Microbes and Infectious Diseases Evaluation of phytochemical constituents and antibacterial efficacy of Chromolaena odorata (Linnaeus) leaf extracts on bacteria associated with patient's wounds

Abstract

Experimental findings on effects of Chromolaena odorata leaf extracts on bacteria isolated from patient's wound.

Full text

Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
Microbes and Infectious Diseases
Journal homepage: https://mid.journals.ekb.eg/
DOI: 10.21608/MID.2023.218543.1547
* Corresponding author: Emmanuel Fisayo Lambe
E-mail address: haywhydotphiz@gmail.com
© 2020 The author (s). Published by Zagazig University. This is an open access article under the CC BY 4.0 license https://creativecommons.org/licenses/by/4.0/.
Original article
Evaluation of phytochemical constituents and antibacterial
efficacy of Chromolaena odorata (Linnaeus) leaf extracts on
bacteria associated with patient’s wounds
Emmanuel Fisayo Lambe*, Muftau Kolawole Oladunmoye
Department of Microbiology, School of Life Sciences, Federal University of Technology Akure, P.M.B. 704, Ondo State, Nigeria.
Introduction
Wounds are a common occurrence in our
daily lives, ranging from minor cuts and bruises to
more severe injuries [1]. They can be caused by
accidents, surgeries, or even diseases [2-4].
Understanding wounds, their types, healing process,
and management is crucial for individuals,
healthcare professionals, and caregivers.
Wound infections refer to infections that
occur in a wound or surgical incision [5]. When a
wound is not properly cleaned, protected, or cared
for, bacteria or other microorganisms can enter the
wound, leading to an infection [6]. Wound
infections can be caused by various types of
bacteria, including Staphylococcus aureus,
Streptococcus pyogenes, and Escherichia coli,
among others [7]. It is however important to note
A R T I C L E I N F O
Article history:
Received 18 June 2023
Received in revised form 12 July 2023
Accepted 14 July 2023
Keywords:
Antibacterial
Wound infection
Phytochemicals
Chromolaena odorata
m
A B S T R A C T
Background: This research was designed to show evaluation of antibacterial activities of
Chromolaena odorata (C. odorata) Linn extracts on bacteria associated with wound
infections. Methods: A total of 127 wound exudate samples were collected from the
patients with chronic wound infections attending the Federal Medical Centre, Owo, First
Mercy Hospital and University of Medical Teaching Hospital, Akure, Ondo State, Nigeria,
between November 2021 and January 2022. Questionnaires were served to each patient in
order to obtain information about the wound. Morphological and biochemical
characterization of bacteria were determined using standard microbiological methods. The
C. odorata extract was subjected to Soxhlet and maceration extraction methods. The
solvents used were cold water, hot water, N-hexane and ethanol. Phytochemical evaluation
was carried out on the extracts. The antibiotic susceptibility patterns of the isolates were
determined by testing against antibiotics and extracts. Qualitative phytochemical analysis
showed the presence of alkaloid, flavonoid, glycoside, phenol, phlobatannin, saponin,
steroid, tannin, and terpenoid in the extracts of C. odorata. Results: A total of 13 bioactive
compounds were observed in the ethanol extract of C. odorata. The highest leakages of
Na+ and K+ were observed in Proteus mirabilis at 12.9 mol/kg and 30.0 mol/kg. There
was higher leakages of Na+ in all the test isolates than K+. Conclusions: The antibacterial
efficacy of leaf extracts of C. odorata has been evidently proven against bacterial isolates
associated with wound infections, suggestive of good alternative treatment materials.

Lambe EF et al / Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
that not all wounds will become infected, and the
severity of an infection can vary. If wound infection
is suspected, appropriate medical attention should
be sought [8].
Wound infections are often characterized
by the invasion of bacteria into open or damaged
skin, resulting in inflammation and impaired healing
[9]. Bacterial infections can significantly delay the
recovery process, increase morbidity, and, in severe
cases, lead to life-threatening complications [10].
Understanding the bacteria associated with wound
infections, their characteristics, and their antibiotic
resistance patterns is crucial for effective diagnosis,
treatment, and prevention strategies. Some of the
bacteria associated with wound infections are
Pseudomonas aeruginosa, Streptococcus pyogenes,
Staphylococcus aureus, Escherichia coli and
Enterococcus species [7].
Large numbers of antibiotics have been
usually combined for the treatment of wound
infection. Narrow and broad spectrums antibiotics
are available for the treatment. However, improper
and irrational use of antibiotics as well as genetic
and non-genetic drug resistant mechanisms of
bacteria may result into antibiotic resistance [11].
The development of antibiotic-resistant bacteria has
greatly limited the effectiveness of conventional
antimicrobial therapy [12]. This is why alternative
therapeutic measures have been sought in the past
years.
Many species of plants and herbs with
wound healing activities have been identified in
Africa and developing countries as a result of ethno-
botanical research advancement. The use of
medicinal plants in wound management and care
includes debridement, disinfection and provision of
suitable environment for natural healing process
[13]. Studies have shown that active ingredients
from medicinal plants are less toxic, with low or no
side effects compared with orthodox therapeutic
agents; hence, the increased and renewed interest in
the use and application of medicinal plants in the
wound healing process both in diabetic and non-
diabetic conditions [13]. In 2015, Rajasree et al.
[14] established the fact that C. odorata had
bioactive therapeutic substances that produced
effects on wound healing. Also, the constituents of
the plant extracts modulate one or more of the
overlapping wound healing stages. Traditional use
of C. odorata leaves involves pasting the ground
leaves topically.
In this study, we evaluated the
phytochemical constituents of leaf extracts of C.
odorata as well as their antibacterial activities
against bacteria isolated from wound exudates.
Materials and methods
Sample size determination
The WHO (2009) formula with ± 10 % precision
level for large sample was applied since the
population was homogenous.
The formula; n = N/ 1 + N (e) 2
Where; n = the sample size; N = size of the
population; e = level of significance (or limit of
tolerable error).
Collection of clinical samples and isolation of
bacteria
A total of 127 clinical samples were obtained by
means of rotation of sterile swabs (transport swab)
from wounds between November, 2021 and April,
2022 from different hospitals which include Federal
Medical Centre, Owo (FMC), University of Medical
Sciences Teaching Hospital (UNIMEDTH) and
First Mercy Hospital, Akure within the duration
from 8 am to 10 am daily and transported in an ice
pack to the Research laboratory FUTA for further
analysis within 1 hour of collection for
microbiological analysis.
Identification of bacterial isolates
Biochemical tests were carried out for the
identification of bacterial species. The following
were the tests carried out; Gram staining, catalase,
citrate, urease, oxidase, indole, voges-proskauer,
and sugar fermentation tests [15].
Standardization of bacterial isolates from wound
swab samples
Standardization of the culture to 0.5 McFarland’s
standard (108 ¬CFU/ml) was done as described by
Isunu et al. [16].
Collection and preparation of leaf samples of C.
odorata
Fresh leaf samples of C. odorata were collected
from Ojajere Quarters at Ilara Mokin, Ifedore Local
Goverment Area of Ondo State between the hours of
6 - 8 am at a prevailing temperature of about 30±2
℃. All the collections were done in the month of
October 2021. The plants were identified and
authenticated by Prof. M. K. Oladunmoye
(Pharmaceutical microbiologist) at the Department
of Medical Microbiology, Federal University of
Technology, Akure (FUTA), Ondo State.

Lambe EF et al / Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
The leaves of C. odorata were allowed to dry at 28
℃ for 4 weeks and then pulverized to a fine powder
with the aid of a Binatone blender (Model BLG-
621). Four solvents were used for the preparation of
the extracts, namely cold distilled water, hot
distilled water, ethanol 60% concentration and n-
hexane. The extracts were prepared according to the
method described by Isunu et al. [16].
Percentage recovery of the leaf extracts of C.
odorata
The percentage yield of the extract of C. odorata
were calculated thus [17]:
Percentage recovery of extract (%)
=Weight of extract recovered after extraction
Initial weight of plant before extraction
×100
1
Evaluation of the phytochemical constituents of
leaf extracts of C. odorata
The plant extracts of ethanol, n-hexane, cold and hot
aqueous were qualitatively screened as described by
Akinmoladun et al. [18]. Plant extracts were
screened for the presence of cardiac glycoside,
terpenoids, steroids, flavonoids, alkaloids, saponin,
tannin, phlobatannin and phenol. The number of
phytochemical constituents in the extracts were also
carried out using methods described by AOAC [19].
Antibacterial activity of leaf extracts of C.
odorata
The assay for the antibacterial activity of C. odorata
extracts was carried out as described by Isunu et al.
[16] with modifications. The reconstitution of the
extracts was done in accordance to the various
concentration intended for use in this study. Positive
control was maintained with 2mg/ml of Ciprotab ®.
The plates were then incubated for 18 hours at 37 ℃
and the diameter of zones of inhibition were
measured in mm.
Investigation of amount of sodium ion leakages
Sodium ion concentration was estimated by
colorimetric method based on the modified Maruna
and Trinder method. Sodium was precipitated
together by magnessium uranyl acetate as uranyl
magnesium sodium acetate salt. Excess uranyl salts
reacts with potassium ferrocynide to produce a
brownish colour. The intensity of the colour is
inversely proportional to the sodium concentration
in the specimen and is measured photometrically at
530 nm (500 nm – 540 nm).
Statistical analysis
Data obtained in this study were subjected to one-
way analysis of variance (ANOVA), and differences
between means were compared by Duncan’s New
Multiple Range Test at 95% confidence interval
using Statistical Package for Social Sciences (SPSS)
version 20.
Results
Distribution of frequency of pathogenic bacterial
from wound source in relation to age and sex
(Figure 1).
A total of 127 wound samples were collected from
the patients chronic wound infections. The highest
frequency was seen among the male patients
between the age of 21 and 30 years while the lowest
frequency was obtained among patients between
ages of 1 and 10 years.
Identification of bacterial isolates from wound
exudates
The biochemical characteristics of the bacterial
isolates from wound samples are shown in table (1).
Both Gram positive and Gram negative bacteria
were identified as the bacteria involved in wound
infections in this study. The bacteria were identified
as Staphylococcus aureus (S. aureus) (N = 55),
Pseudomonas aeruginosa (P. aeruginosa) (N = 42),
Proteus mirabilis (P. mirabilis) (N = 38), Klebsiella
pneumoniae (K. pneumoniae) (N = 24) and
Escherichia coli (E. coli) (N = 9).
Percentage occurrence of bacteria isolated from
wound swabs
The percentage occurrence of the bacteria isolated
from wound samples of patients are; S. aureus
(33%), P. aeruginosa (25%), P. mirabilis (23%), K.
pneumoniae (14%), and E. coli (5%). Gram positive
bacteria especially Staphylococcus species has the
highest percentage occurrence of the bacteria
involved in wound infections (Figure 2).
Percentage yield of the leaf extracts of C.
odorata
The percentage yield of the extracts varied from
solvent to solvent. The recovery rate for Ethanol, hot
water, cold water and N-hexane are 58.57 %, 51.73
%, 46.85 % and 14.59 % respectively (Figure 3).
Phytochemical constituents of the leaf extracts
of C. odorata
Ethanol leaf extract of C. odorata was found to have
the highest quantity of cardiac glycoside
(882.32±20.58 mg/g) while N-hexane leaf extract of
C. odorata (0.00±0.00mg/g) have the least quantity
of cardiac glycoside, tannin and terpenoids

Lambe EF et al / Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
respectively. Hot water and cold water extracts of C.
odorata have the highest (479.31±2.51) and least
(88.57±4.29) quantity of flavonoid respectively.
Phenol had the highest quantity in hot water extract
of C. odorata (479.31±2.51 mg/g). Alkaloid had the
highest quantity (16.81±0.03 mg/g) in N-hexane leaf
extract of C. odorata. The qualitative and
quantitative phytochemical constituents are shown
in tables (2, 3).
Antibacterial activities of leaf extracts of C.
odorata
The antibacterial activities of leaf extracts of C.
odorata at 100 mg/mL showed that the purified
ethanol extract produced the widest zone of
inhibition (17.67±1.76 mm) against K. pneumoniae,
while crude N
-hexane extract produced no zone of inhibition
against S. aureus and P. mirabilis (Table 4).
Leakage of sodium (Na+) and potassium ions
(K+) from bacterial isolates by the ethanol leaf
extract of C. odorata
The leakage of sodium (Na+) and potassium ions
(K+) from bacterial isolates by the ethanol leaf
extract of C. odorata is presented in figure (4). The
highest leakages of Na+ and K+ were observed in P.
mirabilis with values of 12.9 mol/kg and 30.0
mol/kg respectively. There was higher leakage of
K+ in all the test isolates than Na+.
Table 1. Biochemical characteristics of bacteria isolated from wound samples.
S/N
Gram
Reaction
Catalase
Coagulase
Citrate
Oxidase
Motility
Glucose
Fructose
Lactose
Sucrose
Maltose
Organisms
1
-ve rod
-
-
-
-
NM
AG
AG
AG
AG
AG
P. aeruginosa
2
+ve
cocci
+
+
+
-
NM
AG
AG
AG
AG
AG
S. aureus
clusters
3
-ve rod
+
-
+
-
NM
AG
AG
AG
AG
AG
K. pneumoniae
4
-ve rod
+
-
-
-
M
AG
AG
AG
AG
AG
E. coli
5
-ve rod
+ve
-ve
+
-
M
AG
AG
AG
AG
AG
P. mirabilis
Key: NM: Non motile, M- Motile; AG-Acid and Gas production, A- Acid production only, +: present, -: absent, +ve: Gram positive, -ve:
Gram negative

Lambe EF et al / Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
Table 2. Qualitative phytochemical constituents of the leaf Extracts of C. odorata
Phytochemicals
Cold water
Hot water
Ethanol
N-hexane
Tannin
+
+
+
-
Saponin
+
+
+
-
Steriod
+
+
+
-
Terpenoid
+
+
-
-
Phenol
+
+
+
+
Glycoside
+
+
+
+
Alkaloid
-
-
-
-
Phlobatannin
-
-
-
-
Flavonoid
+
+
+
+
Keys: Present +; Absent –
Table 3. Quantitative phytochemical constituents of the leaf extracts of C. odorata
Phytochemicals
Cold water
Hot water
Ethanol
N-hexane
Tannin
32.14±1.99b
47.82±0.43b
57.94±3.86a
0.00±0.00a
Terpenoid
2.16±0.29a
9.90±.54a
13.67±0.71a
0.00±0.00a
Phenol
146.14±.19e
365.34±.61e
283.43±57.58c
128.89±6.33b
Glycoside
194.37±6.75f
289.13±13.19d
882.32±20.58d
359.15±16.78d
Alkaloids
0.00±0.00a
0.00±0.00a
0.00±0.00a
16.81±0.03a
Flavonoid
88.57±4.29d
479.31±2.51f
163.12±2.51b
247.61±11.71c
Saponin
70.84±2.59c
74.14±20.09c
1129.72±17.50e
0.00±0.00a
Steriod
1.52±.06a
2.01±0.10a
3.77±0.07a
0.00±0.00a
Values are presented as Mean±SE of duplicates, values in the same column carrying same superscript are not different significantly
(p<0.05) according to new Duncan’s Multiple Range test.

Lambe EF et al / Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
Table 4: Comparative susceptibility pattern of crude and purified leaf extract of C. odorata at 100 mg/ml on
bacterial isolates from clinical samples
Isolate
PEC
CEC
PNH
CNH
CCW
PCW
PHW
CHW
S. aureus
16.33±0.88a
11.67±0.33a
10.67±0.33ab
0.00±0.00a
10.67±0.33a
11.67±0.33a
13.33±0.33a
11.00±0.58ab
P.
aeruginosa
16.00±1.15a
12.00±0.58a
11.33±0.33b
10.67±0.00b
10.67±0.67a
12.00±0.58a
14.00±0.58a
10.67±0.58a
K.
pneumonia
e
17.67±1.76a
13.67±0.61a
13.00±0.00c
10.67±0.67b
10.33±0.33a
12.33±0.33a
15.33±0.67a
12.67±0.33ab
E. coli
16.33±.88a
12.33±0.88a
12.67±0.58c
11.00±0.58b
11.00±0.58a
13.33±0.88a
15.33±0.88a
13.00±1.00b
P.
mirabilis
16.33±.88a
13.67±0.88a
10.00±0.00a
0.00±0.00a
12.00±0.58a
13.33±0.67a
14.67±.88a
12.00±0.58ab
Values are presented as Mean±SE of duplicates, values in the same column carrying same superscript are not different significantly
(p<0.05) according to new Duncan’s Multiple Range test.
Keys: PEC- Purified Ethanol Chromolaena; CEC- Crude Ethanol Chromolaena; PNH- Purified N-hexane; CNH- Crude N-hexane; CCW-
Crude Chromolaena Cold water; PCW- Purified Chromolaena Cold water; PHW- Purified Hot Water ; CHW- Crude Hot Water
Figure 1. Distribution of frequency of pathogenic bacteria from wound source in relation to age and sex.
0
5
10
15
20
25
1 to 10 11 to 20 21 to 30 31 to 40 41 to 50 51 plus
Frequency
Age range (Years)
Male Female

Lambe EF et al / Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
Figure 2. Percentage occurrence of bacteria isolated from wound swabs.
Figure 3. Percentage yield (%) of extracts of C. odorata
Figure 4. Leakage of potassium ions (K+) and sodium ion (Na+) from bacterial isolates by the ethanol leaf
extract of C. odorata
33%
25%
23%
14%
5%
S. aureus
P. aeruginosa
P. mirabilis
K. pneumoniae
E. coli
14.59
58.57
51.73 46.85
0
10
20
30
40
50
60
70
N- hexane Ethanol Hot water Cold water
Percentage yield
Extraction solvent
0
5
10
15
20
25
30
35
Concentration (mol/kg)
Isolates
Na+(mol/kg)
K+ (mol/kg)

Lambe EF et al / Microbes and Infectious Diseases 2023; Article-In-Press, DOI: 10.21608/mid.2023.218543.1547
Discussion
In this study, it was observed that the rate
of wound pathogenic organisms was most
pronounced among male patients that were within
the third decade of life and it was distributed across
the ages and sex. This is similar to the findings of
Seleh et al. [20] and Tom et al. [21], who asserted
that the predominance among patients in this
category is most likely due to the fact that male
exposure to a possible wound and/or trauma is
greater as they represent majority of the workforce
responsible for hard/risky labour.
Gram positive bacteria especially S. aureus
was the most predominant bacteria observed and has
percentage occurrence of 33 % among the isolated
bacteria involved in wound infections while E. coli
was the least with percentage occurrence of 5%.
Similar study was conducted from different parts of
Nigeria [22].
The ethanol leaf extract of C. odorata has
the highest percentage recovery compared to the
aqueous and N-hexane extraction solvent. The N-
hexane extraction solvent has poor yield of recovery
of the extracts. The differences in polarity of the
solvents could affect the solubility of grinded plant,
percentage recovery and odour of the extracts [23].
Tannins, saponins, terpenoids, phenols,
flavonoid, steroid and alkaloids were present in all
the extracts of C. odorata. This is supported by
Vijayaraghavan et al. [24] who purported that the
medicinal values of plants lie in their phytochemical
constituents.
The findings revealed that the ethanol
extracts of C. odorata could be attributed to the
nature of active compounds and the stronger extract
ion capacity of ethanol could have produced greater
number of active constituents responsible for the
antibacterial activity.
The leaf extracts exhibited antibacterial
activities on the test bacterial isolates, showing
varying zones of inhibitions. Similar findings have
been reported in other studies [25,26]. The ethanol
and aqueous leaf extracts of C. odorata showed
antibacterial effect on both Gram positive and Gram
negative bacteria tested suggesting that its bioactive
components possess broad spectrum antibacterial
activity.
The highest leakages of Na+ and K+ were
observed in P. mirabilis at 12.9 mol/kg and 30.0
mol/kg. There was higher leakage of Na+ in all the
test isolates than K+. It is well known that the
cytoplasm of living cells, generally, contains more
potassium ions than sodium ions [27].
Conclusion
This study has shown that C. odorata leaf
extracts could be effectively used in wound
treatment as it inhibited the in vitro growth of
bacteria isolated from infected wounds. The leaf
extracts of C. odorata if properly harnessed could be
a source of active antibacterial agents for the
development of drugs against the pathogenic
bacteria responsible for wound infections.
Acknowledgments
The authors hereby acknowledge the Ondo
State Health Research Ethics Committee for
providing an ethical clearance for this study. We
also acknowledge the Department of Crop, Soil and
Pest Management, FUTA, for helping to
authenticate the plant material used for the study.
Author’s contributions
Author Oladunmoye, K. M., designed the
study. Author Lambe, E. F., developed the
methodology, acquired the data, analysed and
interpreted the data. Author Lambe, E. F., wrote the
first draft of the manuscript. Both authors read and
approved the final draft of the manuscript.
Conflict of interest
The authors have declared no conflict of
interest.
Funding
No funding received.
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