1

POTENTIAL ANTIMICROBIAL ACTIVITY OF SAMBONG LEAVES EXTRACT

AGAINST ESCHERICHIA COLI

Arceo, Jennifer
De Vera, Dwight
Difuntorum, Kristin
Macagaling, Rocenne
Marzan, Leah
Pangda, Jaceziel
Sumingwa, Jen Ira

University of Baguio High School

ABSTRACT

Gastroenteritis is one of the prevailing problems in primary care. It is one of the leading
cause of death worldwide, and although a vast amount of gastroenteritis case is not fatal,
it still causes a huge amount of morbidity and economic burden. There are various
microorganisms which cause gastroenteritis, but the researchers focused on Escherichia
coli based on the availability of the bacteria. Escherichia coli are gram-negative,
facultatively anaerobic, rod-shaped species belonging to the family Enterobacteriaceae
usually found in the gut flora in the intestines of humans and other mammals. Most
strains of Escherichia coli are harmless. However, Esherichia coli, specifically Shiga-
toxin producing Escherichia coli, along with Staphylococcus aureus are amongst the
leading bacterial cause of food-related diseases. Escherichia coli infections are usually
not deadly as long as proper medication and care is observed. Yet in the Philippines,
about 90 million do not have access to healthcare service while some fail to go for a
checkup because of financial problems, or negligence of the early signs of symptoms.
Because of this, the researchers wanted to find an alternative for treating Escherichia coli
gastroenteritis that is available and affordable to the majority of Filipinos. Blumea
balsamifera (sambong) is perceived as a potential treatment for gastroenteritis because
phytochemical studies reveal that it contains four flavonoids which is associated with
powerful anti-inflamatory and antioxidant activities, and alkaloids which displays
antimicrobial activity against both gram-positive and gram-negative bacteria.
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Phytochemical studies also show that Blumea balsamifera contain the chemicals
Icthyothereol acetate, Cyptomeridiol, lutein and ß-carotene which can potentially fight
against Escherichia coli. In light of this, the researchers hypothesized that Blumea
balsamifera can eradicate Escherichia coli because of its numerous biological activities.
In this paper, the effectiveness of sambong leaves extract at concentrations of 25% (75%
distilled water), 50% (50% distilled water), 75% (25% distilled water), and 100%
sambong leaves extract, against Escherichia coli were tested. The most effective
concentration was compared to the effectiveness of the control group by using
antimicrobial susceptibility test specifically Baur-Kirby disk diffusion. Mean was used in
comparing the effectiveness of each concentration based on the zone of inhibition. The
results show that the 25% (75% distilled water), 50% (50% distilled water), 75% (25%
distilled water), and 100% sambong leaves extract concentrations in the experiment has
an average of 6mm zone of inhibition thus indicating that Escherichia coli is resistant to
sambong leaves extract based on the interpretative criteria given by the European
Committee in antimicrobial testing. Ciprofloxacin also displayed a more significant
activity against Escherichia coli compared to the given concentrations. All 25% (75%
distilled water), 50% (50% distilled water), 75% (25% distilled water), and 100%
sambong leaves extract were ineffective against Escherichia coli, and Ciprofloxacin is far
more effective in eradicating Esherichia coli than all the given concentrations. The pre-
extraction preparation of plant samples, extraction method, and storage of the extract are
the major factors which contributed to the negative results of the study.

Keywords: Blumea balsamifera, Escherichia coli, Gastroenteritis, Diarrhea, Herbal

medicine

INTRODUCTION

Gastroenteritis is one of the leading cause of death worldwide (World Health

Organization, 2018). Although a vast amount of gastroenteritis case is not fatal, it still
causes a vast amount of morbidity, and economic burden wherein noroviral
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gastroenteritis cost 2 billion dollars in healthcare alone, and foodborne disease cost more
than 15.5 billion dollars annually (Marlow, 2016). By the use of replacing lost fluids and
electrolytes, gastroenteritis may be treated. In severe cases, adults treat gastroenteritis by
taking over-the-counter medicines. However, this medicines are unsafe for consumption
of children and infants (Treatment of Viral Gastroenteritis “Stomach Flu”, 2018). Severe
gastroenteritis for infants and children require hospitalization or a doctor’s prescription
(Gastroenteritis in children, 2018), but according to Kenworthy (2017) and Flordeliz
(2015), about 90 million Filipinos do not have access to healthcare service while some
fail to go for a checkup because of financial problems, or negligence of the early signs of
symptoms.

Literature Review

Gastrointestinal infections, although not usually severe, can cause serious

problems on some healthcare settings and are usually found in primary care (Gastro-
intestinal infections, 2018). Gastroenteritis, from its name, means inflammation of the
stomach and small intestine. It is a diarrheal disease classified by frequency of watery
stool (Sattar and Singh, 2019). It is also known as the stomach flu — a common
condition that causes vomiting, stomach pain, cramping, fever, nausea, and headache
(Stomach Flu: What You Need to Know, 2019). Although there are ways to treat
gastroenteritis, another potential alternative treatment is Blumea balsamifera or locally
known as sambong.
Causes of gastroenteritis differ depending on the geographical location or place.
Another factor which influences the cause of gastroenteritis is the immune system of the
area’s inhabitants (Sattar and Singh, 2019). Causes of gastroenteritis include bacterial,
viral, fungal, parasitic infection, and sometimes chemical toxins or drugs (Boyce, 2017).
However, according to Stuempfig and Seroy (2019), viral pathogens are the most
common cause of gastroenteritis. We the researchers, however, focused on the bacterial
cause of gastroenteritis. Sattar and Singh (2018) mentioned that “bacterial causes are
more responsible for severe cases of infectious diarrhea than other infectious etiologies”.
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The same study (Sattar and Singh, 2019), stated that 87% of diagnosed adults with
diarrhea characterized by having four or more watery stools for more than three days
were all caused by bacteria.
Boyce (2017) and Fletcher (2018) listed the common bacteria which causes
gastroenteritis namely Salmonella, Campylobacter, Shigella, and Escherichia coli. Boyce
(2017) also mentioned that Salmonella and Campylobacter are the most common
bacterial cause of gastroenteritis while Sattar and Singh, (2019) stated that Salmonella is
the most common cause in the United States. Shigella is known to be the third common
cause of gastroenteritis associated with bloody diarrhea (Peirano et al., 2018). Although
not as common as Campylobacter, Shigella, and Salmonella, Escherichia coli is the most
common cause of traveler’s diarrhea (Traveler’s Diarrhea, 2018) and is the fourth
common bacterial cause of gastroenteritis.
According to Gastro-intestinal infections (2018), an estimate of 3-6 million
children worldwide die each year from infectious gastroenteritis while diarrhea still
remains as the leading cause of death among young children (Diarrhoeal Disease 2018).
Gastrointestinal-related diseases are also associated with an estimated 2.2 million deaths
and is the second leading cause of death among children from 5 years old below. In the
Philippines, foodborne and waterborne diseases are steadily depreciating wherein a total
of 5,154 cases of diarrhea has been reported in 2017 which is 0.1% lower than the
previous year (2016) (WHO, 2018). January to July, 2018 statistics from the Department
of Health (2018) also shows that bloody acute diarrhea has lowered by 13% compared to
the same time in the year of 2017. However, a total number of 1,763 rotavirus infection,
which usually lead to gastroenteritis, as stated by Rotavirus Infections (2018), were
reported from January 1 to July 28, 2018. In Cordillera, it is said that the number of
cases of foodborne and waterborne diseases increased from 410 in 2005 to 2,082 in 2009,
and 1,938 in 2010. From 2005-2010, gastroenteritis has been the leading food and
waterborne disease in Baguio (National Statistical Coordination Boards, 2011). For the
first forty weeks of this year (2019) it is reported that in the Cordillera, about one
thousand three hundred forty-seven cases of diarrhea, wherein Benguet has 41.2% of the
reported cases (See, 2018).
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According to Questions and Answers (2014), most strain of Escherichia coli are
harmless and keeps the digestive tract healthy. However, some strains, usually shiga
toxin-producing Escherichia coli (STEC) or Escherichia coli O157:H7, cause severe
foodborne disease (WHO, 2018). Escherichia coli infection is usually associated with
gastrointestinal disease. However, it also causes pneumonia and is culprit to 75% to 95%
of urinary tract infections (What is E. coli?, 2018). Escherichia coli, as defined by
Biology Online (2015), are “gram-negative, facultatively anaerobic, rod-shaped species
belonging to the family Enterobacteriaceae” usually found in the “gut flora in the
intestines of humans and other mammals”.
Humans acquire Escherichia coli via lack of hygiene, touching infected animals
or their feces, and consuming contaminated meat and water (Escherichia coli, n.d.).
Although everyone is at risk of acquiring shiga toxic Escherichia coli, some people are
more vulnerable than others. Some risk factors include age, a weakened immune system,
season (usually Escherichia coli breakouts happen during summer), low stomach acid
levels, and consuming contaminated food and water (Pietrangelo, 2017). In terms of
food-related risks, beef accounts for 55% of Escherichia coli infection, vegetables cause
21%, dairy products cause 11%, while other meat causes 6% of infection (Goolsby,
2018). Symptoms of Escherichia coli may occur as early as 24 hours after infection or as
late as one week (Pevzner, 2018) and usually last from 10 hours up to 8 days depending
on the person (Escherichia coli: A-to-Z Guide from Diagnosis to Treatment to
Prevention, 2017). Although some can easily recover from Escherichia coli infection, it
can be life-threatening to people with weakened immune system and infants (Nordqvist,
2017).
The ancient civilizations were known to have relied on the power of herbal
medicines, and even the modern world has adopted some of their practices and herbal
concoctions in utilizing this alternative medicine. The use of herbal medicine expands
rapidly across the globe as people resorts to treating various health problems including
gastrointestinal problems (Tilburt & Kaptchuk, 2008). Currently, the sales of herbal
medicines are rapidly rising and widely consumed by people all over the world.
According to the reports of the World Health Organization (WHO), out of the 252 drugs
in the essential medicine list, 11 percent are exclusively of plant origin. Roughly it makes
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up over 80% of the populations in developing countries that mainly rely on herbs. It is
because herbs are less expensive and are safer than conventional medications (Pang, et
al., 2014).
One herbal medicine that is rising to fame is Blumea balsamifera. Around the
world, using Blumea balsamifera as herbal medicine is frequently observed. The leaves
of Blumea balsamifera are used in traditional Thai and Chinese medicine for the
treatment of septic wounds and other infections (Sakee U, 2011). Moreover, the whole
plant and its crude extracts, as well as its isolated constituents, display numerous
biological activities, such as antitumor, hepatoprotective, superoxide radical scavenging,
antioxidant, antimicrobial and anti-inflammation, anti-plasmodial, anti-tyrosinase,
platelet aggregation, enhancing percutaneous penetration, wound healing, anti-obesity,
along with disease and insect resistant activities (Pang et al., 2014). It is considered as a
carminative, stomachic and antispasmodic agent and has been used to treat conditions
like flatulence, dyspepsia, diarrhea, intestinal colic and dysentery. This is also widely
distributed in Eastern and Southern Asia, having its healing properties ("Sambong |
Blumea balsamifera Herbal Medicine, Health Benefits, Uses, Side Effects", 2019). The
Indian people used a decoction of leaves for the treatment of gas distention and
abdominal colic. Meanwhile, a poultice of fresh pounded leaves is applied locally to treat
hemorrhoids. The roots also have appetite stimulating properties (Globinmed.com, 2018).
In the Philippines where herbal plants species abound, it is customary for Filipinos to use
them in nursing minor sicknesses such as cough, colds, flu, infections, and other skin
infections. Though there are still many Filipinos who still question the efficacy of these
plants, medical researches and studies have already acknowledged their value in the
world of medicine.
Phytochemical studies show that Blumea balsamifera contain the chemicals
icthyothereol acetate, cyptomeridiol, lutein and ß-carotene which can potentially fight
against Escherichia coli (Sambong: Blumea balsamifera Herbal Medicine, 2016).
Another study conducted by Llido et al. (2014), states that the effectiveness of Blumea
balsamifera leaves against Escherichia coli is affected depending on the amount of
concentration. Through the said pieces of literature and studies, the researchers
 7

hypothesized that since sambong leaves have numerous biological activities, then it can
eradicate Escherichia coli.

Significance of the Study

The findings of this study would be beneficial to the community which in fact,
medicine plays an important role towards health (Alana B., 2018). Constant intake of
commercial drug may develop resistant bacteria which, consequentially, would not
respond to antibiotics that may have worked in the past (The Development of Antibiotic
Resistance Bacteria, 2019). Herbal drugs on the other hand, are safer and accessible and
may also strengthen the immune system. Commercial drugs are also not financially
flexible for everyone (International Conference on Herbal & Traditional Medicine, 2018).
Thus, this study can help individuals with gastroenteritis who cannot afford commercial
drugs to have access to a more effective and inexpensive treatment. Furthermore, this
study is essential to the researchers because they would want to determine if the Sambong
leaf extract is more effective or as effective than commercial drugs in treating
Escherichia coli. If the hypothesis was proven true, the Sambong leaf extract would be
safer and would be a more affordable alternative in treating Escherichia coli
gastroenteritis.

Objectives of the Study

This research specifically aims explicitly the following:

 To determine the effectiveness of sambong leaves extract against Escherichia coli

considering the following concentrations: 25%, 50%, 75%, and 100%.
 To compare the effectiveness of Sambong leaves extract against Escherichia coli between
and among the concentrations and Ciprofloxacin.
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MATERIALS AND METHOD

This section shows the study design, sample of the study, materials, procedures,
treatment of data, and ethical considerations that were implemented on the study.

Study Design

The study is quantitative wherein the researchers used true experimental-

descriptive design as their research method. According to Shuttleworth (2008), “true
experimental design is regarded as the most accurate form of experimental research”.
Through the said pieces of literature and studies stated in the introduction, the researchers
came to a hypothesis that sambong leaves can eradicate Escherichia coli since it has
numerous biological activities. Our study aims to prove or disprove the hypothesis by
computing the mean of the zone of inhibitions descriptively. True experimental
experiment has three requirements which were present in this study namely: the control
group (Ciprofloxacin) and experimental group (sambong leaves extract), researcher-
manipulated variable (sambong leaves extract concentration), and random assignment
(random picking of agar plates). Five groups were involved in the experiment namely:

Table 1.
Groups Involved in the Experiment

Randomly Assigned Groups Name and Concentration

Positive Control group Ciprofloxacin

A. Experimental group 1 25% Sambong extract solution
B. Experimental group 2 50% Sambong extract solution
C. Experimental group 3 75% Sambong extract solution
D. Experimental group 4 100% Sambong extract
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Sample of the Study

The researchers used a pure cultured Escherichia coli for testing the effectiveness
of the antimicrobial activity of sambong leaves and Ciprofloxacin. The pure cultured
Escherichia coli was acquired at the University of Baguio. The study only included
sambong (Blumea balsamifera) leaves extract and Escherichia coli. Inclusive criteria in
choosing the sambong leaves are the following: 7 cm to 20 cm long (Sambong, 2018),
and came from sambong plants planted on the same soil with similar condition (Variables
for Beginners, n.d.). The researchers chose to use sambong because it is readily available
and mainly because of its numerous biological activities that can potentially eradicate
Escherichia coli. The researchers gathered the sambong leaves sample from Irisan,
Baguio City.

Materials

The following materials were used for the extraction of sambong leaves, and for
culturing Escherichia coli:

Table 2
Materials and Functions
Materials Functions

Sterile Filter Paper To soak the sambong leaves extract

Vernier Caliper To measure the growth of inhibition
Tweezers Used to get the sterile filter paper
Applicator Stick Used for spreading the bacteria
Biohazard Bag For the disposal of bacteria
Lysol To disinfect the materials that were used
Alcohol Lamp To sterilize the materials used
Reagent Bottle Containers for different concentrations
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Mortar and Pestle For grinding the leaves

Graduated Cylinder For measuring the different concentrations
Blender Cut the leaves into small pieces
Cheesecloth To extract the grinded leaves
Bowl Container for the fresh, leaves extract
Glass bottle Container for the extract

Procedures

The sambong leaves were collected and extracted in Irisan, Baguio City in
accordance to our technical adviser’s instructions. Using a blender, the leaves were first
cut into smaller pieces and then grinded using a mortar and pestle. Once the leaves were
grinded, it was extracted by compressing the grinded leaves inside a cheesecloth. The
collected extract was placed in a glass jar. In the laboratory, with the assistance of a
medical technician, the extract was diluted with distilled water creating four different
concentrations: 25% (75% distilled water), 50% (50% distilled water), 75% (25%
distilled water), and 100% sambong leaves extract. For 24 hours, sterile filter paper along
with the mixture of the extract and distilled water were diluted in an agar plate. The
researchers subcultured three to five colonies of Escherichia coli from a pure cultured
Escherichia coli using trypticase soy broth (TSB). The TSB culture was incubated at
35°C to 37°C until its turbidy of MacFarland Standard was achieved. As for the
inoculation of the plate, the researchers used the Mueller- Hinton agar as a medium of
microbiological growth for antibiotic susceptibility tests. A sterile cotton swab was
dipped into the inoculum suspension to remove the excess moisture by pressing it to the
tube wall. The Mueller - Hinton agar was inoculated by spreading the agar to the entire
plate wherein there must be a consistent 60-degree spread. This was repeated twice for 3
to 5 minutes and not exceeding 15 minutes. The medical technologist conducted the
experiment while the researchers observed.
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Treatment of Data

The effectiveness of different sambong leaves extract at 25% (75% distilled

water), 50% (50% distilled water), 75% (25% distilled water), and 100%, was determined
using mean. The same method was also used in order to determine the effectiveness of
the control, Ciprofloxacin. Mean was computed by measuring the zone of inhibition on
all trials and all concentrations using a vernier caliper. The data was then compared to
one another in order to determine the effectiveness of sambong leaves against
Escherichia coli. Zone size interpretative criteria was given by the European Committee
in antimicrobial susceptibility testing (European Committee on Antimicrobial
Susceptibility Testing, 2019).

Table 3.
Zone size interpretative criteria by the European Committee
Zone Size Effectiveness

26mm or more Very Effective

24mm to 25mm Effective
24mm or less Not Effective

Ethical Considerations

As researchers, understanding the properties of the specimen is a must. There are

many potential hazards when working with Escherichia coli that may result to foodborne
epidemic like gastroenteritis, and diarrhea. Therefore, the medical technologist, who
conducted the experiment, wore protective equipments such as gloves, facemask, goggles
and lab coats to minimize the risk. In addition, proper aseptic techniques in handling the
materials were observed. All the materials used were also sterilized before and after use.
Furthermore, proper waste disposal was implemented and exercised to avoid spread of
disease. The medical technologist was in charge of culturing the bacteria. A technical
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adviser was also present during the experiment in order to ensure the implementation of
the rules stated above. The Sambong leaves extract were disposed of in a non-
halogenated organic compound amber bottles while the Escherichia coli and agar were
disposed in biohazard bags after Lysol treatment. The researchers assure that no Sambong
(Blumea balsamifera) was wasted. No human and animal were harmed during the
experiment. The results were disseminated to the community by publishing a research
paper.

RESULTS AND DISCUSSIONS

This section shows the results obtained through the conducted experiment along
with other literature which supports the results and discussions.

Table 4.
Effectiveness of Blumea balsamifera extract and Ciprofloxacin against Escherichia coli
Percentage Trial 1 Trial 2 Trial 3 Mean Remarks

25% 6mm 6mm 6mm 6mm Not Effective

50% 6mm 6mm 6mm 6mm Not Effective
75% 6mm 6mm 6mm 6mm Not Effective
100% 6mm 6mm 6mm 6mm Not Effective
Ciprofloxacin 35mm 42mm 41mm 39.33mm Very Effective

Table 4 shows the antimicrobial activity of sambong leaves extract and

Ciprofloxacin against Escherichia coli. All 25% (75% distilled water), 50% (50%
distilled water), 75% (25% distilled water), and 100% sambong leaves extract has a 6mm
zone of inhibition. Ciprofloxacin, in contrast, has an average of 39.33 mm zone of
inhibition. According to the European Committee in antimicrobial testing, a
microorganism is susceptible with a zone of inhibition of 26mm or more, intermediate at
24mm-25mm, and resistant at 24mm or less. From this, the data gathered shows that
 13

Escherichia coli is resistant to sambong leaves extract and Ciprofloxacin displayed a

more significant activity compared to the given concentrations. All 25% (75% distilled
water), 50% (50% distilled water), 75% (25% distilled water), and 100% sambong leaves
extract were ineffective against Escherichia coli, and Ciprofloxacin is far more effective
in eradicating Esherichia coli than all the given concentrations.
However, according to Pang et al. (2014), Blumea balsamifera displayed a
significant activity against Escherichia coli while another study conducted by Dano &
Maratas (2017) stated that Blumea balsamifera contains 10%-30% alkaloids which
displays antimicrobial activity against both gram-positive and gram-negative bacteria
(Ayandele and Adebiyi, 2007). In terms of antimicrobial activity of differing
concentrations, Deen (2016) mentioned that concentrations of 50% and 75% are usually
more effective in eradicating bacteria compared to concentrations of 25% and 100%. It is
because a concentration of 25% is too weak to eradicate a microorganism while a 100%
concentration creates a protein layer which protects the membrane of the microorganism
thus causing the microbes to remain in dormant stage. Several factors have contributed to
the negative results of the study.
First, fresh sambong leaves were used for extraction, but according to Simon
(2017), “dried plant materials are common and best to extract phytochemicals”.
Azwanida (2015) supports the aforementioned study by emphasizing that although both
dry and fresh samples showed no significant effect in total phenolics, dry samples contain
more flavonoids. Sunmonu, Orhevba, and Yusuf (2015), also states that dry samples
contain higher nutritional activities compared to fresh samples. Another evidence
supporting the advantage of using dry plant samples is that fresh samples are fragile and
tend to deteriorate faster than dried samples (Azwanida, 2015). Simon (2017) also
emphasized that “wet sample produce more viscosity and lack separation of secondary
metabolites corresponding to polarity of the solvent used”. In other words, fresh samples
may also be used for experiments, however, laboratory extraction is needed to extract the
desired phytochemicals.
This leads to the second factor wherein the researchers failed to do another
extraction method besides grinding. Nichols (2019) enumerated the importance of
laboratory extraction: isolates compounds from plant materials, makes the extract easily
 14

manipulated or concentrated, and enables the selective removal of components in a

mixture. The last factor affecting the results was using a clear, glass jar for storing the
extracted sambong leaves instead of a non-halogenated organic compound amber bottle.
Upadhyay (2015) emphasized that green, fresh leaves contain chloroplast, active
metabolic and protein synthesizing machinery which can possibly form new compounds
in response to light and other factors. Improper handling of the plant extract caused it to
be exposed to sunlight and the clear glass container further added to the damage.

CONCLUSION AND RECOMMENDATION

According to World Health Organization, gastroenteritis is one of the leading

cause of death and although a vast amount of gastroenteritis case is not fatal, it still
causes a massive amount of morbidity and economic burden. This research aims to look
for an affordable and safer alternative in treating gastroenteritis. In order to achieve that,
the researchers investigated the effectiveness of sambong leaves extract against
Escherichia coli considering the following concentrations: 25% (75% distilled water),
50% (50% distilled water), 75% (25% distilled water), and 100% sambong leaves extract.
The study also compared the effectiveness of Sambong leaves extract against Escherichia
coli between and among the concentrations and Ciprofloxacin.
Several studies support the research’s hypothesis, however, the conducted
experiment manifested a negative result. Results show that all four given concentrations
have a 6mm zone of inhibition and in contrast, Ciprofloxacin has an average of 39 mm
zone of inhibition. From the results, it can be concluded that Escherichia coli is resistant
to sambong leaves extract at 25%, 50%, 75%, and 100% concentration based on the
interpretative criteria given by the European Committee in antimicrobial testing.
Ciprofloxacin also displayed a more significant activity against Escherichia coli
compared to the given concentrations. To answer the study’s objectives, the results
indicated that all 25% (75% distilled water), 50% (50% distilled water), 75% (25%
distilled water), and 100% sambong leaves extract were ineffective against Escherichia
coli, and Ciprofloxacin is far more effective in eradicating Esherichia coli than all the
 15

given concentrations. Factors that contributed to the negative results of the study are
namely: pre-extraction preparation of plant samples, extraction method, and storage of
the extract.
From the conducted experiment, the research recommends that future researchers
observe correct pre-extraction preparation of plant samples, use right extraction method,
and properly store the extract in replicating this study. The researchers suggest utilizing
other parts of the sambong plant since leaves were only used in the experiment. It is also
advised to use dried plants sample and rotary evaporation as an extraction method. The
research also suggests using non-halogenated organic compound amber bottles in storing
the extract to avoid the sample from spoiling in reaction to light.
 16

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