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Antimicrobial Property of Hagonoy Plant (Chromolaena odorata) Extract against
Klebsiella Pneumoniae
Thesis · March 2017
DOI: 10.13140/RG.2.2.10548.94087
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Angeles University Foundation
Angeles City
Integrated School
Biological Research
Antimicrobial Property of Hagonoy
Plant (Chromolaena odorata) Extract
against Klebsiella Pneumoniae
Submitted by:
Manaloto, Ysabelle (Principal Author)
Balatbat, Clarence
Garcia, Sam Rose
Lingad, Jester
Magcalas, Jeremiah Earl
Martinez, Regine
Romero, Arielle Rae
Grade 11 – St. Andrew
March 27, 2017
ANGELES UNIVERSITY FOUNDATION
Angeles City
Integrated School
A partial fulfilment for the final requirement for Biostatistics and General Biology II
“Antimicrobial Property of Hagonoy Plant (Chromolaena odorata) against Klebsiella
pneumoniae”
Manaloto, Ysabelle (Principal Author)
Balatbat, Clarence
Garcia, Sam Rose
Lingad, Jester
Magcalas, Jeremiah Earl
Martinez, Regine
Romero, Arielle Rae
Grade 11- St. Andrew
March 27, 2017
Acknowledgement
We would like to thank God for giving us all the strength and knowledge to be able to
perform this study. We would like to acknowledge the presence of our proctors who supported
us throughout the process. We would also like to thank Dr. Annaliza Molina and her students for
providing us assistance for the betterment of the study.
Abstract
The increasing resistance of different infections against the continued use of systemic
and topical antimicrobial agents should be given proper attention. This problem gave
antimicrobials that came from plants, also known as herbal medicines, to receive increasing
attention and be used in formulation of new medicines. The objective of the study is to prove the
antimicrobial activity present within the leaves of Chromolaena odorata. Leaf samples of C.
odorata, also known as hagonoy plant, were collected in Bamban, Tarlac. The ethanolic extract
of C. odorata was tested against gram negative, Klebsiella pneumoniae to identify its
antimicrobial effectiveness. The Kirby-Bauer Agar Disc Diffusion which is oftenly used for test
bacterial susceptibility to antibiotics was used for this experiment. The extract was compared to
two positive controls, piperacillin and ceftriaxone, and one negative control and was measured
in mm after 24 hours. To examine the difference between the inhibition of the extract with the
controls, T-Test Independent was used. In result, the plant extract showed no significant
difference between the positive control (P>0.05) which suggest that the ethanolic plant extract
of C. odorata is an effective source of antimicrobial activity. The mean results of the extract had
surpassed the total growth inhibited by the positive control, piperacillin, while the other positive
control, ceftriaxone, maintained the highest inhibition to the test organism, Klebsiella
pneumoniae. The results against the test organism have proved the presence of antimicrobial
activity within the leaves of C. Odorata which suggest to further evaluation about the contents of
the plant extract.
Chapter I
Introduction
Folk medicine has been using herbal and natural products throughout the world for
centuries because of present biological active compounds present therein that give protection to
them against microbial and insect attack (Kuruvilla, 2002). Herbal plants or also called as
medicinal or traditional plants are known all over the world. In fact, based on the World Health
Organization, there are approximately 60-80% of the world’s population that relies on traditional
medicines for treating common illnesses (WHO, 2002). It is estimated that there are 250,000 to
500,000 species of plants on earth (Borris R. P., 1996). It is also common for us to use
antibiotics in order to cure different illnesses but this practice has side effects not only to our
different body organs but continuous use of antibiotic can lead the bacteria to become resistant
to antibiotics, which is why pharmaceutical companies explore the field of traditional medicine in
order to discover and to obtain new sources of medicine.
Chromolaena odorata, is a Perennial scandent or semi-woody shrub that is used as
cough remedy and as ingredient together with lemon grass and guava leaves to heal malaria
(Doss A et al., 2011). Although it was used for traditional medicine, it’s still considered as a
harmful weed in the recent past for its invasive and allelopathic nature. In order to prove its
importance for traditional medicine, the researchers conducted this study to reveal the
effectiveness of C. odorata which is considered as one of the world’s worst tropical weed (R.
Munniapan et al., 1998), against gram negative, Klebsiella pneumoniae, which is often
associated why healthcare diseases like pneumonia, urinary tract infection, wound infections,
bloodstream infections, surgical infections, and meningitis (NNIS System, 2004). The bacteria to
be used for this study, is a nosocomial bacteria that was gathered from patients of Angeles
University Foundation Medical Center.
Statement of the Problem
This study was conducted to evaluate the effectiveness of antimicrobial property of C.
odorata and to furthermore answer the following questions:
1. Is there significant difference between the zone of inhibition of Hagonoy plant extract
and negative control against Klebsiella Pneumoniae?
2. Is there a significant difference between the zone of inhibition of hagonoy plant extract
and Piperacillin?.
3. Is there a significant difference between the zone of inhibition of hagonoy plant extract
and Ceftriaxone?.
Formulation of the Hypothesis
The researchers hypothesized that C. odorata is not effective for antimicrobial testing.
The researchers also formulated that:
1. There is no significant difference between the zone of inhibition of Hagonoy plant
extract and negative control against Klebsiella pneumoniae.
2. There is no significant difference between the zone of inhibition of Hagonoy plant
extract and Piperacillin against Klebsiella pneumoniae.
3. There is no significant difference between the zone of inhibition of hagonoy plant
extract and Ceftriaxone against Klebsiella pneumoniae.
Significance of the Study
As number of antibiotics became less effective against antibiotic resistant bacteria, the
need of new formulations of possible medications is widely needed in order to continuously
resist against certain bacteria. The significance of this study is to provide new possible source of
antimicrobial property which can be used as an alternative for commercially produced
antibiotics. This study provides evaluation on the importance of Chromolaena odorata for the
society and to prove the presence of antimicrobial property on its leaves. This study will show
comparison between the Hagonoy plant extract and pharmaceutically formulated antibiotics
which are used to heal bacteria inflicted diseases to prove effectiveness of the plant extract as
antimicrobial agent.
Conceptual Framework
Input Process Output
Gathering of Data, A. Experimental Design Difference between the zone
Specimens, and Samples B. Kirby-Bauer Disc of inhibition of plant extract
Diffusion method for against controls.
susceptibility
Testing.
C. Comparison of
effectiveness of
plant extract to
different controls
Scope and Delimitation of the Study
Hagonoy plant extract is to be used for this study against one human pathogen,
Klebsiella pneumonia. This study focuses on the effectiveness of C. odorata as a medicinal
plant by testing it against K. pneumoniae, by using Kirby-Bauer disc diffusion method. The
extract would then be compared to two positive controls and one negative control. The two
positive controls are piperacillin and ceftriaxone while for the negative control, broth solution
would be used. This study wants to contribute knowledge about the effectiveness of medicinal
plants to heal various diseases which can be source to different medications.
Chapter II
Review of Related Literatures
The use of therapeutically made antibiotics, are commonly used up until now in order to cure
different types of diseases. According to S. Ravikumar et al., Treatment with these available
antibiotics which are often used can lead to resistance among pathogenic bacteria that can lead
to much greater treat. In addition to this, Bacanegra-Gracia et al., stated that overuse and
misuse of these antibiotics may cause side effects that can harm vital organs. In order to treat
infections which are often caused by different bacteria, the use of antibiotic drugs which are
superficially formulated to kill infection causing bacteria is needed. But based on Fish’s study in
the year 2002, some bacteria have developed antibiotic resistance, which means that they may
not respond to many common antibiotics. Generally, bacteria has a genetic ability to transmit
and acquire resistance to drugs. That’s why pharmaceutical companies nowadays are
developing new kinds of antibiotics to replace those that are no longer effective (Silbergeld E.
K., 2008).
One way to prevent bacteria from being resistant to antibiotics is by creating new
compounds that are not based on existing synthetic antimicrobial in accordance to the study of
Shah. This has led other pharmaceutical companies to develop varieties of medicines and
antibiotics with the use of traditional herbs which are used as traditional medicine which is
considered as the oldest healthcare known to mankind. According to Cammune et al., in 1992,
the ability of the plants to grow on different types of soils which are rich in microorganisms,
gives potential to the plant to produce wide range of selective anti-bacterial compounds which
enables them to wade off potential microbial invaders. One medicinal plant which showed
different antimicrobial properties and is used as medicinal plant is Chromolaena odorata or also
known as the Hagonoy Plant.
Chromolaena odorata is commonly known in
the south and western
stern part of Nigeria, as well as in
other parts of West Africa, wherein it is considered
as a toxic weed. According to Doss et al. in 2011,
the leaf of C. odorata is used as cough remedy
and an ingredient to cure malaria together with
Figure 1. Chromolaena odorata
lemon grass and guava
va leaves. It was stated n the study of Vital and Wendell in 2009 and the
study of Suksamrarn in 2004, that the other traditional medicinal uses of C. odorata includes
anti-diarrheal,
diarrheal, astringent, antispasdomic, antihypertensive, anti
anti-inflammatory,
inflammatory, diuretic tonic,
antipyretic, and heart tonic. Many researchers also
revealed other ability of the leaves of C. odorata as
antimicrobial (M. M. Iwu et al.,, 1984), antifungal (A.
Ngono Ngane et al.,, 2006), wound healing (T. T. Phan
et al., 2001) and as cure for burns,
burn soft tissue wounds,
and skin infections (Lovet T. Kigigha et al., 2013).
Figure 2. Bush of C. odorata
Studies which were conducted in order to test the antimicrobial and antibacterial property of
C. odorata had shown positive results like for the study of S. Esath Natheer et al. wherein C.
odorata is being tested alongside with two other plants, C. odorata showed maximum
antibacterial activity as compared to the two other plants. It was also concluded in the study of
Udonsi that C. odorata is a highly valuable medicinal plant having
having different compounds with
antioxidant, anti-inflammatory,
inflammatory, and other activities proving the folklore use of this plant by the
people.
Chapter III
Research Methodology
The methodology section of this study includes the following processes: Collection of
Specimen, Collection of Bacteria, Preparation of the Leaves, Preparation of Ethanolic
Extract, Preparation of Media, Decontamination, Preparation of Disc and Susceptibility
Testing. Statistical tool would be used for testing the hypothesis.
Research Design
For this study, experimental design would be used by the researchers in proving the
presence of antimicrobial property of C. odorata by using different controls which will be
compared to its ethanolic extracts.
Flow Chart
Collection of Leaf Collection of
Samples of C. Bacteria on Urine Preparation of the
odorata Samples of Leaves
Patients
Preparation of the Converting Leaf
Media Samples to
Decontamination (Nutrient Agar) Ethanolic Extract
Susceptibility
Testing
Preparation of Disc (Measuring of
Zone of Inhibition)
Research Procedures
Collection of Specimen
The Hagonoy plant commonly known as C. odorata is used as herbal medicine here in
the Philippines. Two sacks of leaf sample were obtained from Bamban, Tarlac, Philippines.
The plant extract of C. odorata was provided by the study of Dr. Annaliza Molina.
Collection of Bacteria
The test bacteria was funded by the study of Dr. Annaliza Molina that was obtained from
Angeles University Foundation Medical Center. Test subject K. pneumoniae (D16-141) is
collected from the urine samples of the patients.
Preparation of the Leaves
The leaves gathered were washed in order to reduce microbial load present in the
samples. After reducing the microbial load of the leaves, it was then air dried until it became
brittle. Dried leaves of hagonoy plant were cut into fine pieces by using a Blender.
Preparation of Ethanolic Extract
Leaves where then soaked with 1L of 98% solution of ethanol. After sealing the mixture,
it was left for 48 hours in order for the leaves to absorb the ethanol. After 48 hours, the
mixture was filtered using a glass funnel, filter paper and beaker. The filtered extract
obtained was evaporated with the use of rotary evaporator until the mixture become
concentrated. The purpose of this is to remove excess ethanol within the solution producing
Preparation of Media
Nutrient Agar was used as media for Klebsiella pneumoniae. The solution was prepared
on an Erlenmeyer flask. 250mL of distilled water was added to 7g of Nutrient agar and was
mixed while heated on a hot plate using a magnetic stirrer until boiling point.
Decontamination
All the equipments to be used were washed. In order to further decontaminate the
equipments, including the ones, that contain the solutions, they were heated inside the
autoclave, but before placing them inside the autoclave, cover the equipment that does not
contain any solutions with paper while the ones that contain solutions, cover the lid with foil
and cotton then seal with an autoclave tape. The autoclave tape will then determine whether
the solution was decontaminated after heating.
Figure 3. Decontamination using Autoclave
Preparation of Disc
Whatman I filter paper with six layers, were perforated using a decontaminated puncher
producing discs with 7mm in diameter. Preparation for Positive Control and Negative Control
Discs: 30 /g of ceftriaxone, piperacillin and broth were impregnated on three different discs.
Ceftriaxone is an antibiotic used to cure gram positive bacteria while Piperacillin is used for
gram negative bacteria. Preparation for Ethanolic Extract Discs: 30µ/g of ethanolic crude
extract of hagonoy leaves were impregnated in three discs. All discs impregnated with
solutions were dried before used.
Figure 4. Impregnated Dics
Susceptibility Testing
Nutrient agar was dispensed on three different petri dishes. The plates were inoculated
with respective test organism using cotton swabs. The ethanol discs, positive control discs,
and negative control discs were then transferred using flamed but cooled forceps into the
surface of the inoculated agar plates with sufficient spaces in order to avoid overlapping of
inhibitions. The plates were incubated at 370C overnight, and the diameter of inhibition of
each discs were measured in mm for every 20th, 22nd, and 24th. Measurements obtained at
the 24th hour would be considered as the final measurement.
Figure 5. Measuring of Inhibition
Statistical Treatment
Analysis Statistical Tool
1. Identification of significant difference
between Hagonoy plant Extract and T-test Independent
Broth against Klebsiella Pneumoniae.
2. Identification of significant
difference between Hagonoy plant T-test Independent
Extract and Piperacillin against
Klebsiella Pneumoniae
3. Identification of siginificant difference
between Hagonoy plant Extract and T-test Independent
Ceftriaxone against Klebsiella Pneumoniae
Chapter IV
Results and Discussion
After the effectiveness of the plant extract of C.odorata was tested against one gram
negative (Klebsiella pneumoniae) by conducting the Kirby Bauer Agar Disc Diffusion Method,
results showed antimicrobial effect of extract against K. pneumonia. Results also contained
differences between the zones (of inhibition) of the extract and the controls used in the study.
According to the T-Test Independent results, the negative control and the plant extract showed
significant difference (P<0.05), which is very statistically significant, between the diameter of
their zone of inhibition against the tested organism while positive control, piperacillin, did not
show any significant difference (P>0.05) between its inhibitory zone compared to the zone of
plant extract. Positive control, ceftriaxone, and plant extract on the other hand showed
differences which are not quite significant (P>0.05).
Extract Piperacillin Ceftriaxone Broth
27mm 6mm 30mm 0mm
21mm 16mm 33mm 0mm
15mm 16mm 30mm 0mm
Table 1.0 Diameter of Discs Inhibitory Zone
Figure 6. Zone of Inhibition of Extract, Piperacillin, and Ceftriaxone
The study showed that there is no significant difference between the effectiveness of C.
odorata plant extract and the two positive controls, piperacillin and ceftriaxone, which are
chemically tested to cure infections and diseases caused by different human pathogens. This
states that the effect of the plant extract is comparable to some formulated antibiotics for
medication. The plant extract showed positive results (mean = 21) against Klebsiella
pneumoniae which had surpassed the total mean of the diameter of inhibition by the control
drug, piperacillin, (mean = 12.6667) which is an antibiotic that aids against gram negative
bacteria. However, the control drug ceftriaxone retained the highest antimicrobial effect (mean =
31).
These results have proved that ethanolic extract of Chromolaena odorata is effective
against Klebsiella pneumoniae and proved the presence of antimicrobial property on its leaves.
The presence of most compounds that act against most micro-organisms like flavonoids,
tannins, steroids, alkaloids, and saponins, were revealed after conducting phytochemical
analysis according to the study of Stanley, M. C.
Chapter V
Conclusion
The ethanolic extract of Chromolaena odorata showed positive results after being tested
against Klebsiella pneumoniae proving the presence of its antimicrobial property and
importance as a medicinal plant. It was proven that the extract of the plant can be used as
possible substitute for some existing drugs in aiding certain types of infections and diseases.
Drugs which were compared to the extract showed no difference between their effectiveness in
inhibiting the growth of bacteria. This showed great possibilities of effective use of the ethanolic
extract of C. odorata for formulation of new antibiotics in the future.
Recommendation
In this study, the plant extract was tested against one gram negative bacteria and gained
positive results. Therefore, in order to solidify and to improve more the knowledge about the
antimicrobial effectiveness of C. odorata as herbal medicine, possible researches in the future
must conduct comparison with the effect of different plant extract using different solutions. The
extract might also be tested against one gram positive and one gram negative bacteria in order
to provide comparison to the two types of bacteria. We also Wanted to push phytochemical
analysis to the plant used obtained in the Philippines to further classify copounds present in the
leaf extract.
Bibliography
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Appendices
Table 2: Results for the comparison of Plant Extract and Negative Control
Data Summary
A B Total
n 3 3 6
63 0 63
- X
1395 0 1395
- X2
SS 72 0 733.5
21 0 10.5
mean
ResultsQ
Meana—Meanb t df one-tailed 0.0018715
21 +6.06 4
P
two-tailed 0.003743
Table 3: Results for the comparison between extract and piperacillin
Data Summary
A B Total
n 3 3 6
63 38 101
- X
1395 548 1943
- X2
SS 72 66.6667 242.8333
mean 21 12.6667 16.8333
ResultsQ
Meana—Meanb t df one-tailed 0.0793415
8.3333 +1.73 4
P
two-tailed 0.158683
Table 4: Results for the comparison between extract and ceftriaxone
Data Summary
A B Total
n 3 3 6
63 93 156
- X
1395 2889 4284
- X2
SS 72 6 228
mean 21 31 26
ResultsQ
Meana—Meanb t df one-tailed 0.051655
10 -2.77 4
P
two-tailed 0.050331
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