European Journal of Medical and Health Sciences, 2(2), 28–38, 2020
Publisher homepage: www.universepg.com, ISSN: 2663-7529 (Online) & 2663-7510 (Print)
https://doi.org/10.34104/ejmhs.020.28038
European Journal of Medical and Health Sciences
Journal homepage: www.universepg.com/journal/ejmhs
Antibacterial Activity of Cissus quadrangularis Stem Extract on the
Pathogenic and Industrial Waste Watered Bacteria
Md. Golam Mosaib1, Md. Abdullah Al Maruf2, Rabiul Islam3, Shahriar Mahmud4, Shaharuq Nahid
Sohana4, Md. Abu Sayeed Imran4, Mehadi Hasan Rony4, Maidul Islam5, Fatema Tuz Zuhora6, and
Shafiqul Islam6
1
Dept. of Biochemistry and Molecular Biology, Gono Bishwabidyalay, Savar, Dhaka, Bangladesh; 2Dept. of Biochemistry
and Molecular Biology, Jahangirnagar University, Dhaka, Bangladesh; 3Divisional DNA Screening Laboratory, Faridpur
Medical College Hospital, Ministry of Women & Children Affairs, Faridpur, Bangladesh; 4Dept. of Biotechnology and
Genetic Engineering, Islamic University, Bangladesh; 5Apex Biotechnology Laboratory, Dhaka, Bangladesh; and 6Dept. of
Microbiology, Gono Bishwabidyalay, Savar, Dhaka, Bangladesh
*Correspondence: gmosaib@gmail.com
ABSTRACT
Cissus quadrangularis (Vitaceae) is a popular climber conspicuous by its flesh quadrangular stem widespread
throughout the Bangladesh. The in vitro antimicrobial activity of C. quadrangularis extracts was studied
against selected pathogenic bacteria, industrial wasted bacteria, and broth dilution assay. The most commonly
used method of microbiological assay is the disc diffusion method. C. quadrangularis stem extracted with four
solvents (Petroleum spirit, methanol, ethyl acetate, and dichloromethane) were tested for antimicrobial activities
against some pathogenic microorganisms Sarcina lutea (002-1), Xanthomonas campestris (004-1), Escherichia
coli (005-1), Klebsiella pneumonia (006-1) and some industrial (Tannery, Tobacco, and Sugar mill) waste
watered bacteria by disc diffusion method. Among the four extracts, ethyl acetate showed moderate
antibacterial activity against X. campestris (004-1) and industrial watered bacteria. But, the commercial disc
Oxicycline doesn’t show any antibacterial activity against the industrial waste watered bacteria. Petroleum
spirit, methanol, and dichloromethane extract were ineffective against all of the tested bacteria.
Keywords: Cissus quadrangularis, Antibacterial effect, Industrial waste, Stem extract, Pathogenic, and Tannery.
INTRODUCTION
Medicinal Plants have been used as a source of
medicine since the dawn of civilization. The plant
designed as medicinal is implied that it is useful as a
drug or therapeutic agent or an active ingredient of a
medicinal preparation. Various medicinal plants have
been applied for years in daily life to treat disease all
over the world (Nair et al., 2004). The stem contains
two unsymmetrical tetracyclic triterpenoids, and two
steroidal principles. The presence of β-sitosterol, δUniversePG I www.universepg.com
amyrin, δ-amyrone, and flavonoids (quercetin) having
different potential metabolic and physiological effects
have also been reported (Jainu and Devi, 2004). The
ulcer protective effect of a methanolic extract of C.
quadrangularis was similar to that of the reference
medicine sucralfate (Jainu and Devi, 2004). Many
imitate agents such as estrogens in hormone
replacement therapy, especially estrogen receptor
modulators have been designed to treat osteoporosis
but each one of them is linked with side effects such
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Mosaib et al., / European Journal of Medical and Health Sciences, 2(2), 28-38, 2020
as hypercalcemia, hypercalciurea, raised risk of
endometrial, and breast cancer, breast tenderness,
menstruation, thromboembolic events, vaginal
bleeding and hot flushes (Ogey et al., 2001; and
Sanyal and Ahmad, 2005).
C. quadrangularis (Linn) has been applied by
common people in Bangladesh for promotion of
fracture healing and well known as Hadjod. It is also
known as Vitis quadrangularis wall which belongs to
family Vitaceae. Some other news on C.
quadrangularis noticed its effectiveness in
management of obesity, and symptoms linked with
metabolic disorders (Oben et al., 2006), as well as its
antioxidant and free radical scavenging pursuit in vitro
(Mallika and Shyamala, 2005).
Phytochemical studies of C. quadrangularis have
shown the presence of different versatile components
such as flavanoids, triterpenoids, Vitamin C, stilbene
derivatives and many others, e.g. resveratrol,
piceatannol, pallidol perthenocissin, and phytosterols.
Out of which ascorbic acid, triterpene, β-sitosterol,
ketosteroid, two asymmetrical tetracyclic triterpenoids
and calcium were identified as major constituents of
this plant (Jainu and Devi, 2004; and Enechi et al.,
2003). The root powder also contain a rich source of
mineral elements (mg/100g dry matter): potassium
67.5, calcium 39.5, zinc 3.0, sodium 22.5, iron 7.5,
lead 3.5, cadmium 0.25, copper 0.5 and magnesium
1.15 (Somova et al., 2003). Fresh stems of C.
quadrangularis develops irritating action on the skin,
which may be attributed to the existence of calcium
oxalate and 31 methyl tritiacontanoic acid along with
taraxeryl acetate, taraxerol and isopentacosanoic acid
(Prajapati et al., 2003).
Bangladesh is based with various kinds of plant and
most of them have medicinal properties. The ash
formed from the C. quadrangularis contains mostly
carbonates and to a smaller extent phosphates of
sodium, potassium, magnesium and calcium. Presence
of potassium tartarate is also reported (Austin et al.,
2004). The extract measured well with Acetyl salicylic
acid (Viswanatha et al., 2006). As it measured well
with acetyl salicylic acid its analgesic responses with
the nature of its chemically active components needs
to be explored (Jainu and Devi, 2004). The ethyl
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acetate fraction of both fresh and dry stem extracts at a
concentration of 100 ppm showed 64.8% antioxidant
activity in the β-carotene linoleic acid system and
61.6% in the 1, 1-diphenyl-2- picrylhydrazyl systems
(Furukawa et al., 2004). It works by preventing the
transformation of arachidonic acid to inflammatory
prostaglandins (Mallika and Shyamala, 2006).
The optimum preservative dose of 500 mg/kg of
extract was applied for the pretreatment of gastric
ulcers with various doses of CQE (250, 500, and 750
mg/kg) for 7 days which significantly attenuated these
biochemical alters caused by aspirin in rats (SanchezFidalgo et al., 2004). So, the plant has been taken into
consideration with a keen interest to investigate the
following aims and objectives: Isolation of the extract
from C. quadrangularis stems using different solvents
such as ethyl acetate, dichloro methane, and methanol
and petroleum spirit; observation of the antibacterial
activity of different extract of C. quadrangularis
against various infectious bacteria and industrial waste
watered bacteria; comparisonal study of commercial
discs and prepared discs of C. quadrangularis stem on
bacterial growth; and determination the MIC values of
using different solvent extracts of C. quadrangularis
stem against different bacterial strain and industrial
waste watered microbes.
MATERIALS AND METHODS
The experiment has been conducted in Microbiology
and Biochemistry laboratory at Gono University,
Savar, Dhaka, Bangladesh for the screening of
antimicrobial activity, is a typical microbiological
assay, which is performed with culture of
microorganisms. The zone of inhibition is compared in
millimeter (mm) unit. This is a measure of
antibacterial activity of the test compound. The
materials and methods applied in this investigation are
described below under the following heading:
Plant Material: Cissus quadrangularis stem.
Collection of Plant Material: The C. quadrangularis
stem was collected from Dhaka District of Bangladesh
in the month of September, 2019. There is usually a
wide choice among liquids to be applied as solvents
for extraction operation. However the following
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Mosaib et al., / European Journal of Medical and Health Sciences, 2(2), 28-38, 2020
solvents were applied in this experiment: Methanol,
Ethyl acetate, Dichloro methane, and Petroleum spirit.
Collection of Industrial Waste Water: Three (3)
samples of waste water were collected from the
following sources - Tannery waste water from
Hazaribagh; Tobacco waste water from leaf factory,
Gazipur; and Sugar mill waste water from Faridpur
sugar mill, Faridpur.
Culture of Waste Watered Bacteria: Two types of
media were applied, viz., MacConkey Agar Media and
Nutrient Agar media. Each of the waste water was
cultured to MacConkey agar media and Nutrient agar
media was applied for spread culture. For stick
culture, the Nutrient agar media was applied. Then
some of the colonies were transferred to conical flask
for liquid culture with the help of Nutrient broth.
Extraction Methods: After collection of the C.
quadrangularis stem were cleaned with rinsed water
and cut down the stem and then air dried and then it
was pulverized into a fine powder (Fig 1).
Extraction from the Powder Sample: Ten (10) gm
of the C. quadrangularis stem powder were weighted
separately with electric balance and 60 ml each of the
solvent (methanol, ethyl acetate, dichloromethane, and
petroleum spirit) were added in each conical flask.
The powder was dissolved separately with methanol,
ethyl acetate, and dichloromethane and petroleum
spirit. The samples with solvent were placed in water
bath shaker for 24 hours at (30-36) oC for proper
extraction.
Extraction of the Extract: The extract of plant
materials was filtered. This was performed by passing
the extracts through Whatman filter paper.
[
Bacterial Species: Gram negative i.e. E. coli (005-1),
K. pneumoniae (006-1) and Gram positive i.e. S. lutea
(002-1) and X. campestris (004-1) were applied in the
present study to determine the antibacterial activity of
the different extract.
Culture of Bacterial Species: For culture of bacterial
species from the stock culture of bacteria, lactose
broth media was applied. For preparation of lactose
media, suspended 0.325 gm of powdered broth in 25
ml distilled water in a conical (100ml) flask. Then the
each species of stock cultured bacteria were
transferred to each conical flask in front of laminar air
flow. Then the flasks were shaked in a shaker machine
about 24 hrs. The major approaches for testing the
antimicrobial activities of extracts were disc diffusion
method, agar dilution method (Luangtongkum et al.,
2007) and the broth dilution method (Kianbakht and
Jahaniani, 2003). In this experiment the disc diffusion
method was applied.
Fig 1: Preparation of stem sample and fine powder.
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Concentrating the Extract and Preparation of Disc:
The extracts were then air dried after filtration to
concentrate. The filter paper was punched with the
punching machine and the disc was made. The discs
were taken into a Petri dish and sterilized in an
autoclave for 15 minutes with 121 ºC and 15 psi
pressure.
Bacterial Culture Media: For cultivation and
maintenance of different bacterial culture and for the
identification and microbial sensitivity, nutrient agar
was applied. Nutrient agar medium; a microbiological
culture medium commonly applied for the routine
cultivation of non-fastidious bacteria. Also, it can
grow on the surface of the Nutrient agar and is clearly
visible as tiny colonies. In nutrient broth, the bacteria
grow in the liquid, and were visible as a soupy
component, not as clearly distinguishable clumps.
Preparation of Media for the Tested Organisms: In
this study, nutrient agar media was applied for
antibacterial screening. For the test, 5.6gm of nutrient
agar was dissolved into 200ml distilled water in 250ml
conical flask. The media was properly dissolved with
the distilled water and then sterilized in an autoclave
for 15 minutes with 121 ºC and 15 psi pressure. After
autoclaving, the media was poured into the autoclaved
Petri dishes in the laminar air flow cabinet.
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Table 1: Composition of the culture media was used.
Composition of Lactose Broth (LB) Media
Ingredients
Composition of Nutrient Agar (NA) media
Amount(gm/l)
Peptone
Meat (beef)
Lactose
Ingredients
5.00
3.00
5.00
Amounts(gm/l)
Peptic digest of the animal tissue.
Sodium chloride
Beef extract
Yeast extract
o
pH ( at 25 C )
6.9±2
Agar
pH ( at 25 C ) 6.8 ± 0.2
Minimum Inhibitory Concentrations (MIC)
Determination of C. quadrangularis Stem Extracts:
In the experiment, Minimum inhibitory concentrations
(MICs); as the lowest concentration of an
antimicrobial that will inhibit the visible growth of a
microorganism after overnight incubation. MICs were
applied by diagnostic laboratories prominently to
confirm resistance, but most often as a study tool to
examine the in vitro activity of new antimicrobials,
and data from such studies have been applied to
evaluate MIC breakpoints. The method gives
information on the depot of standard antibiotic
powder, preparation of stock antibiotic solutions,
media, and production of inocula, incubation periods,
and reading and interpretation of findings. In the
present study, it was determined following the serial
dilution. The lowest concentrations of the extracts,
which did not show any growth of tested organisms
after microscopic evaluation, were determined as MIC
(Rahman et al., 2019).
Sample Solution Preparation: Stock working
solution of the plant stem extract was prepared by
dissolving 0.316 gm dried stem extracts in 10 ml
solvent (ethyl acetate) into a separate flask. So it was
to be 10 times diluted. Then 10 ml of ethyl acetate
solvent was added in the same flasks. Then 162.00 μl
extract solution was transferred into a screw capped
test tube and 838.00 μl of same solvent was added in
the same test tube. Therefore, the final concentration
was reached to 512 μg/ml.
Serial Dilution: For preparing 512 μg/ml to 2 μg/ml,
1ml of the solvent was added to each of the nine
screws capped test tube. 1 ml of the having 512 μg/ml
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5.00
1.50
1.50
1.50
15
o
extracts was added to the 1st test tube containing 1ml
of particular solvent and mixed well in the vortex and
then 1ml of this solvent was shifted to the second test
tube containing 1ml of the same solvent. After mixing
well, 1ml of this mixture was shifted to the third test
tube. This process of serial dilution was continued up
to the last test tube. Finally, the clustered of the last
test tube was 2 μg/ml.
Preparation of Working Disc for Antimicrobial
Test: The disc paper was soaked with each
concentration of extracts and placed at room
temperature for air dry for 15 hours. After completion
of air dry, the disc paper was labeled according to
different concentration and finally the labeled disc
paper was taken into the vial and it was ready for
antibacterial activity and the waste watered (Tannery,
Tobacco, Sugar mill) bacteria.
RESULT AND OBSERVATION
Determination of Antibacterial Activity of Ethyl
Acetate Extract of C. quadrangularis Stem: From
the Table 2 it has been shown that the stem powder of
the C. quadrangularis showed greatest antibacterial
activity against tested bacteria, viz., X. campestris
(004-1). The crude extract of stem powder produced
12 mm zone of inhibition against X. campestris (0041). It produced no zone of inhibition against other
tested bacterial strain.
The ethyl acetate extracts of C. quadrangularis stem
showed inhibitory activity against X. campestris (0041) with 12 mm inhibitory zone. Commercial antibiotic
disc (Penicillin) was applied as a positive control that
showed anti-bacterial activity against X. campestris
(004-1) (Fig 2).
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Table 2: Diameter of the zone of inhibition produced by ethyl acetate extracts of C. quadrangularis against
different bacterial strain.
Name and number
Zone of inhibition (mm)
of bacterial strain
Commercial antibiotic disc Extract of Ethyl acetate
30 (Kanamycin)
S. lutea (002-1)
17
(Penicillin)
+
X. campestris (004-1)
17 (Ampicillin)
E. coli (005-1)
34 (Ciprofloxacin)
K. pneumonia (006-1)
N.B. (-) Not Inhibition, (+) Inhibition
Negative Control
-
40
30
Positive control
20
Negative control
10
Ethyl acetate
extract
0
S. lutea
X.
campestris
E. coli
K.
pneumonia
Fig 2: Comparative antibacterial activity of commercial disc, ethyl acetate extract of C. quadrangularis and ethyl
acetate solvent against selected micro-organisms.
Zone of inhibition of
commercial Penicillin
Zone of inhibition of C.
quadrangularis
Fig 3: Zone of inhibition with ethyl acetate extracts of C. quadrangularis against X. campestris (004-1).
No zone of inhibition of
Crude extract
Zone of inhibition of
commercial Penicillin
Fig 4: No zones of inhibition with dichloromethane extract of C. quadrangularis against X. campestris (004-1).
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Inhibition of
commercial Penicillin
No zone of inhibition
of Crude extract
Fig 5: No Zone of inhibition with methanol extracts of C. quadrangularis against X. campestris (004-1).
No zone of inhibition
of Crude extract
Zone Inhibition of
commercial Penicillin
Fig 6: No zones of inhibition with petroleum spirit extract of C. quadrangularis against X. campestris (004-1).
Table 3: MIC of Ethyl Acetate against X. campestris (004-1)
Microorganisms
1024
S. lutea
+
X. campestris
E. coli
K. pneumoniae
N.B. (-) Not Inhibition, (+) Inhibition
Concentration of Ethyl acetate extract (µg/ml)
512
-
256
-
128
-
64
-
32
-
16
-
Control
-
Zone of inhibition against
X. campestris
No zone of inhibition
against X. campestris.
Fig 7: MIC of ethyl acetate extract against X. campestris (004-1); A, B, C, D, E, F, G disc contain 1024, 512, 256,
128, 64, 32, 16 µg/ml respectively.
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The ethyl acetate extract shows an inhibitory zone at
1024 µg/ml concentration and does not show any
antibacterial activity at the concentration of 512
μg/ml, 256 μg/ml, 128 μg/ml, 64 μg/ml, 32 μg/ml, and
16 μg/ml against X. campestris. So; the MIC value of
ethyl acetate extract is 1024 μg/ml (Table 3).
Determination of Antibacterial Activity against
Waste Watered Bacteria (Tannery, Tobacco, and
Sugar Mill)
From the Table 3 it has been shown that the stem
powder of the C. quadrangularis showed antibacterial
activity against bacteria that lived in waste water. The
crude extract of stem powder produced 18 mm zone of
inhibition against Tobacco waste water, 14 mm zone
of inhibition against Tannery waste water and 16mm
zone of inhibition against Sugar mill waste watered
bacteria.
Table 4: Diameter of the zone of inhibition produced by ethyl acetate extracts of C. quadrangularis again
different industrial waste watered bacteria.
Industrial
Sample of
Microbes
Diameter of zone of inhibition (mm)
Commercial antibiotic disc (Oxicycline)
Tobacco
Tannery
Sugar
N.B. (-) Not Inhibition, (+) Inhibition
Crude extract (mm)
Negative control
-
+
+
-
-
+
-
30
Commercial antibiotic
disc
Crude extract
20
10
Negative control
0
Tobacco Tannery
Sugar
Fig 8: Comparative antibacterial activity of commercial disc, Ethyl acetate extract of C. quadrangularis and Ethyl
acetate solvent against selected waste watered microorganisms.
Zone of inhibition of with ethyl acetate (EA) extract of C. quadrangularis stem against Tannery, Tobacco
and Sugar mill waste water:
Zone of inhibition of C.
quadrangularis stem
No zone of inhibition of
commercial Oxicycline
Fig 9: Zone of inhibition with ethyl acetate (EA) extract of C. quadrangularis stem against Tannery waste water.
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No zone of inhibition of
commercial Oxicycline
Zone of inhibition of C.
quadrangularis stem
Fig 10: Zone of inhibition with ethyl acetate (EA) extract of C. quadrangularis stem against tobacco waste water.
Zone of inhibition of C.
quadrangularis stem
No zone of inhibition of
commercial Oxicyclin
Fig11: Zone of inhibition with ethyl acetate (EA) extract of C. quadrangularis stem against Sugar mill waste
water.
Table 5: MIC of Ethyl Acetate against Tobacco, Tannery and Sugar.
Industrial sources of
microorganisms
Tobacco
Tannery
Sugar
Concentration of Ethyl acetate extract (µg/ml)
1024
512
256
128
64
32
16
Control
+
+
+
-
-
-
-
-
-
-
-
N.B. (-) Not Inhibition, (+) Inhibition
Zone of inhibition of C.
quadrangularis stem
Fig 12: Zone of inhibition with ethyl acetate (EA) extract at A in 1024 μg/ml of C. quadrangularis stem against
Tannery waste water.
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Zone of inhibition of
C. quadrangularis
Fig13: Zone of inhibition with ethyl acetate (EA) extract at 512 μg/ml of C. quadrangularis stem against sugar mill
waste water.
Zone of inhibition C.
quadrangularis stem
Fig 14: Zone of inhibition with ethyl acetate (EA) extract at 512 μg/ml of C. quadrangularis stem against
Tobacco waste water.
The ethyl acetate extract showed antibacterial activity
at the concentration of 512 μg/ml against all of the
waste watered bacteria. But ethyl acetate extract did
not show antibacterial activity at the concentration of
(256 μg/ml, 128 μg/ml, 64 μg/ml, 32 μg/ml, 16 μg/ml,
8 μg/ml, 4 μg/ml, and 2 μg/ml) against all of the waste
watered bacteria. Commercial antibiotic disc
(Oxicycline) was applied as a positive control that did
not show any anti-bacterial activity against waste
watered bacteria.
DISCUSSION
Plants have supplied a major source of inspiration for
novel drug components as plants derived drugs have
made pivotal contribution towards human health.
Phytomedicine can be applied for the therapy of
diseases as is done in case of Unani and Ayurvedic
system of drugs or it can be the base for the maturing
of a medicine. The results of the antibacterial activity
of stem of C. quadrangularis against the investigated
bacterial strains and industrial sample of microbes
were shown in the Table 2 and Table 4. The present
study proved the traditional use of C. quadrangularis
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as an antihemorrhoidal drug in Thai folk medicine
(Murthy et al., 2003).
The increasing social and economic implication
caused by pathogenic bacteria means there is constant
striving to develop new antibacterial agents (Uddin et
al., 2014). Due to the identified and potential toxicity
of chemical antibiotics, there has been an increasing
demand for safe and effective antibacterial from
natural sources (Happy et al., 2018). Thus plants
extracts are promising natural antibacterial agents with
potential applications in pharmaceutical industries for
controlling the pathogenic bacteria. In this experiment,
isolation and characterization of some clinical enteric
bacteria from laboratory of Gono University of Dhaka
and industrial waste water sample from different
industries and examined the effectiveness of a
medicinal plants water bacteria (Sharif et al., 2019).
The crude ethyl acetate extract of C. quadrangularis
showed potentate antibacterial activity against X.
campestris (004-1) strain in compared to commercial
antibiotic disc Penicillin and antibacterial activity
against some industrial waste water in compared to
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Mosaib et al., / European Journal of Medical and Health Sciences, 2(2), 28-38, 2020
commercial antibiotic disc Oxicycline. The presence
of antibacterial substances in the higher plants is well
established (Srinivasan et al., 2001). It also produced
18 mm zone inhibition against Tobacco waste water,
14 mm zone of inhibition against tannery, 16 mm zone
of inhibition against sugar mill waste water. So it has
been observed that ethyl acetate extract of C.
quadrangularis showed moderate antibacterial activity
against pathogenic bacteria and industrial waste
watered bacteria.
CONCLUSION
From the observed result of the research work it can
be concluded that the C. quadrangularis stem extract
inhibit the growth of pathogenic bacteria strain,
possesses the potent antibacterial activity against
pathogenic bacteria strain, antibacterial effect against
industrial (tannery, tobacco, and sugar mill) waste
watered bacteria, and can be applied as a therapeutic
agent. The ethyl acetate, dichloromethane, methanol
and petroleum spirit extract of C. quadrangularis was
tested for their antibacterial activity against selected
pathogenic bacteria (Firoz et al., 2016). The sample
solution is applied on the test plate containing
microorganism. The sample solution diffuses in the
surrounding medium and the plates are kept in an
incubator (37 oC) for 24 hours. The plant extract
possess any antibacterial activity, it will inhibit
bacterial growth in the surrounding area giving a clear
zone of inhibition (Shahen et al., 2019). It can be
concluded that the free radical scavenging activity of
the plant extract may be in charge of for the treating
action against tissue destroy (Gabriel et al., 2005). The
following works can be performed in future:
characterization of the active components of C.
quadrangularis and further characterization of the
microbes that present in industrial waste water.
ACKNOWLEDGEMENTS
The authors acknowledge the laboratory and financial
support from Gono Bishwabidyalay, Savar, and
Dhaka, Bangladesh and co-authors for their help
during the research work.
CONFLICT OF INTEREST
All the authors of this manuscript agreed that they
have no conflict of interest to publish the manuscript.
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