Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

ANTIBACTERIAL
ACTIVITY of DMSO
CRUDE EXTRACTS
of ALOE VERA
(Aloe perfoliata) GEL
AGAINST
Escherichia coli

LIFE SCIENCE-A
Montalban, Ritz Jann R.
Villanueva, Jesse Ray A.
Madrigal, Maribeth S.
Montesa, Frency Hara M
10 - Aristotle
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

CHAPTER I
THE PROBLEM: RATIONALE AND BACKGROUND
Rationale

Aloe Perfoliata commonly known as aloe vera is a succulent plant species of the
genus Aloe. It originates from the Arabian Peninsula but grows wild in the tropical climates
around the world and is cultivated for agricultural and medicinal uses. Aloe Vera gel contains
powerful antioxidants which belong to a large family of substances known as polyphenols.
Polyphenols act as antioxidants. They protect cells and body chemicals against damage caused
by free radicals, reactive atoms that contribute to tissue damage in the body.

Polyphenols can also inhibit the growth of Escherichia Coli. Most Escherichia Coli
strains are harmless but some serotypes can cause serious food poisoning in their hairs, and are
occasionally responsible for product recalls due to food contamination. Other infections or
diseases it can cause are diarrhea, urinary tract infection (UTI) and pneumonia. Diseases due to
E. Coli represent a critical problem to human health. They are one of the main causes of
morbidity and mortality worldwide. The resistance developed by the bacteria to various
antibiotics restricts he choice of antibiotics for therapies.

Antibacterial resistance is a serious threat to mankind because of the infection causing

bacteria has become multidrug resistant. This adds urgency to the research for new infection
fighting strategies. In recent years, the antibacterial properties of the medicinal plants are being
increasingly reported from different parts of the world. Therefore, researchers have turned their
attention to plants to develop better drugs against bacterial infections.

The researchers have decided to use aloe vera that is abundant to tropical countries
such as Philippines to have a proper and sustainable a accessibility while conducting the
research in addition, aloe vera is relatively common in multiple hygienic products; soap,
shampoo and the likes.

This study aims to determine if aloe vera extract has antibacterial properties against
Escherichia Coli.
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

Statement of the problem:

This study will be conducted to assess the antibacterial activity of aloe vera
(Aloe perfoliata) against Escherichia coli.
Specifically the study sought to answer the following questions:
a. Will the gel extract of Aloe Vera be able to kill the bacteria Escherichia coli?
b. At what rate does Aloe Vera extract kill Escherichia coli?
Objectives:
 To find the effectiveness of aloe vera extract as an antibacterial based on the rate
at which it extinguishes bacteria
 The study aims to examine the antibacterial activity of the extracts of Aloe
perfoliata gel against Escherichia coli.
Research Objectives:
This study will aim to determine the following objectives:
 To find the effectiveness of aloe vera extract as an antibacterial based on the rate at which
it extinguishes bacteria.
 To examine the antibacterial activity of DMSO crude extracts of Aloe perfoliata gel
against Escherichia coli.

Hypothesis:
Alternative Hypothesis:
There will be significant difference between the antibacterial agent and agent
causing bacteria
Null Hypothesis:
There will be no significant difference between the antibacterial agent and agent
causing bacteria

Significance of the Study:

The findings of this study will result to the benefit of society considering that people use
hygienic products have antibacterial properties of their daily use. The greater demand for said
products implies that more resources will be needed to create such products, Specifically the
antibacterial agent in each product. Thus, factories or companies that may adopt the findings
derived from the results of the study may use the undistinguished effectiveness of Aloe Vera
(Aloe perfoliata) as an alternative antibacterial agent for their products. For the researchers, the
study may help them uncover the critical potential of aloe vera that the other researchers may not
have been able to explore.

Scope and Limitations

The study is limited in specific parameters such as the study’s:
Content – The study focuses on the antibacterial properies of aloe vera as an antibacterial
agent.
Subject – the study will use the succulent plant, aloe vera.
Time – the study will be conducted in a week period.
Place – the production will be carried out in the laboratory of the researcher.
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

Definition of terms
 Antibacterial
A property of a substance that could suppress the growth and reproduction of a
bacterium.
 Aloe Vera
A succulent plant species of the genus Ale. A gelatinous substance obtained
from a kind of aloe, used especially in cosmetics as an emollient and for the treatment of
burns.
 DMSO
Dimethyl sulfoxide is an organosulfur compound with the formula (CH3)2SO.
This colorless liquid is an important polar aprotic solvent that dissolves both polar and
nonpolar compounds and is miscible in a wide range of organic solvents as well as water.
 Escherichia Coli
A gram-negative, rod shaped, colitorm bacterium that is commonly found in
the intestines of warm blooded organisms.
 Zone of Inhibition
It is the celar area without any presence of growth surrounding the filter
paper and correlates to the sensitivity of bacterium to an antibiotic

Theoretical Framework

Independent Variable Dependent Variable

The DMSO crude extracts of Aloe The effect of DMSO extracts of Aloe Vera
Vera (Aloe Perfoliata) gel applied to gel to E. Coli
Escherichia Coli

Control Variable

The different extract concentration

applied to the pathogen

Experimental Variable
The zone of inhibition of Escherichia Coli

According to R.J. Malar et al. (2012) reported intheir study that aloe vera is used to treat stomach
ailments, gastrointestinal problems, skin disease, constipation, radiation injury in flammating
effect and diabetes.
(Davis, 1997) concentrated extracts of Aloe Vera are used as laxative and as a
hemmorhoid treatment.
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

Chapter II
Review of Related Literature
A. Antibacterial properties on plants
Plants and plant products have been used as medicines since the star of history. Many
researchers have conducted research on the plant products to check their antimicrobial effects
(Abu-shanab et al., 2004). The use of plant extracts and phytochemicals both with known
antimicrobial properties is of great significance, in the past few years a number of investigations
have been conducted worldwide to prove antimicrobial activities from medicinal plants.
(Alonzo-Paz et al., 1995) ( Nascimento et al., 1990).

The extensive utilization of plants in the medical field has been very beneficial to the
constant development of effective medicines particularly those which are mainly derived from
plants. The bioactive compounds present in various extracts of plants are continuously being
studied all across the globe for more acquisition of therapeutic treatments against a wide array of
diseases principally on disease causing bacteria. In line with this, numerous discoveries have
been made. For instance, studies have revealed the antimicrobial activities of the gel and leaf
of Aloe vera against a wide range of bacteria (Agarry et al., 2005). Similarly, bearberry and
cranberry juice have been widely utilized for their ability in treating urinary infections. Plant
species such as lemon balm, garlic and tea tree are recognized as broad-spectrum antimicrobial
agents (Rios and Recio, 2005). The aforementioned findings are only some of the cases that
exhibit the potent potentials of plants against several bacterial infections.

With the feasible results of the use of plants against different diseases, World Health
Organization has tightened its policy and its support even more on the use of medicinal plants
and products. The discovery of penicillin paved the way to the discovery and production of other
antibiotics such as streptomycin, aureomycin and chloromycetin. The same goes with the
discovery of the antibacterial properties found in plants which led to the intensive interest to
further study the phytochemicals present in plants in order to develop plant derived antibiotics.
Furthermore, the main groups of antimicrobial compounds from plants which are called
secondary metabolites such as alkaloids, steroids, tannins, and phenol compound (Ciocan et al,
2007) have been the most effective phytochemicals against bacteria .

B. Aloe Vera (Aloe perfoliata) as an antibacterial succulent plant

Aloe Perfoliata, sabila as it is locally known, is a succulent plant species of the genus
Aloe. It originates from the Arabian Peninsula but grows wild in the tropical climates around the
world and is cultivated for agricultural and medicinal uses. Aloe Vera gel contains powerful
antioxidants which belong to a large family of substances known as polyphenols. Polyphenols
act as antioxidants. They protect cells and body chemicals against damage caused by free
radicals, reactive atoms that contribute to tissue damage in the body.
Polyphenols can also inhibit the growth of Escherichia Coli. Most Escherichia Coli
strains are harmless but some serotypes can cause serious food poisoning in their hairs, and are
occasionally responsible for product recalls due to food contamination. Other infections or
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

diseases it can cause are diarrhea, urinary tract infection (UTI) and pneumonia. Diseases due to
E. Coli represent a critical problem to human health. They are one of the main causes of
morbidity and mortality worldwide. The resistance developed by the bacteria to various
antibiotics restricts he choice of antibiotics for therapies. (Berger, A. et al., 2017)
R.J. Malaret al. (2012) reported in their study that aloe vera I used to treat stomach
ailments, gastro intestinal problems, skin disease, constipation, radiation injury, inflammatory
effect and diabetes. Concentrated extracts of aloe vera are used as laxative and as a hemorrhoid
treatment. (Davis, 1997)
C. Escherichia coli
The genus Escherichia coli belongs to the bacterial family Enterobacteriaceae, class
Gamma Proteobacteria which are considered to be unicellular microorganisms. E.coli is a Gram
– negative rod-shaped bacterium with an outer membrane which primarily comprises
lipopolysaccharides. (NODC Taxonomic Code,1996). Escherichia coli or simply referred as E.
coli is characterized as the most studied free-living organism. It was named after Theodor
Escherich, who discovered the gram negative bacteria which was the cause of infant diarrhea that
was common at their time. Furthermore, most type of this bacteria does not usually cause
diseases instead they were beneficial of regulating the small intestine’s activities. Some E. coli
that yields Shiegan toxins caused multiple dysenteries, thousands of hospitalizations and
hundreds of casualties. Shigan toxin-producing E. coli, was the most notorious type that induced
various forms of illnesses. (Marler Clark, 2018). E. coli has been reported worldwide. Treatment
for E. coli infection has been increasingly complicated by the emergence of resistance to most
first-line antibacterial agants. Over the years, resistance to cephalosporins among members of
enterobacteriaceae has increased mainly due to the spreading of extended-spectrum β-Lactamase.
(M.U. Rasheed, N Thajuddin, P. Ahamed, Z.Teklemariam, and K. Jamil, 2014).

D. Antibiotic Sensitivity Testing

As characterized by Dr. T.V. Rao, it is a laboratory test that measures the effectiveness
of antibiotic rectifier upon a bacterial infection. It is used in determining the local pattern of
prescribing antibiotic and it is mainly for presenting the change trend in the local isolates.
Methods include the following: obtaining a bacterial sample, selecting antibiotics to be
prescribed based on the type of the bacteria and lastly, obtaining a control sample from bacterial
sample. After the World Health Organization committee had the standardized disk diffusion
susceptibility test it was then called Kirby-Bauer disk diffusion test. The Clinical Laboratory
Standards Institute (CLSI) updates and modifies the original procedure of Kirby-Bauer through a
global consensus process in order to maintain the uniformity of the technique and reproducibility
of results. Interpretative guidelines for zone sizes are included in their publications. This
represents the standard for performing susceptibility testing a of American Society for
Microbiology. The pathogenic organism is grown on sterile agar in the presence of various
antibacterial impregnated filler paper disks. Through this, zone of inhibition is determined and
basically the only standard test that will be utilized for this research study.
E. DMSO (Dimethyl sulfoxide) crude Extract of Aloe Vera
Bandaru Narasinga Rao et al (2012) reported in their study that Dimethyl sulfoxide is an
organosulfur compound. It is an important polar aprotic solvent that dissolves both polar and
non-polar compounds and is miscible in a wide range of organic solvents as well as water. It is
extensively used as an extractant in biochemistry. In the present study, aimed to examine the
antimicrobial activity of DMSO aloe gel extract against some selected microbial pathogen.
 Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

Chapter III
Methodology
A. Research Locale
The research study will be done within the premises of Mandaue City Science
High School and around Mandaue City

B. Research Design
This research will be an Experimental quantitative study. This will deal with the
effectiveness of antibacterial properties of Aloe Vera (Aloe Perfoliata) against Escherichia Coli
in which will be performed at Mandaue City Science High School.
Collection/
Preparation of
Concentration Materials
1

Experimental Validating the

Extract Concentration Data Collected
Group (Aloe
perfoliata) 2

Analysis and
Concentration
Interpretation of
3
Data

Drawing of
conclusion

Collection/
Replication Preparation of
1 Materials

Positive Control Extract Replication Validating the

(Amoxicillin) 2 Data Collected

Replication
Analysis and
3
Interpretation of
Data

Drawing of
conclusion
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

C. Materials and Methods

C.1.1 Producing the Aloe Vera Gel Extract
The materials that will be needed to produce DMSO extracts of Aloe Vera gel
are Aloe Vera Leaves, this can be found in the neighborhood of Mandaue and many other places
in Cebu, Philippines. Other materials are distilled water for cleansing, sterile knife, mortar and
pestle.
C.1.2 Culturing the E. Coli Bacteria
The materials that will be used to culture E. Coli bacteria are inoculating loop
for cultivating microbes, bunsen burner for sterilization, nutrient agar or other alternatices like
old coconut, petri dishes for storage, an incubator and an autoclave for the disposal of the culture
medium.
C.1.3 Preparation of Different Concentrations.
The materials needed will be 3 vials for storage, 50 mL bottle of distilled water,
graduated cylinder for measuring the Aloe Vera gel extract.
C.1.4 Preparation of Inoculum
The material that will be neededare sterile saline solution and the inoculating
loop.
C.1.5 Paper Disc Method
The materials needed will be agar plates, sterile disks and incubator
C.1.6 Disposal of Culture Medium
The material needed will be an autoclave for sterilizing the nutrient agar.
C.1.7 Determination of Antibacterial Activity
The materials needed will be glass ruler for measuring, refrigerator for storage
and the different concentrations of aloe vera gel extract.

C.2. Procedures
C.2.1 Collection of Aloe Vera Leaves
Leaves of Aloe Vera will be collected from the houses of in and around Mandaue City,
Cebu, Philippines.
C.2.2. Extraction of Aloe Vera Gel
Mature, healthy and fresh leaves of A. Vera will be washed in running tap water for 5
minutes and rinsed with sterile distilled water, then will be dissected longitudinally and the
colourless parenchymatous tissue (Aloe Vera Gel) will be scraped using a sterile knife without
the fibbres. The gel will be gounded with DMSO (DMSO is dangerous, it can cause vomitting,
nausea and diarrhea when taken) using the mortar and pestle. The extrac will be centrifuged at
5000 rpm for 5 minutes. The resulted fluids will be named DMSO crude extract of Aloe Vera
gel.
C.2.3. Culturing of bacteria
Escherichia Coli, E. Coli is a bacterium that grows in the lower intestines of mammals.
This bacteria is the most widely used organism in molecular genetics. Part of the reason E. Coli
is commonly used in scientific research is that it is easy to grow in a lab.
First, the researchers have to sterilize the inoculating loop by placing it in the flame of
the bunsen burner. Pass the lower half of the loop through the flame until it glows red. Allow the
loop to cool. You can touch it to the sterile agar on the plate to make sure it has cooled down. Do
not place the loop on the table or let it contact anyting other than sterile agar or the decided
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

culture. Now that it has been sterilized. Dip the loop into the E. Coli culture and then remove it.
Open the agar plate and gently glide the loop back and forth across the surface of one section of
the agar. Take care to not scratch through the agar with the loop. The agar provides the nutrients
the E. Coli need to grow.
Place the loop in the bunsen burner flame again to sterilize it. Once the loop rod cools,
touch it to a sterile section streak. This second streak is a divided version of the first streak.
Repear this sterilization and gliding process until several sections of the agar plate have been
glided over. The reason to do this is so the researcchers can later pick from single, clonal
colonies- not to much concentrated(which will have too much growth, and not allow you to pick
from a single colony) and not too dilute (Which would give no colonies).
Sterilize the loop in the flame one last time before putting it aside in the work area. Put the
top back onto the agar plate. Turn the plate upside down and place it into the incubator for set to
37 degrees Celsius. This ideal incubation temperature simulates the temperature of the human
body where E. Coli reside. Whithin 24 hours to 48 hours, visible colonies of E. Coli bacteria will
appear in the agar plate.
C.2.4 Preparing the Inoculum
24hrs culture of E.coli will be obtained. The microorganism will be inoculated to the
sterile saline solution on tubes using inoculating loop until it becomes comparable to the 0.5
McFarland Standard. The bacterial suspension will be performed using aseptic techniques inside
the biological safety cabinet level 2
C.2.5 Paper Disc Method
Nutrient agar will be cooled to 50-55C. The cooled medium will be poured to the
individual sterile petri dishes. Inoculum of test bacteria which is comparable to the standard will
be swabbed uniformly on solidified sterile agar plates using sterile cotton swab. Different
concentration of extracts (25%,50% and 75%) will be prepared by diluting the crude extracts
with distilled water. The sterile disks of 6 mm diameter will be soaked to the prepared extract.
The soaked disks will be placed on the swabbed plates aseptically using sterile forceps. Forcep
will be sterilized by soaking it in 80% ethanol and will be passed unto flame. The inoculated
plates will be incubated at 37°C for 24 h for bacteria in upright position and the zones of
inhibition that will be formed around will be measured using a glass ruler. Known antibacterial
such as Amoxicillin was introduced to the plates after the preceding procedure. Diameters of the
inhibition zone by the extract and the known drug will be compared.
All procedures will be done in three trials with three replicates at the Senior High
Science Laboratory, Mandaue City Science High School, Mandaue City, Cebu, Philippines.
C.2.6 Disposal of Culture Medium
All inoculated plates were then disposed by decontamination process. It was conducted
by sterilizing the nutrient agar with bacteria in autoclave for 121 degrees Celsius for 15 minutes.

C.2.7 Determination of Antibacterial Killing

The supernatant will be conducted and stored in refrigerator at 4 degrees Celsius.
Different concentration of A. Vera gel extract will be subjected to antibacterial studies. Pure
bacterial culture will be obtained from Mandaue City Science School Laboratory, Mandaue City.
A bacterial culture of E. Coli will be maintained in nutrient agar medium at room temperature
and will be subcultured into newly prepared nutrient agar slants. Antimicrobial activity will be
carried out by disc diffusion method against selected pathogen. The crude DMSO extracts were
used for bioassay against baceria. Sterile discs with 6mm diameter will be loaded with 1, 2, and 4
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

mL of gel DMSO extracts and will be introduced into the sterile medium with the test organisms.
The plans will be incubated at 37 degrees Celsius for 24 hours. Antimicrobial activity will be
evaluated by measuring the zone of inhibition. A glass ruler will be used to measure the zone of
inhibition. All the process will be repeated thrice and results will be recorded.

C.3 Related Study’s Data

Table 1; Anti-Bacterial Activity of the DMSO Aloe Vera gel Extracts
Name of Pathogen Conclusion of DMSO Aloe Vera gel extract in mL and zone of
inhilation in mm.
25% 50% 75%
Escherichia coli 10 12 13

DMSO gel extracts of Aloe Vera were screened for the antibacterial activity against a
human pathogen and the result is given in Table 1. The antibacterial activity has been observed
in the DMSO gel extracts of Aloe Vera against the tested bacteria with varied concentration. The
maximum zone of inhibition of 13mm for E.Coli was observed. All three different
concentrations (25 % 50% and 75%) of DMSO gel extracts of Aloe Vera showed the inhibitory
effect on the pathogen with the maximum zone of inhibition in the highest concentration.

D. Analysis of Data
One-way ANOVA test will be used to determine the significant difference between
the antibacterial agent and agent-causing bacteria.
Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli

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Kedarnath, Kaveri, M , Vishwanath , B (October 2013) Antimicrobial Activity of Aloe Vera

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Malar, R.J., Johnson ,M, Beaulan, N, Laju, R.S., Anupriya, G, et al. (2012, March 03) Anti-
Bacterial and Anti-fungal Activity of Aloe Vera Gel Extract. Retrieved from International
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Ribnicky DM, Komarnytsky S, Ilic N, Poulev A, Borisjuk N, Brinker A, et al. (December

20, 2002) Plants and human health in the twenty-first century. Retrieved from NCBI.

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Antibacterial Activity of DMSO Crude Extracts of Aloe Vera (Aloe perfoliata) Gel Against Escherichia coli