PERPETUAL HELP COLLEGE OF MANILA

124O V. CONCEPCION ST., SAMPALOC, MANILA

BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

Effectiveness of Orange (Citrus sinensis) Zest Antibacterial

Properties as an Alternative antiseptic solution to Povidone-
Iodine (PVP-I)
Kristal Kate Corpuz, Princess Daniela Majadora, Neobe Kherin Martinez, Anthony Orata,
Alma Orbita, Demi Raine Sanchez
Abstract

Wounds are often infected due to invasion of various microorganisms, which slows
down the healing process of the wounds. Several antiseptic solutions such as the Betadine
(Povidone-iodine) are being used to cleanse wounds to accelerate the wound healing
process. Although Povidone-iodine is widely used and available, some studies found that
PVP -I have corrosive properties which might damage the skin surface. Concerning this,
orange has been found to have antimicrobial bioactive compounds. This study aims to
determine the effectiveness of orange zest against microorganisms as an alternative
antiseptic wound solution to Povidone Iodine and other existing antiseptic solution. The
researchers extracted orange peels using ethanol extraction and olive oil infusion. Both
extracts underwent a Zone of inhibition test to determine the antibacterial potential of
orange peel. Findings showed that both solutions have been found to have an average of 6.8
mm bacterial inhibition against skin microorganisms. Properties of orange zest are
therefore a potential antiseptic agent for wounds due to its antibacterial properties.

Keywords: Orange zest, antiseptics, wounds, antibacterial

INTRODUCTION

Wounds are often regarded as one of the common injuries that could appear of a sudden.
Wounds can be classified into two types: Opened and Closed wounds. The Makati Medical
Center (2020) explained that open wounds cause an external or internal break in the skin’s
tissues. Encyclopedia Britannica (2019) has explained that closed wounds do not cause the skin’s
exterior tissues to be damaged and can heal faster than an open wound. Moreover, Onyekwelu et
al. (2017) added that Class 4 wounds are dirty-infected wounds that are derived from
inappropriate medical intervention. Wounds normally heal themselves, a normal biological
process in our body (Guo & Dipietro, 2010). The wound healing process is achieved in unified
and organized phases, which are homeostasis, inflammation, proliferation, and remodeling
(Gosain & Dipietro, 2004; & Mathieu et al., 2006). Mathieu et al. (2006) and Guo & DiPietro
(2010) presented the functions of the phases of wound healing: Homeostasis (Vascular
constriction, Platelet aggregation, and fibrin formation); Inflammation (neutrophil infiltration,
monocyte infiltration, and lymphocyte infiltration), Proliferation (re-epithelialization,
angiogenesis, collagen synthesis, and ECM formation), and Remodeling (collagen remodeling,
vascular maturation, and regression). Mathieu et al. (2006) added that these phases and their
functions must occur in a proper series, given in a specific time and duration to avoid improper
tissue repair. In some cases, wounds delay the normal healing process as they failed to proceed
through the different phases of healing at a specific time, often called Chronic wounds
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

(FrykbergRobert, 2015 & Nunan et al., 2014). Oftentimes, Chronic wounds would stop in the
inflammatory phase of the wound healing, which is derived from the imbalance healing process
(FrykbergRobert, 2015; Bjarnsholt et al., 2008; & Kirketerp-Møller et al., 2008), and the
presence of the bacteria influence wound healing imbalance (Kirketerp-Møller et al., 2008). The
most common bacteria that can be detected in a bacterial wound are the Pseudomonas
aeruginosa and Staphylococcus aureus, which are also found on the skin’s surfaces. Several
researchers (Colsky et al., 1998; Gjødsbøl et al., 2006; & Valencia et al., 2004) believe that the
presence of Staphylococcus aureus in wound infection. Staphylococcus aureus, a Gram-positive
bacteria (Taylor & Unakal, 2022), is considered a major bacterial virus that can cause a lot of
infections, which are normally found on the skin, most especially in the nasal area (Lowy, 1998).
Lowy (1998) added that Staphylococcus aureus might not be harmful to healthy skin, but
infectious when entered the bloodstream.

Inhibiting bacteria and other foreign materials can be done using wound cleansers and
antiseptic solutions. It is assumed that any type of wound should be treated with antimicrobial
solution and wound cleansers that help to decrease the wound infection (Bianchi, 2000). One of
the antiseptic solutions that are widely used for self-medication is Iodine. Iodine is acclaimed as
the most effective antiseptic solution for any wound infections (Atiyeh et al., 2009). Povidone-
Iodine (PVP-I) is a water-soluble iodine-releasing agent composed of complex iodine and
polyvinylpyrrolidone (Lachapelle et al., 2013 & McDonnell et al., 1999). Lachapelle et al.
(2013) and McDonnell et al. (1999) added that the small molecules of PVP-I invade bacterial
wounds and suddenly oxidize proteins, nucleotides, and fatty which cause the cell to die.
Moreover, several researchers (Hoekstra et al., 2017; Oduwole et al., 2010; Johani et al., 2018; &
Capriotti et al., 2018) have observed that PVP - I also act against biofilms created in the wounds,
both in-vitro and ex-vivo observations. Although there were no signs of side effects of Povidone-
Iodine in most clinical studies, there are rare cases of anaphylaxis and skin irritation due to
exposure to PVP-I. Velasquez et al. (2009) and Masse et al. (2006) have stated that rare cases
such as skin irritation or iododerma-like eruption could have the possibility of side effects, that
might be derived from the oxidation of iodine and hypersensitivity. In a medically-reviewed blog
article written by Kerr (2018), several reactions might be seen due to exposure to any mixtures
containing iodine such as itchy rashes, hives (urticaria), and anaphylaxis.

Aside from any laboratory and factory-produced antiseptic solutions, extracts from
plants, fruits, and vegetables are also helpful to any infectious injuries. Aminov (2010) has stated
in his study that herbal medicines are primitively used against infectious illness and are found to
be effective equally to pharmaceutical medications. Salem & Saeed (2020) emphasized that
organic medicines derived from leaves, flowers, fruits including peels, and plant stems can be
used for developing antibacterial agents, as they provide a protective barrier against bacteria.
Salem & Saeed (2020) explained various researchers have found that fruit and vegetable peels
are important constituents of pharmaceutical medicine. Salem & Saeed (2020) added that fruits
and vegetable peels are being thrown into waste instead of using them as a pharmaceutical
ingredient. Several researchers (Ferhat, 2008; & Berna et al., 2000) have pointed out that orange
waste can cause problems with water pollution, as the essential oil in orange (limonene) and
pectin might contaminate aqueous solutions. Most of the peels are found to have anti-microbial,
anti-inflammatory, and antioxidant properties. Specifically, the pulp found on orange and lemons
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

peels has antibacterial properties (Parashar et al., 2014). The orange (Citrus sinensis) is one of
the important species belonging to the genus Citrus and part of the Rutaceae Family (Ibri et al.,
2017). Orange contains important nutrients needed for health maintenance such as vitamin C and
E, carotenoids, and flavonoids (Di Majo et al., 2005).

Escobedo-Avellaneda et al. (2014) stated that oranges also have bioactive properties,
nutraceuticals, and functional compounds that can be found on peels. Moreover, citrus fruits such
as oranges and lemons have flavanones (a small group of polyphenols present in citrus fruits),
flavanol glycosides, and flavones (a class of flavonoids) which contain primary bioactive
compounds such as naringin and hesperidin (Escobedo-Avellaneda et al., 2014). Moreover,
Escobedo-Avellaneda et al. (2014) emphasized that flavonoids are known to have antioxidant,
anti-inflammatory, antimicrobial, and antiallergenic activities. Some citrus species have
antibacterial activities that could eliminate clinically significant bacterial strains. For instance, a
study conducted by Min et al. (2014), found that peel extract from Satsuma Mandarin (Citrus
unshiu) can slow down the propagation of certain bacteria such as the Bacillus cereus,
Staphylococcus aureus, and Listeria monocytogenes. In another study by Dubey et al. (2011),
Hydromethanolic extract of orange peel can act as an anti-bacterial agent from specific bacteria
such as Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermis, E. coli, Shigella
flexineri, Pseudomonas aeruginosa. Aside from the bacterial mechanisms of oranges, some
studies found some medicinal properties that can be derived from the fruit. According to Shetty
(2016) and Alabaka et al. (2006), essential oils of oranges can be used for different ailments such
as antiseptic, astringent, deodorant, disinfectant, insect repellent, immune system, and skin.
Alabama et al. (2006) added that oranges are used to inhibit vitamin deficiencies.

Problems/Gaps in the Knowledge

Throughout the review of related literature about the subject matter, the researchers have
found to have gaps relating to experimental research about the antimicrobial activities of citrus
fruits. Although there are numerous studies have been on the antibacterial properties of Oranges,
there are only limited studies about the antimicrobial properties of Orange zest in the Philippines.
In a survey made by Tan (2017) at the St. Paul University Quezon City, there were no findings of
inhibiting activities of several fruit peels such as Orange to Staphylococcus aureus, Micrococcus
luteus, and Serratia marcescens which were expected to be. These findings by Tan (2017) might
be a reference that oranges’ antibacterial compounds are ineffective, depending on the species of
citrus used and the procedures of the observation done. Contrary to this, in another study by
Penecilla & Magno (2011) from the Western Visayas State University, several Citrus species
such as Orange (Citrus sinensis) and Lemon Rind (Citrus aurantifolia) have found an active
zone of inhibition against gram-positive bacteria with a diameter of 9mm to 15 mm. There were
no scientific studies that found that the zest of oranges had been used as an antiseptic solution or
as an alternative solution to wounds. These gaps of knowledge and the scarcity of local studies
about oranges will lead to a research opportunity.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

Objectives

Behind the numerous studies about the anti-microbial activities of citrus species, the
effectiveness of the antibacterial properties of orange zest as an alternative antiseptic solution to
Povidone-Iodine has not yet been widely reported in a local setting. The objective of this
research is to gain an understanding of the study of the efficiency of treating wounds by
converting to an innovative way using the common Povidone-Iodine by substituting with an
organic compound known as the zest of an orange. The following are the main objectives of the
study: (1) To determine the inhibition potential of extract from orange peels from any common
bacteria that can be found in our surroundings, particularly microorganisms found on the skin;
(2) To examine the range of factors of using an alternative route if the utilization of orange zest
may be capable enough to potentially be of use as a replacement for Povidone-Iodine in treating
wounds and the like; (3) To determine the influence on the varying components between the two
different methods especially the application of orange zest and its competency with medical
purposes; (4) To create an organic, affordable, and low maintenance antiseptic solution; and (5)
To compare the style, quality, and accuracy of the clinical use of orange zest and to recognize the
positive and negative impacts whether improved or not that it brings on surgical outcomes and
wound healings.

Purpose/ Significance

Browsing through the related literature, the researchers have found to have limited local
studies about the antibacterial properties of oranges. These gaps in knowledge led the proponents
to the founding purpose of this study, which is to find organic and alternative solutions for
wound and injury management apart from the side effects of the existing antiseptic solution
being used, which have been reported to have adverse effects. Other than that, this study also
aims to maximize the utilization of Orange peels by determining their medical properties,
especially as a wound-healing and cleanser agent, instead of being thrown into waste. The
following are the sectors that will benefit from the results of the study: (1) Health practitioners
- The health practitioners can benefit from this study by gaining an alternative organic antiseptic
solution that they can use for their patients who have known iodine allergies or with
contraindications to iodine use; (2) Pharmaceutical manufacturers. Pharmaceutical
manufacturers can benefit from this study by gaining an innovative idea of a product that can be
launched locally in the market that can be made as a wound cleanser alternative to the usual
antiseptic solutions found in the market; (3) Consumers. Consumers can benefit from this study
by having a choice for an antiseptic product that is both organic and can be used by consumers
with known contraindications to iodine use; (4) future researchers. Future researchers can
benefit from this research project by acquiring additional knowledge that is related to this study
which they can use for their future studies. The researchers aim to broaden future researchers’
knowledge and to serve as a springboard for future researchers to discover other potentials of
orange peel.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

Scope and Limitations

Although other fruits and vegetable peels have been found to have antibacterial properties
other than the oranges, the proponents will be only focusing on the discovery of the orange peels
due to their availability in the market and bioactive compounds. The study will only undergo
limited laboratory experiments to be done at home and the production of the organic antiseptic
solution made of the extracted orange peels. The whole study including the experimentation,
production, and framing of the results will be required to be accomplished in 3 months as per the
requirement of the curriculum. With the short period, the proponents will maximize the possible
time in studying the components and activities of orange and the production of the output.
However, due to the restrictions of Covid-19 and the implementation of distance learning, the
proponents will conduct such experiments and productions to be done at home and individually,
although laboratory assessment is highly recommended to conduct this study. The availability of
apparatuses, tools, materials, and equipment might be limited and the duration of the
investigation might consume more time due to home-based experiments.

METHODS

This section will focus on the overall production of the conversion of orange zest in an
antiseptic solution. Moreover, the proponents will be focusing on the extraction of oranges zest
and will undergo experimentation to obtain its effectiveness. Apart from the methodologies to be
done throughout the study, existing antiseptic solutions will be presented and analyzed in this
section.

I. PRODUCT ANALYSIS

There is a wide variety of clinical and topical antiseptic solutions that are being used to
cleanse wounds. The antiseptics solutions that are being used are the biguanides (chlorhexidine
gluconate and polyhexanide), halophenols, bisphenols, silver compounds, hydrogen peroxide,
Alcohol, Lugol’s solution, and Povidone-Iodine. Among all of these particulars, chlorhexidine,
alcohol, and povidone-iodine are commonly used for wound cleansing, especially for emergency
use. Although these antiseptic solutions are readily available in pharmacies being sold at a
reasonable price, some consumers are most likely to utilize organic compounds from fruits and
vegetables due to their availability in the environment and they are cheaper than any antiseptics,
such as citrus fruits and leafy vegetables.

Chlorhexidine

Chlorhexidine is a biguanide antiseptic solution that is commonly used for sanitary,

wound cleaning, and dental cleansing and healing (Thomas et al., 2009 & McDonnell et at.,
1999). Chlorhexidine is produced in two solutions: a 0.05% dilution as a wound cleanser and a
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

4% solution for skin surgical preparation and disinfection (Atiyeh et al., 2009). It has been used
in the clinical setting because of its low toxicity and its ability of it to combine with other
substances for fastening to the epidermis, teeth, oral mucosa, and mucous membrane (Jackson,
2005). Such as the other topical antiseptic solutions, Chlorhexidine has the potential to kill gram-
positive bacteria (such as Staphylococcus aureus, which is commonly present in the skin and
upper respiratory tract) and is more effective than any antiseptics, as it surpasses antiseptic
preparations of povidone-iodine, triclosan, hexachlorophene, or chloroxylenol (Jackson, 2005).
Although Chlorhexidine has low toxicity, it should not be eyes, the middle ear, or the meninges
(Atiyeh et al., 2009). Chlorhexidine-based mouthwashes are also efficient for dental care. Certain
chlorhexidine mouthwashes contain ethanol (Lyon, 1994). However, it imposes several collateral
effects such as taste impairment, tooth, and mucosal staining. (Moshrefi, 2002; Flötra et al.,
1971; & Lang et al., 1988). Moreover, chlorhexidine can also impose effects such as Cancer
(Lyon, 1994), increased risk of oral & oropharyngeal cancers (McCullough & Farah, 2008 &
Cole et al., 2003), Burning mouth syndrome, dry mouth feeling, and dysgeusia (Pelaez et al.,
2004).

Alcohol

Types of alcohol such as ethyl (ethanol), isopropyl (isopropanol, propane-2-ol), and n-

propanol are known to be effective antibacterial agents and are mainly used for skin sanitizing,
disinfectant, and antiseptic solutions (Jackson, 2005; McDonnell & Russell, 1999). These
alcohols are effective in vitro bacterial activity against gram-positive and gram-negative bacteria,
including fungi, enveloped bacteria, and Mycobacterium tuberculosis. Although ethyl alcohol is
not used for regular use, too much exposure to it might cause harmful effects. According to the
New Jersey Department of Health (2016), repeated exposure of skin to alcohol (ethanol and
isopropyl) might cause dryness and cracks in the skin and is often associated with peeling,
redness, and itching. Moreover, it can impose several effects such as headache, drowsiness,
nausea, vomiting, unconsciousness, confusion, loss of coordination, and irritation (nose and
throat). It might also affect the liver and nervous system. Worst, alcohol might cause fire
accidents as it is a flammable liquid.

Povidone-Iodine (PVP-I)

Lastly, is the Povidone-Iodine (PVP-I). A type of iodophor, PVP-I is composed of a

complex of iodine and a solubilizing carrier that acts as a reservoir of free active iodine
(McDonnell & Russell, 1999). The betadine that is widely available in pharmacies has a wide
variety of antiseptic formulations such as aqueous solution (10% PVP-I), alcoholic solution
(10% PVP-I), and surgical scrub (7.5% PVP-I) (Zamora, 1986). The polyvinylpyrrolidone
component of PVP-I increases the antibacterial potentialities of the Iodine content, in which the
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

polyvinylpyrrolidone directs the Iodine to bacteria and penetrates it (Zamora, 1986; McDonnell
& Russell, 1999). Some studies report the variety of uses with the PVP-I, such as Handwashing,
hand antiseptics, and surgical scrubbing (Larson, 1994 & Raybrouck, 1986). Velasquez et al.
(2009) and Masse et al. (2006) have stated that rare cases such as skin irritation or iododerma-
like eruption could have the possibility of side effects, that might be derived from the oxidation
of iodine and hypersensitivity. In a medically-reviewed blog article written by Kerr (2018),
several reactions might be seen due to exposure to any mixtures containing iodine such as itchy
rashes, hives (urticaria), and anaphylaxis.

Orange Essential Oil

For this study, extracts from orange peels called limonene will be the primary component
for producing an alternative antiseptic solution. Essential oils are byproducts extracted from
plants and fruits that contain intricate mixtures of metabolites and derivatives that are responsible
for the distinctive scent of some plants and fruits, including citrus fruits. Essential oils also
exhibit antimicrobial activity against a broad variety of bacteria which includes antibiotic-
resistant strains and fungal organisms (Edogbanya et al., 2019). A study was conducted by the
Department of Plant Science and Biotechnology in Nigeria in 2019 with citrus essential oil
extract quantifying the microbiological activity of citrus essential oil in microbial strains such as
Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Among the varieties of
citrus fruit tested, the orange variety (Citrus sinensis) exhibited the most inhibitory effect on both
Gram-positive and Gram-negative bacteria (Frassinetti et al., 2011)

Furthermore, citrus peel essential oils extract is noted to be a rich source of bioactive
ingredients and essential micronutrients particularly vitamin C and E, carotenoids, and
flavonoids which are important to sustain a human’s physical state (Taiwo et al. 2019). For
centuries, the essential oils that are extracted from Citrus sinensis are found to possess numerous
medicinal properties and are used extensively as an antiseptic, disinfectant, insect repellant, and
immune booster among many others. It is also known to treat various illnesses such as colds, flu,
and scurvy, and aid in combating viral and bacterial infections. The efficacy of orange essential
oil is attributed mainly to its high Vitamin C content which is regarded to stimulate the body to
produce white blood cells, mainly neutrophils that fight off harmful antigens such as bacteria and
viruses (Yashaswini & Arvind, 2018).

Likewise, an article (New Directions Aromatics, 2018) listed the benefits of orange peel
essential oil such that its chemical components of Limonene which exhibit anti-oxidant and
detoxification properties; Monoterpene Hydrocarbons (β- Myrcene and α-Pinene) which aids in
the anti-inflammatory process and can be a potent analgesic, antiseptic and antibiotics;
Citronellol Geraniol and Linalool which are both believed to demonstrate an antibacterial and
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

sedative effect; Neral which has an apoptotic effect (which plays a major role in preventing
cancer cell growth). Thus, orange essential oil’s powerful antibacterial properties can be
advisable to use on cuts, wounds, and abrasions to prevent the growth and spread of bacterial
infection from occurring. On the other hand, its antiseptic properties prevent the wound from
being purulent consequently avoiding the occurrence of tetanus (Firdous, 2020; Nagdeve, 2021).
The orange essential oil also has found some side effects during usage. According to Dosoky &
Setzer (1966) and Healtine (n.d), several citrus essential oils are phototoxic, which can cause
skin painful reactions when exposed to sunlight. Healthline (n.d) also stated that citrus essential
oils that is old and oxidized can cause dermal sensitization. With the consequences of each
antiseptic solution and the essential oils of oranges, the proponents aim to generate an organic,
non-irritable, and less toxic antiseptic wound solution out of the orange essential oil. The
challenge in the study’s product is to preserve the freshness of the generated orange antiseptic
solution for longevity.

II. MATERIALS

For making the product:

Method 1 (Extracting Orange Essential Oil using Grain or Ethyl Alcohol): Oranges,
zester/grater, glass jar, paper towel, coffee filter or cheesecloth, bowl, warm water, and ethyl
alcohol/grain alcohol (e.g. vodka)

Method 2 (Infusing Orange Essential Oil using Extra Virgin Olive Oil): Orange, zester, extra
virgin olive oil/olive oil, and bowl

In Obtaining results:

Zone of Inhibition Test (Kirby-Bauer Test/Disc Diffusion Antibiotic Sensitivity Test): Paper
filter disc (6mm in diameter), petri dish (100 ml), agar, cotton swabs, bacteria (Staphylococcus
aureus, which can be found on skin), and extracted and infused orange essential oils

METHODS AND PROCEDURES

For making the product:

Method 1: Extracting Orange Essential Oil using Grain or Ethyl Alcohol

Using a zester or a grater, grind off the orange peels. The number of oranges used may
depend on the desired amount of orange extract oil needed. After removing the peels, place them
on a paper towel and let them dry under the rays of the sun, but try not to over-dry your peels.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

After drying, grind the dried orange peel using a food processor until you achieve a coarse
consistency. Side aside.

Place the grain alcohol or ethyl alcohol in a bowl filled with warm tap water to speed up
the extracting process of the orange zest. It should not be too hot (exceeding 32 degrees). Let it
soak for about 15-20 minutes. Place the ground orange peel into a clear glass jar or container.
Pour in enough alcohol that is sufficient enough to soak the orange peels. After pouring enough
alcohol, cover the jar tightly with the lid and vigorously shake the jar for a couple of minutes.

Allow the mixture to sit for two to three days. While the mixture is set for two to three
days, make sure to shake it three times a day to get more oil from the mixture. After a few days,
use a coffee filter or cloth and a bowl to strain the mixture to isolate the ground peels from the
fluid. Be sure that all liquids are squeezed into the bowl. Cover the bowl of the liquid mixture
with a cloth or paper towel and let it settle again for a few days. It will allow the excess alcohol
in the mixture to evaporate, and you will be left with the orange oil. Once the alcohol has
evaporated, put the oil in a tight glass container with a lid

Method 2: Infusing Orange Essential Oil using Extra Virgin Olive Oil

Using a zester or a grater, grind off the orange peels. The number of oranges used may
depend on the desired amount of orange extract oil needed. After grinding the peels, place them
in a small saucepan and pour some olive oil on the cover of the orange zest. Allow the mixture to
heat over medium heat for five minutes or until the olive oil starts to bubble. After heating, let
the mixture cool for a few minutes, and remove the orange zest from the mixture by using the
filter method.

To Obtain results:

Zone of Inhibition Test (Kirby-Bauer Test/Disc Diffusion Antibiotic Sensitivity Test) for Anti-
Microbial Activity of Extracted Orange peels

The Zone of Inhibition Test (Kirby-Bauer Test/Disc Diffusion Antibiotic Sensitivity

Test) is used to determine the effectiveness of an antibiotic solution to fight against bacteria
(Bhargav et al., 2016 & Hudzicki, 2009). It was usually done in a petri dish, wherein a paper
filter disc soaked in an antibiotic solution is placed on the agar surface with bacteria. An
antibiotic is said to be effective against certain bacteria if the surrounding of a soaked paper filter
disc has no bacterial invasion. It can be seen as a clear and colorless surface around the disc
(Bhargav et al., 2016)

The Oranges were purchased from the market. Before proceeding with the Inhibition test,
essential oils from the Orange zest were extracted using Ethyl alcohol or Grain alcohol (refer to
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

Method 1) or infused with olive oil (refer to Method 2). Both solutions (Infused and extracted
orange oil) will undergo the Zone of Inhibition test. Prepare your agar by dissolving agar powder
into a bottle with water. Heat the agar solution until it is completely clear. After heating, cool the
agar solution and pour it into the petri dish. The agar will hold the bacteria and the soaked filter
disc for inhibitory activity. Soak the paper filter disc (6mm in diameter) with the orange essential
oil solutions. Once the agar solution on the petri dish has been hardened, use a swab to spread the
bacteria on the surface of the agar and stick the soaked paper filter disc on the agar surface as
well. Leave the culture for about 4 - 6 days and observe if there is an inhibition zone around the
soaked disc.

RESULTS AND DISCUSSION

Extraction of Orange peels

This study utilized the orange essential oil extract in determining the effectiveness of
orange zest as an antiseptic solution. Orange zests were grounded into pieces and extracted oil
using ethyl alcohol and olive oil. Ethyl alcohol is a more effective extracting agent for most
vegetable and fruit peels but consumes more time to extract oil. The ethyl alcohol needed to
evaporate so that essential oil will be left out. Olive oil was easy to dissolve the component of
the essential oil while it was heated on the stove. The duration of the process of extraction takes
about 3 - 6 days.

Table 1-2. Inhibitory activity of extracted orange essential oil (using ethyl alcohol and extra
virgin olive oil) against skin flora

Trial 1

Method of Zone of Bacterial Inhibitory (millimeters in diameter)

extracting/infusing orange
essential oil 1 - 2 days 3 - 4 days

Extraction using Ethyl 6.2mm 7 mm

Alcohol

Infusion using Extra Virgin 6 mm (-) 6.5 mm

Olive Oil
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

Trial 2

Method of extracting Zone of Bacterial Inhibitory (millimeters in diameter)

orange essential oil
1 - 2 days 3 - 4 days

Extraction using Ethyl 6.9 mm 7 mm

Alcohol

Infusion using Extra Virgin 7 mm 8 mm

Olive Oil
Legend: (-) no bacterial inhibitory

Zone of Inhibition test

Both solutions (extracted and infused with orange oil) have undergone antibacterial
testing using the Zone of Inhibition (Kirby-Bauer Test). Tables 1 and 2 show the measurements
of the inhibition zones of both solutions observed for about an average of 3 days. Based on the
results, both solutions have been found to have inhibition zones from both solutions in the first
trial. Extracted orange essential oil using ethyl alcohol has found 6.2 mm - 7 mm of inhibitory
activity against skin flora - which is the main reservoir of staphylococcus, corynebacterium, and
propionibacterium (Grice et al., 2009). The infused orange essential oil was found to have lesser
inhibitory activity than the extracted orange extract used by ethyl alcohol. The infused orange
essential oil was found to have a 6 mm - 6.5 mm inhibition zone against skin flora.

Both solutions increased the measure of bacterial inhibitory against skin bacteria for the
second trial (refer to trial 2). Filter paper discs were soaked in both solutions for 1 minute, which
is longer than the first trial. For this time, infused orange essential oil inhibited more bacterial
growth than the extracted orange essential oil. The extracted orange essential oil has shown
bacterial inhibition of 6.9 mm to 7 mm in 4 days. On the other hand, the infused orange essential
oil has shown bacterial inhibition, for about 7 mm to 8 mm in 4 days, 1mm larger than the
extracted one.

Comparing the results from other studies, orange peel and essential oil have also been
found to have larger antibacterial activity against various microorganisms. Saleem and Saeed
(2020) have found that orange peel extract using ethanol has bacterial inhibition of 12 ± 0.2 mm
against Staphylococcus aureus, 17 ± 0.4 mm against Escherichia coli, and 24 ± 1.1 mm against
Klebsiella pneumonia. The ethanol extract yielded 2.64 g of orange peel. In a study by Ekwenye
and Edeha (2010), ethanolic extract orange peel has been found to have a smaller diameter of
bacterial inhibition; 2 mm against Staphyloccocus aureus around and 3 mm against Escherichia
coli and Klebsiella pneumonia.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

Local studies have also obtained studies about the antimicrobial activity of orange oil
essential extracts. A study by Tan (2017) from St. Paul University Quezon City has found that
orange extracts have no bacterial inhibition (0mm) against skin microorganisms (Staphylococcus
aureus, Micrococcus luteus, and Serratia marcescens). Contrary to Tan's (2017) findings,
Penicilla and Magno (2010) found that ethanolic extracts of orange peel have a 10 mm of
bacterial inhibition against Staphylococcus aureus and Bacillus subtilis and a 12 mm zone of
inhibition against Pseudomonas aeruginosa.

Conclusion

No studies have been conducted about the zone of inhibition of infused orange essential
oil using olive oil, although utilization of olive to infused orange peel is widely used. Based on
the statistical results, both solutions obtained from this study (extracted and infused with orange
essential oil) have found antibacterial effectivities against wound microorganisms and would
possibly be utilized as wound antiseptic solutions. The extracted orange essential oil has an
average of 6.8 mm zone of inhibition in the span of 2 trials and 8 days, while infused orange
essential oil has an average of 6.9 mm zone of inhibition.

Recommendations

Although effectiveness had successfully shown obtained from the study, some problems
were encountered during the testing. Experiments were done at home, which makes it difficult
for the researchers to provide more laboratory apparatuses to identify the specific bacteria used
in the testing and to undergo phytochemical analysis for bioactive components present in the
solutions made. For further related studies, phytochemical analysis is highly recommended to
determine the constituents present in extracts and reveal the bioactive compounds that are
beneficial for medical uses (Okoli et al., 2009). Certain analyses about other beneficial properties
of orange zest such as Insecticides activity, weight loss, relaxation, and acne cure are also
recommended. Ezeonu et al. (2001) have found that sweet orange has insecticidal properties
against mosquitoes, houseflies, and cockroaches.

REFERENCES

[1] Different types of open wounds and how to treat them. (2020, December 10). Makati Medical
Center. https://www.makatimed.net.ph/blogs/different-types-of-open-wounds-and-how-
to-treat-
them/#:~:text=An%20open%20wound%20is%20a,it%20at%20risk%20of%20infection.
[2] Britannica, T. Editors of Encyclopaedia (2019, February 12). wound. Encyclopedia
Britannica. https://www.britannica.com/science/wound
[3] Onyekwelu, I., Yakkanti, R., Protzer, L., Pinkston, C. M., Tucker, C., & Seligson, D. (2017).
Surgical wound classification and surgical site infections in the orthopedic patient.
Journal of the American Academy of Orthopaedic Surgeons. Global Research &
Reviews, 1(3).
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

[4] Guo, S., & Dipietro, L. A. (2010). Factors affecting wound healing. Journal of dental
research, 89(3), 219–229. https://doi.org/10.1177/0022034509359125.
[5] Gosain, A., & DiPietro, L. A. (2004). Aging and wound healing. World journal of surgery,
28(3), 321-326.
[6] Mathieu, D. (Ed.). (2006). Handbook on hyperbaric medicine (Vol. 27). New York::
Springer.
[7] FrykbergRobert, G. (2015). Challenges in the treatment of chronic wounds. Advances in
wound care.
[8] Nunan, R., Harding, K. G., & Martin, P. (2014). Clinical challenges of chronic wounds:
searching for an optimal animal model to recapitulate their complexity. Disease models
& mechanisms, 7(11), 1205-1213.
[9] Bjarnsholt, T., Kirketerp‐Møller, K., Jensen, P. Ø., Madsen, K. G., Phipps, R., Krogfelt, K.,
... & Givskov, M. (2008). Why chronic wounds will not heal: a novel hypothesis. Wound
repair and regeneration, 16(1), 2-10.
[10] Kirketerp-Møller, K., Jensen, P. Ø., Fazli, M., Madsen, K. G., Pedersen, J., Moser, C.,
Tolker-Nielsen, T., Høiby, N., Givskov, M., & Bjarnsholt, T. (2008). Distribution,
organization, and ecology of bacteria in chronic wounds. Journal of clinical
microbiology, 46(8), 2717–2722. https://doi.org/10.1128/JCM.00501-08
[11] Colsky, A. S., Kirsner, R. S., & Kerdel, F. A. (1998). Microbiologic evaluation of cutaneous
wounds in hospitalized dermatology patients. Ostomy/wound management, 44(3), 40-2.
[12] Gjødsbøl, K., Christensen, J. J., Karlsmark, T., Jørgensen, B., Klein, B. M., & Krogfelt, K.
A. (2006). Multiple bacterial species reside in chronic wounds: a longitudinal study.
International wound journal, 3(3), 225-231.
[13] Valencia, I. C., Kirsner, R. S., & Kerdel, F. A. (2004). Microbiologic evaluation of skin
wounds: an alarming trend toward antibiotic resistance in an inpatient dermatology
service during 10 years. Journal of the American Academy of Dermatology, 50(6), 845-
849.
[14] Taylor, T. A., & Unakal, C. G. (2021). Staphylococcus aureus. StatPearls [Internet].
[15] Lowy, F. D. (1998). Staphylococcus aureus infections. N. Engl. J. Med.
[16] Bianchi, J. (2000). The cleansing of superficial traumatic wounds. British Journal of
Nursing, 9(Sup3), S28-S38.
[17] Atiyeh, B. S., Dibo, S. A., & Hayek, S. N. (2009). Wound cleansing, topical antiseptics, and
wound healing. International wound journal, 6(6), 420-430.
[18] Lachapelle, J. M., Castel, O., Casado, A. F., Leroy, B., Micali, G., Tennstedt, D., &
Lambert, J. (2013). Antiseptics in the era of bacterial resistance: a focus on povidone-
iodine. Clinical Practice, 10(5), 579.
[19] McDonnell, G., & Russell, A. (1999). Denver. Antiseptics and disinfectants: Activity,
action, and resistance. Clin Microbiol Rev, 12(1), 147-79.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

[20] Hoekstra, M. J., Westgate, S. J., & Mueller, S. (2017). Povidone‐iodine ointment
demonstrates in vitro efficacy against biofilm formation. International wound journal,
14(1), 172-179.
[21] Oduwole, K. O., Glynn, A. A., Molony, D. C., Murray, D., Rowe, S., Holland, L. M., ... &
O'Gara, J. P. (2010). Anti‐biofilm activity of sub‐inhibitory povidone‐iodine
concentrations against Staphylococcus epidermidis and Staphylococcus aureus. Journal
of orthopedic research, 28(9), 1252-1256.
[22] Johani, K., Malone, M., Jensen, S. O., Dickson, H. G., Gosbell, I. B., Hu, H., ... & Vickery,
K. (2018). Evaluation of short exposure times of antimicrobial wound solutions against
microbial biofilms: from in vitro to in vivo. Journal of Antimicrobial Chemotherapy,
73(2), 494-502.
[23] Capriotti, K., Pelletier, J., Barone, S., & Capriotti, J. (2018). Efficacy of dilute povidone-
iodine against multi-drug resistant bacterial biofilms, fungal biofilms, and fungal spores.
Clin Res Dermatol Open Access, 5(1), 1-5.
[24] Velázquez, D., Zamberk, P., Suárez, R., & Lázaro, P. (2009). Allergic contact dermatitis to
povidone-iodine. Contact Dermatitis, 60(6), 348-349.
[25] Massé, M., Falanga, V., & Zhou, L. H. (2008). Use of topical povidone‐iodine resulting in
an iododerma‐like eruption. The Journal of Dermatology, 35(11), 744-747.
[26] Kerr, M. (2018). Iodine Allergy. Healthline.
https://www.healthline.com/health/allergies/iodine?fbclid=IwAR3rA-
ZxYC7W8ew348zCrjIlxCwgiDNYSRQdbjZB0eXEyan3LsyXbCaDelI#symptoms
[27] Aminov, R. I. (2010). A brief history of the antibiotic era: lessons learned and challenges for
the future. Frontiers in microbiology, 1, 134.
[28] Saleem, M., & Saeed, M. T. (2020). Potential application of waste fruit peels (orange,
yellow lemon, and banana) as a wide range of natural antimicrobial agents. Journal of
King Saud University-Science, 32(1), 805-810.
[29] Ferhat, M. A., Meklati, B. Y., Visinoni, F., Vian, M. A., & Chemat, F. (2008). Solvent-free
microwave extraction of essential oils: green chemistry in the teaching laboratory.
Chimica Oggi, 26(2), 48-50.
[30] Berna, A., Tárrega, A., Blasco, M., & Subirats, S. (2000). Supercritical CO2 extraction of
essential oil from orange peel; effect of the height of the bed. The Journal of
Supercritical Fluids, 18(3), 227-237.
[31] Parashar, S., Sharma, H., & Garg, M. (2014). Antimicrobial and antioxidant activities of
fruits and vegetable peels: A review. Journal of Pharmacognosy and Phytochemistry,
3(1)
[32] Ibri, K., Bouhadi, D., Hariri, A., Meddah, B., Tir Touil, A., & Ould Yerou, K. (2017). THE
USE OF ORANGE (CITRUS SINENSIS) PEEL AS ANTIMICROBIAL AND
ANTIOXIDANT AGENTS.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

[33] Escobedo-Avellaneda, Z., Gutiérrez-Uribe, J., Valdez-Fragoso, A., Torres, J. A., & Welti-
Chanes, J. (2014). Phytochemicals and antioxidant activity of juice, flavedo, albedo, and
comminuted orange. Journal of Functional Foods, 6, 470-481.
[34] Min, K. Y., Kim, H. J., Lee, K. A., Kim, K. T., & Paik, H. D. (2014). Antimicrobial activity
of acid-hydrolyzed Citrus unshiu peel extract in milk. Journal of Dairy Science, 97(4),
1955-1960.
[35] Dubey, D., Balamurugan, K., Agrawal, R. C., Verma, R., & Jain, R. (2011). Evaluation of
the antibacterial and antioxidant activity of ethanolic and hydroethanolic extract of sweet
orange peels. Recent Research in Science and Technology, 3(11).
[36] Shetty B (2016) Assessment of antioxidant, antibacterial, and phytochemical analysis peel
of citrus sinensis. Pak J Pharmaceutical science, 28, 231-239.
[37] Abalaka, M. E., & Bello, A. O. (2006). Antibacterial activity of Citrus sinensis (Orange)
peels on bacterial isolates from the wound. UMYU j Microbiol Res, 1, 1-8.
[38] Tan, C. (2017). Anti-Microbial Properties of Fruit Peeling Extracts on Skin Bacteria.
International Journal on Social Innovation & Research, 8(1), 1-1.
[39] Penecilla, G. L., & Magno, C. P. (2011). Antibacterial activity of extracts of twelve
common medicinal plants from the Philippines. Journal of Medicinal Plants Research,
5(16), 3975-3981.
[40] Thomas, G. W., Rael, L. T., Bar-Or, R., Shimonkevitz, R., Mains, C. W., Slone, D.
S., ... & Bar-Or, D. (2009). Mechanisms of delayed wound healing by commonly used
antiseptics. Journal of Trauma and Acute Care Surgery, 66(1), 82-91.
[41] McDonnell, G., & Russell, A. D. (1999). Antiseptics and disinfectants: activity,
action, and resistance. Clinical microbiology reviews, 12(1), 147-179.
[42] Atiyeh, B. S., Dibo, S. A., & Hayek, S. N. (2009). Wound cleansing, topical
antiseptics, and wound healing. International wound journal, 6(6), 420-430.
[43] Jones, C. G. (1997). Chlorhexidine is still the gold standard?. Periodontology 2000,
15, 55-62.
[44] Jackson, M. M. (2005). Topical antiseptics in healthcare. Clinical Laboratory
Science, 18(3), 160.
[45] Lyon, F. (1994). IARC monographs on the evaluation of carcinogenic risks to
humans. Some industrial chemicals, 60, 389-433.
[46] Moshrefi A. (2002). Chlorhexidine. J West Soc Periodontol Periodontal Abstr,
50(1),5-9
[47] FLÖTRA, L., Gjermo, P. E. R., RÖLLA, G., & WAERHAUG, J. (1971). Side effects
of chlorhexidine mouthwashes. European Journal of Oral Sciences, 79(2), 119-125.
[48] Lang, N. P., Catalanotto, F. A., Knöpfli, R. U., & Antczak, A. A. (1988). Quality‐
specific taste impairment following the application of chlorhexidine digluconate mouth
rinses. Journal of clinical periodontology, 15(1), 43-48.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

[49] McCullough, M. J., & Farah, C. S. (2008). The role of alcohol in oral carcinogenesis
with particular reference to alcohol‐containing mouthwashes. Australian dental journal,
53(4), 302-305.
[50] Cole, P., Rodu, B., & Mathisen, A. (2003). Alcohol-containing mouthwash and
oropharyngeal cancer: a review of the epidemiology. The Journal of the American Dental
Association, 134(8), 1079-1087.
[51] Pelaez, C., & GC, E. G. (2004). Alcohol-containing mouthwashes and oral cancer.
Critical analysis of literature. Medicina Oral: Organo Oficial de la Sociedad Espanola de
Medicina Oral y de la Academia Iberoamericana de Patologia y Medicina Bucal, 9(2),
120-3
[52] McDonnell, G., & Russell, A. D. (1999). Antiseptics and disinfectants: activity,
action, and resistance. Clinical microbiology reviews, 12(1), 147-179.
[53] Inhaling Ethyl Alcohol - Hazardous Substance Fact Sheet. (2016). New Jersey
Department of Health. https://www.nj.gov/health/eoh/rtkweb/documents/fs/0844.pdf
[54] Isopropyl Alcohol, Hazardous Substance Fact Sheet. (2016). New Jersey Department
of Health. https://nj.gov/health/eoh/rtkweb/documents/fs/1076.pdf
[55] McDonnell, G., & Russell, A. D. (1999). Antiseptics and disinfectants: activity,
action, and resistance. Clinical microbiology reviews, 12(1), 147-179.
[56] Zamora, J. L. (1986). Chemical and microbiologic characteristics and toxicity of
povidone-iodine solutions. The American journal of surgery, 151(3), 400-406.
[57] Larson, E. L., & 1994 APIC Guidelines Committee. (1995). APIC guidelines for
handwashing and hand antisepsis in health care settings. American journal of infection
control, 23(4), 251-269.
[58] Reybrouck, G. (1986). Handwashing and hand disinfection. Journal of Hospital
Infection, 8(1), 5-23.
[59] Velázquez, D., Zamberk, P., Suárez, R., & Lázaro, P. (2009). Allergic contact
dermatitis to povidone-iodine. Contact Dermatitis, 60(6), 348-349.
[60] Massé, M., Falanga, V., & Zhou, L. H. (2008). Use of topical povidone‐iodine
resulting in an iododerma‐like eruption. The Journal of Dermatology, 35(11), 744-747.
[61] Kerr, M. (2018). Iodine Allergy. Healthline.
https://www.healthline.com/health/allergies/iodine?fbclid=IwAR3rA-
ZxYC7W8ew348zCrjIlxCwgiDNYSRQdbjZB0eXEyan3LsyXbCaDelI#symptoms
[62] Edogbanya, P., Suleiman, M. O., Olorunmola, J. B., & Oijagbe, I. J. (2019).
Comparative study on the antimicrobial effects of essential oils from peels of three citrus
fruits. MOJ Biology and Medicine, 4(2), 49-54.
[63] Frassinetti, S., Caltavuturo, L., Cini, M., Della Croce, C. M., & Maserti, B. E.
(2011). Antibacterial and antioxidant activity of essential oils from Citrus spp. Journal of
Essential Oil Research, 23(1), 27-31.
 PERPETUAL HELP COLLEGE OF MANILA
124O V. CONCEPCION ST., SAMPALOC, MANILA
BASIC EDUCATION DEPARTMENT - SENIOR HIGH SCHOOL

[64] Taiwo, M. O., Olisemeke, J. K., Obero, O. J., & Abibu, W. A. (2019). Qualitative
properties and antibacterial activity of essential oil obtained from Citrus sinensis peel on
three selected bacteria.
[65] Yashaswini, P. (2018). Arvind: Antimicrobial Properties of Orange (Citrus reticulata
var. Kinnow). Peel Extracts against Pathogenic Bacteria. Int J Curr Microbiol App Sci,
7(3), 737-746.
[66] Firdous, D. (2020). Orange Oil Health Benefits, Uses, And Its Side Effects.
https://www.lybrate.com/topic/benefits-of-orange-oil-and-its-side-
effects?fbclid=IwAR1IUrgi6NRt8Ur3AlbOrETgFWluDwEToYTaGcu230GAc-
enDt8wqe2w-IQ
[67] Nagdeve, M. (2021). 13 Amazing Benefits Of Orange Essential Oil.
https://www.organicfacts.net/health-benefits/essential-oils/orange-essential-
oil.html?fbclid=IwAR0AhOA4JpISuKRjVTwrgVm1u0nJnI51j_o0Nr1AvpMNWWYZ2
pYx56qEJ6A
[68] Dosoky, N. S., & Setzer, W. N. (2018). Biological activities and safety of Citrus spp.
essential oils. International journal of molecular sciences, 19(7), 1966.
[69] The Benefits of Orange Essential Oil and How to Use. (n.d). Healthline.
https://www.healthline.com/health/orange-essential-oil-
uses?fbclid=IwAR2_A_v8lqJFoZtctfKpgD34i0tHUiNH9fxgcfmVZtuCN66Doprvxq_nq
74#safety
[70] Bhargav, H. S., Shastri, S. D., Poornav, S. P., Darshan, K. M., & Nayak, M. M.
(2016, February). Measurement of the zone of inhibition of an antibiotic. In 2016 IEEE
6th International Conference on Advanced Computing (IACC) (pp. 409-414). IEEE.
[71] Hudzicki, J. (2009). Kirby-Bauer disk diffusion susceptibility test protocol. American
society for microbiology, 15, 55-63.
[72] Grice, E. A., Kong, H. H., Conlan, S., Deming, C. B., Davis, J., Young, A. C., ... &
Segre, J. A. (2009). Topographical and temporal diversity of the human skin microbiome.
science, 324(5931), 1190-1192.
[73] Saleem, M., & Saeed, M. T. (2020). Potential application of waste fruit peels (orange,
yellow lemon and banana) as wide range natural antimicrobial agent. Journal of King
Saud University-Science, 32(1), 805-810.
[74] Ekwenye, U. N., & Edeha, O. V. (2010). The antibacterial activity of crude leaf
extract of Citrus sinensis (sweet orange). International Journal of Pharma and Bio
Sciences, 1(4), 743-750.
[75] Okoli, R. I., Turay, A. A., Mensah, J. K., & Aigbe, A. O. (2009). Phytochemical and
antimicrobial properties of four herbs from Edo State, Nigeria. Report and opinion, 1(5),
67-73.
[76] Ezeonu, F. C., Chidume, G. I., & Udedi, S. C. (2001). Insecticidal properties of
volatile extracts of orange peels. Bioresource technology, 76(3), 273-274.