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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …

OUR LADY OF FATIMA UNIVERSITY

BACHELOR IN MEDICAL LABORATORY SCIENCE

Cytotoxic and Antioxidant activity of Origanum vulgare (Oregano plant) Essential

Oil against Diethylnitrosamine induced Esophageal cancer in Mus musculus

A Research Presented to

Dean Jose Jurel M. Nuevo, RMT, MSMT, PhD and

To the faculty of Bachelor of Medical Laboratory Science

OUR LADY OF FATIMA UNIVERSITY

In Partial Fulfillment

Of the Requirements for the Introduction to

Medical Laboratory Science Research

Lacanlale, John David


Mendoza, Lyka P.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
TABLE OF CONTENTS

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

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …

RESEARCH ABSTRACT

Thymol and carvacrol in Oregano vulgare (oregano plant) are the substances

that have strong antioxidants properties, which could protect organism against

oxidative stress. In this paper, we introduced an experimental method to know if the

Origanum vulgare can inhibits cell proliferation against esophageal cancer.

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

Oregano vulgare essential oil (OVEO) as an antioxidant against Esophageal cancer

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

to determine the potential antioxidant property of OVEO. The researchers aim to

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

proliferation inhibition and DPPH assay to determine its scavenging activity.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …

CHAPTER 1

THE PROBLEM AND ITS BACKGROUND

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

Origanum vulgare (oregano plant) against Diethylnitrosamine induced EC to reduce its

growth in the early phase of Esophageal cancer among Mus musculus.

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

herbs considered and in comparison to vegetables, oregano has 42 times more

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

disarranges, acid reflux, and rheumatoid joint pain.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
Oregano has been utilized as a culinary and therapeutic herb for thousands of

years. It encompasses a useful effect upon the stomach related and respiratory systems

and is additionally utilized to promote menstruation. It ought to not be utilized medicinally

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,

carminative, cholagogue, diaphoretic, emmenagogue, expectorant, stimulant, stomachic

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

several bioactive properties including anti-oxidant, anti-microbial, anti-inflammatory, and

analgesic properties (Veenstra and Johnson, 2019). Oregano EO is broadly utilized in

nourishment innovation, due to its antimicrobial, antioxidant (additive), and flavoring

properties.

In traditional medicine, oregano has been utilized for respiratory conditions (i.e.

asthma, bronchitis, hack), gastrointestinal (i.e. the runs, indigestions, stomachache), anti-

bacterial, anti-inflammatory, menstrual disarranged, and diabetes. Greek oregano

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
(Origanum vulgare) all through the world is the foremost recognized herb as being bona

fide or unique “oregano” (Veenstra and Johnson, 2019).

The antibacterial and antioxidant properties of oregano have been attributed

mainly to carvacrol and thymol, which are the major components of its essential oil.

Antibacterial effects have been reported for oregano against Clostridium perfringens,

Pseudomonas aeruginosa, and Staphylococcus aureus. Studies comparing the

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

pathological conditions, including cancer, cardiovascular diseases, neurodegenerative

disorders, and drug toxicity (John Coccimiglio et al., 2016).

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)

In this paper, we introduced a method, the experimental method to know if the

Origanum vulgare had effective antioxidants against esophageal cancer. This study

hopes to prove that oregano vulgare essential oil can treat and use against esophageal

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
cancer. With these problems brought by the undesirable abundance of cancer cells, the

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

to provide breakthroughs for future researchers to be able to construct ways on the

possible uses of the Origanum vulgare and ways to utilize it.

STATEMENT OF THE PROBLEM

This research aims to prove the effectiveness of the essential oil in Origanum

vulgare in neutralizing free radicals and prevention against Esophageal cancer.

Specifically, this research aims to answer the following questions:

1. What is the mean IC50 of Origanum vulgare essential oil against esophageal
cancer?

2. What is the mean percentage inhibition against esophageal cancer


2.1 Origanum vulgare essential oil
2.2 Positive Control

3. What is the scavenging activity against esophageal cancer


3.1 Positive Control
3.2 Experimental group

4. Is there a significant difference in the scavenging activity among positive


control and experimental group?
5. Is Origanum vulgare effective in inhibition of cell proliferation against
esophageal cancer

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …

HYPOTHESES OF THE STUDY

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

level of significance of 0.05.

1. There is no significant difference in the scavenging activity among positive

control and experimental group

2.Origanum vulgare is not effective in inhibition of cell proliferation against

esophageal cancer

SIGNIFICANCE OF THE STUDY

The purpose of the study was to determine the potential Origanum vulgare as

prevention in strains of esophageal cancer introduced to Mus musculus. The findings of

this study will be useful to the following:

Esophageal cancer patients. This research will serve as an avenue for the prevention

of cancer cells spreading among patients diagnosed with esophageal cancer.

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

the aid of Origanum vulgare antioxidants.

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

cells throughout the body and to keep it under control.

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Pharmaceutical Scientists. This will give an idea for scientists to formulate a

maintenance drug out of Origanum vulgare.

Students. This research will serve as a reference in doing research, related to the use of

Origanum vulgare or aid in Esophageal cancer.

Educator. This research will serve as a material for teaching research and as well as

basis for research paper writing.

Future researchers. This research will serve as a reference for future studies that

includes esophageal cancer and uses of Origanum vulgare.

SCOPE AND LIMITATIONS

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

Minumum Lethal Dose (MLD) or minimum dose of Diethylnitrosamine capable of

inducing esophageal carcinogenesis (4) 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.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
The study was presided at Our Lady of Fatima University, City of Valenzuela,

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

(ACEF) of the Our Lady of Fatima University.

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

resulting in esophageal cancer.

DEFINITION OF TERMS

Antioxidants. Substances that are used in preventing or slowing the damage to cells

caused by free radicals.

TP53. The gene commonly elevated in Esophageal Cancer.

MTT Assay. Used in the study to measure the total number of TP53 gene present to

exposed Mus musculus induced wth diethylnitrosamnine.

Carcinogenesis. The production of cancer cells that results in cancer build up.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
Free radicals. Scavenge the body to seek out other electrons so they can become a pair,

this may cause damage to cells, proteins and DNA.

Mus musculus model. This refers to the model organism used in the study and

introduced with the RHBDF2 cell line of esophageal cancer.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
Chapter 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents the review of related literature and studies considered relevant to

the understanding of this study

RELATED LITERATURE

Botanical Descriptions

Origanum vulgare, commonly known as oregano, could be a bushy, rhizomatous,

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

Coccimiglio et al., 2016).

Oregano or marjoram (Origanum vulgare Linn.) is the most variable species of the

genus Origanum, characterized by morphological and chemical diversity. Used worldwide

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since ancient times in traditional and popular medicine, oregano is spread all over 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

its action (Beltrán J. M. G., Espinosa C., et al. 2018).

Origanum species grow copiously on mountainous areas and hilly areas with

extensive ranges of altitudes. The immense variability in volatile as well as nonvolatile

fractions of the genus Origanum that can thrive in varied climatic belts gives them a strong

utility in agriculture, medicine, and cosmetics, as a flavoring and aromatic agent. In

addition to relevance in medicine and agriculture, essential oil from O. vulgare can be

used as food disinfectant. (Jan, S., Rashid, M., et al. 2020)

Constituents

Origanum vulgare L. is an aromatic enduring herb that belongs to the Lamiaceae

family. The bioactive constituents of this herb, such as carvacrol and thymol possess

several medicinal properties, such as antioxidant, antidiabetic, anti-inflammatory,

antimicrobial, antiviral, antiparasitic, anti-neoplastic, and immune modulatory. Moreover,

it is considered a standard natural, less toxic, and residue free feed additive, that is

successfully used in livestock and fish. Additionally, in humans, Origanum vulgare 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

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
activities in ruminants and fish, data that will be possibly useful for physiologists,

nutritionists and veterinarians (Alagawany, Abd El-Hack et al., 2020).

Chemical preservatives have been used to avoid food spoilage. However, since

chemical preservation leads to constraints in consumer acceptability and other health-

related issues, researchers have begun to investigate the use of organic and natural

preservatives (Davidson P. M., Critzer F. J., et al. 2013)

Carvacrol Property

Oregano essential oils obtained from the genera Origanum, Thymus,

Coridothymus, Thymbra, Satureja and Lippia are rich in carvacrol, a monoterpenic phenol

isomeric with thymol. Carvacrol is responsible for the biological activities of oregano.

Many diverse activities of carvacrol such as antimicrobial, antitumor, antimutagenic,

antigenotoxic, analgesic, antispasmodic, antiinflammatory, angiogenic, antiparasitic,

antiplatelet, AChe inhibitory, anti elastase, insecticidal, antihepatotoxic and

hepatoprotective activities and uses such as feed additive, in honeybee breeding and in

gastrointestinal ailments have been shown. (Baser K. H. C. 2008)

Carvacrol acetate was identified as the major compound of oregano essential

oil. Oregano essential oil presented antioxidant activity by the DPPH and ABTS

methods. Antimicrobial activity of essential oil was effective against A. acidoterrestris.

The use of this essential oil can be indicated as a natural preservative (Dutra, T. V.,

Castro, J. C., et al. 2019)

Carvacrol has been reported for analgesic and anti-inflammatory activity by

cyclooxygenase inhibition but it could induce gastrointestinal toxicity because of its non-

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
selective inhibition. Transdermal microemulsion from Origanum vulgare essential oil

delivers carvacrol into and through the skin which would overwhelm the gastrointestinal

problems. Origanum vulgare oil microemulsion was suggested to be used for the

transdermal delivery and anti-inflammatory activities enhancement of carvacrol.

(Laothaweerungsawat, N., Neimkhum, W., et al. 2020)

Thymol Property

Thymol, chemically known as 2-isopropyl-5-methylphenol is a colorless crystalline

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

various pharmacological properties including antioxidant, free radical scavenging, anti-

inflammatory, analgesic, antispasmodic, antibacterial, antifungal, antiseptic, and

antitumor activities. It reveals the pharmacological properties of thymol has multiple

therapeutic actions against various cardiovascular, neurological, rheumatological,

gastrointestinal, metabolic, and malignant diseases at both biochemical and molecular

levels. The noteworthy effects of thymol are largely attributed to its anti-inflammatory

(via inhibiting recruitment of cytokines and chemokines), antioxidant (via scavenging of

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

low density lipoprotein cholesterol in the circulation and membrane stabilization)

(via maintaining ionic homeostasis) effects (Nagoor Meeran, M. F., Javed, H., et al. 2017).

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Higher concentrations of thymol produced lower values for IC 50 according to

DPPH technique, indicating an increase in antioxidant properties. ( Borgarello, A. V.,

Mezza, G. N., et al. 2015).

RELATED STUDIES

Antioxidants Activity

In recent years, the efficiency of essential oils from Oregano species has been

reported in many studies. Antioxidant activity of Oregano is associated with its

constituents rosmarinic acid (RA) and carvacrol (CA). Essential oils containing CA are the

subject of increasing scientific interest because of their multiple biological effects,

including antioxidant activity that is associated with the prevention of various degenerative

diseases. HPLC postcolumn assay is highly promising to investigate natural antioxidants

with various derivatization techniques. (Baranauskaite, J., Kubiliene, A., et al 2017)

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

have a potent antioxidant capacity. It has a significant relationship between phenolic

content and antioxidant enzyme activity. Therefore, the higher concentrations of

supplementation on superoxide dismutase SOD and glutathione peroxidase GPx, due to

the addition of DOP, may provide a more efficient scavenging of free reactive radicals in

ducks (Park, J. H., Kang, S. N., et al. 2015).

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Aging-related diseases can be triggered by multiple factors such as oxidative stress.

Oxidative stress is an imbalance between free radicals and antioxidants, so today,

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.

However, when administered orally, gastrointestinal digestion can modify some of

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.

vulgare hydroalcoholic extracts. Compared to powder, the encapsulation can protect

physically the components of the extracts from the digestion process, favouring the

maintenance of their antioxidant activity. Thus, two capsules of 250 mg of a

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

Oregano (Origanum vulgare) is a phytobiotic and is known to contain

antimicrobial compounds such as carvacrol (CAR), thymol and their precursors, p-

cymene and γ-terpinene, generally equating to 80% of essential oil contents. CAR and

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
thymol have consistently demonstrated antimicrobial properties at low doses, 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

therapeutic treatments are required. (Bauer, B. W., Gangadoo, S., 2019)

Natural antimicrobials extracted from oregano have an inhibitory activity against S.

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.

(Lofa, A., Velasco, V., Gerding, M., et al. 2019)

Anti-proliferative Activity

Oregano extracts showed antiproliferative activity in vitro. The extracts were

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

mutations and epigenetic alterations. (Baranauskaite, J., Kubiliene, A., et al 2017)

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

the essential oil for 72 h. Interestingly, OOEO exhibited a dose-dependent antiproliferative

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
activity against all human cancer cell lines tested. However, the different cell lines

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

the essential oil for 72 h. Interestingly, OOEO exhibited a dose-dependent antiproliferative

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

Inflammation is a normal biological response of the body to tissue damage,

infections and chemical or physical agents. During inflammation, the production of

inflammatory mediators is triggered. Examples of these mediators are cytokines,

prostaglandins, enzymes, nitric oxide (NO) and reactive oxygen species (ROS), among

others. If inflammation is not controlled the pro-inflammatory mediators are overproduced

which might induce pathologic processes related to diseases such as arthritis,

atherosclerosis, and cancer to name a few. Consequently, inhibition of the mediators is

an imperative goal to treat inflammatory diseases. The individual components of EOs of

oregano have also been studied to better understand their effect on inflammation. For

example, (Lima et al.) demonstrated that carvacrol exerts anti-inflammatory activity on a

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
typical mice inflammation model. When carvacrol was administered to mice (at 50 and

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

cyclooxygenase-2 (COX-2) might be responsible for the effects mentioned (Leyva-López,

N., Gutiérrez-Grijalva, E., et al. 2017)

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

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
composed of carvacrol, thymol, cymene, and terpinene. The growing interest in its use in

food preservation is mainly due to consumers’ demands for a substitution of synthetic

preservatives in food by natural substances with a similar effect. (Bedoya-Serna, C. M.,

Dacanal, G., et al 2018)

The antifungal activity of the experimental herb O. vulgare was confirmed by

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

inhibition as 30 mm for O. vulgare as compared to 22 mm for nystatin. This observation

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

the Candida species.

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

highest. The occurrence of esophageal cancer is affected by multiple factors, including

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

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
is a common chemotherapeutic drug used to treat esophageal cancer. However, drug

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

syndrome, which predisposes to a wide spectrum of early-onset cancers. Most mutations

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

well as targets for pharmacological intervention.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
Research Flow Chart

This flowchart below is a schematic diagram in determining the effectiveness of

the Origanum vulgare essential oil in fighting against Esophageal cancer (RHBDF2 gene)

in Mus musculus.

PRE-EXPERIMENTAL PHASE

Collection of Origanum vulgare Submission of the research


in San Ildefonso, Bulacan protocol for the approval of the
IACUC

Secure research animal permit


Quantification and drying of the from the Bureau of Animal
plant sample Industry

Purchase of Mus musculus mice


model from DOST/FDA

Oregano was brought to


Department of Science and
technology in Taguig City for the
isolation of essential oil Acclimatization of test animals
through hydro-distillation for seven (7) days

EXPERIMENTAL PHASE

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
EXPERIMENTAL PHASE

4 groups of model organism were


given different treatments

Control

Given water, seven days a week

Treated but not exposed Exposed but not treated

Given OVEO (20 mg), seven Given NDEA, three days a week
days a week and water during the other four
days.

Exposed and treated

Given NDEA, three days a week


and OVEO (20, 60, 80 mg) during
the other four days.

MTT Assay
DPPH Assay

POST EXPERIMENTAL PHASE

Evaluation and Record

Conclusion
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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
CHAPTER 3

METHODOLOGY

Research Design

The researchers will use quantitative approach experimental design. A quantitative

research aim is to determine the relationship between one thing and another in a

population in which it may be a descriptive or experimental (Hopkins 2000).

Experimental research design is an attempt by the researchers to maintain control over

all factors that may affect the result of an experiment. The research entailed six (6) phases

of experimentation: (1) Collection Origanum vulgare (2) Hydrodistillation of dried

Oreganum vulgare leaves (3) Establishment of the Minumum Lethal Dose (MLD) or

minimum dose of Diethylnitrosamine capable of inducing esophageal carcinogenesis (4)

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

are one of their major industries (Bulacan.gov)

The phase two (2) of the study the hydro-distillation of dried Origanum vulgare

leaves took place at Department of Science and Technology, Taguig City.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
The other phases of the study: The phase 3 (3) Establishment of the Minumum Lethal

Dose (MLD) or minimum dose of Diethylnitrosamine capable of inducing esophageal

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

highway Valenzuela city.

Population and Sample of The Study

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.

Data Gathering Procedure

The following procedures were carried out to identify the effectiveness of Origanum

vulgare essential oil as an antioxidant in fighting against esophageal cancer induced in

Mus musculus mice models.

1. Collection and sampling

The sample of Oregano leaves (Origanum vulgare) was collected at San

Ildefonso, Bulacan.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
2. Preparation and Extraction of Essential Oil

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.

Hydrodistillation of 5 kilograms of dried Origanum vulgare leaves produced 20 mL of

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.

3. Diethylnitrosamine induced esophageal cancer in Mus musculus

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

of corporal weight, based on a mean consumption of water of 40ml per animal.

The Origanum vulgare essential oil was obtained through hydro distillation done at

Department of Science and Technology, Taguig City and administered orally to the

animals in a dosage of 20, 40, 60 mg/animal/day.

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.

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Sixty Mus musculus rats aged 5 to 8 weeks, were divided into four different groups

according to the drinking drugs:

Group I - controls: only water, seven days a week

Group II - only OVEO (20 mg), seven days a week

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),

60 (GVI) mg / animal / day, dissolved in drinking water.

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

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
use of radioactive isotopes. The MTT assay involves the conversion of the water soluble

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble

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.

● Quantification of cell growth and viability.

● Measurement of cell proliferation in response to growth factors, cytokines and

nutrients.

● Measurement of cytotoxicity.

5. Additional Materials Required

• 10μL, 100-200μL and multi-channel pipettes

• 96 well plates

• CO2 incubator

• DMSO

• PBS

• Plate reader capable of reading absorbance at 570nm

6. MTT Assay Protocol

6.1 Collect cells at logarithmic phase, count the cells.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
Note: Add 2000 cells per well for cell proliferation assay and 5000 cells per well for cell

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

10μL of the MTT labeling reagent added to 100 µL of medium alone.

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

of Various diseases and health impairment. Consequently, interest in antioxidant rich

foods has been recently increasing. Shimamura et al. improved the 2,2-diphenyl-1-

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
picrylhydrazyl (DPPH) assay, a procedure for evaluating antioxidant Capacities, and

reported it may be applicable as a standard method to evaluate the antioxidant capacity

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

for the simultaneous analysis of multiple samples

Preparation of DPPH working solution

1. Add approximately 1 mL of ethanol to a tube of DPPH Reagent and sonicate for

60 seconds.

2. Transfer all of the solution prepared in step 1 to a 10 mL measuring flask.

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

2 and 3 until no color is observed.

* Please use sonication to dissolve the DPPH reagent because it is difficult to

dissolve.

* Any undissolved DPPH may impact the assay. Please make sure that all of the

DPPH is dissolved.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
* If air remains in the hollow of the tube it is hard to dissolve the DPPH Reagent

completely by sonication. Please remove the air-by. sinking the tube of -the

DPPH Reagent diagonally into a sonicator.

5. Make up to a final volume of 10 mL with ethanol.

*Prepare the DPPH working solution fresh each day.

Preparation of the Trolox Standard solution

1. Add approximately 1 mL of ethanol to the Trolox Standard tube and completely

dissolve the contents by vortexing or sonication.

2. Transfer all Of the solution prepared in step 1 to a 10 mL measuring flask and

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

fresh each day.

Determination of the optimum concentration range

1. Prepare four or more concentrations for each sample with a 10-fold dilution from

the highest concentration

2. Add 20 uL of each sample prepared in step 1 to the appropriate wells.

3. Add 20 of solvent used for sample extraction or dilution to the wells of Blank 1

and Blank 2

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
*to avoid any concentration change because of volatilization, move to step

4 immediately.

4. Add 80 uL of Assay Buffer to each well.

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

and mix well by pipetting.

*Because the reaction starts immediately after the addition of the DPPH

working solutions, it should be dispensed with a multichannel pipette to minimize

any time lag in pipetting.

*DPPH working solution is also used for the antioxidant capacity assays

described later. To avoid any concentration change because of volatilization,

dispense only the required volume of DPPH working solution into a reservoir.

7. Incubate the microplate at 25C for 30 min in the dark

8. Measure the absorbance at 517 nm using a microplate reader.

*If the 517 nm filter is not available measure the absorbance at 500 nm or

greater (as close as possible to 517 nm.

9. Calculate the inhibition ratio of the samples from the following equation:

Inhibition ratio of sample (%) = (Ace - As)/Acs * 100

ACS. Blank 1 •Blank 2

As. Absorbance of samples - Blank 2 or sample blank (in the case where the

sample is highly colored)

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
10. Plot the inhibition ratio (Y) against the sample concentration (x) and draw a

regression line (y=ax+b).

11. Determine the optimum concentration range encompassing the 50% scavenging

concentration for DPPH-radicals from the regression line drawn in step 10.

(1) Measurement of the DPPH-radical scavenging ratio of Trolox and unknown

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.

2. Add 20 ul of the sample solution at four or more concentrations (using the

optimum concentration range that was determined from a preliminary

experiment), 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.

* To avoid any concentration change because of volatilization, move to step 4

immediately.

4. Add 80 uL of Assay Buffer to each well.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
5. Add 100 uL of ethanol to the wells of Blank 2 and Blank 3 and mix the wells by

pipetting.

6. Add 100 uL of DPPH working solution to the wells of Trolox, samples and

Blank 1, and mix well by pipetting.

7. Incubate the microplate at 25C for 30 minutes in the dark.

8. Measure the absorbance at 517 nm with a microplate reader.

* If a 517 nm filter is not available, measure the absorbance at 500 nm or greater


(as close as possible to 517 nm).

9. Calculate the inhibition ratio of the samples from the following equation:

Inhibition ratio of Trolox (%) = (Ac -AR)/Ac x 100

Ac: Absorbance of 0 pg/mL Trolox Standard solution - Blank 3

AR: Absorbance of 40 to 80 pg/mL Trolox Standard solution - Blank 3

Inhibition ratio of sample (%) = (ACS - As)/Acs x 100

Acs: Blank 1- Blank 2

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).

(2) Calculation of the Trolox equivalent antioxidant capacity (TEAC)

1. Calculation TEAC of the from following equation:

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
TEAC = (Trolox)/ IC50(sample)

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.

Statistical Data Analysis

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).

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
RESEARCH BUDGET
Item Amount Price

Reagents and chemicals

Diethynitrosamine 25 ml = Php 8,750


OVEO 20 ml = Php 2,500

Estimated Total:

Php 10,000

Organisms

Mus musculus 60 organisms = Php 7,200

Estimated Total: Php 7,200

Laboratory Tests

MTT Assay kit = Php 2,250


DPPH Assay Kit = Php 2,750

Estimated Total: Php 5,000

Transportation

Muntinlupa City = Php 150


Getting the necessary things for = Php 200
experimentation

Our Lady of Fatima University


Valenzuela Campus = Php 100

Estimated Total: Php 450

Others

Printing of the Manuscript = Php 100


Capsule & Folder = Php 350

Estimated Total: Php 450

GRAND TOTAL Php 23,100

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …

GANNT CHART

Activities JULY AUG SEP OCT NOV DEC JAN FEB MAR

Finalsubmission of
Research proposal

Proposal Defense

Approval of Research
topic

Meeting with adviser

Buying of research
equipments

Collection of the plant

Experimentation

Data collection

Data analysis

Data interpretation

Research report writing

Series of revisions

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …

Curriculum Vitae

John David D. Lacanlale

390 San Vicente, Macabebe, Pampana

[email protected]

+63 935 611 0279

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.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …

Lyka P. Mendoza

15 Verde street Lapnit, San Ildefonso, Bulacan

09276526072

[email protected]

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

she achieved various awards at academic, sports and public speaking.

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CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ORIGANUM …
BIBLIOGRAPHY

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