The Effectiveness of Virgin Cocos nucifera Oil and Artemisia dracunculus Oil

as Supposed Antibacterial Agents against

Staphylococcus aureus 

By

Xavier John P. Esperanza

Allyssa Beatrice G. Meer

Shem Jazeel A. Angeles

Aaron Vince M. Deunida

A Capstone Research Project Submitted to the
Senior High School Department
Colegio San Agustin
City of Biñan

In Partial Fulfillment of the Requirements

For Senior High School
Science, Technology, Engineering, and Mathematics Strand
March 2018
ABSTRACT
Acne, also known as Acne Vulgaris, a skin infection caused by abnormal activity of the

sebaceous gland found at the bottom of the hair follicles. Thus, this study expounds the supposed

1
antimicrobial activity of coconut oil and tarragon oil on Staphylococcus aureus since the said

bacterium causes the stated skin complication, having the two oil extracts as primary home

remedies.

Cocos nucifera oil or coconut oil, when applied to skin allows microbes to convert the

acid Monocaprin and Monolaurin which replaces the acid layer that is usually removed through

regular washing and wiping. Artemisia dracunculus oil or tarragon oil is also an acne remedy.

The distillation process of tarragon oil allows the accumulation of steamed leaves residue which

is an anti- oxidant that clears skin impurities.

Cocos nucifera was acquired from a local market while Artemisia dracunculus was

acquired from a local landscape shop located in Binan., Laguna. Cocos nucifera oil was acquired

through the boiling method. The Artemisia dracunculus oil extract was obtained through rotary

evaporation using 500mL of Hexane as an initial amount of the said extraction solvent.

Based on the research conducted , the virgin coconut oil extract inhibits the growth of S.

aureus, negating the conclusion of a verified literature that it had no Minimum Inhibitory

Concentration (MIC) against S. aureus. On the other hand, the MIC of Tarragon oil again S.

aureus is greater than its already concluded MIC against the stated bacterium according to an

established literature. Therefore, Virgin coconut oil is a more effective antibacterial agent against

S. aureus compared to Tarragon oil.

CHAPTER 1

INTRODUCTION

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Background Information

Acne Vulgaris, generally distinguished as Acne, is a skin infection instigated by the

abnormal activity of the sebaceous glands found at the bottom of a cluster of hair follicles

(Nordqvist, 2015). Sebum, an oily substance that is also a factor in obstructing the skin from

drying out, is produced by the said glands (Oakley, 2014). Aberrantly large amount of sebum

which develops a plug with dead skin cells and impedes the hair follicles is a major component

that characterizes a person with acne (Aslam et al., 2015). The hormonal alteration during

adolescent teenage year is the principal reason of acne (James, 2005). Levels of circulating

androgen hormones undergo a significant upsurge during puberty which radically steers to

increased sebum amount and severer abundance of skin cells (Youn et al., 2005).

Despite the fact that drugs operative through ingestion are recommended by doctors to

neutralize acne, painful irritation of skin including blistering, burning, clustering, severe redness,

itching or swelling and skin rash are the proven side effects of the said products (Ogbru, 2016).

On average, the drug prices have quintupled since 2009 according to Rosenburg’s study

of dermatology drug prices published in JAMA Dermatology. Tazorac which costs Php6,278.75,

Noritate which charges Php2,511.5, MimyX which requires Php2,009.2, Pandel which amounts

to Php1,758.05 and Duac at Php502.3 are only some of the numerous acne drug being retailed at

the market (Pollack, 2015).

There is a substantial evidence which a possible pathogenetic role for Staphylococcus

aureus (S. aureus) in acne vulgaris. Staphylococcus aureus bacteria, is the most general root of

the complication known as folliculitis where small white-headed pimples grop when hair

3
follicles are infected with bacteria. Folliculitis is caused by S. aureus is marked by itchy, white,

pus-filled bumps, known as pimples.

As an alternative which is also the source of potent microbial agents namely Capric Acid

and Lauric found in breast milk which keep newborn babies protected from infections, virgin

Cocos nucifera oil or coconut oil, when applied on skin, allows microbes to convert the said

acids into Monocaprin and Monolaurin which assist in replacing the protective acid layer on the

skin usually removed through regular washing and wiping of teenagers. Fundamentally, the

nonexistence of microbial infection prevents acne development (“Coconut Oil for Acne,” n.d.).

Aside from coconut oil, Artemisia dracunculus oil or tarragon oil can also be an efficient

acne remedy. The distillation process can lead to the accumulation of steamed tarragon leaves’

residue which is an antioxidant that clears skin impurities. Potassium in tarragon facilitates

bacterial extermination (“8 Amazing Benefits Of Tarragon”, n.d.). Tarragon contains terpinen-4-

ol ( Pandey & Singh, 2017). Terpinen-4-ol eliminates acne bacteria by inhibiting bacterial

strains including Staphylococcus aureus and Propionibacterium acnes (Wolfstein, 2015).

Objectives of the study

The study aims to determine the possible antibacterial activities of virgin Cocos nucifera

oil and Artemisia dracunculus oil against Staphylococcus aureus . Specifically, the study aims

to:

1. examine the supposed efficacy of virgin Cocos nucifera oil as an antibacterial agent

against Staphylococcus aureus  by determining its minimum inhibitory concentration

(MIC) and comparing it to its confirmed MIC according to an established literature ;

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 2. examine the supposed efficacy of Artemisia dracunculus oil as an antibacterial agent

against Staphylococcus aureus  by determining its minimum inhibitory concentration

(MIC) and comparing it to its confirmed MIC according to an established literature; and

3. determine which between the extra-virgin coconut oil and the tarragon oil has the smaller

minimum inhibitory concentration (MIC)

Significance of the study

The only Specialty Society in Dermatology recognized by the Philippine Medical

Association and the Philippine College of Physicians teenagers from 10-18 years old are the

persons commonly affected by acne (“The Philippine Dermatological Society”, n.d.).

A cleanser, toner and a non-prescription medicated lotion enclosed by a fixed acne

treatment cost from Php1,440 to Php2,880 per month. Prescription antibiotics, topical creams,

ointments and hormone therapy collectively costs from Php2,260.35 to Php10,046 per month.

Facials or chemical peels typically cost from Php 3,757.25 to Php10,046 per session (“Acne

Treatments Cost”, n.d.).

Despite the said drug prescriptions clinically proven to counteract acne situations

reviewed to relieve the ailments of such sufferers, there are consequences represented by side

effects namely joint pain, back pain, feeling dizzy, drowsy or nervous, dryness of lips, nose, skin

or mouth, cracking or peeling skin, itching , rash , fingernails or toenails alteration. Isotretinoin,

as most usually recommended by doctors, brings a host of unsafe side effects from clinical

depression, dry skin, hair loss, birth defects and even erectile dysfunction (“Accutane”, n.d.).

5
Additionally, the costs concomitant with the treatment of acne mostly occupied by acne drug-

acquisitions exceed $3 billion (“Acne”, n.d.).

Since the instances wherein acne-treating drugs bear adverse outcomes are evident in

spite of being costly, a low-cost, safe and effective acne treatment must be distinguished and

established not only focusing on the fiscal aspect of being economical but also concentrating on

a harmless way of remedying and eliminating the said skin illness for the benefit of many.

Home therapies are the paramount resolution contradicting the deleterious and expensive

nature of customary acne drugs. Brimming with medium fatty acids like like lauric acid and

caprylic acid working to fight the underlying causes of acne such as candida overgrowth,

autoimmunity and inflammation, coconut oil will provide antibacterial, antiviral and

antifungal agents in the body, contradicting the nature of lavish acne drugs with almost the

similar functions (Enig, 1996).

On the other hand, tarragon can be used to remove damaged skin cells as an

antiseptic. Its 1,8-cineole property may serve multiple therapeutic purposes such as anti-

inflammatory, antibacterial, airborne antimicrobial and antioxidant (“1,8-Cineole”,n.d.) Hence,

the study will provide a substantial underpinning to produce actual results generated by

gradual experimentations that may shrink the number of acne sufferers and back the said

individuals with skin anomalies-medications coupled with significant diminishment to

treatment expenses due to the context of home remedies.

6
Scope and limitations
Only two home remedies namely coconut oil and tarragon oil were examined through

different trials to scrutinize the said substances’ reaction to separate samples of

Staphylococcus aureus.

The minimum inhibitory concentrations of coconut oil and tarragon oil against

Staphylococcus aureus were recorded. Concentrations of coconut oil and tarragon oil varying in

terms of milligrams per ml were released on the wells of a 96-well plate which already contained

50µl.

The responses of Staphylococcus aureus to the two oil samples were observed. The

corresponding MIC of the two oil samples as displayed by the selected wells of the 96-well

plates were compared to their respective MIC according to published literatures.

The study was only regulated with two primary medicinal subjects which were evaluated

based on successive analyses and surveillance on the bacteria that was supposed to be destroyed.

No common clinical prescription was used as a final point of contrast in settling valid likeness

and divergence among the two home remedies and a proven medical treatment agent. The tools

to be used for the series of experimentations were limited by the school laboratory’s capacity.

CHAPTER 2

REVIEW OF RELATED LITERATURE

Nomenclature of S. aureus

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 Alexander Ogston (1844-1929) was a Scottish surgeon who determined the main

source of pus in 1880. Ogston became rational about the significance of antisepsis

supported by Joseph Lister (1827-1912) after reviewing the high rate of post-operative

mortality and death as a possible aftermath of surgery. Ogston discarded the modern

philosophy denoting suppuration as a mandatory phase in wound treatments and adopted

the antiseptic techniques of Lister, provided with the lack of inflammation in the injuries of

Lister's post-operative patients (“The Discovery and Naming of Staphylococcus aureus “,

n.d.).

Conversely, it was Ogston who verfied the account. Lister supposed that air was the

cause of putrefaction. When applying carbolic acid (phenol) dressings to surgical wounds, air

was eliminated, thus preventing suppuration, a type of putrefaction. For Ogston, however, this

explanation was inadequate. Ogston opened the abscess of a patient, made a stained smear of the

pus and used a microscope to inspect it (Elek, 1959).

Ogston conjectured that the micrococci tiggered acute abscesses. Ogston

demonstrated the formation of new abscesses, followed by septicemia inidcations after

injecting pus into guinea pigs and mice from acute abscesses. Ogston noticed the

micrococci by studying the blood of the septic animals. Abscess formation would be averted if

Obston had pretreated the pus with carbolic acid or heat. Moreover, Ogston devised a micrococci

culture scheme by inoculating hens' eggs with pus and incubating them. The substances of

the infected eggs were observed to have comparable pyogenic activity to that of the original

pus (Ogston , 1880, 1881).

The pus in chains had already been specified Streptococci by Billroth in 1874

although Ogston was not the earliest to examine the pus microscopically and designate

8
micrococci. Ogston called the the cluster micrococci “"staphylococci” in 1882, from the

Greek “staphyle” which refers to a bunch of grapes (Rosenbach, 1884).

Anton J. Rosenbach (1842-1923), a German surgeon, isolated two strains of

staphylococci in 1884. The two strains were termed according to the pigmented manifestation of

their colonies: Staphylococcus aureus. The term was derived from the Latin aurum for gold, and

Staphylococcus albus, which is now called epidermidis, from the Latin albus for white

(Rosenbach, 1884).

Morphological Attributes

Staphylococcus aureus on Columbia agar with 5% defibrinated sheep blood will display

distinct colonies that are round, convex, and 1-4 mm in diameter with a sharp boundary. Clear

beta-hemolysis zones recurrently bound colonies of Staphylococcus aureus on blood agar plates.

The etymological origin of the bacteria's name; aureus meaning "golden" in Latin, is the golden
(“Staphylococcus aureus”,n.d.).
appearance of colonies of various strains. Only weak or nonexistent beta-hemolysis and special

cultivation media with oxacillin, mannitol and sodium chloride frequently characterize

methicillin-resistant strains of Staphylococcus aureus (i.e. MRSA) due to isolation utilization.

MRSA growth and colony production of certain color depending on the pH indicator used are

possible on the said media

Acne

There is still is a deficiency of recognized evidence reinforcing that P. acnes causes acne

despite the fact that the said bacterium was first isolated from acne vulgaris. This is primarily

9
caused by the lack of good models of acne and the fact that P. acnes is present on all persons as a

part of the normal flora (Unna, 1979).

Different models have been assessed and some efforts have been done to mimic the

pathogenesis of acne (De Young et al., 1984; Holland et al., 2008; Nakatsuji et al., 2009;

Nakatsuji et al., 2008). The complex nature of the disease attributes the difficulty of addressing

if P. acnes is involved in the progress of acne vulgaris. The reader is recommended three reviews

for further reading of this topic by (Bojar & Holland 2004; Toyoda & Morohashi, 2001;

Dessinioti & Katsambas, 2010).

The relationship between acne vulgaris and P. acnes is not distrusted even though the

contribution of P. acnes to acne vulgaris is queried. P. acnes was the most enzymatically active

and also the most recurrently isolated Propionibacteria from acne vulgaris (Höffler , 1977) but

also that P. granulosum only was isolated from patients with acne (Gehse et al., 1983).

Staphylococcus and Malassezia are also frequently isolated, depicting the fact that

Propionibacteria are not the only bacteria found in acne lesions (Leeming et al., 1988).

Moreover, Bek-Thomsen et al. discovered that only P. acnes colonize all follicles from normal

skin, while S. epidermidis and a few other bacteria infect follicles from patients with acne (Bek-

Thomsen et al., 2008).

Additionally, Höffler et al. exhibited that the secretion of unalike enzymes contrasted

between isolates from patients with normal skin and with acne, with the first producing more

sialidases (Höffler et al., 1981) and more DNAse and lecithinase, even though less proteolytic

enzymes were produced by these isolates (Höffler et al., 1985).

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 Several papers explain the contribution of P. acnes in the growth of acne vulgaris

(Webster, 2002) and putative virulence factors (Farrar et al., 2000) that are supposed to facilitate

the inflammatory reaction. It has however recently been proven that severe acne is associated to

some clones of P. acnes, while other clones are connected to the normal flora (Lomholt & Kilian,

2010).

Types of Acne

Acne is segmented to different types discerned by appearances and growth processes.

Bacteria, dead skin cells and oil trigger pore blockages and diminutive bumps identified as

blackheads or whiteheads. A blackhead occurs when a blocked pore stays open whereas a

whitehead results from the closing of a blocked pore leading to whitish bump top. Papules are

denoted by the phase beyond whiteheads where the manifestation of sebum, bacteria and dead

skin cells beneath the skin have initiated inflammation Swelling, redness and the absence of pus,

a viscous yellowish or greenish opaque liquid produced in infected tissue indicate the existence

of papules (Cole, 2015).

Pustules, on the other hand, are analogous to papules but pustules are typified by

containing pus. Severe types of acne are signified by nodules and cysts. Firm acne lesions lodged

deep within the skin refer to nodules while cysts are considered as the softer form of nodules due

to confined pus (“Acne Treatments,”n.d.).

Mild, moderate or severe type of acne are the bases of acne medication (Dawson &

Dellavalle, 2013). Amalgamation of treatments intended for the most effectual results is

sometimes prescribed by a physician to preclude acquiring of drug-resistant bacteria (“Acne

Treatment-Overview,” n.d.).

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 Means of alleviating mild acne including whiteheads and blackheads may involve gentle

cleansing with lukewarm water and a soap such as Cetaphil designed for dry, sensitive skin,

leaving skin feeling soft, smooth and hydrated. Applying of benzoyl peroxide, an organic

compound that eradicates bacteria, reduces irritation and congested blocked pores used as

ingredients in Brevoxyl or Triaz and utilizing salicylic acid, a monohydroxybenzoic acid aimed

for skin and plantar warts treatment are the other modes of resolving mild acne (“Acne

Treatment-Overview,” n.d.).

Based on the researchers’ observations, atypical reactions among some of the P. acnes strains

with the antibodies used were evident. Conversely, it was shown and proven through the recA

sequencing that they belonged to type I, and they were later determined to be parts of a seperate

type, IB (Valanne et al., 2005). Recently, a fourth P. acnes type, type III was discovered

(McDowell, 2008).

Since the P. acnes strains grow cells with long filaments that aggregates rather than

forming single coryneformed bacteria, they are morphologically diverse from other known P.

acnes types (McDowell, 2008). However, there is still minor data on the medical relevance of the

different types even though the stated types are well known (Brüggemann, 2010).

S. aureus
83insuccessive
Acne Pathogenesis
agreeing patients with clinically diagnosed acne who were regularly

undergoing treatment at the University of Pennsylvania, Philadelphia, were recruited. A visual

examination was executed to analyze acne presence and severity; all patients had oropharyngeal

and nasal swab cultures performed for S aureus. The wide-ranging survey form assessed any past

skin or soft-tissue infection attributed to S. aureus, indication and duration of any antibiotic use,

12
current or recent antibiotic use, history of acne, history of acne treatment, age, sex and race

(Fanelli et al., 2011).

Specimens were acquired with a pre-moistened, sterile, flocked, polyester, fiber-tipped

swab. The swab was rotated five times in each nostril, lightly scraping each quadrant of the nasal

mucosa to take samples from the anterior nares. A different swab was used to obtain a separate

set of samples from the oropharynx by swabbing each tonsil, as well as the posterior pharynx. A

transport solution was used to handle the specimens. A homogeneous suspension was produced

by when the swab in transport was vortexed (Fanelli et al., 2011).

A selective and differential S. aureus plate was used for transferring 100 µL of the

suspension and a medium for the detection of  methicillin-resistant S. aureus (MRSA) was used

for the transfer of another 100 µL of the similar suspension. A temperature of 37°C for up to 48

hours was used to incubate the plates which were initially streaked. The plates examined for the

presence of S. aureus and MRSA organisms after the incubation process (Fanelli et al., 2011).

Susceptibility testing was accomplished using disk diffusion testing with minocycline,

doxycycline, ciprofloxacin, trimethoprim-sulfamethoxazole, erythromycin and clindamycin for

S. aureus-positive samples. Detection of inducible clindamycin resistance was performed,

preceded by the usage of standard methods for disk diffusion susceptibility for antimicrobial

susceptibility testing (Fanelli et al., 2011).

The survey forms linked to the patient reports and chart documentations of at least one

month of current topical or antibiotic practice (eg, tetracyclines, erythromycin, clindamycin)

intended for acne treatment were used to define the primary exposure. The confirmed

confounding variables referred to assessed separate exposures (Fanelli et al., 2011).

13
 S. aureus colonization was determined in a total of 36 of the 83 participants (43%).

Methicillin-resistant S. aureus (MRSA) was detected in two of the 36 patients (6%); 20 (56%)

had their throat as the sole site of S. aureus; 9 (25%) had their nose as the sole site of S. aureus;

and 7 (19%) had both their nose and throat as the sites of S. aureus. The prevalence odds ratio

for the colonization of S aureus was 0.16 (95% confidence interval [CI], 0.08–1.37) after 1 to 2

months of exposure and augmented to 0.52 (95% CI, 0.12–2.17) after 2 months of exposure

(P =.31) when antibiotic-using patients with acne were compared with nonusers (Fanelli et al.,

2011).

Resistance to treatment with clindamycin and erythromycin (40% and 44%, respectively),

was displayed by many of the S. aureus plates, principally the nasal isolates. Resistance rates (<10%)

to treatment with tetracycline antibiotics were exhibited by a small number of plates. An extended

antibiotic use from the tetracycline class which is a common acne treatment reduced the

prevalence of S. aureus colonization; resistance to the tetracycline antibiotics was not heightened

(Fanelli et al., 2011).

Antibiotic Utilization

Medical practices suggest that Staphylococcal infections are ordinary but considerable

clinical problems. Resistance to penicillin is displayed by most strains of S. aureus. On the other

hand, communal emergence is being indicated by methicillin-resistant strains of S. aureus

(MRSA) that are common in hospitals (Rayner & Munckhof, 2005).

The management of serious methicillin-susceptible S. aureus (MSSA) infections still

prefer penicillinase-resistant penicillins (flucloxacillin, dicloxacillin) as its antibiotics.

Conversely, less serious MSSA infections such as skin and soft tissue infections or in patients

14
with penicillin hypersensitivity consider first generation cephalosporins (cefazolin, cephalothin

and cephalexin), clindamycin, lincomycin and erythromycin to have vital therapeutic roles,

although patients with immediate penicillin hypersensitivity (urticaria, angioedema,

bronchospasm or anaphylaxis) are characterized by cephalosporins contra-indication (Rayner &

Munckhof, 2005).

Parenteral vancomycin must be used to treat all serious MRSA infections. However, if

the patient is vancomycin allergic, treatment with teicoplanin must be employed. Treatment with

a combination of two oral antimicrobials, typically rifampicin and fusidic acid, must be used for

nosocomial strains of MRSA are typically multi-resistant (mrMRSA), and mrMRSA, since

utilization as individual agents will result to rapid resistance development (Rayner & Munckhof,

2005).

The preferred antibiotics for less serious nmMRSA infections such as skin and soft tissue

infections are lincosamides (clindamycin, lincomycin) or cotrimoxazole. Australia and New

Zealand are the sites of most community-acquired strains of MRSA which are non multiresistant

(nmMRSA) (Rayner & Munckhof, 2005).

Good antistaphylococcal activity distinguishes new antibiotics such as linezolid and

quinupristin/dalfopristin. However, the said antibiotics are also typified by high costs. Patients

with highly resistant strains and failure or intolerance to standard therapy must receive antibiotic

reservations (Rayner & Munckhof, 2005).

Virgin Coconut oil as a remedy

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 Coconut oil, also known as copra oil, is a comestible oil obtained from the kernel or meat

of mature coconuts harvested from the coconut palm (Cocos nucifera). It is slow to oxidize

because of its high saturated fat content. Thus, it is resistant to rancidification which is the

process involving a product gradually having an unpleasant smell or taste. It can last up to six

months at 24 °C (75 °F) (Yong, 2016).

Acne breakouts are fundamentally infections in the skin pores, the identical way a wound

can become disease-ridden when bacteria is introduced. Lauric acid which has natural

antibacterial properties is contained by coconut oil. Anti-microbial, anti-fungal, and anti-

inflammatory properties were proven comprise the said fatty acid. Careful treading must be

performed when executing the stated method since coconut is assessed at a 4-distinction on the

comedogenicity scale which measures the pore-clogging activity of an ingredient, rating 0-5 with

5 being the description for the most pore-clogging component (Yong, 2016).

Not all skin types are positively responsive to coconut oil’s reviewed effects after

application, specifically when talking about the skin on an individual’s face which is the most

expected to experience a breakout despite the fact that people with characterized by very dry or

very oily skin may be the best beneficiaries (Yong, 2016).

Tending to aggressively use astringent products to combat acne-prone skin can have the

adverse effect of exacerbating acne even more resulting from the stimulation of the skin to

compensate by producing more oil after stripping the skin of its natural oils. Coconut oil

nourishes the user’s skin with vitamin E, omega-3 fatty acids, and lauric acid and bind with all

the excess sebum and lipid-soluble surface dirt which is able to be dislodged and lifted with oil,

eliminating impurities from the pores. (Yong, 2016).

16
 Massaging a slight amount of coconut oil using the hands to liquefy it if it’s in its solid

balm state and massaging onto the face are the best prime means. Following with a gentle

cleanser such as Cetaphil Gentle Skin Cleanser and Glossier’s new Milky Jelly Cleanser that will

facilitate oil removal without over-stripping the skin is the next phase (Yong, 2016).

A patch test is recommended for coconut oil-treatment novices. Coconut oil may not be

applied for speedy acne treatment but the said ingredient is a great acne medication-alternative

and moisturizer (Yong, 2016).

Tarragon oil as a remedy

Antioxidant capability forms a connection with antibacterial activity as well as the total

phenolic content of herb extracts or essential oils (Sharafati-Chaleshtori et al., 2011; Albayrak et

al., 2010). Antioxidant and antimicrobial activities were exhibited by oxygenated monoterpenes

(Ayoughi et al., 2011). Antioxidant activity and linoleic acid oxidation inhibition less than

butylated hydroxytoluene were displayed by Tarragon oil due to high level of methyl chavicols

as represented by the total phenolic content and oxygenated monoterpenes. Antioxidant activities

were also displayed by other Tarragon compounds like limonene, linalool, eugenol and

spathulenol (Ayoughi et al., 2011; Kirchner et al., 2010).

Diverse degrees of growth inhibitory effect on tested bacterial strains were displayed by

the essential oil. The highest level of sensitivity was exhibited by the Gram negative bacteria

namely, S. marcescens and Sh. Dysenteriae while the highest level of resistance was exhibited by

Gram positive bacteria namely, L. monocytogenes and S. aureus. The antibacterial activity of

Tarragon oil against E. coli and S. aureus was already reported by prior studies (Bonyadian &

Karim, 2002). Tarragon oil also inhibits the growth of Salmonella typhimurium, L.

17
monocytogenes, Yersinia enterocolitica and Bacillus cereus (Bonyadian & Moshtaghi, 2008).

The results showed the higher degree of sensitivity of E. coli and Y. enterocolitica compared to

that of other bacteria. S. aureus had a higher recorded sensitivity than E. coli to Tarragon oil

(Raeisi et al., 2012). Conversely, the restricted diffusion of the hydrophobic compounds through

the hydrophilic cell wall structure leads to a higher level of resistance is exhibited by Gram

negative bacteria than Gram positives (Amensour et al., 2010).

18
 CHAPTER 3

METHODOLOGY

1.0 Acquisition of Staphylococcus aureus 

The bacteria contained in a 42.82g petri dish bottom was provided by Colegio San

Agustin-Binan - Senior High School Department’s Laboratory.

2.0 Obtaining of the Independent Variables

Virgin Coconut oil and Tarragon oil were extracted using two different methods namely,

boiling method and rotary evaporation, respectively.

2.1 Coconut Oil Extraction

Three full-sized coconuts separately weighing 1.41 kg (3.11 lbs), 1. 44 kg

(3.17 lbs), and 1.43 kg (3.15 lbs) were bought at Carmona Public Market. Three separate

0.1692kg (0.373 lbs), 0.1728 kg (0.381 lbs), and 0.1716kg (0.378 lbs) shells were

obtained from the said coconuts using a machete (“How to Make Virgin Coconut

Oil” , n.d.).

Three separate collections of coconut meat were obtained from the shells,

weighing 0.145kg (0.32 lbs), 0.167kg (0.368lbs) and 0.159kg (0.351 lbs), respectively. All

of the gathered coconut meats were cut into small fragments using a knife. The said small

fragments were divided into three groups since the blender cannot grind all of the

meats in one blending phase. The groups contained coconut meat fragments weighing

19
 0.150kg (0.331 lbs), 0.150kg (0.331 lbs) and 0.171kg (0.38 lbs), respectively

(“How to Make Virgin Coconut Oil” , n.d.).

946. 352 ml of water was heated in a saucepan which was placed on top of a

burner. The burner was turned to medium-high at a temperature of 330 °C. The first

group of coconut meat was grinded together with approximately 300ml of heated water

using a 650W blender. The lid of the blender was held in place while the blending

process took approximately 30 seconds for the two variables to become a uniform mixture. A

sieve was used to separate the coconut milk from the coconut chaff. The coconut chaff was

pressed hardly using the hands to accumulate all its liquid content. The second and third

groups of coconut meat fragments followed the similar procedure (“How to Make Virgin

Coconut Oil”, n.d.).

All of the collected liquid content was poured into a tray and kept in a freezer

for 48 hours. After 48 hours, it was observed that the oil has risen as denoted by a

white solid layer floating on top of the water. The white solid layer was taken from the tray

and was put into a frying pan for heating, starting from low (275 °C) to medium (300

°C) heat. The said layer was initially measured at 451.5g equivalent to 500mL. After 45

more minutes, the liquid substance greatly diminished in volume, indicating the presence of

pure oil containing charred solid coconut bits (“How to Make Virgin Coconut Oil”,

n.d.).

2.1 Tarragon Oil Extraction

10 Tarragon plants were acquired from a local landscape shop located along the

streets of Silmer Village, Biñan, Laguna. 100 g of leaves were gathered from 10

20
Tarragon plants. The leaves were put inside a 500 Erlenmeyer flask. 500mL of Hexane

was measured by transferring the said chemical compound into a 250mL graduated cylinder

twice for accuracy. Only 470mL of Hexane was poured into the 500mL Erlenmeyer flask

containing the Tarragon leaves since the said flask could not anymore contain more volume.

The mixture was left still for 28 hours (Subramaniam, 2015).

The liquid residue was separated from the leaves using a glass funnel with a filter

paper. The said residue was transferred into a 400mL beaker. 420mL of liquid residue,

weighing 275, 100mg , was accumulated since approximately 50mL was absorbed by

the leaves. The liquid residue was processed in the rotary evaporator for 5 hours

(Karayannakidis, 2015).

3.0 Broth Microdilution Test

The susceptibility of bacteria to multiple antibiotics can be assessed at once by means of

the broth microdilution method (Lee, 2013). Broth microdilution is substantially accurate. Agar

dilution, which is the gold standard susceptibility testing, is analogous to the accuracy of broth

microdilution’s results (Engelkirk & Duben-Engelkirk, 2008).

3.1 Preparation of Broth Medium

.  2.1 grams of Müeller-Hinton broth was suspended in 100mL of distilled

water. Frequent agitation was applied when the suspension was being mixed

and dissolved by heating on a hot plate. The suspension was boiled for

one minute until complete dissolution. The solution was dispensed into a

500mL Erlenmeyer flask. The said solution was sterilized in autoclave at 121°C

for 15 minutes (“Müeller-Hinton Broth”, n.d.).

21
 3.3 Dissolution of Oil Extracts
100mg of virgin coconut oil and 100mg of Tarragon oil were separately

dissolved in 2mL of dimethyl sulfoxide (DMSO) (Mariano & Gloriani, 2017).

3.2 Preparation of Bacterial Inoculum

The tip of a sterile cotton swab was dipped into the provided

Staphylococcus aureus bacteria placed on a 42.82g petri dish bottom.

The similar swab was dipped into the sterilized Müeller-Hinton broth.

The bacterial inoculum was first prepared in a cuvette . The cuvette was inserted to a

Vernier spectrophotometer. The bacterial inoculum was prepared at an optical density

of 0.072 at 625 nm using the said instrument. (Mariano & Gloriani, 2017).

3.3 Application

50 µl of the bacterial inoculum was released into a total of 56

wells that were contained by a 96-well plates. Virgin coconut oil and

tarragon oil was released into the first three and second three columns of the 96-

well plates, respectively, using a micropipette observed at a horizontal manner.

Each column contained 8 wells. A two-fold dilution method was performed,

reducing the concentration of the oil extracts, separately, by one half

in every well. The first horizontal set of wells of the six columns

contained 25, 000 µg/ml. The second horizontal set of wells contained 12,500 µg/ml.

The third horizontal set of wells contained 6, 250 µg/ml. The fourth horizontal set

of wells contained 3, 125 µg/ml. The fifth horizontal set of wells contained 1,563

µg/ml. The sixth horizontal set of wells contained 781.25 µg/ml. The seventh

22
 horizontal set of wells contained 391 µg/ml. The eighth set of wells contained 195

µg/ml. The seventh column of the 96-well plates consisted of four wells which

jointly refer to the positive control. All of the four wells at the said column contained

50 µl of S. aureus. The ninth column also consisted of four wells

which only contained 50 µl of the bacterial inoculum without any added oil extract or

bacteria, jointly referring to the negative

The 96-well plates were incubated at 37 °C for 39 hours and 50

minutes (“Minimum Inhibitory Concentration”, n.d.).

3.5 Measurement of the Minimum Inhibitory Concentrations (MIC)

The turbidity of the wells were examined. The minimum

inhibitory concentrations of the virgin coconut oil and Tarragon oil were

measured by determining the concentrations of the clearest wells in their respective

columns. The concentrations were added and divided by the number of columns

occupied by the separate oil extracts (Mariano & Gloriani, 2017).

4.0 Statistical Analysis

T-test was used to determine and compare the means of two sets of data referring to the

concentrations of the clearest wells of the six columns occupied by virgin coconut oil and

Tarragon oil, respectively. The said type of analysis was also used to conclude whether the

two oil extracts have a significant difference according to their respective data.

23
 CHAPTER 4

RESULTS AND DISCUSSION

1.0 Characterization and Yield of Extracted Virgin Coconut Oil

1.1Color
1.1 Color

The virgin coconut oil extracted through the use of boiling method was partially

clear and predominantly yellowish in color. High quality virgin coconut oil manufactured by

licensed producers is white in color at solid form and clear at liquid form. Inferior quality

is signified by any discoloration (“Coconut Research Center”, n.d.).

1.2 Yield of Extract

The 451.5g white layer equivalent to 500mL diminished its volume to

110mL pure virgin coconut oil after 45 minutes of heating. The coconut oil was

poured into a 220g container. The coconut oil weighed 99.33g.

Extraction method Volume before Volume before extraction Percent Yield

Table 1. Summary of the virgin coconut oil extraction procedure
extraction
Boiling 500mL 110mL 22%

2.0 Characterization and Yield of Extracted Tarragon Oil

2.1 Color

24
 After processing the liquid residue in the rotary evaporator for 5 hours. Tarragon

oil extract with a color ranging between green and yellow was produced (“Tarragon

Essential Oil”,n.d.).

2.2 Yield of Extract

420mL of the liquid residue equivalent to 275,100mg reduced its weight to only

125mg pure Tarragon oil.

Extraction method Weight before Weight before Percent yield

extraction extraction
Rotary evaporation 275,100mg 125mg 0.05%
Table 2. Summary of the Tarragon oil extraction procedure

3.0. Analysis of the Wells

Figure 1. 96-well plates after
incubation
8

7
6
5
4
3
2
1
1 2 3 4 5 6 7 8 9

25
 The blue and red frames comprise of the first six columns of the 96-well plates which

held 48 wells, each containing 50µL of bacterial inoculum and varying concentrations of the

solution of virgin coconut oil and DMSO for the first three columns, and varying concentrations

of the solution of Tarragon oil and DMSO for the second three columns. The first column had

the first well containing the clearest result, indicated by the concentration of 25,000 µg/ml. The

second column also had the first well containing the clearest result, indicated by the

concentration of 25,000 µg/ml. The third and last column occupied by the solution of virgin

coconut oil and DMSO had the third well containing the clearest result, indicated by the

concentration of 6,250 µg/ml.

The fourth column had the first well containing the clearest result, indicated by the

concentration of 25,000 µg/ml. The fifth column also had the first well containing the clearest

result, indicated by the concentration of 25,000 µg/ml. The third and last column occupied by the

solution of Tarragon oil and DMSO had all its wells being turbid, automatically assigning a

concentration of 50,000 µg/ml.

The yellow frame comprises of the seventh column which contained four wells and

referred to the positive control. The said column served as the basis of turbidity where bacterial

growth occurred. On the other hand, the purple frame comprises of the ninth column which also

contained four wells and referred to the negative control. The said column served as the basis of

clarity where bacterial growth did not occur.

4.0. Computation of the Minimum Inhibitory Concentrations (MIC)

Table 3. Components in solving for virgin coconut oil’s MIC

26
 Column Well Concentration MIC

1 1 25,000 µg/ml 0.05%

18, 750 µg/ml
2 1 25,000 µg/ml
3 3 6,250 µg/ml

Column 1 Column 2 Column 3 Sum/Dividen Number of Quotient

d
Columns
Well 1 Well 1 Well 3 Total Divisor MIC

Addend 1 Addend 2 Addend 3

25,000
Table 3. µg/ml 25,000
1 Computation 6,250 µg/ml
of virgin coconut 56, 250
oil’s MIC 3 18, 750
µg/ml
µg/ml µg/ml

Table 4. Components in solving for Tarragon oil’s MIC

Column Well Concentration MIC
4 1 25,000 µg/ml
5 1 25,000 µg/ml
6 (All wells are turbid) 50,000 µg/ml 33,333.33333 µg/ml

Table 4. 1 Computation of Tarragon oil’s MIC

Column 4 Column 5 Column 6 Sum/Dividen Number of Quotient

d
Columns
Well 1 Well 1 (All wells are Total Divisor MIC

turbid)
Addend 1 Addend 2 Addend 3
25,000 µg/ml 25,000 50, 000 100, 000 3 33,333.3333
3 µg/ml
µg/ml µg/ml µg/ml

27
 Based on the tables displayed above, the MIC of virgin coconut oil against S. aureus is
18, 750 µg/ml while the MIC of Tarragon oil against S. aureus is 33,333.33333 µg/ml.

5.0. Comparison of the Minimum Inhibitory Concentrations (MIC)

28
 Figure 1: Mean Minimum Inhibitory Concentration (in ten
thousands)
Same superscript in each graph denotes no significant difference in T-test

3.5
33,333. 33333
3 µg/µl

2.5
a
Mean MIC (µg/ml)

1.5 Virgin Coconut Oil

a Tarragon Oil

0.5

0
Category 1
Plant Extract

Based on the graph displayed above, the MIC of virgin coconut oil against S. aureus

which is 18, 750 µg/ml is lower than the MIC of Tarragon oil against S. aureus which is

33,333.33333 µg/ml.

5.0. Interpretation of the difference in the Minimum Inhibitory Concentrations (MIC)

The Smaller minimum inhibitory concentration (MIC) which is the lowest

concentration of a chemical that averts visible growth of bacterium was

determined by the clarity of the wells in every column, wherein a stronger antibiotic

corresponds to a clearer well because a lower concentration of the antibiotic is

adequate to prevent the growth.

5.0. Comparison of the Minimum Inhibitory Concentrations (MIC) to concluded MIC

based on established literatures

29
 The virgin coconut oil extract has an MIC of 18, 750 µg/ml which denies its

studied MIC of represented by NI, denoting no inhibition (Tangwatcharin & Khopaibool, 2012)

while the Tarragon oil extract’s MIC of 33,333.33333 µg/ml is higher than its proven MIC of

1250 µg/mL (Raeisi et al., 2012).

CHAPTER 5
CONCLUSION AND RECOMMENDATIONS

Acne vulgaris is the most customary malady of human skin that where

approximately to 80% of adolescents and young adults encounter in the span of their

lives (Nakatsuj et al., 2009). Acne vulgaris is characterized by a role of S. aureus in its

30
 pathogenesis, joining S. epidermidis and P. acnes as acne-causing bacteria (Hassanzadeh,

et al., 2008).

Virgin coconut oil eliminates bacteria that trigger the development of acne due to

its lauric acid content (McDonell, 2016). However, the said oil extract does not inhibit the

growth of S. aureus which causes acne (Tangwatcharin & Khopaibool, 2012). Tarragon oil, on

the other hand, has an antibacterial effect on pathogen bacteria involving S. aureus (Raeisi

et al., 2012), supporting its antioxidant property (Shruti, 2015).

Virgin Cocos nucifera oil or virgin coconut oil was extracted by the means of

boiling method while Artemisia dracunculus oil or Tarragon oil was extracted using

rotary evaporation. The plant oil extracts were tested to determine its supposed

antibacterial activity against S. aureus using the broth microdilution method.

The results revealed that both the virgin coconut oil and Tarragon oil extracts

inhibit the growth of S. aureus with the similar effects since there was no significant

difference according to the T-test statistical analysis performed.

Based on the results, the following were concluded:

1. the virgin coconut oil extract inhibits the growth of S. aureus, contradicting the claim

of an established literature that it had no MIC against the stated bacterium

2. the MIC of Tarragon oil against S. aureus is higher than its already concluded MIC

against the stated bacterium according to an established literature ; and

31
 3. the virgin coconut oil is a more effective antibacterial agent against S. aureus

compared to Tarragon oil.

Upon conducting the study, the following are recommended:

1. collect more Tarragon leaves for extraction since only 100 grams of leaves can be

accumulated from 10 Tarragon plants, resulting to only 125mg of oil;

2. use another extraction method to accumulate virgin coconut oil such as the soxhlet

extraction procedure since the boiling method used resulted to oil discoloration;

3. determine the minimum bactericidal concentrations (MBC) of the two oil extracts

against S. aureus

4. utilize more wells provided by the 96-wells plate to apply more concentrations which

will show a broader range in turbidity difference ; and

5. apply an indicator dye to the wells to compare the bacterial growth inhibiting-activities

more efficiently and determine the MIC more effectively and; and

6. perform more trials of the broth microdilution method to assure the accuracy of the

resulting data.

32
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 Appendix A
Summary of Coconut Oil Extraction
I. Cut the coconut into smaller pieces.

43
 II. Boil more than 900Ml of water

III. When the water is ready pour it into

the blender then add enough coconut

IV. When the coconut is shredded , take it

out from the blender then separate the
juice from the meat.

44
V. After getting all the juice, pour it into
a hot pan then wait until the oil is
starting to produce.

VI. Separate the oil from the coconut oil

crumbs.

45
 Appendix B
Summary of Tarragon Oil Extraction
I. Remove all the leaves from the
branch.

II. Put 500ml of Hexane into the

Erlenmeyer Flask .

III. Separate the Hexane from the leaves.

46
IV. Put it into the rotary evaporator.

Appendix C
Summary of Coconut and Tarragon Oil Dilution
I. 100mg of Tarragon Oil and Coconut
Oil were weighed

II. Dilute it to 2mL (DMSO)

Appendix D

47
 Summary of Muller Hinton Broth
I. Dissolve Mueller Hinton Broth with
distilled water then place it inside the
Autoclave machine

II. Swab a small amount of S.aureus then

dip it into the Mueller Hinton Broth.

Appendix E
Summary of Antimicrobial Testing
I. On 56 wells put 50µL of Mueller
Hinton Broth with S. aureus. On the
first 3 columns , add virgin coconut
oil. On the next 3 columns, add
Tarragon oil. The 6th column will be
the positive control while the 9th
column will be the negative control.

II. Incubate for 24 hours

Appendix F
Results of Antimicrobial Testing

48
 Coconut Oil Tarragon Leaves Oil
25,000 25,000
25,000 25,000
6,250 50,000

T-Test: Paired Two Sample for Means

  Variable 1 Variable 2
Mean 18750 33333.33333
Variance 117187500 208333333.3
Observations 3 3
Pearson Correlation -1
Hypothesized Mean Difference 0
df 2
t Stat -1
P(T<=t) one-tail 0.211324865
t Critical one-tail 2.91998558
P(T<=t) two-tail 0.422649731
t Critical two-tail 4.30265273  
NO SIGNIFICANT
DIFFERENCE

49
50