0 Introduction 1
1.1 Statement of the problem 4
1.2 Hypotheses of the Study 5
1.3 Significance of the Study 5
1.4 Scope and limitation of the Study 6
1.5 Definition of Terms 7
2.0 Review of Related Literature 9
3.0 Research Method 22
3.1 Research Design 22
3.2 Research Locale 22
3.3 Sample and Population of the Study 23
3.4 Data Gathering Procedure 23
3.4.1 Collection and Sampling 23
3.4.2 Preparation and Extraction of Essential Oil 24
3.4.3 Diethylnitrosamine 24
3.4.4 MTT Assay 25
3.4.5 DPPH Assay 27
3.5 Research Ethics 33
3.6 Statistical Data Analysis 33
Bibliography 38
Fig. 1 Research flowchart 20
Appendix A: Research Budget 34
Appendix B: Gannt Chart 35
Appendix C. Curriculum Vitae 34
RESEARCH ABSTRACT
Thymol and carvacrol in Oregano vulgare (oregano plant) are the substances
that have strong antioxidants properties, which could protect organism against
Esophageal cancer (EC) is the eighth most common cancer and is the sixth leading
cause of death worldwide. This study was conducted to evaluate the effectiveness of
cell line. According to studies, Evidence has shown that OVEO had an anti-tumor
effect. OVEO have shown anti-tumor activity in in vivo assays. The researchers aim
identify the potential anti-cancer effect of Origanum vulgare, it also seeks to answer
if this study can develop antioxidant agent strong enough to eliminate or reduce the
risk of acquiring esophageal cancer. Using MTT assay to determine its cell
CHAPTER 1
INTRODUCTION
Esophageal Cancer occurs in the esophagus, it is a long, hollow tube that runs
from your throat to your stomach. Your esophagus helps move the food you swallow from
the back of your throat to your stomach to be digested. Esophageal cancer usually begins
in the cells that line the inside of the esophagus. This study shows the utilization of
Studies examining the antioxidant activities of different forms of oregano (new, dry,
and ready-to-use herb mix glues) appeared that oregano holds its solid antioxidant
capacity in both new and dry form (John Coccimiglio et al., 2016). Fresh oregano on a
weight for weight basis had three to 20 times higher antioxidant capability than the other
antioxidant activity than apples, 30 times more than potatoes, 12 times more than oranges
and four times more than blueberries (Zheng and Wang, 2001). The clears out and dried
herb of oregano as well as its essential oil are customarily utilized for respiratory
years. It encompasses a useful effect upon the stomach related and respiratory systems
by pregnant ladies in spite of the fact that it is perfectly safe in little amounts for culinary
purposes. The leaves and flowering stems are strongly antiseptic, antispasmodic,
and mildly tonic. It is extremely sedative and should not be taken in large doses, in spite
of the fact that mild teas have a soothing effect and aid restful sleep. It should not be
prescribed for pregnant women. Remotely, oregano is utilized to treat bronchitis, asthma,
joint pain and muscular pain. The plant can be utilized new or dried - gather the total plant
(but not the roots) in late summer to dry and store for winter use.
Oregano is often used in the form of an essential oil that is distilled from the leaves
and stem. A few drops of the essential oil, put on cotton wool and placed in the hollow of
an aching tooth, frequently relieves the pain of toothache. This plant is one of the best
natural antiseptics because of its high thymol content (L., 2005). Oregano (Origanum
vulgare) is an herb prominent in the Mediterranean diet, and has been shown to possess
properties.
In traditional medicine, oregano has been utilized for respiratory conditions (i.e.
asthma, bronchitis, hack), gastrointestinal (i.e. the runs, indigestions, stomachache), anti-
mainly to carvacrol and thymol, which are the major components of its essential oil.
Antibacterial effects have been reported for oregano against Clostridium perfringens,
antioxidant properties of Mediterranean food spices and common food additives have
shown that extracts from oregano were more effective than butylated hydroxyanisole
(BHA) and butylated hydroxytoluene (BHT) in inhibiting lipid peroxidation. The use of
synthetic antioxidants to prevent free radical damage can involve questionable nutritional
value and toxic side effects while natural antioxidants present in many plants reduce
oxidative damage and help in preventing mutagenesis, carcinogenesis, and aging due to
their radical scavenging activities. The role of free radicals has been implicated in several
The bioactive constituents of O. vulgare leaves extract are rosmarinic acid, linalool,
thymol, carvacrol, tannins, flavonoids, triterpenes, phenol carvacrol, and thymol. Oregano
also contains iron, vitamins, calcium, copper, niacin, magnesium, and thiamine. Oregano
has been used in the treatment of cancer, muscular degeneration, and heart disease. (V.
Kumar, 2016)
Origanum vulgare had effective antioxidants against esophageal cancer. This study
hopes to prove that oregano vulgare essential oil can treat and use against esophageal
researchers conducted a study which aims to know the antioxidants effectiveness through
the use of Mus musculus as a model organism. Through this, the researchers also intend
This research aims to prove the effectiveness of the essential oil in Origanum
1. What is the mean IC50 of Origanum vulgare essential oil against esophageal
cancer?
The researcher opted for the 5% level of probability with 95% level of reliability to
determine the degree of significance of findings. The hypotheses below established the
esophageal cancer
The purpose of the study was to determine the potential Origanum vulgare as
Esophageal cancer patients. This research will serve as an avenue for the prevention
Individuals with Family History of Esophageal Cancer. This research will help those
at risk in acquiring esophageal cancer through neutralizing free radicals in the body with
Medical Doctors. This study will give an opportunity to make development of new
treatments with the help of Origanum vulgare in slowing the further progression of cancer
Students. This research will serve as a reference in doing research, related to the use of
Educator. This research will serve as a material for teaching research and as well as
Future researchers. This research will serve as a reference for future studies that
This research was conducted to determine the validity of using Origanum vulgare
in neutralizing free radicals and as a prevention from acquiring Esophageal cancer. This
involves the use of five (5) weeks old to eight (8) weeks old Mus musculus as the model
organism to test for the effectiveness of Origanum vulgare in reducing free radicals.
The research entailed six (6) phases of experimentation: (1) Collection Origanum
vulgare (2) Hydrodistillation of dried Oreganum vulgare leaves (3) Establishment of the
Introducing Origanum vulgare essential oil orally to GI, GIV, GV and GVI of Mus
musculus subject organism with different dosage (6) MTT assay for cell proliferation and
Metro Manila during the academic year 2019-2020. The study attempted to determine the
antioxidant properties of the Oregano that was collected in San Ildefonso, Bulacan. The
establishment of this study was conducted at the Animal Care Experimentation Facility
On the other hand, the test animals were bred for experimental purposes coming
from the Department of Science and Technology and secured approval from the
Institutional Animal Care and Unit Committee (IACCU) of the Our Lady of Fatima
University and submitted to Bureau of Animal Industry (BAI) for the approval and issuance
of Animal Research Permit of the Animal Review and Ethics Committee of BAI.
This experimentation was limited to the use of Origanum vulgare only and did not
include other species of the family Lamiaceae. Furthermore, it was limited to the study of
its antioxidants that greatly contribute to the prevention of carcinogenic cells build up
DEFINITION OF TERMS
Antioxidants. Substances that are used in preventing or slowing the damage to cells
MTT Assay. Used in the study to measure the total number of TP53 gene present to
Carcinogenesis. The production of cancer cells that results in cancer build up.
Mus musculus model. This refers to the model organism used in the study and
This chapter presents the review of related literature and studies considered relevant to
RELATED LITERATURE
Botanical Descriptions
woody-branched perennial which ordinarily develops to 1-3’ tall and to 2’ wide on square
stems clad with fragrant, glandular-spotted, adjusted to applaud takes off (to 1 1/2” long)
which are as a rule whole but in some cases have somewhat toothed edges. Little, two-
lipped, pinkish-purple, or white blooms (ordinary mint family), each with 4 protruding
stamens and leafy purple-toned bracts, sprout in axillary or terminal corymb-like spikelets
which rise over the foliage in summer. Plants are local from Europe to central Asia, but
escaped gardens and naturalized in parts of the eastern and far western U.S. and
Canada. Oregano, could be a perpetual bush local to the dry, rough calcareous soils
within the precipitous region of the Mediterranean and Euro/Irano-Siberian districts but it
is additionally developed for its employments as a herb and helpful properties (John
Oregano or marjoram (Origanum vulgare Linn.) is the most variable species of the
world and is particularly abundant in the Mediterranean, Eurasian, and North African area.
Several studies have shown that the plant has a wide variety of secondary metabolites,
most of them phenolic compounds such as flavonoids, terpenoids, phenolic acids and
alkaloids, and fatty acids, among others, which are the main components responsible for
Origanum species grow copiously on mountainous areas and hilly areas with
fractions of the genus Origanum that can thrive in varied climatic belts gives them a strong
addition to relevance in medicine and agriculture, essential oil from O. vulgare can be
Constituents
family. The bioactive constituents of this herb, such as carvacrol and thymol possess
it is considered a standard natural, less toxic, and residue free feed additive, that is
extensively used with promising health benefits against respiratory, digestive, and urinary
disorders. This review casts light on description, chemical composition and structure of
Origanum vulgare, as well as its therapeutic applications in human and its biological
Chemical preservatives have been used to avoid food spoilage. However, since
related issues, researchers have begun to investigate the use of organic and natural
Carvacrol Property
Coridothymus, Thymbra, Satureja and Lippia are rich in carvacrol, a monoterpenic phenol
isomeric with thymol. Carvacrol is responsible for the biological activities of oregano.
hepatoprotective activities and uses such as feed additive, in honeybee breeding and in
oil. Oregano essential oil presented antioxidant activity by the DPPH and ABTS
The use of this essential oil can be indicated as a natural preservative (Dutra, T. V.,
cyclooxygenase inhibition but it could induce gastrointestinal toxicity because of its non-
delivers carvacrol into and through the skin which would overwhelm the gastrointestinal
problems. Origanum vulgare oil microemulsion was suggested to be used for the
Thymol Property
monoterpene phenol. It is one of the most important dietary constituents in thyme species.
For centuries, it has been used in traditional medicine and has been shown to possess
levels. The noteworthy effects of thymol are largely attributed to its anti-inflammatory
free radicals, enhancing the endogenous enzymatic and non-enzymatic antioxidants and
chelation of metal ions), antihyperlipidemic (via increasing the levels of high density
lipoprotein cholesterol and decreasing the levels of low density lipoprotein cholesterol and
(via maintaining ionic homeostasis) effects (Nagoor Meeran, M. F., Javed, H., et al. 2017).
RELATED STUDIES
Antioxidants Activity
In recent years, the efficiency of essential oils from Oregano species has been
constituents rosmarinic acid (RA) and carvacrol (CA). Essential oils containing CA are the
including antioxidant activity that is associated with the prevention of various degenerative
The substances have strong antioxidant properties, which could protect organisms
against oxidative stress. In the present study, similar to the in vitro 1,1-diphenyl-2-picryl-
hydrazyl (DPPH) radical scavenging activity of oregano extract, in vivo study showed that
dried oregano powder (DOP) also possess a significant radical scavenging activity and
the addition of DOP, may provide a more efficient scavenging of free reactive radicals in
compounds capable of reducing or neutralizing free radicals are being studied for a
therapeutic use. Origanum vulgare L. is a traditional medicinal plant used for a wide
number of health problems due to its antimicrobial, carminative and antioxidant activities.
therapeutic properties. To avoid this, two different solid oral formulations have been
designed for an O. vulgare extract evaluating their antioxidant behaviours in vitro and in
vivo after a simulation of gastrointestinal digestion. In this sense, the stability of the
reaction does not have an important role in the differences observed between the
digested powder and the encapsulated extract. However, the encapsulated form always
shows a higher activity, so we consider that it could be the optimal oral formulation for O.
physically the components of the extracts from the digestion process, favouring the
hydroalcoholic extract of Origanum vulgare could be a potentially minimum daily dose for
antioxidant oral treatment. (de Torre, M. P., Vizmanos, J. L., Cavero, R. Y., & Calvo, M.
I. 2020)
Antimicrobial Activity
cymene and γ-terpinene, generally equating to 80% of essential oil contents. CAR and
reduced cell membrane potential which can lead to cell death. This antimicrobial activity
may have the potential to protect broilers from enteric pathogens and consumers from
food borne diseases. Oregano powder has the potential to be used as a low dose
subtherapeutic feed additive, preserving antibiotics for situations when high dose
aureus strains from swine origin, with no effect using maqui extract. The characteristics
of S. aureus strains of swine origin, and about the potential use of NAs from oregano to
enhance the control of antibiotic‐resistant S. aureus strains in the pork supply chain.
Anti-proliferative Activity
about 2.1–2.9 times more active against MDA-MB231 cell line ( < 0.05) compared to U87
cell line.This different activity could be due to the fact that glioblastoma cells are usually
more resistant to chemo- and radiotherapy; they may contain accumulated gene
Origanum onites essential oil OOEO was examined for its antiproliferative activity
against a panel of human cancer cell lines from four different tumor types. In particular,
melanoma cells (A375), breast cancer cells (MCF-7), hepatocellular carcinoma cells
(HepG2), and colon cancer cells (HT-29) were treated with increasing concentrations of
exhibited different sensitivity to the OOEO, evident by the different IC50 values . The
strongest antiproliferative effect (lowest IC50 value for 72 h) was observed in the HT-29
colon cancer cell line (0.35 ± 0.2 μg/mL) followed by A375 skin melanoma (8.90 ± 0.7
μg/mL), MCF-7 breast carcinoma (10.0 ± 1.7 μg/mL), and HepG2 hepatocellular
carcinoma cells (23.0 ± 4.2 μg/mL). OOEO was examined for its antiproliferative activity
against a panel of human cancer cell lines from four different tumor types. In particular,
melanoma cells (A375), breast cancer cells (MCF-7), hepatocellular carcinoma cells
(HepG2), and colon cancer cells (HT-29) were treated with increasing concentrations of
activity against all human cancer cell lines tested. However, the different cell lines
exhibited different sensitivity to the OOEO (Spyridopoulou, K., Fitsiou, E., et al. 2019).
Anti-Inflammatory Activity
prostaglandins, enzymes, nitric oxide (NO) and reactive oxygen species (ROS), among
oregano have also been studied to better understand their effect on inflammation. For
100 mg/kg), presenting paw edema, the levels of IL-1β and prostaglandin E2 (PGE2)
prostaglandins were diminished. The reduction on the mRNA expression of IL-1β and
Antitumor Activity
Evidence has shown that EOO had an antitumor effect. EOO have shown
antitumor activity both in in vivo and in vitro assays. An in vivo study reported that low
doses of EOO in a three-month period exerted preventive action by decreasing the sizes
of tumors by 1.5 times in diseased animals. It was suggested that the EOO could possibly
affect the development and progression of the tumor process via the activation of
regulator cell molecules. The in vitro antitumor activity of EOs of O. onites, was analyzed
against rat adipose tissue endothelial cells and c-H-ras transformed rat embryonic
fibroblasts (5RP7) cells. EOO at 125, 250 and 500 µg/mL resulted in significant inhibition
of cell viability. In addition, EOO induced apoptosis of 5RP7 cells and blocked in vitro tube
formation which accounts for its angiogenic activity. (Bostancioglu R.B., Kurkcuoglu M.,et
al 2012)
Antifungal properties
Oregano essential oil is among the most investigated essential oils due to its
antifungal properties, as it can prevent the growth of a broad spectrum of fungi, such
as Aspergillus sp., Fusarium sp., and Penicillium sp.Oregano essential oil is mainly
comparing it with the known antifungal agent like nystatin. Antifungal susceptibility testing
was carried out using agar well diffusion method. Antifungal activity showed a zone of
proved that the experimental herb O. vulgare is antifungal in nature and also it is superior
in its activity when compared to nystatin (Bhat, V., Sharma, S. M., Shetty, V., et al. 2018).
The effect of O. vulgare against reference strains of Candida was found to be all
susceptible to the essential oil of O. vulgare. (Rosato et al. and Souza et al.) evaluated
and observed that the essential oil O. vulgare appeared to be effective, inhibiting all
Esophageal cancer
Esophageal cancer is the eighth most common tumor in the world. Esophageal cancer
patients in China account for more than half of the total number of esophageal cancer
patients in the world, and here the mortalities of both male and female patients are the
genetics, living environment, bad habits (such as smoking and drinking) and others. It is
not easy to detect esophageal cancer at an early stage, and esophageal cancer in the
middle and advanced stages is generally treated with chemotherapy and radiation. As a
broad-spectrum antitumor drug, cisplatin mainly causes DNA damage in tumor cells, and
resistance generated by esophageal cancer cells is a decisive factor that affects the
chemotherapeutic effects.
TP53 gene
Somatic mutations in the TP53 gene are one of the most frequent alterations in
human cancers, and germline mutations are the underlying cause of Li-Fraumeni
are single-base substitutions distributed throughout the coding sequence. Their diverse
types and positions may inform on the nature of mutagenic mechanisms involved in
cancer etiology. TP53 mutations are also potential prognostic and predictive markers, as
the Origanum vulgare essential oil in fighting against Esophageal cancer (RHBDF2 gene)
in Mus musculus.
PRE-EXPERIMENTAL PHASE
EXPERIMENTAL PHASE
Control
Given OVEO (20 mg), seven Given NDEA, three days a week
days a week and water during the other four
days.
MTT Assay
DPPH Assay
Conclusion
OUR LADY OF FATIMA UNIVERSITY COLLEGE OF MEDICAL LABORATORY
SCIENCE
22
CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
CHAPTER 3
METHODOLOGY
Research Design
research aim is to determine the relationship between one thing and another in a
all factors that may affect the result of an experiment. The research entailed six (6) phases
Oreganum vulgare leaves (3) Establishment of the Minumum Lethal Dose (MLD) or
Diethylnitrosamine induce to Mus musculs (5) Introducing Origanum vulgare essential oil
orally to GI, GIV, GV and GVI of Mus musculus subject organism with different dosage
(6) MTT assay for cell proliferation and viability (7) DPPH Assay for scavenging activity.
Research Locale
The phase one (1) of the study includes the collection and extraction of Origanum
vulgare was done at San Ildefonso, Bulacan where Farming and vegetable production
The phase two (2) of the study the hydro-distillation of dried Origanum vulgare
carcinogenesis, and phase (6) MTT assay for cell proliferation and viability (7) DPPH
Assay for scavenging activity was done at Medical Technology Laboratory 3 and the
phase (4) Diethylnitrosamine induce to Mus musculs, phase five (5) Introducing Origanum
vulgare essential oil orally to GI, GIV, GV and GVI of Mus musculus subject organism
with different dosage was done at the animal care and experimental facility of the Our
Lady of Fatima University located at the old allied medicine building at 120 MacArthur
The study utilized 60 mice models aged five (5) to eight (8) weeks old. The animals
were bred for experimental purposes coming from the Food and Drug administration
(FDA) in Muntinlupa City, Philippines. The test animals were housed in the Animal Care
and Experimental Facility (ACEF) of the Our Lady of Fatima University Valenzuela
Campus.
The following procedures were carried out to identify the effectiveness of Origanum
Ildefonso, Bulacan.
The leaves of Origanum vulgre were collected from a local area of San Ildefonso,
Bulacan, and were washed thoroughly with tap water. The leaves were brought to the
Department of Science and Technology, Taguig City for the extraction of essential oil.
essential oil for a percentage yield of 0.4% (v/w). Anhydrous sodium sulfate was added
to the oil to remove traces of moisture. The extracted oil was stored in refrigerator freezer
at 4oC.
Diethylnitrosamine (NDEA) was acquired from the Sigma (Sigma Chemicals Co.
St. Louis, Millstone - USA), flask with 100ml, density of 0.95 g/ml, molecular weight of
102.1 g/mol and chemical formula C4H10N2O. The compound was diluted in drinking
water, and administered to the animals for 3 days of the week, in the dosage of 10mg/Kg
The Origanum vulgare essential oil was obtained through hydro distillation done at
Department of Science and Technology, Taguig City and administered orally to the
The animals were divided into six groups (I, II, III, IV, V, VI) with ten rats, which
received water, OVEO and NDEA in alternate days, and observed for 180 days.
Group III - NDEA, three days a week and water during the other four days
Group IV - NDEA, three days a week and OVEO (20 mg) during the other four
days
Group V - NDEA, three days a week and OVEO (40 mg) during the other four
days
Group VI - NDEA, three days a week and OVEO (60 mg) during the other four
days
The dosages of NDEA were: - 10 mg. / Kg / day and OVEO - 20 (GII,GIV), 40 (GV),
4. MTT Assay
Determination of cell growth rates is widely used in the testing of drug action,
cytotoxic agents, and screening other biologically active compounds. Several methods
can be used for such determinations, but indirect approaches using fluorescent or
chromogenic indicators provide the most rapid and large-scale assays. The assay is
designed for the spectrophotometric quantification of cell growth and viability without the
purple formazan crystal. The formazan crystals formed are then solubilized, and the
concentration of resulting colored solution determined by optical density at 570 nm. The
result is a sensitive assay with excellent linearity up to approximately 106 cells per well.
nutrients.
● Measurement of cytotoxicity.
• 96 well plates
• CO2 incubator
• DMSO
• PBS
cytotoxicity assay. Cells number of each well depends on cell density and growth rate.
6.2 For adherent cells, remove the medium and replace it with 100 µL of fresh culture
medium. For non-adherent cells, centrifuge the 96 well microplate, pellet the cells,
carefully remove as much medium as possible and replace it with 100 µL of fresh medium.
6.3 Add 10 μL of the MTT labeling reagent to each well. Include a negative control of
6.4 Incubate the microplate at 37°C for 4 hours in a humidified chamber (5% CO2 ). At
high cell densities (>100,000 cells per well) the incubation time can be shortened to 2
hours.
6.5 Add 100 μL of Formazan solubilization solution to each well and mix thoroughly.
6.6 Incubate the microplate at 37°C for 4-18 hours in a humidified chamber (5% CO2 ).
6.7 Check for complete solubilization of the purple formazan crystals and measure the
spectropho- tometrical absorbance of the samples using a microplate reader at 570 nm.
Note: The wavelength between 560 and 600 nm can be chosen if 570nm is unavailable.
DPPH ASSAY
Recent findings suggest that a decline in internal antioxidant capacity causes the onset
foods has been recently increasing. Shimamura et al. improved the 2,2-diphenyl-1-
of antioxidants.
The DPPH Antioxidant Assay Kit is based on the DPPH assay improved by Shimamura
and enables quick and easy measurements of the antioxidant capacity of a sample.
Using this kit, the antioxidant capacity is expressed as the Trolox equivalent antioxidant
capacity (TEAC), a value calculated from the IC50 of the antioxidant and the IC50 of
Trolox. The DPPH Reagent and Trolox Standard included in this kit only need to be
dissolved prior to Moreover, the protocol for this kit supports a microplate assay format
60 seconds.
3. Add another aliquot of approx. 1 mL of ethanol to the tube from step 1 sonicate
for 60 seconds.
4. Check the color of the solution and if the solution has a purple color, repeat steps
dissolve.
* Any undissolved DPPH may impact the assay. Please make sure that all of the
DPPH is dissolved.
completely by sonication. Please remove the air-by. sinking the tube of -the
add ethanol to 10 mL
3. Dilute the 100 ug/mL Trolox Standard solution prepared in step 2 with ethanol to
make 80, 60, 40, and 0 ug/mL solutions * Prepare the Trolox Standard solution
1. Prepare four or more concentrations for each sample with a 10-fold dilution from
3. Add 20 of solvent used for sample extraction or dilution to the wells of Blank 1
and Blank 2
4 immediately.
5. Add 100 uL of ethanol to the wells of Blank 2 and mix well by pipetting.
6. Add 100 uL of DPPH working solution to the wells of the samples and Blank 1
*Because the reaction starts immediately after the addition of the DPPH
*DPPH working solution is also used for the antioxidant capacity assays
dispense only the required volume of DPPH working solution into a reservoir.
*If the 517 nm filter is not available measure the absorbance at 500 nm or
9. Calculate the inhibition ratio of the samples from the following equation:
As. Absorbance of samples - Blank 2 or sample blank (in the case where the
11. Determine the optimum concentration range encompassing the 50% scavenging
concentration for DPPH-radicals from the regression line drawn in step 10.
samples
*Please follow the order of this protocol because it is optimized for an antioxidant
assay.
1. Add 20 uL of 0, 40, 60, and 80 ug/mL of Trolox Standard solution to each well.
3. Add 20 uL of ethanol to the wells of Blank 3 and add 20 uL of the solvent that
was used for sample dilution to the wells of Blank 1 and Blank 2.
immediately.
pipetting.
6. Add 100 uL of DPPH working solution to the wells of Trolox, samples and
9. Calculate the inhibition ratio of the samples from the following equation:
As: Absorbance of samples - Blank 2 or sample blank (in case the sample is
highly colored)
10. Plot the inhibition ratio (y) against the sample concentration (x) and draw a
regression line (y= ax+b).
Research Ethics
The study utilized 60 mice models aged five (5) to eight (8) weeks old. The animals
were bred for experimental purposes coming from the Food and Drug Administration
(FDA) in Muntinlupa City, Philippines. The researchers submitted for the approval of the
experimental protocol to the institutional animal care and use committee of the ACEF of
Our Lady of Fatima University and upon the approval of the experimental protocol it was
submitted to the Bureau of Animal Industry for the issuance of animal research permit.
The Kruskal Wallis Test (nonparametric variance analysis) was used for the
statistical analysis followed by multiple comparisons using the DUNN Test for number of
tumors and the Fischer's Exact Test for comparison of proportions, with significance of 5%
(p <0.05).
Estimated Total:
Php 10,000
Organisms
Laboratory Tests
Transportation
Others
GANNT CHART
Activities JULY AUG SEP OCT NOV DEC JAN FEB MAR
Finalsubmission of
Research proposal
Proposal Defense
Approval of Research
topic
Buying of research
equipments
Experimentation
Data collection
Data analysis
Data interpretation
Series of revisions
Curriculum Vitae
John David D. Lacanlale first born son of Roel of the house Lacanlale and Relebeth of the house
Dagundong. He was born on March 1, 1999 at Batasan Birthing center. He finished primary and secondary
level of study at San Vicente San Francisco High School where he receives multiple award both curricular
and extracurricular. He was a consistent honor student during his elementary days and he also excel in
extracurricular when he is already in high school. He is now a 3rd year student currently taking Bachelor
of Medical Laboratory Science at Our Lady of Fatima University Valenzuela City Campus.
Lyka P. Mendoza
09276526072
Lyka Mendoza is the middle-born child of Mr. and Mrs. Ariel Mendoza. She was born on February
21 year 2000 at Castro Maternity Hospital in Baliuag, Bulacan, and currently residing at San Ildefonso,
Bulacan. A 3rd year Medical Laboratory Science student, a 20-year-old female aspiring to be a medical
doctor and be an inspiration to all the of the student that also aspire to be a doctor. She finished her
Elementary at San Ildefonso Elementary school and finished her high school at Saint John School where
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