PHOTOCHEMICAL

SCREENING AND ANTIBACTERIAL ACTIVITY OF Cassia occidentalis

(Fabaceae L.) LEAVES EXTRACTS AGAINST ENTERIC BACTERIA
A
B.Sc. RESEARCH FINDINGS
BY

ALIYU UMAR
NAS/BIO/16/1036
UNDER THE SUPERVISION OF

MALAM MUJAHEED ABUBAKAR

DEPARTMENT Of BIOLOGICAL SCIENCES, FACULTY OF NATURAL AND APPLIED SCIENCES,

SULE LAMIDO UNIVERSITY KAFIN HAUSA JIGAWA STATE, NIGERIA.
MAY, 2021
 INTRODUCTION/LITERATURE REVIEW

MEDICINAL PLANT

PHYTOCHEMISTRY

Cassia occidentalis

ENTERIC BACTERIA

Escherichia coli

Salmonella typhi

Shigella spp.
 STATEMENT OF RESEARCH PROBLEM

A bacterial gastrointestinal infection (GIT) is a common illness worldwide and has a

considerable effect on the public health of communities. In developing countries, it is a major
cause of death claiming about two million lives each year among children's <5years of age
(Moorin et al., 2010).

Multiple resistance to currently available antibiotics by pathogens including bacteria

(Salmonella typhi and some Shigella spp.) are responsible for various diseases, in man is
increasing at an alarming rate and therefore the need to source for antimicrobial agent of
natural origin especially from enormous plant around us (Taiwo et al., 2013).

Also, synthetic medicines (antibiotics) exhibit more side effects than natural products. This
study is therefore one of the approach to solve such problems.
 JUSTIFICATION

Some clinical isolates (enteric bacteria) are capable of causing diseases towards our health.
These organisms are; Escherichia coli, which is responsible for causing most of diarrhea
especially in children, Salmonella typhi responsible for causing typhoid fever and Shigella spp.
responsible for causing shigellosis/dysentery (Prevost et al., 1995).

The increase use of antibiotics has induced microorganisms to acquire resistance factor which
have become a burning predicament as a result there's an urgent need to find the alternative of
chemotherapy drugs in diseases treatment particularly those of plants origin which are easily
available and have considerably less side effects (Khulbe and Sati, 2009).
 AIM OF THE STUDY

The study is aimed at investigating the phytochemicals present and the antibacterial
activity of Cassia occidentalis L. (leguminosae) leaves extracts against enteric
bacteria.  
 OBJECTIVES OF THE STUDY

To identify the phytochemicals present in the Cassia occidentalis leaves extracts.

To determine the antibacterial activities of Cassia occidentalis leaves extracts against enteric
bacteria (clinical isolates).

To determine the minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) of Cassia occidentalis leaves extracts.
 HYPOTHESIS

There's no antibacterial activity on the extract of Cassia occidentalis leaves

against the enteric bacteria.

There's no antibacterial activity on the extract of Cassia occidentalis leaves on the

MIC and MBC.
 MATERIALS AND METHODS

MATERIALS:
Pestle and mortar, Petri dishes, test tubes, test tube racks, spirit lamps, microscope,
autoclave, hand gloves, wire loop, what man filter, incubator, cork borer,
transparent ruler, micropipette, weighing balance, measuring cylinder, beakers,
droppers, media solvents and all other reagents were collected from the Laboratory
Unit, Department of Biological Sciences.
 METHODS

Plant sample collection and identification:

Cassia Occidentalis leaves was collected in March, 2021 from an uncultivated

farmland located in Kafin Hausa town Jigawa State Nigeria. It was then properly
cleaned from extraneous matter, shade dried was employed at room temperature
(25°C) for 3-4 days. The dried leaves material was ground to fine powder using
clean wooden pestle and mortar and stored for further use in an air-tight container
to prevent oxidation.

Identification of the plant materials was done in Biological Sciences Department

Laboratory, Sule Lamido University Kafin Hausa by Dr. Yusuf Nuhu and
accession number also was given.
 CONT’D

PREPARATION OF THE EXTRACTS:

The extracts were prepared by consecutive extraction method with little modifications using
an organic solvents with increasing of polarity order (hexane and methanol).

The leaves powder (100g) was macerated in n-hexane (500ml) at room temperature (25±5°C)
the content was mixed thoroughly and left to stand for about 72 h with an occasionally shaken
to increase the extraction capacity and then filtered using muslin cloth.

The marc (residue) obtained after extraction in hexane was further extracted consecutively
with methanol. The marc obtained in previous extraction step was extracted in the next
solvent and the extracts obtained in each extraction step were collected, dried and weighed for
calculation of yield percentage and later stored in an air tight container until required (Haq et
al., 2020).
CONT’D
PHYTOCHEMICAL SCREENING:
C. occidentalis leaves extracts were subjected to phytochemical screening using standard techniques as
described by (Taiwo et al., 2013; Harborne, 1998). The metabolites tested for includes alkaloids,
anthraquinone, tannins, flavonoids, saponins and steroids

TEST FOR ALKALOIDS

Wagner’s test: To 3ml of the sample extract, few drops of Wagner’s reagent was added along the side of the test
tube. The appearance of reddish-brown precipitate indicates the presence of Alkaloids.
(Wagner’s reagent = Aqueous solution of Iodine and Potassium Iodide)

TEST FOR SAPONINS

Frothing test: To 5ml of the sample extract, 10ml of distilled water was added and then shake vigorously for
about 30seconds, and then it allowed standing for 30min. A honey comb-like froth formed indicates the
presence of saponins.
CONT’D
TEST FOR TANNINS
Ferric Chloride Test: To 2ml of the sample extract, 5ml of distilled water was added and then filtered. To the
filtrate 2 drops of Ferric Chloride solution was added. The formation of a blue-black precipitate indicates the
presence of hydrolysable tannins.
TEST FOR FLAVONOIDS
NaOH Test: five drops of aqueous NaOH was added to 5ml of the plant extract, the appearances of yellow
coloration indicates the presence of Flavonoids
TEST FOR STEROIDS
Two ml of acetic anhydride was added to 2ml of each extract in a test tube. 1ml of conc. Sulphuric acid was
added down the side of the test tube. A colour change observed immediately and later of blue-green colour
indicates the presences of steroid
TEST FOR ANTHRAGUINONES
Borntrager test: About 1.0 g of the plant extract was dissolved in petroleum ether and filtered. Aqueous
ammonia was then added to the filtrate, formation of pink colouration was taken as indication for the presence
of anthraquinones in the plant extract.
CONT’D
TEST ORGANISMS CONFIRMATION:

Escherichia coli, Salmonella typhi, and Shigella spp. were collected from febrile patients
with gastrointestinal tract infection from Federal Medical Center Laboratory Clinic
Nguru, Yobe State.

The identification of the bacteria was based on their biochemical characteristics using
procedure described by Cheesbrough, (2018) (Coagulase test, Catalase test, Indole and
methyl red test).

Further, the pure culture was subcultured on nutrient agar slants and preserved in the
refrigerator at 4°C until required for study.
 CONT’D
Antibacterial screening test:
The antimicrobial activities of the extracts were determined using agar-well diffusion method
with little modifications as described by Taiwo et al. (2013)
 One hundred microliter (100 µL) of the standardized bacterial suspension was evenly spread
on nutrient agar medium using a sterile wire loop.
Wells were then bored into the agar medium using a sterile 6 mm cork borer and were filled
with the solution of the extracts taken care not to allow spillage of the solution to the surface
of the agar medium.
The plates were allowed to stand on the laboratory bench for 1 h to allow for proper
diffusion of the extract into the medium.
The plates were later incubated in an incubator at 37°C for 24 h after which they were
examined for zones of inhibition.
The effects of the extracts on the test isolates were compared with that of the standard
antibiotic, ciprofloxacin.
CONT’D
Determination of minimum inhibitory concentration (MIC):
Minimum inhibitory concentration of plant extracts was determined by broth dilution method,
a standard method of Hena et al. (2010).
Nutrient broth was prepared and sterilzed using autoclave. 1ml of the prepared broth was
dispensed into tubes numbered 1-9 using sterile syringe and needles. A stock solution of
6.25mg/ml of plant extracts was prepared. Then 1ml of the solution was dispensed into tube
numbered 1. Subsequently, from the tube 1, serial dilution was carried out where 1ml from
tube 1 was transferred on to tube 7 and 1ml from tube 7 was discarded.
The concentrations of the extracts after dilution was 6.25, 3.13, 1.56, 0.78, 0.39, 0.19 and
0.09mg/ml. Tube 8 was a control for sterility of the media (negative control) and tube 9 for
viability of the organisms (positive control). Then 1ml of the inoculum was transferred in to
each tube starting from tube 1 to tube 9 with exception of tube 8 to which another sterile
broth will be added. The tubes was incubated at 37°C for 24h and bacterial growth was
examined by observing the turbidity. The lowest concentration of the extract that inhibited
the growth of bacteria was detected by the lack of visual turbidity and was taken as minimum
inhibitory concentration (MIC).
CONT’D
Determination of minimum bactericidal concentration (MBC):
The MBCs of the extracts was determined using the method described by Adegboye et al.
(2008). Samples were taken from tubes with no visible growth in the MIC assay and sub-
cultured onto freshly prepared nutrient agar medium and later incubated at 37oC for 48 hours.
The MBCs were taken as the lowest concentration of extract that did not allowed any bacterial
growth on the surface of the agar plates.

Statistical Analysis:
The results are presented as mean ± standard deviation (SD). Comparisons between the groups
were performed by the analysis of variance (ANOVA) (using SPSS 20.0 for Windows Computer
Software Package). Significant differences were compared by Duncan’s Multiple Range test; a
probability level of less than 5% (P˂ 0.05) was considered significant (Duncan, 1955).
 RESULTS

Table 1: Percentage yield of extracts recovered.

_________________________________________________________________
Extracts Yield (%) Appearance
_________________________________________________________________
Methanol 12.7 Black gummy mass
n- Hexane 10.5 Green powdery mass
_________________________________________________________________
 CONT’D

Table 2: Phytochemical screening results obtained from C. occidentalis leaves extracts

__________________________________________________________________________
Phytochemical tested Test Methanol n-Hexane
__________________________________________________________________________
Alkaloids Wagner’s - -
Saponins Frothing + +
Tannins Ferric chloride + -
Flavonoids NaOH + -
Steroids Alkaline + +
Anthraquinones Borntrager + -
_________________________________________________________________________
Keys: + = positive, - = negatives
 CONT’D

Table 3: Biochemical test results for the test organisms.

_____________________________________________________________________
S/N Organisms Catalase Coagulase Indole MR
_____________________________________________________________________
1 Salmonella typhi + - - +
2 Escherichia coli + - + +
3 Shigella spp. nt nt + +
_____________________________________________________________________
Keys: + = positive, - = negative, MR = methyl red, nt = not tested
 
 CONT’D
Table 4: Antibacterial screening test result
__________________________________________________________________________________________
S/N Extracts/Tested Organisms Concentrations/zones of inhibition Ciproflaxacin (Positive control)

__________________________________________________________________________________________
25mg/ml 12.5mg/ml 6.25mg/ml 50mg/ml
Methanol
1 Escherichia coli 25mm 18mm 12mm 27mm
2 Salmonella typhi 30mm 24mm 20mm 38mm
3 Shigella spp. 14mm 11mm 9mm 32mm
n-Hexane
1 Escherichia coli 20mm 15mm 10mm 27mm
2 Salmonella typhi 22mm 19mm 11mm 38mm
3 Shigella spp. 12mm 9mm 7mm 32mm
__________________________________________________________________________________________
 40

35
ZONE OF INHIBITION (mm)

30

25

20 Series1
S. typhi
15 Shigell spp.

10

0
M 25mg/ml 12.5mg/ml 6.25mg/ml n 25mg/ml 12.5mg/ml 6.25mg/ml C 50mg/ml

EXTACTS CONCETRATONS (mg/ml)

Figure 1: Zones of inhibition of the extracts at different concentrations

 CONT’D

Table 5: Minimum Inhibitory Concentration (MIC) of C. occidentalis leaves extracts

___________________________________________________________________________
S/N Test Organisms MTHE (mg/ml) NHNE (mg/ml)
___________________________________________________________________________
1 Escherichia coli 0.39 1.56
2 Salmonella typhi 0.19 0.78
3 Shigella spp. 1.56 3.13
___________________________________________________________________________
Key: MTHE = Methanol , NHNE = n-hexane
 
 CONT’D

Table 6: Minimum Bactericidal concentration (MBC) of C. occidentalis leaves extracts.

_______________________________________________________________________
S/N Test Organisms MTH (mg/ml) NHN (mg/ml)
__________________________________________________________________________
1 Escherichia coli 1.56 6.25
2 Salmonella typhi 0.39 3.13
3 Shigella spp. 3.13 6.25
__________________________________________________________________________
Key: MTH = Methanol, NHN = n-hexane
 
 DISCUSSION
Among the solvents used, methanol was found to have the highest percentage yield of 12.7% and n-
hexane with 10.5%. Compare with the study of pedpriya et al. (2010). Showed that equeous and methanol
was found to have maximum extractive yield than petroleum ether, chroloform and benzene. This showed
that the plant materials were more soluble in polar solvents as compared to the non-polar solvents.

Investigation on the phytochemicals compounds of C. occidentalis leaves extracts revealed the presence of
saponins, tannins, flavanoids, steroids and anthraquinones with absence of alkaloids in the methanol and
n-hexane extracts and the presence of these phytochemicals varied to the one another. Similarly in study of
pedpriya et al. (2010). Reported the same with our finding with absence of alkaloids and in contrast with
the study of Nnma et al. (2018). Which revealed the presence of saponins, tannins, alkaloids, flavanoids
and anthraquinones. The medicinal properties of plant materials results from the secondary metabolites
present in the plant which maybe attributed to a single compounds or a combination of the metabolites.
Phytochemicals exert antimicrobial activity through different mechanisms, tannins for instance act by iron
deprivation, hydrogen bounding or specific interact-tions with vital proteins such as enzymes in microbial
cells (Scalbert, 1991).
CONT’D
presence study of antibacterial potential of the crude extracts represented by the mean of zone
of inhibition (mm) of different concentration (25, 12.5, and 6.25mg/ml) against the test
bacteria are represented in. The crude extracts showed the appreciable antibacterial activity.
Plant extracts are potential sources of novel antimicrobial compounds especially against
bacterial pathogens (Abdulsalami et al., 2019).

The present investigation showed that the crude extracts inhibited the growth of
gastrointestinal track pathogens but their effectiveness varied with the extracts. The methanol
extract showed the best antibacterial activity with zone of inhibition ranging from 9 - 30mm,
while that of n-hexane ranging from 7 - 22mm, indicating the synergetic activity of the
phytochemicals present. Similarly in the study of Vedpriya et al. (2010). Methanol extract was
found to be best active against six tested bacteria (P. aeruginosa, K. pneumoniae, P. mirabilis,
E. coli, S. aureus, S. epidermidis). Also in previous studies, for leaves extract, E. coli was
found to be sensitive (Jain et al., 1998). and in some experiments resistant (Abo et al., 1998).
CONT’D
Saganuwan and Gulumbe (2006). Observed that the E. coli was sensitive to
methanol, hexane, chloroform and aqueous extract of leaves of C. occidentalis
(collected from Kacha town, Niger state) at a concentration range 900-1000
mg/mL. Therefore, is work support my work. All the organisms test showed
different form of susceptibility but S. typhi are found to be more sensitive to the
extracts at all concentrations ranging from 20 - 30mm, followed by E. coli ranging
from 12 - 25mm and last shigella spp. with less effect ranging from 9 - 14mm.
Also the n-hexane exhibited a good activity on all the organisms at all
concentrations S. typhi ranging from 11 - 22mm followed by E. coli ranging from
10 - 20mm and Shigella spp. with 7 - 12mm against the bacterial isolates .

Therefore n-hexane has less effect compared to methanol extract. This could be
due to the fact that the active compounds present in the plant were more soluble in
polar solvents than the non polar solvents.
CONT’D

Generally, the diameter zone of inhibition increased with increased extract

concentrations. Similar results have also obtained by other researchers, Taiwo et
al. (2013) reported that the dichloromethane and n-hexane fractions of the plant
extract of C. occidentalis leaves possess biocidal property against some
pathogenic bacterial isolates or organisms of clinical origin. Moreover, the study
carroborates with the work of Vedpriya et al. (2010) who demonstrated in-vitro
antibacterial effect of C. occidentalis leaves extract on various human pathogenic
microbes incriminated in gastrointestinal track infection.
CONT’D
However the reference standard antibiotic showed better inhibitory property (P<0.05) on the
test isolates than the leaves extracts . This is confirmed by the report of Oluduru et al.
(2011) that conventional antibiotic drugs are more active than the plant extract seemingly
due to the chemical synthesis of the pure compounds of the antibiotics.

E. coli, S. typhi and Shigella spp. which are all known to cause ailments in man we're also
susceptible to these extracts (Taiwo et al., 2013). Thus, the study support the usefulness of
C. occidentalis in folklore remedies for the treatment of disease caused by these pathogens.

The analysis of variance one way ANOVA showed that there's significance different between
the groups (solvents) and between the E. coli, S. typhi and Shegella spp in their zones of
inhibition (P<0.05).
CONT’D

The MICs exhibited by methanol and n-hexane extracts against the susceptible
bacterial isolates ranging from 0.19 to 1.56mg/ml and 0.78 to 3.13mg/ml , showed
that Shigella spp. has the highest MIC from both methanol and n-hexane extracts
of (1.56 and 3.13mg/ml) followed by E. coli (0.39 and 1.56mg/ml) and S. typhi
with least MIC of (0.19 and 0.78mg/ml) respectively. And this showed that S.
typhi is more effective than E. coli and Shigella spp. However, this is an indication
that the two extracts exhibited different antibacterial effect against the susceptible
isolates.
The MBC exhibited by methanol extract against the susceptible organisms
ranging from 0.39 to 3.13mg/ml. In the other hand, the MBC exhibited by n-
hexane extract against the isolates was between 3.13 to 6.25mg/ml. This is a
reflection of what was observed for the MICs results.
 CONCLUSION
Conclusively, the presence of these bioactive compounds as revealed by the
phytochemicals screening of leaves extracts of C. occidentalis leaves, maybe
responsible for potent antibacterial activity of the plant. Escherichia coli, Salmonella
typhi and Shigella spp. are susceptible to Cassia occidentalis leaves extracts. The
activity of the extracts on the test organisms increased with increase in concentrations
The extracts exhibited bacteriostatic effects on the test organisms at lower
concentrations (MICs) and bactericidal effects at higher concentrations (MBCs) It
also showed that polar solvents are more effective than the non-polar solvents.

This study therefore provide the scientific backings and justifies the traditional claim
of the plant and it's immense potential in the treatment of bacteria that are implicated
in either cholera, diarrheoa, dysentery or gastrointestinal disorder. It also concluded
that the potency of the herbal mixture is dependent on the dose administered, which
may vary with target organisms or infections.
 RECOMMENDATION
The antibacterial assay should be carried out at higher concentrations of the
methanol and n-hexane extracts of Cassia occidentalis leaves to assess their
activity on Shigella spp.

The Cassia occidentalis leaves extracts should also be tested on other micro-
organisms to ascertain their activity on other disease-causing agents.
 It is necessary to determine the toxicity of the bioactive constituents and their
side effects, thereby establishing their pharmacokinetics.

The ministry of health should make it mandatory that all herbal products be
subjected to scientific verifications before being sold and use as remedy
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APPENDIX , APPENDIX A AND B
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