Flavonoids in Citrus paradisi as an Alternative for Chemical
Disinfectant
Aleera Asanza, Ashley Bautista, Aaron Carreon, Mingmae Co, Rio Dizon, Shane Gedaro
Table of Contents
CHAPTER I
THE PROBLEM AND ITS BACKGROUND.......................................................................................... 3
Introduction ............................................................................................................................................. 3
Theoretical Framework .......................................................................................................................... 4
Disinfectant .......................................................................................................................................... 4
Environmental Ethicality ................................................................................................................... 4
Conceptual Framework .......................................................................................................................... 5
Statement of the Problem ....................................................................................................................... 5
Hypotheses ............................................................................................................................................... 6
Significance of the Study ........................................................................................................................ 6
Scope and Delimitation ........................................................................................................................... 6
Definition of Terms ................................................................................................................................. 6
CHAPTER II
REVIEW OF RELATED LITERATURE AND STUDIES .................................................................... 8
Related Literature................................................................................................................................... 8
Foreign ................................................................................................................................................. 8
Local ..................................................................................................................................................... 9
Related Studies ........................................................................................................................................ 9
Foreign ................................................................................................................................................. 9
Local ................................................................................................................................................... 10
Synthesis of Reviewed Literature and Studies ................................................................................... 10
CHAPTER III
METHODOLOGY ................................................................................................................................... 12
Research Design .................................................................................................................................... 12
Materials ……......................................................................................................................................... 12
Procedure ............................................................................................................................................... 12
I.
Pre-Experimentation ................................................................................................................ 12
II.
Experimentation .................................................................................................................... 13
III.
Post-Experimentation ........................................................................................................... 13
CHAPTER IV
PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA ............................................ 14
CHAPTER V
SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS................................ 19
Summary of Findings ........................................................................................................................... 19
Conclusions ............................................................................................................................................ 22
Recommendation................................................................................................................................... 23
BIBLIOGRAPHY ..................................................................................................................................... 24
........................................................................................................................................................................
CHAPTER I
THE PROBLEM AND ITS BACKGROUND
Introduction
In today’s society, people are facing a situation that requires them to use hand sanitizer and
chemical disinfectants. Disinfectant is a chemical agent that exterminates microorganisms;
(bacteria, viruses, and fungi). Hospitals use this on a day–to–day basis while it may provide
disposing of bacteria and viruses, it also has harmful side effects; for one, Health Care Without
harm (2019) states when hospitals use chemical disinfectants, it often contributes to poor indoor
air quality and may contain harmful chemicals that can cause reproductive disorders, respiratory
ailments (includes occupational asthma), etc. Some chemical disinfectants may include persistent,
bioaccumulation, and toxic chemicals (PBTs), which are classified as hazardous waste, in other
words, they can contribute to environmental pollution. The environment of a person is one of the
main ways one’s health can improve; Nightingale’s theory states the environmental settings of
one’s person can affect both the biological and physiologic processes. Flavonoids are diverse
secondary metabolites in plants, with a multitude of different functions such as pigmentation and
UV protection, it is also well known as an antibacterial agent against a large range of pathogenic
microorganisms. Flavonoids when discovered in the 1800s – 1940s, the pigments in the plants,
which were later found as flavonoids, ancient times their chemical structure was not identified
until it was at the end of the 19th century. The 20th-century flavonoids and related substances were
chemically characterized in multiple plants. Healthline (2019) states; that flavonoids can regulate
cellular activity and fight radicals that can cause oxidative stress in the body. The fruits in this
study will be using grapefruit, which has the highest in flavonoids. National Library of Medicine
flavonoids is enriched with flavonoids, alkaloids, poly-phenolic compounds that carry antiseptic,
disinfectant, and antimicrobial activities. During old eras, they were used as antiseptics and
disinfectants.
In the study of Huda Ahmed Alghamdi (2021), they studied the herbal disinfection
techniques, formulations, and preparations of human health-friendly hand sanitizers. The
researcher wanted to create a product that was a human health-friendly hand sanitizer, since most
of the available hand rubs are composed of isopropyl alcohol, H2O2, and ethanol in different
combinations. They had 3 different herbs they could use one was Guava leaves, the second was
the German Chamomile (flowers of the Chamomile), the last was Azadirachta indica L. (highly
medical plant with the purposes e.g., antiviral, anticancer, immune-modulator, skin disorders, liver
function improvements, etc.), their main objective was to find a way where they could make a
hand sanitizer with indigenous medicinal plants, in this study the researchers are going to be
making a citrus paradisi herbal disinfectant if it is possible to make it an alternative in the chemical
disinfectant use today.
Theoretical Framework
Disinfectant
Andersen (2019) defines sterilization as “the destruction of all forms of microbial life and
that anything is sterile when the likelihood of a living microbe is equal to or less than 10^-6.” The
definition of Blunt (1871) is slightly different from that of Andersen: “Disinfectants are a mere
deodorizer—not assailing the virus of disinfected substances, but rather masking their poisonous
character by precipitating their offensive gasses.” Traditional disinfectants are known to be
effective, but also harmful in a way it can cause various health issues: chronic respiratory
problems, skin irritation, headaches, and vomiting. As Brooke Jackson said, “While disinfectants
are intended to protect us from getting sick, they’re a bit of a double-edged sword.” Seema Sarin
builds on this, stating that many of our traditional disinfectants contain volatile organic
compounds. These traditional disinfectants are commonly chemicalized, for example alcohol as it
is a powerful germicide, which means it has the ability to kill a wide variety of germs, including
bacteria, viruses, and fungi. But as established, alcohol per se is harmful. According to Ben-Erik
van Wyk and Michael Wink (2017, p 7), they say that the use of alcoholic extracts instead of
traditional watery extracts may result in ineffective treatment or even harmful side-effects.
Diversely, together with chemical germicides are herbs that contain medicinal properties that have
been extensively useful and popular in earlier times. Many of these herbs contain antimicrobial
properties. On a theoretical standpoint, “plants can be used in very dilute form to treat illnesses
associated with the symptoms produced by high doses of the same plants” (van Wyk & Wink,
2017, p 14). Scarfalloto, in his theory, states that “the wealth of phytonutrients found in fruit,
provided powerful regulatory roles that allowed the primate brain to grow faster, culminating with
the exponential brain growth that transformed early hominids into modern humans. Naturally,
based on the literature discussed, assume that since chemical disinfectants are harmful per se, and
herbs have been used extensively as treatment due to their medicinal properties, it is reasonable to
assume that the researchers can use herbs as an alternative disinfectant for those that are
chemicalized. Hence, based on the argument, it is proposed that there is a potential for plants to
expand and compete with chemicalized disinfectants, while also being a safer option for people to
use.
Environmental Ethicality
A correlation observed between a person and their environment had shown significant
difference in terms of recovery rate. Nightingale (1860) believes air, pure water, sufficient food,
efficient drainage, light, and cleanliness of the person and environment are what affects a person’s
recovery rate or health in general. These factors greatly contribute to the emotional and physical
well-being of a person thus, if one or more is absent, then a person may experience diminished
health. Chemical disinfectants, per se, are harmful for our health. The World Health Organization
adds on this, stating that spraying disinfectants can be harmful. The Homeopathy Theory supports
the theory that “unsanitary environments contribute greatly to ill health, and that the environment
can be altered in order to improve conditions for a patient and allow healing to occur” (Nightingale,
1860), as disinfectants promotes personal cleanliness and aims to stop microbial activity within
the body with a minimized risk of harming yourself. To add on this, “it is very likely that
phytochemicals will find their way into arsenal of antimicrobials which may be used as
disinfectants” (Khanam & Afsar, 2013). Now, according to the literature arguments, assume that
the personal cleanliness and sanitary environment contributes positively on our physical wellbeing, and that traditional disinfectants fused with herbs can stop microbial activity without the
risk of long-term health issues, then it is within reason to presume that traditional disinfectants
fused with herbs can support the Environmental Theory. Therefore, it is proposed that plants with
medicinal properties fused with traditional disinfectants can help a person achieve overall
cleanliness and ultimately minimize the adverse effects of chemical disinfectants.
Conceptual Framework
Figure 1 depicts a schematic representation of the conceptual framework of this study. As
follows, it would be efficacy, length of exposure, condition with different temperatures, ph level
and availability. Data gathering will then be collected to show an accurate result and
representation. Flavonoids will not be extracted as a whole as Citrus paradisi has antimicrobial
activities. After these processes, photoionization will be commenced to measure the amount of
Volatile Organic Compounds indoors. With chemical disinfectant and the proposed output of a
flavonoid-based disinfectant, comparing both into different sections whilst limiting the Volatile
Organic Compound(VOC) emissions.
Statement of the Problem
The flavonoids in Citrus paradisi, also known as the grapefruit, will be observed in this
study to determine if it is a good alternative for disinfectants. This will also aid in determining the
impact of the disinfectant in other aspects such as our environment. The researchers will be guided
by the study's objectives when answering these questions, which includes the following:
1. Is it a good herbal disinfectant based on these terms:
1.1. Efficacy;
1.2. Length of exposure;
1.3. The disinfecatant’s condition with different temperatures;
1.4. pH Level;
1.5. Availability
2. What are the harmful effects of disinfectants containing VOCs to the environment and people?
3. How can flavonoid extractions be a substitute for chemical disinfectants?
Hypotheses
The researchers hypothesized that there is no significant difference in the use of Flavonoids
in Citrus paradisi as an alternative for chemical disinfectant.
Significance of the Study
Poor Community. This option could assist with relaxing the costs of every single family
that will purchase this since each of the fixings being utilized are less expensive. This is another
option, but there won't be an adverse consequence on the local area.
Future Researchers. There are potential understudies that would be searching for data
regarding this flavonoids as an elective sanitizer later on. This exploration would be a major
assistance for them to upgrade their own.
Medics. These people are working in front of many ill people, this can lead to the
possibility of bacteria, germs, and viruses. They always have a chemical disinfectant in hand.
Students. since the face–to–face classes are nearing; students need the basic necessities to
protect themselves from getting different types of illness.
Scope and Delimitation
The main goal of this study is to develop a viable herbal disinfectant through the use of
flavonoids while successfully decreasing indoor Volatile Organic Compounds (VOCs). Although
disinfection is necessary to address the coronavirus, the increasing use of disinfectant products has
exacerbated indoor air pollution, especially for housekeeping crews who are exposed to these
chemicals on a daily basis. This research involved a series of observations involving pH
evaluation, organoleptic tests, culturing of bacteria, and laboratory tests. Those formulations that
passed the screening would then be tested for a potential disinfectant.
A photoionization detector was used to count the amount of volatile organic compounds
(VOCs) to see if there were any changes after conducting the experiment. As Citrus paradisi is
the only fruit used in this study, other citrus genera cannot be compared in terms of performance,
regardless of its flavonoid level. In various aspects and dimensions, the results of flavonoids
extraction from Citrus paradisi was compared to a commercial chemical disinfection.
Definition of Terms
Aloe Vera. A succulent plant species of the genus Aloe, having some 500 species, Aloe
is widely distributed and is considered an invasive species in many world regions.
Antimicrobial properties. These kill or slow down the spread of microorganisms,
microorganisms include bacteria, viruses, fungi, etc.
Disinfectant. The term disinfection refers to an action that uses antimicrobial agents to
apply to non-living objects in order to destroy microorganisms that live on the objects. It is also
known as disinfection medicine because it is used to kill microorganisms for disinfection (Chen
2004).
Essential Oil. Concentrated hydrophobic liquid containing volatile chemical compounds
from plants, essential oils are also known as volatile oils, ethereal oils, petroleum, or simply as the
oil of the plant from which they were extracted.
Ethyl Alcohol. A member of a class of organic compounds that are given the general name
alcohols; its molecular formula is C2H5OH.
Flavonoids. It is a diverse group of plant chemicals found in almost all fruits and
vegetables.
Gram Positive Cocci. Bacteria that give a positive result in the Gram stain test, which is
traditionally used to quickly classify bacteria into two broad categories according to their type of
cell wall.
Isopropyl Alcohol. This is a colorless liquid that has a major component of rubbing
alcohol, within regular household items such as disinfectants and hand sanitizers. (Ivan N. Co,
Kyle J. Gunnerson 2019)
Organoleptic Test. Involves the assessment of flavor, odor, appearance, and mouthfeel
of a food product. The organoleptic testing of food products is essential in ensuring products
comply with organizational and customer requirements.
Pathogenic bacteria. Bacteria that can cause disease, most species of bacteria are harmless
and are often beneficial but others can cause infectious diseases. The number of these pathogenic
species in humans is estimated to be fewer than a hundred.
pH Level. The measure of how acidic/basic water is in its range goes from 0 - to 14, with
7 being neutral.
Phytochemicals. Compounds that are produced by plants mostly found in fruits,
vegetables, grains, beans, etc.; it also has great antioxidant potential, with beneficial effects on
human health by reducing the different chances of diseases (cancer, cardiovascular diseases, and
inflammatory diseases).
Respiratory ailments. These are the different types of respiratory diseases including
asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, pneumonia, and lung
cancer.
Volatile Organic Compounds. A large group of chemicals that are found in many
different products that are used in people’s daily lives, most common are Aerosol sprays, Cleaners,
and room deodorizers.
CHAPTER II
REVIEW OF RELATED LITERATURE AND STUDIES
The content of this chapter contains different studies and literature that are utilized to
achieve the purpose of this study. To elaborate, studies and literature regarding the potential of
flavonoids for herbal disinfectants that originated both locally and overseas are discussed in this
review. This is to build a stronger foundation for the relationship of the literature and studies with
the objective of the present research.
Related Literature
Foreign
The state of Washington reported on January 19, 2020, their first confirmed case of
coronavirus disease. Three months after the report, a total of 720,630 COVID-19 cases, along with
37,202 deaths had been reported to CDC from all America (Chang et al., 2020). Naturally, this led
the media to spread false information like wildfire regarding disinfection methods that claimed to
be effective against the virus. It wasn’t long until people started falling for this ridiculous
misinformation that eventually diminished their health (Rai et al., 2020). Furthermore, according
to Chang et al. (2020), in early March 2020, it was found that there was a significant increase in
the daily number of calls received by poison centers regarding chemical exposure. To make matters
worse, a large percentage of the total calls regarding chemical exposure was represented by
children aged 4 or less.
People of older ages are also victims of chemical exposure. According to Chang et al.
(2020, as cited by Rai et al. 2020), once there was an adult woman who soaked her groceries in a
sink composed of 10% bleach solution, vinegar, and hot water after hearing from the news
suggesting to clean all recently purchased goods. Very soon the woman noted a noxious smell that
eventually developed into respiratory difficulties. These measures can lead to an even more serious
health issue like seizures, blindness, coma, or worse death as these chemicalized disinfectants are
harsh on the skin and can easily cause problems in different parts of the body.
From the past to the present, there is an underlying issue that hospitals are facing, and that
is the relentless increase of infections (Khanam and Afsar, 2013). It has been found that the
transmission of multidrug-resistant pathogens and infections are largely represented by health care
workers as they are often exposed to transmissible diseases based on Vos (2018) and Khanam and
Afsar (2013). On the bright side, a routine for hand hygiene can help prevent the spread of
microorganisms, especially those that came from hospital resources making the use of safe
disinfectants for hands a necessity, as stated by Khanam and Afsar. However, as discussed, the use
of chemicalized disinfectants has many adverse effects, especially when used frequently.
Fortunately, the search for phytochemicals as an antimicrobial is on and there have been
numerous tests and studies that found plant phytochemicals with antimicrobial properties (Khanam
and Afsar, 2013). It has been found that consumers tend to reject the use of chemically obtained
additives with antimicrobial activity; therefore, additives derived from plants such as flavonoid
can be a valid and preferred option (Cowan, 1999, as cited by Patra et al, 2012). There are a number
of functions that can be found in flavonoids that benefit the human body with its protective
properties (Panche et al., 2016). Flavonoids are phenolic compounds that have been found to be
effective against a wide array of microorganisms with its antimicrobial properties, as stated by
Khanam and Afsar. Additionally, flavonoids are now widely used in nutrition and health care due
to it being recognized by researchers when they isolated its structure and discovered that it has
antiviral, antifungal and antibacterial properties (Panche et al., 2016).
It has been found that flavonoids are abundant in vegetables, fruits, seeds, nuts, spices, and
grains making it almost unavoidable in human and animal diets (USDA Database, 2007, as cited
by Patra et al., 2012). It has been found that there are a number of subgroups found in plants:
aurones, chalcones, anthocyanins, proanthocyanidins, isoflavones, flavandiols, flavonols, and
flavones. Isoflavones are almost only found in legumes, anthocyanins in berries, catechins in liquid
form such as tea and wine, flavones in celery, and lastly are flavanones which are primarily found
in citrus fruits (Panche et al, 2016 and Patra et al., 2012).
Local
Through the development of traditional and alternative healthcare and its integration into
the national health care system, the quality and delivery of both will be improved for the Filipino
people (Article 1 Section 2 of Republic Act No. 8423, 1997). Scientific research into the
development of traditional and alternative healthcare systems which has a direct impact on public
health care would be encouraged. And to promote and advocate for the use of traditional,
alternative, preventive, and curative health care modalities that have been shown to be safe,
effective, and cost-effective, as well as to be compliant with government medical practice
requirements (Article 1 Section 3 of Republic Act No. 8423, 1997).
The Philippines and alternative medications have been around for a long time that have been
passed from one generation to another. These folkloric treatments weren’t taken too seriously and
weren't popular due to the lack of scientific evidence, but there were some alternative medicines
that came out from the market such as, Vitex negundo (lagundi) and Blumea balsamifera
(sambong). The country’s Department of Science and Technology even launched a program called
“Tuklas Lunas” (Tupas et al. 2020) to have drug discovery and development by using the country’s
biodiversity (PCHRD 2019).
Related Studies
Foreign
Children under the age of 12 who were exposed to hand sanitizer and reported to the
National Poison Data System (NPDS) between 2011 and 2014. The study was divided into two
age groups (0–5 years old and 6–12 years old). Approximately 70,669 people in this age group
have been exposed to hand sanitizers, with 92 percent exposed to alcohol-based disinfectants and
the remaining 8 percent exposed to non-alcoholic disinfectants (Santos et al., 2017). However,
misuse of these preparations may be hazardous to both human health and the environment.
Excessive use of these disinfectants can be fatal. These chemicals will have toxic and hazardous
effects on the environment if they are released through evaporation (Slaughter et al., 2014).
Frequent use of disinfectants has also been linked to an increased risk of other viral diseases and
antimicrobial resistance (Mahmood et al., 2020).
In a study by Steinemann et. al (2020) Australia’s government labeled as fragranced
pandemic disinfecting products such as government-issued public health recommendation for a
product type to be used more frequently and extensively for anti-coronavirus purposes. Mainly
focusing on the Volatile Organic Compounds (VOCs) composition, 27 of the 399 VOCs
(occurrences) emitted by the 26 products are classified as potentially hazardous, accounting for
approximately 30% of all VOC ingredients. All of the products emitted between one and four
VOCs that were classified as potentially hazardous. Ironically, disinfectants were heavily sprayed
to reduce the possibility of contracting COVID-19.
Citrus species are among the world's most popular fruit crops, and they are farmed all over
the world for their economic and nutritional value. Citrus, like other fruits and vegetables, is an
important source of antioxidant molecules (polyphenols, ascorbic acid, and carotenoids) that can
inhibit the harmful effects of free radicals on the human body; Citrus species are valued fruits not
only in the agri-food industry, but also in the pharmaceutical industry, due to their functional
values and health-promoting properties. Flavonoids are significant polyphenol components found
in many citrus fruit locations (skin, peels, seed, pulp membrane, and juice). Flavonoids possess a
wide range of biological features (antiviral, antifungal, and antibacterial activities). Several studies
have also shown that citrus flavonoids have health benefits, particularly antioxidant, anticancer,
anti-inflammation, anti-aging, and cardiovascular protective properties. The current study aims to
look at the most recent advancements in flavonoids research in diverse Citrus species.
Local
Hand sanitizer containing the extract containing the same antibacterial compounds as other
plants. The study's goal is to see how effective the newly designed product's bactericidal potential
is. A topical medication containing an ingredient that can inhibit the formation of germs,
particularly bacteria.
In the study of Estel, Dimple et al. (2019), the researchers conducted an experiment about
Aloe vera as a hand sanitizer. The formulation contains Aloe vera, calamansi, ethanol, glycerin,
and coconut oil. The purpose of the study was to produce an alternative hand sanitizer and the
researchers were able to achieve this goal. The hand sanitizer wasn't observed for its antimicrobial
activity yet the active ingredient, ethanol, made the hand sanitizer antiseptic.
Laarni M. Villanueva-Magsombol and Carina R. Magbojos-Magtibay. (2020). The
researchers formulated an extract from common local fruits (orange, mango, and papaya) with its
peelings and added dimethyl sulfoxide (DMSO). The formulated extracts are tested for its
antimicrobial activity against S. aureus and E. coli, it is observed that the mango has the most
effective antimicrobial activity. It also found in the study that a combination of fruit extracts has a
greater bacterial inhibition against S. aureus and E. coli.
Synthesis of Reviewed Literature and Studies
In the many different studies, the researcher has collected a few studies that will be
beneficial in the study, studies from foreign literature to local literature, while each study has a
different purpose, it correlates the study that the researchers are studying. Disinfectants nowadays
are being used in different places and used in objects when bought online, it has been stated before
that chemical disinfectants have a large negative effect on human health and the environment.
While chemical disinfectants can remove microorganisms, there have been studies that stated that
herbal disinfectants can be used as an alternative disinfectant.
In the different studies of Chang et al., (2020), in Washington, January 19, 2020, their 1st
confirmed case of COVID – 19, three months of a report there was an increase of cases of the same
virus a total of 720,630 cases, along with 37,202 diseased had been reported to CDC from all of
America. Studies by Vos (2018) and Khanam and Afsar (2013), found that the transmission of
multidrug-resistant pathogens and infections are widely transmittable to Health Care Workers as
they are more often to be exposed to transmissible diseases, a routine of hand hygiene can help
prevent the spread of microorganisms, Khanam and Afsar stated that Flavonoids are phenolic
compounds that are effective against a large variety of microorganisms with their antimicrobial
properties. In the study of Cowan, (1999), as cited by Patra et al, (2012), It has been found that
consumers tend to reject the usage of chemical additives with antimicrobial activity, additives from
plants such as flavonoids can be a valid option to consumers. In another study of USDA Database,
(2007), as cited by Patra et al., (2012), It had been found that flavonoids are abundant in vegetables,
fruits, seeds, nuts, spices, and grains making it almost unavoidable in humans and animal diets. As
studied by Panche et al., (2016), additional, flavonoids are now being used in nutrition and health
care due to them being recognized by researchers, isolating their structure, and having discovered
that they had antiviral, antifungal, and antibacterial properties, It has been found that there are
several subgroupings in plants, aurones, chalcones, anthocyanins, proanthocyanidins, isoflavones,
flavanols, flavonols, and flavones, Isoflavones are commonly found in legumes, catechins in liquid
form such as tea and wine, Flavones are in celery, flavanones are primarily found in citrus fruits.
Slaughter et al., (2014), The usage of chemical disinfectant can be hazardous to both human health
and the environment, the excessive use of disinfectants can be fetal chemicals will have toxic and
hazardous effect in the environment if they are being released through evaporation.
In other studies, Steinemann et. al (2020), the main focus on the Volatile Organic
Compounds (VOCs) composition, 27 of the 399 VOCs (occurrences) that are emitted by the 26
products are classified as potentially hazardous, accounting for approximately 30% of all VOC
ingredients. With the global trend of chemical disinfectants and VOCs increasing indoors, herbal
disinfectants are now starting to be introduced into the market as a prevention measure, an
alternative, and an environmentally friendly solution, some herbal disinfectants also irritate the
same way as chemical disinfectants would. Plants are now gaining more popularity as a natural
antibacterial agent since they do not create antibiotic resistance, which is frequent with synthetic
antibiotics, in different countries like Iran, India, and Bulgaria, Deg. Securigera securidaca (L.)
Dorf & Co. (Fabaceae) one of the species of this genus are found in Iran, Mill, Rosa damascena
(Rosaceae) it is a tiny shrub with scented flowers that bloom in the spring, Currently, R.
Damascena is the primary species grown for rose water and attar in central Iran (Kashan), in India
and Bulgaria, Tripleurospermum disciforme (C.A. Mey) Schultz Bip is an Asteraceae genus that
is endemic to Europe and Western Asia. It is being cultivated in many different parts of Iran, three
plants have various traditional and folk applications in Iran, there are only a few studies on its
antibacterial properties. Citrus like other fruits and vegetables are an important source of
antioxidant molecules that can inhibit the harmful effects of free radicals on the human body, citrus
species are not only valued fruits in the agri-food industry, it is also in the pharmaceutical industry,
due to their health-promoting properties. The significance of polyphenol components found in
many citrus fruit locations, Flavonoids can also possess a wide range of biological features, citrus
flavonoids have a health benefit, particularly antioxidant, anticancer, anti-inflammation, antiaging, and cardiovascular protective properties.
CHAPTER III
METHODOLOGY
This chapter discusses the research design and methods that will be used in this study. This
section also includes information about the respondents, the materials, the procedures to be
followed, and the study's recommendations.
Research Design
The researchers utilized the quasi-experimental design as it is examined to be suitable to
achieve the purposes of this study. A quasi-experimental research design aims to test hypotheses
based on quantitative data (White and Sabarwal, 2014). This design is suggested to have an
important goal for a causal description rather than a causal explanation. (Cook, 2015). The
advantage of this design is its potential to generalize the results of quasi-experiments by examining
the results of patients who may not participate in randomized trials (Maciejewski, 2018). The
researchers will be using a nonequivalent group, pretest-posttest design.
Below are the important phases in conducting this experiment effectively (White and
Sabarwal, 2014):




Constructing Comparison Groups. The researchers will be constructing a comparison
group of individuals that can be matched with the individuals from the experimental group.
Pretest. The researchers will be carefully assessing the differences of the control and
treatment group.
Data Analysis. The researchers will be comparing the results of the control group
(comparison) with the results of the treatment group (experimental) at a single point in time
after the intervention.
Posttest. The researchers will be evaluating which group performed better, thus allowing
the researcher to use initial differences as an explanation for the current differences.
In this study, the researchers compared the effectiveness of herbal disinfectants with
chemicalized disinfectants in terms of antimicrobial activity, toxicity level, environmental
ethicality, and volatile organic compound level. Also, the researchers identified external and
biological factors of the citrus fruits and participants that influence its capacity as a herbal
disinfectant.
Materials
Petri dishes, Ph paper, Beaker (1000 ml), Stirring rod, and Ethyl alcohol were all purchased
from medical and laboratory stores in Bambang, Metro Manila, for our experiment. For the
ingredients, the researchers used Aloe Vera pure extract from an online store, Aloe Vera gel,
Grapefruit essential oil as the extract and the source of flavonoids, Lemon essential oil for scent,
and gelatin bought from a local department store, as well as a spray bottle.
Procedure
I.
Pre-Experimentation
Ethanol was used for disinfecting the glasswares such as the rod and the beaker. For the
inner layer of the glasswares, warm water and soap was used. To avoid fibers from entering the
glasswares, the equipment was immediately dried using acetone. Several materials were used to
prepare the disinfectant in significant amounts, including aloe vera (gel and pure), 70% ethyl
alcohol, grapefruit extract for flavonoids, and lemon oil.
II.
Experimentation
Each formulation was prepared in a 1000 mL beaker and mixed with gentle stirring at room
temperature. Until all ingredients were well incorporated, the researchers gradually stirred the
solution.
.
III.
Post-Experimentation
pH Evaluation
Using a pH test strip ranging from 1-14, the substance was assessed whether it was acidic, basic
or neutral.
Organoleptic Tests
The texture, odor, and color of the gels in colloidal suspension conditions were visually checked
on the prepared samples. After that, it was tested on how long each formulation would be able to
hold up. Occasional tests of temperature movement were also considered.
Culturing of bacteria with Laboratory Testing
Chemists and pathologists were involved in the study, checking the efficacy and fusion of
materials. Microbes were also tested if it was a zone for inhibition.
Total Volatile Organic Compounds
With the help of professional scientists, equipment was acquired and tested the significance of the
solution.
CHAPTER IV
PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA
This chapter presents the findings, analysis and interpretation of the gathered data. Data
was gathered through a series of observations and laboratory analysis.
4.1. Table 1; shows what each percentage was used in the formulations in trials 1 to 6:
Formulation no.
Ethyl Alcohol
Aloe Vera
Lemon Essential Oil
Flavonoids
1
60%
30%
5%
5%
2
39%
39%
11%
11%
3
49%
24%
7%
20%
4
61%
4%
9%
26%
5
61%
21%
—
18%
6
65%
18%
—
17%
4.2. pH evaluation
A pH paper was used to measure the pH of the formulated disinfectants. The study was
carried out to test the neutralization of various prepared formulations. To avoid skin
inflammation and irritation, the ideal pH value for a topical dosage form should be within the
skin's broad pH range, i.e., 4.0 to 7.0. It has been reported that the neutral pH range is the best
environment for the growth of several pathogenic bacteria that can infect the skin. Normal flora,
on the other hand, is more likely to settle in the skin if the pH is slightly acidic.
Table 2. The pH levels of the different disinfectant formulations.
Formulation No.
pH Range
Denomination
1
6
Moderately Acidic
2
5.5
Strongly Acidic
3
6.5
Slightly Acidic
4
6.5
Slightly Acidic
5
7
Neutral
6
6.5
Slightly Acidic
4.2.1 Organoleptic Test - Appearance, Applicability and Scent.
The organoleptic tests of the said disinfectants were inspected in terms of physical
appearance, applicability and smell. Trial 1, 2, and 3 had a translucent formulation. Trial 4, 5,
and were leaning towards opaque. While trial 6 remained clear throughout the entire time period.
The colors were all off-white. Essential oils were tested differently as Formulation 1, 2, 3, and 4
showed a strong scent to handle. In Trials 5 and 6, essential oil was removed as the scent had
overpowered the smell. In addition to that, fragrances can cause more harm such as irritation of
eyes, nose, nausea, and asthma attacks.
4.2.2. Organoleptic Test - Length of Exposure
Table 3.
Formulation No.
Length of Exposure
1
1 hour until spoiled
2
2 hours until spoiled
3
5 hours until spoiled
4
4.5 days until moisture appeared
5
5 days until solidified
6
Observed for 7 days
Picture 1. Formulations after observation.
Trials 1,2, and 3 had conceptualization of a pure aloe vera to retain its availability. Trial 1
formulation can be seen that the mixture turned dark purple, indicating that the aloe vera had
already been spoiled. After three hours of observation, it changed its odor. Trial 2 took two hours
to spoil. It also changed its fragrance after two hours. Identical as Trial 1, the color also turned
purple. Trial 3 which took the longest to spoil in this set of trials. Five hours later, the
formulation also tainted itself. Although it did not change its color, it was a habitable zone for
microbial activity, algae, and fungi. The amount of microbial activity had heavy growth and
covered the petri dish. Thereafter, the microbes multiplied after five hours.
Trials 4, 5, and 6 had preservatives added on the aloe vera as prior experiments had a
short period of efficacy. Trial 4, on the contrary, had zero microbes. It did not decay after 2 days.
However, the mixture was vague. The pH level of this trial was slightly acidic. It can be also
noted that the difference between percentages of each trial had shown a different outcome of
each trial. Trial 5 induced a pH level where it became neutral. It had also been a zone for
microbial activity to flourish. Although not as present as Trial 3, the moisture can cause a creamy
white colony. Trial 6 was observed for a week. The formulation was consistent throughout the
entire observation. Not to mention, it did not cloud unlike the previous mixtures.
4.3. Culture of Bacteria through Laboratory Tests
The results which garnered results from the above tests with the longest efficacy,
appropriate organoleptic tests were transferred to Dr. Jose N. Rodriguez Memorial Hospital and
Sanitarium. Trial 6 passed the tests and was listed with moderate growth of gram positive cocci.
Gram positive cocci include Staphylococcus, Streptococcus and Streptococcus. Although not
necessarily hazardous, normal flora like these can cause harm on open wounds. Half of the plate
had been covered as it was a moderate growth of microorganisms.
Although Gram Positive Cocci was formed, Gram Negative Cocci was killed throughout
the whole incubation period of 24 hours. It can be noted that the latter is developing dangerous
resistance against disinfectants. Its cell wall is more compact, making it harder to remove.
However, it did not culture any gram negative cocci.
Picture 2. Laboratory Tests Results.
4.4. Testing of VOCs
There is no significant difference from the formulation and Volatile Organic
Compounds. Although effective, its maximum time potency is only 10 minutes, same as a
chemicalized low-grade disinfectant. Chemicalized disinfectants would also take less risks
compared to this substance.
CHAPTER V
SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS
This chapter presents the summary of findings, conclusions, and recommendations offered
based on the gathered results and insights during the conduct of the study. These recommendations
will be helpful in creating a more reliable and effective herbal disinfectant.
Summary of Findings
The aim of the study is to create a disinfectant from flavonoids in Citrus paradisi and assess
its potential as an alternative for chemical disinfectants according to the efficacy, length of
exposure, condition with different temperatures, pH level, and availability of the product. It also
aimed to assess the perceived effectiveness of the disinfectant’s ability for reducing the amount of
volatile organic compounds found in homes. Likewise, the primary goal of the study is to find out
if herbal disinfectants can be an alternative while being as effective and accessible as chemical
disinfectants.
Specifically, the study aimed at:

determining if the efficacy, length of exposure, condition with different temperatures, pH
level, and availability of the herbal disinfectant will qualify as a good disinfectant.

determining the harmful effects of disinfectants containing VOCs to the environment and
people.

assessing the ability of flavonoid extract as an alternative disinfectant.
Overall, there were 4 ingredients present in the formulation of the herbal disinfectant with
each of the content’s percentage varied throughout the 6 trials. Specifically, the ingredients of the
herbal disinfectant are ethyl alcohol as the carrier or the majority ingredient, aloe vera as the
soother, lemon essential oil for the fragrance, and flavonoid for the antimicrobial properties.
The researchers followed a 6-step procedure in gathering the data essential to finishing the
objectives. The formulation of trials was done first, creating 6 different trials with the percentage
of each ingredient varied. The pH level of each trial was then evaluated to see which formulation
is the most acidic and the least acidic. The organoleptic test, following the evaluation of pH levels,
was evaluated to inspect the physical appearance, applicability, and smell of each trial. Moreover,
the researchers observed the length of exposure of each trial. The trials were then transferred to
Dr. Jose N. Rodriguez Memorial Hospital and Sanitarium to identify the culture of the bacteria.
Lastly, is the testing of VOCs to determine the disinfectant’s maximum time potency.
1. Evaluation of pH Level, Organoleptic Properties, Length of Exposure, and Efficacy
1.1 pH Level
A pH paper was utilized to measure the pH level of each trial to identify which is
the safest for the skin. Based on the evaluation, trial 1, 2, 3, 4, and 6 had a denomination
range of moderately acidic to strongly acidic. On the other hand, trial 5 had a pH level of
7, where the denomination is neutral. The ideal pH level for the skin is 7.0, making trial 5
the best for the skin. However, neutral is the best environment for pathogenic bacterial
growth.
1.2 Organoleptic Properties
The organoleptic properties of the trials were inspected based on their appearance
and smell. Trails 1, 2, 3, 4, and 5 were showing an odd color, while trial 6 was clear
throughout the inspection. The fragrance or the lemon essential oil was removed for trial 5
and 6 as it had a powerful, intolerable smell.
1.3 Length of Exposure
Trial 1, 2, 3, 4, 5, and 6 was observed thoroughly throughout to see minor to major
developments. Trial 1, 2, and 3 spoiled in the span of 1-5 hours. Trial 4 took 4 days and a
half before moisture started to appear. Trial 5 took 5 days before it solidified, but as for
trial 6, after 7 days of observation, it didn’t spoil.
1.4 Efficacy
After going through multiple tests, trial 1, 2, 3, 4, and 5 was not put through
laboratory testing as each of them have failed or spoiled in a way it changed color or
became a zone for bacteria to flourish. However, trial 6 retained its availability allowing
the researchers to put it through laboratory testing. It was found that trial 6 had a moderate
growth of gram-positive cocci, which is not hazardous unless interacted with an open
wound.
2. Harmful Effects of Herbal Disinfectants.
The ideal pH level of disinfectants for the skin is 4.0 to 7.0. If the pH level of a
disinfectant is below 4.0, it may be too acidic. If the pH level is above 7.0, it can cause
tissue damage. The fragrance of a disinfectant may also be harmful if it’s too strong. Based
on trial 1, 2, 3, and 4, the fragrance was too strong to the point it was intolerable. Strong
fragrance may cause irritation to the eyes and nose and may also trigger asthma attacks.
3. Flavonoid Extractions Substitute for Chemical Disinfectants.
The herbal disinfectants from flavonoid extractions are not as effective as chemical
disinfectants; therefore, it cannot be an alternative. It was reported that the maximum time
potency is only 10 minutes, similar to low-grade, chemicalized disinfectants.
Conclusions
Disinfectant is a term referring to an action that uses antimicrobial agents, mostly on
living objects to destroy or hinder microbial activities. Due to the emergence of COVID-19,
disinfectants have caused high rates of Volatile Organic Compounds. The study's goal is to
develop a disinfectant from Citrus paradisi flavonoids and evaluate its viability as a chemical
disinfection alternative. In order to obtain the data needed to complete the objectives, the
researchers conducted a 6-step approach. In this study, an alternative disinfectant has been
proposed using flavonoids from Citrus paradisi, aloe vera, essential oil, and 70% ethyl alcohol.
Trials 1, 2, 3, 4, and 5 had a strange hue to them, however trail 6 remained clear
throughout the examination. For trials 5 and 6, the fragrance or lemon essential oil was
eliminated since it had a strong, unpleasant odor. Trial 5 took five days to solidify, whereas trial
6 did not spoil after seven days of monitoring. Chemical disinfectants are more effective than
herbal disinfectants derived from flavonoid extractions. It has been concluded that results from
Trial 6 showed potential through admissible pH level compatible with the skin, appropriate
organoleptic test results, suitable length of efficacy, and moderate intake on several types of
bacteria. The antimicrobial tests showed varying activities of Trial 6 against numerous bacteria.
The results provided evidence that it can effectively kill Gram Negative Cocci, which is
more hazardous compared to a normal flora Gram Positive Cocci. Although the disinfectant
possesses these qualities, in contact with an open wound, it may be harmful. The VOCs showed
no significant difference in comparison to the commercialized chemical disinfectant and
flavonoid-based disinfectant.
Recommendation
1. Since the findings of this study showed that there is a need for advanced laboratory
equipment, the school should provide such lab equipment for the researchers as the
experiment might cost much.
2. Because the researchers used an alternative method, it is advisable to use the original
procedure. Use sugarcane alcohol instead of ethyl and grapefruit extract instead of essential
oil to achieve the objective of making an alternative disinfectant.
3. The researchers recommend performing the experiment inside the laboratory to avoid
unnecessary chemical reactions.
4. For our study we used Essential Oils for our extracted Grapefruit, ideally using a real
extraction could help getting the expected results.
5. For the soothing component we used Aloe Vera, through the process of our
experimentation the Aloe Vera was a factor for making the disinfectant acidic. Other
soothing extracts such as Lavender could make the disinfectant not acidic.
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