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American Journal of  
Environment and Climate (AJEC)

Antibacterial Activity of  Cassava Manihot Esculenta Leaves 
Extract Against Escherichia coli

Neil John L. Buendia1, Angel Lhi D. Alcalde1*, Rikka Bianca J. Condes1

Volume 1 Issue 2, Year 2022
ISSN: 2832-403X (Online)

DOI: https://doi.org/10.54536/ajec.v1i2.484
https://journals.e-palli.com/home/index.php/ajec

Article Information ABSTRACT

Received: August 09, 2022
Accepted: August 15, 2022
Published: August 18, 2022

The purpose of  this research is to determine the antibacterial activity of  Cassava (Manihot 
esculenta) leaf  extract against Escherichia coli. The experiment involved the determination 
of  plants, extraction using the maceration method, and antibacterial testing of  the fresh and 
dried cassava (Manihot esculenta) leave extracted with concentrations of  5%, 10%, 20%, 
and 30% ethanol against the bacterial growth of  (Escherichia coli) and culture media using 
Merck Nutrient Agar. A caliper was used to measure the size of  (Escherichia coli) growth in 
millimeters. The results showed that extracts of  fresh and dried cassava (Manihot esculenta) 
leaves extract with ethanol concentrations of  5%, 10%, 20%, and 30% have antibacterial 
activity against Escherichia coli. This study examined the efficacy of  fresh and dried cassava 
(Manihot esculenta) leaves extract against (Escherichia coli) in the 18th and 24th hours. The 
mean of  fresh cassava (Manihot esculenta) leaves extract is 4.4, which differs considerably 
from the mean of  dried Cassava (Manihot esculenta) leaves extract, which is 3.9. The fresh 
cassava (Manihot esculenta) leaves extract was the most potent therapy against (Escherichia 
coli) in the 18th hour, according to the findings of  this investigation. Furthermore, at the 
24th hour, the fresh cassava (Manihot esculenta) leaves extract was the most potent therapy 
against (Escherichia coli). Fresh cassava (Manihot esculenta) leaves extract has a mean of  
4.4, which differs significantly from dried (Manihot esculenta) leaves extract, which has a 
mean of  3.9. According to the findings of  this study’s testing, the 20 percent concentration 
of  fresh cassava (Manihot esculenta) leaves extract with an average of  5.75 was the most 
active treatment against (Escherichia coli). It has been proposed that cassava (Manihot 
esculenta) leaves could be turned into standardized antibacterial herbal. Future research 
should investigate the effects of  cassava (Manihot esculenta) leaves used as an antibacterial 
agent on human health. 

Keywords
Escherichia coli. Manihot 
esculenta, Leaves Extract, 
Antibacterial, Cassava

1 Notre Dame of  Midsayap College, Midsayap, Cotabato, Philippines.
* Corresponding author’s e-mail: delacruzangellhi@gmail.com

INTRODUCTION
Plants have long been utilized to treat people and are 
now recognized as a source of  medicine in conventional 
health care systems (Meilawaty et al., 2019).
According to recent research (Shah et al., 2013), some 
indigenous plants offer interesting biological features that 
may be of  interest to people all over the world. The plants 
include medicinal compounds, antibiotics, and microbial 
components that can benefit people. Many ancient herbal 
uses are being investigated as potential sources of  natural 
components for novel medications. People have known 
for a long time that some plant-based medications can 
treat ailments and kill microorganisms. Many plant parts 
used in traditional medicine can be found in rural regions 
for about the same price as contemporary treatment. 
Rural locations have more medicine and it is less 
expensive than in cities. Plants produce a large number 
of  secondary metabolites, which are essential sources 
of  microbicides, pesticides, and many pharmaceuticals. 
Pharmaceutical substances used in traditional medicine 
are still primarily derived from plants. People are getting 
increasingly interested in a variety of  traditional organic 
components.
E. coli lives and grows in the gastrointestinal tracts of  
many animals, including humans (ClevelandClinic, 2020) 
the majority of  the time, this bacteria is absolutely 
harmless. It aids in the digestion of  food. However, these 

are E. coli strains that could enter a human’s stomach and 
produce diarrhea, stomach ache, cramps, and a low-grade 
fever.
Natural resources from the Philippines are extensively 
used in everyday life as a culinary item in tropical countries. 
Cassava leaves can be used to treat ulcers, rheumatism, 
gout, diarrhea, fever, headache, night blindness, intestinal 
worms, and “beriberi” disease, in addition to consuming 
them. Furthermore, Cassava (Manihot esculenta) leaves 
extract demonstrated antibacterial activity against 
clinical isolates of  Staphylococcus epidermidis as well as 
Propionibacterium acnes, and the findings of  a related 
study indicated that Cassava leaves extract was effective 
in treating pimple-causing bacteria. (Mustarichie at. al., 
2020)
Treatment for E. coli is entirely dependent on the 
administration of  antibacterial agents, particularly 
antibiotics, which may result in more disease-causing 
germs, new strains of  microbial resistance to antibiotics, 
and even renal failure. As a result, it is critical to seek 
out alternative methods of  assistance, one of  which is the 
usage of  herbal plants, notably the cassava plant, which 
has phytochemical qualities such as flavonoid and rutin 
E. Coli - Diagnosis and Treatment - Mayo Clinic, 2020).
Flavonoids, according to (Kumar & Pandey, 2013), are 
a class of  plant metabolites that are hypothesized to 
have antioxidant and antibacterial properties via cell 



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signaling pathways. Flavonoids are powerful antioxidants 
that promote a variety of  health benefits. Aside from 
antioxidant action, these compounds have anti-viral, anti-
cancer, anti-inflammatory, and anti-allergic properties. 
Rutin, on the other hand, is a pigment and a strong 
bioflavonoid component found in the cassava plant that 
is responsible for its antibacterial properties (Ullah et al., 
2020).
There hasn’t been a lot of  research done on the usefulness 
of  Cassava leaf  extract as a treatment for disease-causing 
bacteria. This study compares the antibacterial activity 
of  Cassava (Manihot esculenta) leaves extract against S. 
epidermidis and P. acnes (Mustarichie et al., 2020). As 
a result, our study to assess the antibacterial activity of  
cassava leaf  extract is both contemporary and pertinent. 
The study examined and assessed the effectiveness of  
fresh and dried cassava (Manihot esculenta) leaves extract 
with ethanol concentrations of  5%, 10%, 20%, and 30% 
in inhibiting the growth size of  (Escherichia coli). Finally, 
the goal of  this study was to give scientific evidence that 
cassava (Manihot esculenta) leaf  extract has the ability to 
treat germs (Escherichia coli).     
                
What is Cassava (Manihot esculenta)?
Cassava is a tuberous food plant that is native to the 
tropics of  the American continent. It is a member of  
the spurge family (Euphorbiaceae) and is also known 
as manioc, mandioca, and yuca. According to the 
research, the cassava leaf  contains a high concentration 
of  phytochemical components like tannins, alkaloids, 
steroids, flavonoids, and saponins (Babalola & Ahmed, 
2005). The leaves of  the cassava plant were the source 
of  these antibacterial and antioxidant chemicals. Cassava 
leaf  extract was studied for its potential antibacterial 
effects against Staphylococcus epidermidis and 
Propionibacterium acnes Mustarichie et al. (2021)The 
results of  this study indicated that the cassava plant 
(Manihot esculenta) had antibacterial activity against 
Staphylococcus epidermidis as well as Propionibacterium 
acnes. In addition, it was determined that ethanol extracts 
of  cassava leaves had antibacterial activity against both of  
the bacteria that were tested, with ethyl acetate being the 
most active component of  the extract. The treatment that 
was found to be most successful against Staphylococcus 
epidermidis was a concentration of  5 percent with an 
average of  5.0. On the other hand, a concentration of  2.5 
percent against Propionibacterium acnes was found to be 
the most effective treatment. The average concentration 
was 4.1.
According to (Landers et al., 2012) antibiotic 
development leads to bacterial resistance due to a 
variety of  circumstances. These circumstances include 
inappropriate antibiotic usage in human and animal 
health and their continuous use as growth promoters at 
sub-clinical levels in poultry and cattle production. In 
addition, improper antibiotic usage in human and animal 
health can lead to antibiotic resistance. This research 
looked at the effectiveness of  an extract made from 

cassava leaves as an antibacterial agent in the treatment of  
diarrhea brought on by E. coli infections in both humans 
and animals. According to the findings of  the study, the 
extract of  cassava leaf  was effective in treating diarrhea 
brought on by E. coli. 
Similarly, a study that was carried out by Saad et al. 
(2014) investigated the antibacterial efficacy of  five 
medicinal plants against five clinical pathogens. These 
plants were Moringa oleifera, Cymbopogon citrates, 
Cynodon dactylon, and Manihot esculenta. Plectranthus 
ambonicus was also included in this study (Escherichia 
coli, Klebsiella pneumonae, Pseudomonas aeruginosa, 
Staphylococcus aureus, and Bacillus subtilis). It was 
shown that an extract of  the leaves of  the cassava plant 
(Manihot esculenta) has antibacterial activity against five 
clinical isolates of  pathogens. These pathogens included 
Escherichia coli, Klebsiella peumoniae, Pseudomonas 
aeruginosa, Staphylococcus aureus, and Bacillus subtilis.
However, research conducted by Sari (D. B. 2019) 
evaluated the antibacterial activity of  three different 
varieties of  cassava leaf  (Manihot esculenta Crantz.) 
against the germs Bacillus cereus, Shigella dysenteriae, and 
Vibrio cholerea. Maceration with varying concentrations 
of  ethanol, a positive control, and negative control was 
used in an experiment to investigate the antibacterial 
properties of  an ethanol extract of  three different kinds 
of  cassava leaves. Based on the findings, it was determined 
that an ethanol extract of  cassava (Manihot esculenta) 
leaves was the antibacterial substance that was the most 
efficient against Bacillus cereus.

What is Escherichia coli?
According to Wikipedia, E. coli, sometimes known as 
Escherichia coli, is a type of  bacteria that can be found 
in people’s intestines (Felson, 2020). It has also been 
found in the gastrointestinal tracts of  other mammals. In 
addition to having food poisoning from E. coli, a person 
may develop pneumonia or a urinary tract infection. In 
fact, E. coli is the causal culprit in 75 to 95 percent of  
all instances of  urinary tract infections. Because E. coli 
is a common colon dweller, it frequently travels through 
the bowel to enter the urinary tract. Several strains of  
E. coli have been related to diarrhea, including the kind 
that causes watery travelers’ diarrhea. Antibiotics have 
been demonstrated to lessen the amount of  time spent 
experiencing symptoms and may be recommended to 
people with moderately severe illnesses. Inadequate 
antibiotic use may expedite the development of  germs 
resistant to the drugs used to treat them.
However, (Riley, 2014) emphasized that extraintestinal 
pathogenic E. coli, the specialized strains of  E. coli that 
cause the bulk of  extraintestinal E. coli infections, pose a 
significant yet underappreciated hazard to public health. 
These E. coli strains cause the majority of  extraintestinal 
E. coli infections. Despite the fact that the roots of  
their evolution are unknown, E. coli has a unique ability 
to cause disease in places of  the host body other than 
the intestinal tract. The many virulence characteristics 



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that E. coli possesses illustrate this potential. Effective 
preventive interventions against the morbid and costly 
infections caused by E. coli may be possible if  there is 
a better understanding of  the existence and significance 
of  E. coli, as well as the distinct virulence mechanisms, 
reservoirs, and transmission pathways associated with 
these bacteria.
According to Gomes et al. (2016) study, E. coli causes 
digestive diseases, the most prevalent of  which is 
diarrhea. Diarrhea is a major cause of  morbidity and 
mortality in disadvantaged countries. Hemorrhagic colitis 
is caused by E. coli cytotoxins or Shiga-like toxins, which 
are responsible for the disease. It is currently relevant to 
public health since it is easily isolated from waste items left 
behind by humans and animals, both of  which contribute 
to the pollution of  the environment (Hernadez-Cortez et 
al., 2017). Because of  the frequency of  food and water 
contamination in the environment, people, particularly 
newborns and the elderly, pose a substantial risk to their 
health. Human and animal feces can contain contaminants 
as well as infectious organisms such as E. coli.
Tannins, flavonoids, and rutin are just a few phytochemical 
compounds that can be found in a variety of  plant 
extracts. These chemicals are capable of  treating bacterial 
infections. This study aimed to provide antimicrobial 
effects by utilizing a natural antibacterial agent with 
suitable extraction parameters and concentrations. The 
antibacterial activity of  cassava (Manihot esculenta) 
leaves extract against Escherichia coli will give future 
researchers baseline data, changing it into a standardized 
antibacterial herb plant (Mujeeb, et al., 2014).

Phytochemical Compounds of  Cassava (Manihot 
esculenta) Leaves
The presence of  tannins, alkaloids, steroids, flavonoids, 
and saponins in M. esculenta leaves extract was discovered 
by a phytochemical investigation (Clemen-Pascual et 
al., 2022). Plant phytochemical compounds assess the 
medicinal value of  plant leaves. Antimicrobial and 
antioxidant effects of  flavonoids and tannins Alkaloids, 
on the other hand, have significant physiological effects, 
particularly on the neurological system. 
The presence of  these three phytochemicals in the leaves 
of  M. esculenta revealed that this plant is physiologically 
active and has the capacity to inhibit germs that cause 
diseases. This investigation supported the antibacterial 
activity of  M. esculenta leaves extract. Future research 
should focus on isolating, identifying, and purifying these 
phytochemicals, as well as determining their antibacterial 
potencies and toxicity evaluation in order to formulate 
antibiotics.
Research from Chaiareekitwa et al. (2022) investigated the 
antioxidant components and chlorophyll content, as well 
as the antioxidant activity of  cassava leaf  flour extract from 
different plant ages and groups. The antioxidant content 
(vitamin C, polyphenols, and carotene) was considered 
high and increased as the plants grew. The presence of  
these antioxidant compounds on the Cassava leaf  flour 

extract demonstrates its efficacy as an antibacterial agent. 
The mature the leaf  is, the larger the level of  antioxidants. 
According to the findings of  this study, Cassava leaf  flour 
extract is high in antioxidants, particularly flavonoids. 
Cassava leaf  flour extract’s flavonoid level suggests its 
antibacterial activity.
According to (Shrestha et al., 2021, Kabra et al., 2019, and 
Saini et al., 2013)) M. esculenta leaves were reported to 
have antibacterial action, and anti-oxidant, anti-tyrosinase, 
anti-inflammatory, and hepatoprotective. The high 
flavonoid content of  M.esculenta leaves was responsible 
for these actions. According to a recent study (Chahyadi 
& Elfahmi, 2020), seven glycoside flavonoids are present 
in cassava leaves, with Rutin being the most abundant. 
According to the findings, M.esculenta leaves are a rich 
source of  Rutin, making extraction the best method for 
obtaining a high yield of  bioflavonoids. According to this 
study, the maceration procedure with ethanol was the 
best extraction method for obtaining rutin.
According to the evaluations linked to this work, the 
antibacterial efficacy of  Cassava (Manihot esculenta) 
leaves extract can deter Escherichia coli. Cassava leaves’ 
phytochemical characteristics enable researchers to create 
antibacterial drugs from their extract. Fresh and dried 
Cassava (Manihot esculenta) leaves were macerated with 
95 percent ethanol. In this study, varying percentages 
of  concentration (5 percent, 10%, 20%, and 30%) were 
observed to assess the level of  effectiveness of  fresh 
and dried Cassava (Manihot esculenta) leaves and their 
concentration. Concentrated extracts of  fresh and dried 
Cassava (Manihot esculenta) leaves were tested for 
antibacterial activity against Escherichia coli, a bacteria 
often found in human and animal intestines. This study 
will give baseline data on the efficacy of  Cassava (Manihot 
esculenta) leaves extract against E. coli infections. This 
study also found that cassava (Manihot esculenta) leaf  
extract has antibacterial activities that are useful not 
only in treating pimple-causing bacteria (Staphylococcus 
epidermidis and Propionibacterium acnes), but also in 
infections caused by Escherichia coli.

MATERIALS 
The present study was conducted to evaluate the 
antimicrobial activity of  Cassava (Manihot esculenta) 
leaves extract against clinical isolate of  Escherichia 
coli. This experimentation tested the effectiveness of  
Treatment-A using fresh Cassava (Manihot esculenta) 
leaves extract and Treatment-B using dried Cassava 
(Manihot esculenta) leaves extract.
Research Design. This study manipulates clinical isolates 
of  Escherichia coli where procedures and experimentation 
were done in the Biological Laboratory of  Notre 
Dame of  Midsayap College. Thus, this study adopts 
an experimental research design using a randomized 
complete block design (RCBD).
Instrumentations. The experimental method was used 
in this study to test the antibacterial activity of  Cassava 
(Manihot esculenta) against Escherichia coli. Randomized 



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Complete Block Design was the method used in this 
study, in which experimental units were grouped into 
fresh and dried setups. RCBD was the method used since 
experimental units are grouped into blocks or replicates. 
A caliper was used in measuring the bacterial growth 
size and was measured in millimeters. The treatment of  
the extracts was done between 18 and 24 hours at room 
temperature. 
The results of  the experimentation were recorded and 
analyzed using ANOVA and t-test tools.
Beaker, Erlenmeyer flasks, graduated cylinder, stirring 
rod, inoculating loop, alcohol lamps, petri-dishes, mortar 
and pestle, electric oven, pressurized cooker, inoculating 
chamber, and the caliper was used in this study.
Manihot esculenta leaves were obtained at San Mateo, 
Aleosan, Cotabato and determined using PictureThis- 
Plant Identification Application.
Chemicals. Distilled water, and 95% ethanol were used in 
the experimentation.
Medium for Bacterial Growth. Merck Nutrient Agar was 
used.
Microbial Test. Clinical isolates of  E. coli from the 
University of  Southern Mindanao were used in this study.

Procedures 
Collection of  Cassava (Manihot esculenta) leaves. 
The study began with material collection by selecting 
Cassava (Manihot esculenta) leaves, which were not 
affected by pests, collected and sorted into 2 setups. 
Setup 1 was freshly picked cassava leaves, while Setup 2 
was dried Cassava (Manihot esculenta) leaves. Both were 
macerated and extracted at room temperature for 48 
hours.

Preparation and Sterilization of  Media
28g of  Merck Nutrient Agar was dissolved in a beaker 
with 1 liter of  distilled boiling water. Then, the medium, 
petri-dishes, beaker, and flask were sterilized using an 
autoclave at 121°C psi for 15 minutes. The autoclave 
process will eliminate unwanted microorganisms to 
ensure decontamination of  the culture media. The 
sterilized medium and laboratory apparatus was placed 
on a disinfected incubator to cool off.

Extraction
Fresh and Dried Manihot esculenta leaves were macerated 
using 95%. This method was selected to prevent the 
occurrence of  damage to the thermolabile chemical 
compounds contained in the Cassava (Manihot esculenta) 
leaves. The samples were mashed using mortar and 
pestle, then soaking it in the petri dishes with different 
concentrations. In each dish, 30g of  mashed Cassava 
(Manihot esculenta) leaves was soaked with concentrations 
of  5% (95ml distilled water and 5ml ethanol), 10% (90ml 
distilled water and 10ml ethanol), 20% ( 80ml distilled 
water and 20ml ethanol), and 30% (70ml distilled water 
and 30ml ethanol) of  95% ethanol. The maceration was 

carried out for 48 hours at room temperature.

Antibacterial Activity Test
The clinical isolate of  E. coli was suspended using 
inoculating loop into the sterile petri-dishes with 
20ml of  sterile Merck nutrient agar (solidified at room 
temperature). In each of  the dishes, fresh and dried 
Cassava (Manihot esculenta) leaves extract were added 
with concentrations of  5%, 10%, 20%, and 30% using 
a sterile dropper. The test media were then incubated for 
18-24 hours. In the 18th hour, the diameter of  the test 
bacteria was measured using a caliper. And in the 24th 
hour, the diameter of  the test bacteria was measured 
using a caliper. 

Data Gathering Procedures
Extraction
The efficiency of  Cassava (Manihot esculenta) 
leaves extract against E. coli was investigated in this 
experimentation. In this design, Cassava (Manihot 
esculenta) leaves were collected and assigned to the two 
groups (Fresh and Dried Cassava (Manihot esculenta) 
leaves) and was extracted with concentrations of  5%, 
10%, 20%, and 30% of  95% ethanol. The varying 
concentration of  Fresh and Dried Cassava (Manihot 
esculenta) was done to test its level of  effectiveness 
against E. coli. The responses of  the growing size of  E. 
coli to Fresh and Dried Cassava (Manihot esculenta) on 
the 18th and 24th hours were the data to be gathered and 
analyzed. 

Antibacterial Treatment
Fresh and dried Manihot esculenta leaves extract with 
concentrations of  5%, 10%, 20%, and 30% of  95% 
ethanol were applied against the colony formed by 
cultured E. coli for 18 and 24 hours of  treatment. The 
diameters of  the treated E.coli was measured in the 18th 
and in the 24th hour respectively using a caliper. After 
close observation, both groups were tested to measure 
the degree of  change in the growth size of  E. coli. 
Statistical Tools and Treatment of  Data
Using a t-test, both fresh and dried Manihot esculenta 
leaves extract were compared according to their level 
of  effectiveness against E. coli during 18 and 20 hours 
of  treatment. ANOVA test was used in analyzing the 
effectiveness of  the concentrations between 5%, 10%, 
20%, and 30% of  both Fresh and Dried Manihot 
esculenta leaves extract. A conclusion was drawn after the 
analysis of  the results.

RESULTS
Table 1 presents the bacterial growth response of  Escherichia 
coli to the Fresh Cassava (Manihot esculenta) with 
concentrations of  5%, 10%, 20%, and 30% ethanol. Caliper 
was used in measuring the diameter of  treated Escherichia 
coli in the 18th and 24th hours. The data gathered in this 
study was analyzed using a t-test and ANOVA test. 



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Table 1: Fresh Cassava (Manihot esculenta) leaves extract with concentrations 5%, 10%, 20%, and 30% ethanol 
treatment against Escherichia coli
Diameters (millimeters)
Time Control 5% 10% 20% 30%
18th  hour 4 mm 4 mm 6 mm 4 mm 9 mm
24th  hour 2 mm 3.5 mm 5.5 mm 3 mm 8 mm

Table 2: Dried Cassava (Manihot esculenta) leaves extract with concentrations 5%, 10%, 20%, and 30% ethanol 
treatment against Escherichia coli.
Diameters (millimeters)
Time Control 5% 10% 20% 30%
18th  hour 4 mm 4 mm 4 mm 6 mm 5 mm
24th  hour 3 mm 4 mm 3 mm 5.5mm 4 mm

Table 2 present the s bacterial growth response of  
Escherichia coli to the Dried Cassava (Manihot esculenta) 
with concentrations of  5%, 10%, 20%, and 30% ethanol. 
The diameter was measured in millimeters using a caliper. 
Bacterial growth sizes were measured in the 18th and 
24th hours. The data gathered in this study were analyzed 
using a t-test and ANOVA test. 

Table 3: The t-test result for fresh Cassava Manihot 
esculenta extracts in comparison to 18 and 24 hours 
t-test paired two samples for means.
Time Observations Mean df t-value
18 hours 5 5.4 4 0.01*
24 hours 5 4.4
*Significant at α=0.05

Table 3 compares the effects of  fresh Manihot esculenta 
leaves extract after 18 and 24 hours of  therapy. Fresh 
Manihot esculenta leaves exhibit a significant difference, 
with the 18-hour having a mean of  5.4 and the 24-hour 
having a mean of  4.4. The fresh Manihot esculenta 
leaves extract is most active around the 18-hour mark of  
treatment, according to this finding. Table 4 compares 

Table 4: The t-test for dry Manihot esculenta extracts 
in comparison to the 18 and 24 hours t-test paired two 
samples for means
Time Observations Mean df t-value
18 hours 5 4.4 4 0.05ns
24 hours 5 3.9
*Not significant at α=0.05 

the effects of  dried Manihot esculenta leaves extract 
after 18 and 24 hours of  therapy. There is no significant 
difference in the dried Manihot esculenta after 18 and 
24 hours of  treatment. According to this finding, dry 
Manihot esculenta leaves were effective for both 18 and 
24 hours.
In 18 hours of  therapy, Table 5 compares the effectiveness 
of  fresh and dried Manihot esculenta leaves extract. With 
a mean of  5.4, the fresh Manihot esculenta leaves extract 
differs significantly from the dried Manihot esculenta 
leaves extract, which has a mean of  4.4. Fresh Manihot 

Table 5: The t-test result for fresh and dried Manihot 
esculenta leaves extracts in 18 hours in t-test paired two 
samples for means.
Type of  
leaf  extract

Observations Mean df t-value

Fresh 5 5.4 4 0.19*
Dried 5 4.4
*Significant at α=0.05 

esculenta leaves extract was the most effective treatment 
against Escherichia coli after 18 hours.
In 24 hours of  treatment, the efficiency of  fresh and 
dried Manihot esculenta leaf  extracts against Escherichia 
coli is compared in Table 6. With a mean of  4.4, the fresh 
Manihot esculenta leaves extract differs significantly from 
the dried Manihot esculenta leaves extract, which has a 
mean of  3.9. After 24 hours, fresh Manihot esculenta 
leaves extract was the most effective against Escherichia 
coli.
Table 7 examines the effectiveness of  fresh Manihot 

Table 6: The t-test result for fresh and dried Manihot 
esculenta extracts in 24 hours t-test paired two samples 
for means.
Type of  
leaf  extract

Observations Mean df t-value

Fresh 5 4.4 4 0.19*
Dried 5 3.9
*Significant at α=0.05 

Table 7: The ANOVA result for the concentration 
between the control, 5%, 10%, 20%, and 30% for Fresh 
Manihot esculenta leaves extract.
Source of  
Variation

SS df MS F P-value

Rows 73.57 2 36.78 21.71 0.00*
Columns 22.12 3 7.37 4.35 0.05*
Error 10.16 6 1.69
Total 105.86 11   
*Significant at α=0.05 



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esculenta leaves extract at concentrations of  5%, 10%, 
20%, and 30%. With a P-value of  0.00, the fresh Manihot 
esculenta leaves extract demonstrates a significant 
difference. With an average of  0.16, the control extraction 
of  fresh Manihot esculenta leaves extract is the least active 
therapy against Escherichia coli. With an average of  5.75, 
the 20% concentration is the most effective therapy 
against Escherichia coli, while the 5% concentration is 
the second most effective treatment against Escherichia 
coli, with an average of  5. Table 8 examines the 

Table 8: The ANOVA result for the concentration 
between the control, 5%, 10%, 20%, and 30% for dry 
Manihot esculenta leaves extract.
Source of  
Variation

SS df MS F P-value

Rows 46.20 2 23.10 52.23 0.00
Columns 5.57 3 1.85 4.19 0.06
Error 2.65 6 0.44
Total 54.43229 11
*Significant at α=0.05 

effectiveness of  dry Manihot esculenta leaves extract 
at concentrations of  5%, 10%, 20%, and 30%. With a 
P-value of  0.00, the dry Manihot esculenta leaves extract 
shows a significant difference. With an average of  0.16, 
the control extraction of  dry Manihot esculenta leaves 
extract was the least active treatment against Escherichia 
coli. The 20% concentration is the most active treatment 
with an average of  4.5, while 10% was the second most 
active treatment with an average of  4.125.

Findings
Based on the results and findings of  this study, the Fresh 
and Dried Cassava (Manihot esculenta) leaves extract 
with concentrations 5%, 10%, 20%, and 30% of  ethanol 
had effective antibacterial activity against the bacterial 
growth size of  Escherichia coli. 
In the 18th and 24th hour mark of  treatment, both Fresh 
and Dried Cassava (Manihot esculenta) leaves extract 
with concentrations 5%, 10%, 20%, and 30% of  ethanol 
treatments were active against Escherichia coli.  The Fresh 
Cassava (Manihot esculenta) leaves extract exhibited 
a significant difference and was most active treatment 
against Escherichia coli on both 18th and 24th hour mark 
of  treatment. In addition, according to the results of  
the study the Fresh Cassava (Manihot esculenta) leaves 
extract with concentrations 5%, 10%, 20%, and 30% 
of  ethanol was most active on the 18th hour mark of  
treatment against Escherichia coli.  
On the other hand, Dried Cassava (Manihot esculenta) 
leaves extract with concentrations 5%, 10%, 20%, and 
30% of  ethanol treatments against Escherichia coli had 
no significant difference in the changes of  bacterial 
growth size. According to the findings of  this study, the 
Dried Cassava (Manihot esculenta) leaves extract was 
least active on the 18th and 24th hour mark of  treatment.
The findings of  this study concluded that Fresh Cassava 

(Manihot esculenta) leaves extract with concentration 
of  20% of  ethanol was most active treatment against 
the bacterial growth size of  Escherichia coli. This 
concentration was most active in both 18th and 24th 
hour mark of  treatment. According to (Santos, 2013), 
the Fresh Cassava leaves extract contains rich amount of  
tannins, bioflavonoids and rutins. The presence of  this 
phytochemicals indicates its active antibacterial ability 
in suppressing the bacterial growth size of  Escherichia 
coli. On the other hand, the most active concentration 
of  Dried Cassava (Manihot esculenta) leaves extract 
was 20% of  ethanol concentration. The results of  the 
study affirmed that the optimal concentration added to 
the Cassava (Manihot esculenta) leaves extract was 20% 
ethanol. It was supported by (Chatterjee, 2006) that 20% 
ethanol was a high concentration and was bacteriostatic. 
This concentration prevents the growth of  bacteria. 
Summary
The primary objective of  this research was to examine 
the antibacterial activity of  Fresh and Dried Cassava 
(Manihot esculenta) leaves extract against the Escherichia 
coli colonies. The antibacterial efficiency of  Fresh and 
Dried Cassava (Manihot esculenta) leaves extract was 
added with varying concentrations of  5%, 10%, 20%, 
and 30% ethanol. These concentration was added to 
test the Fresh and Dried Cassava (Manihot esculenta) 
leaves extract level of  effectiveness in treatment against 
Escherichia coli.
The maceration method was used in the extraction 
process, which required utilizing 95 percent ethanol and 
keeping it at room temperature. This method was selected 
to prevent the occurrence of  damage to the thermolabile 
chemical compounds contained in the cassava leaves 
(Mustarichie, 2021). The maceration method was done by 
soaking the powdered cassava leaves into the macerator 
with concentrations of  5%, 10%, 20%, and 30% ethanol 
and was leaf  for 48 hours with occasional stirring.
The bacterial growth size of  Escherichia coli that was 
treated with Fresh and Dried Cassava (Manihot esculenta) 
leaves extract with concentrations of  5%, 10%, 20%, 
and 30% ethanol, was measured in the 18th and 24th 
hour using a caliper. The data results were gathered and 
analyzed using t-test and ANOVA-test tools. 

CONCLUSION  
The results of  this study concluded that Fresh and Dried 
Cassava (Manihot esculenta) leaves extract demonstrated 
antibacterial activity against Escherichia coli. This study 
compares the effectiveness of  Fresh Manihot esculenta 
leaves extract at concentrations of  5%, 10%, 20%, 
and 30%, as well as Dried Manihot esculenta leaves at 
concentrations of  5%, 10%, 20%, and 30%. The varying 
concentrations added to the Fresh and Dried Cassava 
(Manihot esculenta) leaves extract will examine the level 
of  efficiency as an antibacterial agent against Escherichia 
coli.
The fresh Cassava (Manihot esculenta) leaves extract at 
a concentration of  20% has the most active treatment in 



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Am. J. Environ. Clim. 1(2) 23-30, 2022

suppressing the colony size of  Escherichia coli, with an 
average of  5.75. In addition, in the 18th hour of  treatment, 
the 20% concentration of  fresh Cassava (Manihot 
esculenta) leaves extract was the most active. Taking the 
control out of  the evaluation, the 5% concentration of  
fresh Manihot esculenta leaves was the least effective 
treatment in reducing the bacteria’s growth size. In the 
18th and 24th hours of  treatment, the dried Cassava 
(Manihot esculenta) leaves extract exhibits no significant 
difference. The dried Cassava (Manihot esculenta) leaves 
extract was found to be efficacious in both the 18th and 
24th hours. The 20 percent concentration of  dry Cassava 
(Manihot esculenta) leaves extract was the third most 
active therapy against Escherichia coli, with an average 
of  4.5. Then there’s a 10% concentration with an average 
of  4.125.
As a result, it can be stated that a 20 % concentration 
of  fresh Cassava (Manihot esculenta) leaves extract was 
most effective in reducing Escherichia coli growth size.
 
RECOMMENDATION
With the results presented, the cassava plant can be 
used not only as a good source of  food, but also in 
microbiology and medicine. Future research should 
look into the impact of  cassava leaves on human health 
after they’ve been used as an antibacterial agent. Future 
researchers also could conduct a study regarding to 
effectiveness of  Cassava (Manihot esculenta) in treating 
infections and diseases caused by Escherichia coli.
In addition, recommendations have been made to educate 
the community with the antibacterial efficiency of  Cassava 
leaves to provide immediate response and treatment on 
common bacterial infections. Additional research into 
the Cassava plant’s nutrient content and application as 
an antibacterial agent against various microbial infections 
that cause disease is highly recommended. Future 
researchers especially in the field of  medicine must further 
evaluate the effectiveness of  Cassava (Manihot esculenta) 
leaves in treating bacterial infections and developed it as a 
standardized antibacterial agent.

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