Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2022, Article ID 5436476, 15 pages
https://doi.org/10.1155/2022/5436476
Research Article
GC-MS Analysis, Antibacterial and Antioxidant Potential of Ethyl
Acetate Leaf Extract of Senna singueana (Delile) Grown in Kenya
Paul Ochieng Nyalo ,1,2 George Isanda Omwenga ,1 and Mathew Piero Ngugi
1
2
1
Department of Biochemistry, Microbiology and Biotechnology, Kenyatta University, P.O Box 43844-00100, Nairobi, Kenya
Penda Health (K) Ltd, Medical Laboratory Department, P.O Box 22647-00100, Nairobi, Kenya
Correspondence should be addressed to Paul Ochieng Nyalo; ochipaul@gmail.com
Received 17 May 2022; Accepted 4 July 2022; Published 17 August 2022
Academic Editor: Olufunmiso Olusola Olajuyigbe
Copyright © 2022 Paul Ochieng Nyalo et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Bacterial diseases are a leading cause of mortality and morbidity globally. During bacterial diseases, an elevation of host immune
response occurs, which involves the production of free radicals in response to the bacterial infection. The overproduction of free
radicals in excess of the antioxidants leads to oxidative stress. Conventional antibiotics are linked to side effects such as hypersensitivity reactions in addition to bacterial pathogens developing resistance against them. Artificial antioxidants are said to be
carcinogenic. This study sought to confirm folklore use and validate the antibacterial and antioxidant potential of Senna singueana
which has been widely used in the Mbeere community. The in vitro antibacterial potentials of the plant extract were investigated
on Bacillus subtilis ATCC 21332, Escherichia coli ATCC 25922, Salmonella typhi ATCC 1408, and Staphylococcus aureus ATCC
25923. Ciprofloxacin (100 µg/ml) drug was used as a standard reference, whereas 5% DMSO was used as a negative reference. The
antibacterial tests included disc diffusion and minimum inhibitory and bactericidal concentrations. S. singueana ethyl acetate
extract showed broad-spectrum potential against tested bacterial microbes producing mean zones of inhibition (MZI) from
07.67 ± 0.33 to 17.67 ± 0.33 mm. The extract demonstrated a greater effect on Gram-positive than Gram-negative bacterial
pathogens. Antibacterial properties of ciprofloxacin were significantly greater in comparison to plant extract in all the dilutions
(p < 0.05), while 5% DMSO was inactive against all the tested bacteria. MBC values were greater than MIC values. Antioxidant
properties of the extract were determined through scavenging effects of DPPH and hydroxyl radicals (•OH) as well as ferric
reducing antioxidant potential (FRAP) assay. S. singueana demonstrated effects against all radicals formed. Additionally, the
extract exhibited ferric reducing abilities. The extract also contained various phytocompounds with known antibacterial and
antioxidant properties. This study recommends the therapeutic use of S. singueana as an antibacterial as well as an
antioxidant agent.
1. Introduction
Since the ancient time, medicinal plants have been used to
treat various infections. According to the World Health
Organization (WHO), 80% of the global population uses a
variety of plant fractions and their dynamic components as
traditional therapies [1–4]. Phytochemicals produced by
plants including tannins, phenolic compounds, and phytosterols have been demonstrated to have positive and
significant benefits to human health [2]. Flavonoids and
phenolic compounds, for example, have anticancer, antioxidant, antidiabetic, and anti-inflammatory potentials [5].
Medicinal plants are most commonly used in nonindustrialized and traditional cultures, owing to their ease of
availability and lower cost when compared to modern
medicines [6]. Many developing and developed countries
use herbal medicine to maintain human well-being and
personal health and treat bacterial diseases [7]. Medicinal
plants are regularly used in rural societies where pharmaceuticals are unavailable or impossible to obtain. In contrast,
in Westernized societies, medicinal plants are typically used
as an alternative or supplement to prescribed medicine [8].
In Africa, for example, various communities have their
unique approach to health and disease, even down to
2
ethnopathogenic notions of diseases and therapeutic
behavioural patterns [9].
In this study, we evaluated the antibacterial and antioxidant properties of the Kenyan grown Senna singueana
medicinal plant which belongs to the family Fabaceae. The
plant family Fabaceae includes legumes which are fruits of
plants and plant is known as plant of bean or pea. Plants
belonging to this family have various pharmacological
benefits. For example, Acacia catechu which belongs to the
Acacia genus in the Fabaceae family has traditionally been
utilized to cure a variety of illnesses, particularly gastrointestinal as well as stomach-related issues [10]. Similarly,
Glycyrrhiza glabra belonging to the Glycyrrhiza genus within
this family has traditionally been used to treat a variety of
bacterial ailments, including tonsillitis, sore throat, coughs,
and diseases [5].
The genus Senna is categorized in the Fabaceae family,
which contains over 300 species of tree shrubs and subshrubs that can be found in a variety of habitats and in
continents like Africa, America as well as, to a lesser extent,
Pacific islands and Asia [11]. Senna species have exhibited
antimicrobial, anti-inflammatory, antidiabetic, and antimalarial properties [12]. Senna is widely used for a variety
of purposes including decoration, building, nutrition,
poisons, rituals, and medicine [11]. Senna alata bark decoction for example is used in East African communities to
treat cuts after tribal mark incision and tattooing [12].
Senna alata leaves have been used in Thailand to treat
wounds, constipation, and inflammation [13]; Senna
alexandrina leaves and fruits have been used in Sudan to
treat constipation and GIT disorders [14]; Senna occidentalis leaves, roots, and seeds have been used in India to
treat respiratory diseases, malaria, diabetes, and urinary
disorders [15, 16]; Senna sophera has been used to treat
respiratory disorders in India [17]; and Senna tora has been
utilized in China to treat liver illnesses, stomach disorders,
and poor eyesight [18].
S. singueana, which is also known as scrambled egg, is a
deciduous shrub with a light, open crown; it can grow to
1–15 meters tall. It has a spectacular flowering display which
often takes place in the dry seasons [19]. It is an African
traditional medicinal plant with many medicinal uses
throughout the African continent [20, 21]. It is used as a
therapy for diarrhea, conjunctivitis, bilharziasis, and coughs
in different communities [20, 22]. The Mbeere community
calls it Mukengeta and they use it to treat anthrax and elephantiasis [23]. It is also used in both humans and animals
as a purgative and a lactation stimulant [24].
Previous studies on S. singueana reported several biological activities such as hepatoprotective and antiapoptotic
properties of methanol bark extracts [21], antimalarial and
antioxidant properties of ethanol leaf extracts [22, 25],
antinociceptive effects for methanol leaf extracts [19], hypoglycemic capability of aqueous leaf extracts [26], and
antibacterial activities of aqueous leaf extracts [27, 28].
Previous experiments have also demonstrated that both
aqueous and methanol extracts of S. singueana are relatively
nontoxic, and thus safe for use [29, 30]. It has been shown to
have anticancer and antimalarial effects [25].
Evidence-Based Complementary and Alternative Medicine
The plant has also been used traditionally to treat inflammatory conditions, convulsions, constipation, gonorrhea, and heartburn [31]. S. singueana leaves are used to treat
a variety of poultry ailments in Zimbabwe, including coccidiosis, coughing, and flu-like illnesses [32]. The plant is
also said to be used as food and fodder. Its leaves, pods, and
seeds are fed to animals [33].
Bacterial pathogens and increase in antibacterial resistance have continued to rise, leaving patients with few or no
alternative treatment options and an increase in diseases and
deaths globally [34]. Bacterial infections cause almost half of
human deaths in developing countries [35], a situation that
may worsen due to misuse, overuse, or underuse of antibiotics leading to antibiotic resistance [36]. Globally,
Escherichia coli, Staphylococcus aureus, and Klebsiella
pneumoniae are the major causes of community and hospital-acquired bacterial diseases [37]. Bacterial infections
also cause discomfort and suffering among infected individuals, thereby lowering their productivity [38].
Due to the lack of resources, infectious disease reports
from developing countries are not well documented [39]. In
Kenya, microbial pathogens that cause the majority of
human diseases are generally those with high antibiotic
resistance. The top five killers in Kenya are infectious diseases although the data on bacterial infections are not well
documented because a majority of ailments and deaths occur
outside the hospitals [40] with the high prevalence among
Kenyans from poor communities [41].
During bacterial infections, the enzyme nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase catalyzes
activated immune cells like neutrophils to undergo “respiratory burst” to produce ROS such as superoxide (O2−) [42].
Superoxide dismutase catalysis converts superoxide radicals
to hydrogen peroxide (H2O2) [43], which is responsible for
bacterial autolysis at the stationary phase [44]. Hydrogen
peroxide can also be converted into a more toxic hypochlorous acid or react with superoxide to form hydroxyl
radicals which in combination with the two ROS (hydrogen
peroxide and superoxide) can kill any bacteria within the
neutrophil [43]. As the bacterial infection persists in the
body, more ROS as well as RNS are formed by activated
immune cells. When these free radicals are produced in
excess of available natural antioxidants, they contribute to
oxidative stress leading to direct damage of cells [45].
Considering the adverse effects of the conventional
antibacterial and antioxidant drugs, high cost, and the increased pathogenic resistance to these drugs, efforts have
been and are being made to find alternative medicines from
plants that are more effective with fewer side effects [9].
Traditional healers have indigenously used medicinal plants
to cure several diseases including bacterial infections [46].
However, their indigenous uses lack scientific validation.
The Mbeere community uses S. singueana as a remedy to
cure a variety of diseases. This study sought to confirm its
folklore use and validate antibacterial as well as antioxidant
properties of ethyl acetate extract of S. singueana. This study
also determined the phytochemical compositions of ethyl
acetate extract of S. singueana to determine the basis of its
medicinal potentials.
Evidence-Based Complementary and Alternative Medicine
2. Materials and Methods
2.1. Plant Material Collection and Preparation. Fresh leaves
of S. singueana were gathered from Gikuyari village, Thura
Sub Location, in Embu County, Kenya in May 2021 with the
assistance of a local practicing traditional herb doctor. GPS
coordinates for the plant collection site are 0°35′39″ N and
37°38′12″ E. The plant sample was transported to Kenyatta
University, where it was identified by a recognized taxonomist and a specimen preserved at Kenya National Museum’s herbarium for future reference. Voucher number for
the specimen was allocated as PN/001/27698/2018. The
leaves were well washed using running tap water, rinsed
using distilled water (DH2O), and chopped into small pieces.
They were then shade dried for 28 days, finely ground into
powder prior to storage at room temperature in airtight
vessels ready for the extraction process.
2.2. Extraction Procedure. Four hundred grams of dry
powder of S. singueana leaves were soaked in 1.2 L of ethyl
acetate for 72 hours. The solution was occasionally swirled to
achieve complete dissolution. After 72 hrs, the solution was
decanted and vacuum filtered with the help of a Buchner
funnel and Whatman’s filter paper No. 1. Thereafter, using a
rotary evaporator, the filtrate was concentrated to evaporate
the solvent at 90 rpm at 60°C under vacuum. The extract
yield of the plant was determined according to the following
equation:
Percentage Yield �
K1
× 100%,
K2
(1)
where K1 is the mass of concentrated plant extracts and K2 is
the dry mass of the powdered plant before extraction [47].
The resultant extract was placed in airtight sterile clean
glass containers and stored at 4°C awaiting bioassay studies.
2.3. Experimental Design. This study utilized a completely
randomized study design.
2.4. Bacterial Test Organisms and Controls. Bacterial isolates
used for antibacterial assays were sourced from Kenyatta
University’s Microbiology Laboratory, Biochemistry, Microbiology, and Biotechnology (BMB) Department. They
comprised of B. subtilis ATCC 21332, E. coli ATCC 25922,
S. aureus ATCC 25923, and S. typhi ATCC 1408. Ciprofloxacin was utilized as a positive reference (reference antibiotic), whereas DMSO (5%) was utilized as a negative
reference.
2.4.1. Maintenance of Bacterial Stock Cultures. To obtain
fresh bacterial colonies, the bacterial stock pathogens were
streaked on Mueller Hinton Agar (MHA) prior to 24 hours
at 37°C [48]. Thereafter, 3 to 4 colonies were picked with a
sterile wire loop and transferred in sterile glass tubes containing 10 ml of sterile Mueller Hinton Broth (MHB) followed by 24 hours of incubation at 37°C to obtain freshly
grown bacterial suspensions which were kept at 4°C [48].
3
2.5. Preparation of Extract Concentrations and Disc
Impregnation. One hundred milligrams of S. singueana
extract were weighed and then placed in a sterile 2-ml
microcentrifuge tube. One milliliter of 5% DMSO was
added, and the blend properly vortexed thereafter sonicated
to ensure complete dissolution to achieve a 100 mg/ml stock
solution concentration [49]. Twofold serially diluted dilutions were prepared by taking 500 µl of extract’s stock solution and mixing with 500 µl of 5% DMSO to attain
concentrations beginning from 50 mg/ml to 3.125 mg/ml.
Fifteen microliters of serially diluted extract was used to
impregnate sterile discs. The discs were left in the biosafety
cabinet to air dry for about 20 minutes before being placed
on the surface of inoculated media. Ciprofloxacin powder
(100 µg) dissolved in 1000 µl of sterile normal saline [50] was
applied as the positive reference, whereas DMSO (5%) was
applied as the negative reference.
2.6. Antibacterial Sensitivity Tests. Antibacterial sensitivity
assays were conducted using the disc diffusion technique in
triplicates as explained by Benkova et al. [51] and Wolde
et al. [52]. Sterile cotton swabs were dipped in the bacterial
inocula and rotated on the tube’s sides to eliminate surplus
fluid. After which, they were streaked all over the already
prepared Mueller Hinton Agar media. To guarantee the
inocula’s even distribution, the plates were rotated approximately 60 degrees each time. The inoculated plates were
then left to dry for about 5 minutes in a biosafety cabinet
before placing the discs on the surface. Using sterile forceps,
the 6-mm paper discs impregnated with various dilutions of
S. singueana extract, 5% DMSO (negative reference), and
Ciprofloxacin (positive control) were then placed on the agar
surface, one at a time. The plates were placed in sterile
condition at normal room temperature (RTP) for around
15 mins to allow for infiltration of the extract, 5% DMSO,
and Ciprofloxacin into the Mueller Hinton Agar media, then
incubated at 37oC for 24 hours [53], after which the clear
zones around the discs were determined in millimeters
(mm) using a ruler and recorded in spreadsheets. Based on
criteria detailed by Mwitari et al. [54], the antibacterial
potential of the studied extract and the positive control was
determined as follows;
(i) Zones of inhibition <7 mm were considered not to
have any activity,
(ii) Zones between 8 and 11 mm were considered active,
and
(iii) Zones >11 mm were considered very active.
2.7. Minimum Inhibitory Concentration (MIC). To determine the minimum inhibitory concentration, a broth dilution experiment was done in triplicates following the
protocols as performed by Manandhar et al. [53]. The extract
was double diluted to concentrations (conc) from 100 mg/ml
to 1.5625 mg/ml in sterile 96-well plates containing MHB.
This was done by adding equal volumes (100 µl) of the
extract to MHB. After dilution, 20 µl of each test bacterial
suspension, adjusted to standard turbidity (0.5 McFarland),
4
Evidence-Based Complementary and Alternative Medicine
was pipetted to the wells prior to 24-hour 37°C incubation.
Finally, 1% of resazurin solution (50 µl) was added to every
well as an indicator. Thereafter, the plates were re-incubated
at 37°C for 30 mins [53]. The minimum concentration that
inhibited visible blue to pink resazurin color change was
considered minimum inhibitory concentration [55].
Ciprofloxacin powder (100 µg/ml) was diluted the same way
the extracts were diluted, while 5% DMSO was used as a
negative reference.
2.8. Minimum Bactericidal Concentrations (MBCs). Using a
sterile cotton swab, 10 μl of the materials from every well
having concentrations at and above the MICs of studied
antibacterial agents was spread all over the surface of the
MHA plate followed by 37°C incubation for 24 hours [48].
MBC was documented as the minimal concentration with no
visible bacterial growth on MHA [56]. Bacterial growth on
the MHA plates was recorded as bacteriostatic effects of the
extracts, whereas a lack of bacterial growth on the MHA
plates was considered as bactericidal effects of the investigated extracts. This was done in triplicates.
2.9. Determination of In Vitro Antioxidant Activities
2.9.1. In Vitro DPPH Radical Scavenging Capability. S.
singueana extracts’ ability to mop 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals was done in triplicates as
conducted by Arika et al. [57], with little modifications. The
plant extract and ascorbic acid (reference) were prepared at
various concentrations beginning from 15.625 µg/ml to
500 µg/ml. DPPH (1 mM) solution was prepared in methanol. One milliliter of each dilution of the test extract and the
standard was separately placed in clean test tubes, after
which DPPH (0.5 ml) and methanol (3 ml) solutions were
added. The blend was thoroughly vortexed for 5 minutes;
thereafter, it was set aside in a dark cupboard for 30 mins at
ambient temperature. A blank solution containing 3 ml
methanol and 0.5 ml DPPH solutions was also prepared.
Using a spectrophotometer, the solutions’ absorbances were
measured at 517 nm against blank. The plant extracts’ %
DPPH free radical quenching properties were computed as
% DPPH Radical Scavenging activity
�
Abs Blank − Abs Sample
× 100.
Abs Blank
(2)
Half maximal inhibitory concentration (IC50), representing the concentration at which 50% of the DPPH
radicals were mopped, was analyzed using linear regression
analysis [58].
2.9.2. Determination of Ferric Reducing Antioxidant
Capacity. Plant extracts’ ferric reducing potential was determined following the protocol used by Park et al. [59], with
little modifications. Approximately 2.5 ml solution of test
extracts and reference (Ascorbic acid) at varying
concentrations starting from 7.8125 µg/ml to 500 µg/ml were
separately blended with 2.5 ml phosphate buffer (pH 6.6,
0.2 M) along with 2.5 ml potassium ferricyanide (1%) followed by 20 minutes incubation at 50oC. Thereafter, 2.5 ml
of 10% trichloroacetic acid was added into the blend and
vortexed before being centrifuged for 10 minutes at 3000
rotations per min (rpm). The supernate (5 ml), DH2O (5 ml),
and 0.1% ferric chloride (1 ml) were mixed, incubated at
normal room temperature (RTP) for 10 minutes, after which
a spectrophotometer set at 700 nm was used to read the
absorbance against blank. Blank solution comprised all the
reagents other than the plant extract and ascorbic acid. This
was done in triplicates.
2.9.3. Determination of In Vitro Hydroxyl Radical Scavenging
Effects. The extracts’ ability to quench hydroxyl radicals was
done based on protocols carried out by Arika et al. [57] and
Zhang et al. [60]. A blend solution of extract/control (Gallic
Acid) at varied dilutions (15.625 µg/ml to 500 µg/ml) 500 µl,
28.0 mM 2-deoxy-2-ribose dissolved in KH2 PO4–KOH
buffered solution (20.0 mM, pH 7.4) 100 µl, EDTA
(1.04 mmol L−1) 100 µl, ascorbic acid (1.0 µM) 100 µl, FeCl3
(200 mM) 100 µl, and hydrogen peroxide (1.0 mM) 100 µl.
This followed incubation of the blend at 37°C in a water bath
for 60 minutes then 1% cold thiobarbituric Acid (TBA)
1000 µl along with 2.8% trichloroacetic Acid (TCA) 1000 µl
solutions were added before heating the blend at 100°C for
15 minutes where a noticeable pink color developed;
thereafter, the mixture was cooled in cold water. Absorbance
was recorded against blank using a spectrophotometer set at
532 nm. Assays were run in triplicates. The % radical
quenching capability was computed as [57]
%Hydroxyl Radical Scavenging activity
�
Abs Blank − Abs Sample
× 100.
Abs Blank
(3)
2.10. Quantitative Phytochemical Activities. A clean microcentrifuge tube (2.0 ml) was labeled as SS for S. singueana. To
the labeled tube, 1 mg of the test extract was added followed
by 1000 µl of ethyl acetate to liquefy the sample. The sample
was vortexed for 1 min then sonicated for 15 minutes after
which it was centrifuged at 1,400 rotations per min for
5 mins. The resultant supernate (1 mg/ml) dried over anhydrous Na2SO4 was used to prepare experimental solutions
in triplicates at a concentration of 100 ng/µL [57].
GC-MS was done on 7890 A Gas-Chromatograph joined
to a 5975°C mass selective detector (Agilent Technologies),
which consists of an HP-5 MS low bleed capillary column
(30 m long, 0.25 mm wide, as well as 0.25 μm film thick).
Operating parameters of the mass spectrometer included:
relative detector gain mode, 70 eV of ionization energy,
3.3 mins of filament delay time, 1666μ/sec of scan speed,
40–550 m/z of scan range, 230 C ion source temp, and 180°C
quadrupole temp. Helium gas (99.9%) was applied as a carrier
gas at a steady flow speed rate of 1.25 ml per min. Mass
Evidence-Based Complementary and Alternative Medicine
transfer temperature was programmed at 200°C while injector
line transfer temperature was programmed at 250°C, with 1 μl
injection volume. The oven temperature was programmed at
35°C for 5 mins followed by a 10°C/min increase to 280°C for
24.5 mins and then raised at a rate of 50°C per min to 285°C
for 20.5 mins and a total run period of 50 mins. To identify the
phytocompounds found in the extract, a comparison of the
obtained data was matched with mass-spectral library search
reports from the National Institute of Standards and Technology (NIST) 08 and 11, where each unique peak represented a particular chemical substance.
2.11. Data Management and Statistical Analysis. This study’s
data were tabulated in a Microsoft Excel spreadsheet and
organized before being imported into Minitab software
version 17.00, where descriptive statistics values were
conveyed as mean ± STD (standard) error of mean (SEM).
One-way analysis of variance (ANOVA) for inferential
statistics and Tukey’s post hoc test for pairwise comparison
as well as separation of means were used. A statistically
significant p value of <0.05 was used. Comparison of the
plant extract and standard antibacterial and antioxidant
properties were done using unpaired Student’s T-test.
Graphs and tables were used to present the findings.
For GC-MS data, the various compounds were recognized primarily on their retention time (RT) and fragmentation pattern in comparison with the NIST 08, 11
library search report. For identification of the compounds,
an identity match of above 60% with the library phytocompounds was required. The compound names, molecular
weights, and structures were established. The components’
concentrations were expressed in µg/g.
3. Results
3.1. Yield of the Plant Extract. The percentage yield of the
extract was 4.99%, producing a dark green solid extract.
3.2. Antibacterial Sensitivity of the Extract. S. singueana
extract exhibited notable antibacterial effects against all the
tested bacteria in this study. This was manifested by the
visible inhibitory zones surrounding the paper discs impregnated with various dilutions of the extract (Figure 1).
Generally, S. singueana extract demonstrated a greater
potential effect against tested Gram-positive (+ve) pathogens (B. subtilis and S. aureus), recording greater than (>)
12 mm of mean zones of inhibition (MZI) at extract concentration ranges from 25.00 mg/ml to 100.00 mg/ml. At
12.5 mg/ml concentration, the extract had a MZI >12 mm on
S. aureus (Table 1). The extract showed activity at all tested
concentrations against the tested Gram-positive pathogens.
However, S. singueana extract displayed no effect against
tested Gram-negative (-ve) pathogens at concentration
ranging from 3.125 mg/ml to 12.5 mg/ml (Table 1).
At 100 mg/ml concentration, S. singueana extract displayed antibacterial activities against both S. aureus and
B. subtilis that were significantly different from the activities
recorded by Ciprofloxacin, DMSO, and extract
5
concentrations ranging from 3.125 mg/ml to 50.00 mg/ml
(p < 0.05; Table 1). S. singueana extract’s concentration at
100 mg/ml recorded MZI on E. coli and S. typhi that was
statistically similar to 50 mg/ml concentration (p < 0.05;
Table 1) but significantly distinct from concentration
ranging from 3.125 mg/ml to 25 mg/ml (p < 0.05; Table 1).
S. singueana extract at 50 mg/ml concentration showed
activities against all pathogens tested, although the activities
were not significantly distinct from the activities of extract
concentration 25 mg/ml on E. coli and S. aureus (p < 0.05;
Table 1). Similarly, S. singueana extract effect at concentration
25 mg/ml was significantly different from activities of extract
concentrations ranging from 3.125 mg/ml to 12.5 mg/ml
against all pathogens except against S. aureus whose activity
was statistically similar with concentration 12.5 mg/ml
(p < 0.05; Table 1). At concentration 12.5 mg/ml, S. singueana
extract had antibacterial effects against S. aureus and
B. subtilis pathogens only even though the effects were not
significantly different from activities of concentration
6.25 mg/ml (p < 0.05; Table 1). Similar activities were seen
with concentration 3.125 mg/ml, whose effect was statistically
similar to that of 6.25 mg/ml (p < 0.05; Table 1).
In vitro antibacterial potential of S. singueana extract was
dose dependent with recorded MZI against all the tested
bacterial pathogens, increasing with an increase in extract
concentrations (Table 1). Dimethylsulfoxide (DMSO) was
inactive on all the tested bacterial pathogens (MZI 6 mm).
Ciprofloxacin (standard antibiotic) demonstrated significantly higher antibacterial activity against all studied
pathogens producing significantly larger zones of bacterial
growth inhibition than all extract concentrations used in the
study (p < 0.05; Table 1).
3.3. Minimum Inhibitory Concentrations. The tested extract
showed bacterial growth inhibitions against all tested bacterial pathogens and thus subjected to MIC and MBC tests.
The mean MIC means ranged from 1.30 ± 0.26 to
20.83 ± 4.17 mg/ml (Table 2). S. singueana extract showed
statistically similar inhibitory properties on B. subtilis,
S. aureus, and E. coli (p < 0.05; Table 2). Similar activity was
seen in the inhibitory effects of S. singueana extract on
S. typhi and E. coli (Table 2). Ciprofloxacin demonstrated
statistically similar inhibitory effects against all the tested
pathogens (p < 0.05; Table 2).
In comparison to Ciprofloxacin, the ethyl acetate leaf
extract of S. singueana exhibited inhibitory effects at significantly higher concentrations than Ciprofloxacin on all
the tested bacterial pathogens (p < 0.05; Table 3).
3.4. Minimum Bactericidal Concentrations (MBCs).
Generally, the tested extract had higher MBC values than
MIC values against each of the tested bacterial pathogens
(Tables 2 and 4). Mean MBC ranged from 12.50 ± 0.00 to
100.00 ± mg/ml (Table 4). The ethyl acetate extract of
S. singueana exhibited significantly higher bactericidal effects against S. aureus than B. subtilis (p < 0.05; Table 4).
However, its bactericidal effects on S. typhi and E. coli were
statistically similar (p < 0.05; Table 4). Ciprofloxacin
6
Evidence-Based Complementary and Alternative Medicine
Figure 1: Inhibition zones caused by Senna singueana extract on S. aureus.
Table 1: Antibacterial properties of ethyl acetate leaf extracts of S. singueana.
Treatment
5% DMSO
Ciprofloxacin (100 µg/ml)
S. singueana extract
(mg/ml)
100
50
25
12.5
6.25
3.125
S. aureus
06.00 ± 0.00g
26.33 ± 0.33a
Zones of inhibition (mm)
B. subtilis
E. coli
06.00 ± 0.00g
06.00 ± 0.00d
29.67 ± 0.33a
32.67 ± ±0.33a
S. typhi
06.00 ± 0.00d
28.33 ± 0.33a
17.67 ± 0.33b
15.33 ± 0.33c
13.67 ± 0.33cd
12.67 ± 0.33de
10.67 ± 0.67ef
09.33 ± 0.88f
17.67 ± 0.33b
14.33 ± 0.33c
12.33 ± 0.33d
10.67 ± 0.33e
09.67 ± 0.33ef
08.33 ± 0.33f
10.67 ± 0.33b
09.67 ± 0.33b
07.67 ± 0.33c
06.00 ± 0.00d
06.00 ± 0.00d
06.00 ± 0.00d
09.67 ± 0.33b
08.67 ± 0.33bc
07.67 ± 0.33c
06.00 ± 0.00d
06.00 ± 0.00d
06.00 ± 0.00d
The values of mean zones of inhibition (MZI) are conveyed as mean ± std error of mean. Values having similar superscripts within a particular column are
insignificantly distinct after one-way analysis of variance and Tukey’s post hoc (p < 0.05).
Table 2: Minimum inhibitory concentration of S. singueana
extract.
Bacterial strain
S. aureus
B. subtilis
S. typhi
E. coli
Concentration (mg/ml)
S. singueana extract
Ciprofloxacin (µg/ml)
1.30 ± 0.26b
0.16 ± 0.03a
b
3.13 ± 0.00
0.16 ± 0.03a
a
41.67 ± 8.33
0.13 ± 0.03a
20.83 ± 4.17ab
0.05 ± 0.00a
Values were conveyed as mean ± std error of mean. Values having similar
superscript letters within a particular column are insignificantly distinct
(p < 0.05) (two-sample T-test). p < 0.05.
demonstrated statistically similar bactericidal effects on
S. aureus, B. subtilis, and S. typhi (p < 0.05; Table 4).
In comparison to Ciprofloxacin, the bactericidal effects
of S. singueana extract against all tested bacterial pathogens
were at significantly higher concentrations than Ciprofloxacin (p < 0.05; Table 5).
3.5. In Vitro Antioxidant Properties of S. singueana Ethyl
Acetate Extract
3.5.1. In Vitro DPPH Radical Scavenging Properties of
S. singueana Extract. The tested plant extract displayed
DPPH radical scavenging effect across all concentrations
in a dose-dependent trend. As plant extracts’ concentration decreased, DPPH radical scavenging capacity also
decreased (Figure 1). S. singueana extract and ascorbic
acid each showed significantly different DPPH radical
quenching properties at all the dilutions (p < 0.05; Figure 1) except at concentrations between 250.00 µg/ml and
500.00 µg/ml where they each exhibited statistically
similar DPPH free radical scavenging effects (p < 0.05;
Figure 1). The reference, ascorbic acid, demonstrated a
significantly greater scavenging potential of DPPH radicals with an IC50 value of 20.54 ± 2.24 µg/ml in comparison to the studied plant extract which had an IC50
value of 47.97 ± 0.69 µg/ml (p < 0.05).
Evidence-Based Complementary and Alternative Medicine
7
Table 3: Comparison of MICs of the studied ethyl acetate extract and Ciprofloxacin.
Treatment
Ciprofloxacin (µg/ml)
S. singueana extract
S. aureus
0.16 ± 0.03b
1.30 ± 0.26a
Minimum inhibition concentration (mg/ml)
B. subtilis
S. typhi
0.16 ± 0.03b
0.13 ± 0.03b
3.13 ± 0.00a
41.67 ± 8.33a
E. coli
0.05 ± 0.00b
20.83 ± 4.17a
Values were conveyed as mean±std error of mean. Values having similar superscript letters within a particular column are insignificantly distinct (p < 0.05)
(two-sample T-test).
Table 4: Minimum bactericidal concentration of S. singueana extract.
Bacterial strains
S. aureus
B. subtilis
S. typhi
E. coli
Concentration (mg/ml)
S. singueana extract
12.50 ± 0.00c
41.67 ± 8.33b
100.00 ± 0.00a
100.00 ± 0.00a
Ciprofloxacin (µg/ml)
1.30 ± 0.26a
0.65 ± 0.13ab
0.78 ± 0.00ab
0.26 ± 0.06b
Values were conveyed as mean ± std error of mean. Values having similar superscript letters within a particular column are insignificantly distinct after oneway analysis of variance and Tukey’s post hoc (p < 0.05).
Table 5: Comparison of MBC of the studied ethyl acetate extract and Ciprofloxacin.
Treatment
Ciprofloxacin (µg/ml)
S. singueana extract
Minimum bactericidal concentration (mg/ml)
S. aureus
B. subtilis
S. typhi
1.30 ± 0.26b
0.65 ± 0.13b
0.78 ± 0.00b
12.50 ± 0.00a
41.67 ± 8.33a
100.00 ± 0.00a
E. coli
0.26 ± 0.06b
100.00 ± 0.00a
Values were conveyed as mean ± std error of mean. Values having similar superscript letters within a particular column are insignificantly distinct (p < 0.05)
(two-sample T-test).
3.5.3. In Vitro Hydroxyl (•OH) Radical Scavenging Potential
of Ethyl Acetate Extract of S. singueana. The studied plant
extract displayed an efficient hydroxyl free radical scavenging capability which occurred in a dilution-dependent
trend (Figure 3). As illustrated in Figure 3, the hydroxyl
(•OH) radical scavenging potential of gallic acid was significantly greater than that of S. singueana extract in all the
tested dilutions (p < 0.05; Figure 3). There was a significantly
different hydroxyl radical scavenging activity among all the
100
a
b
80
(%) Inhibition
3.5.2. In Vitro Ferric Reducing Antioxidant Potential of Ethyl
Acetate Extract of S. singueana. The seven tested dilutions of
ethyl acetate leaf extracts of S. singueana showed dilutiondependent ferric reducing potential. All the extract concentrations exhibited a significantly lower ferric reducing
activity than ascorbic acid (reference control) (p < 0.05;
Figure 2) except at the lowest concentration of 7.8125 µg/ml
where the ferric reducing potential of the extract exhibited
statistical similarity to that of reference (ascorbic acid)
(p < 0.05; Figure 2). The ferric reducing capability of ascorbic
acid’s concentrations were significantly distinct, with the
highest concentration exhibiting the highest effect (p < 0.05;
Figure 2). However, S. singueana extract showed statistical
similarity in ferric reducing activity at concentrations
ranging from 7.8125 µg/ml to 31.25 µg/ml and concentration
between 125 µg/ml and 250 µg/ml (p > 0.05; Figure 2), although the effect of extract concentration of 500 µg/ml was
significantly greater than all the other concentrations
(p < 0.05; Figure 2).
c
d
60
40
e
a
a
a
b
c
d
e
20
0
15.625
31.25
62.5
125
Conc (µg/ml)
250
500
S. singueana extract
Ascorbic acid
Figure 2: In vitro DPPH radical quenching properties of
S. singueana extract. Bar graphs having identical letters across the
tested concentrations are statistically similar (p < 0.05) (one-way
ANOVA and Tukey’s post hoc tests). Within the same concentration, bar graphs without asterisks (∗ ) are significantly distinct
(p < 0.05) (two-Sample T-Test).
tested concentrations of S. singueana extract (p < 0.05;
Figure 3). As the extracts’ concentrations decreased, its •OH
radicals’ quenching capability also decreased with the lowest
concentration demonstrating significantly the lowest effect
(p < 0.05; Figure 3). Additionally, our findings showed that
gallic acid had a significantly lower IC50 value of
35.33 ± 0.88 µg/ml as compared to the extract whose IC50
value was 67.84 ± 1.34 µg/ml (p < 0.05), indicating that gallic
8
Evidence-Based Complementary and Alternative Medicine
Absorbance at 700 nm
2.5
a
2.0
b
1.5
c
d
1.0
0.5
d* g*
d
c
e
f
a
b
b
d
0.0
7.8125 15.625 31.25 62.5
125
Conc (µg/ml)
250
500
S. singueana extract
Ascorbic acid
Figure 3: Ferric reducing potential of S. singueana extract. Bar
graphs with identical letter/s across the tested concentrations are
statistically similar (p > 0.05) (one-way ANOVA and Tukey’s post
hoc tests). Within the same concentration, bar graphs without
asterisks (∗ ) are significantly distinct (p < 0.05) (two-sample TTest).
(%) Inhibition
100
c
80
d
60
e
f
40
20
f
b
b
a
a
c
d
e
0
15.625
31.25
62.5
125
Conc (µg/ml)
250
500
S. singueana extract
Gallic acid
Figure 4: In vitro •OH radical quenching potential of ethyl acetate
leaf extract of S. singueana. Across the tested concentrations, bar
graphs with identical letter/s are statistically similar (p > 0.05) (oneway ANOVA and Tukey’s post hoc tests). Within the same concentration, bar graphs without asterisks (∗ ) are significantly distinct
(p < 0.05) (two-Sample T-Test).
acid had a greater hydroxyl radical scavenging effect than the
extract.
3.6. Quantitative Phytochemical Composition of Ethyl Acetate
Extract of S. singueana. The S. singueana extract displayed
the presence of a total of 51 compounds, out of which 33
compounds have known biological activities. Based on the
obtained results, oxazolidine, 2-ethyl-2-methyl-, an Oxazoline compound, had the least concentration of
0.02 ± 0.00 µg/g, whereas Squalene, a triterpenoid, had the
highest concentration of 5.24 ± 0.07 µg/g (Table 6). The
findings also revealed a composition of 51.84% hydrocarbons, 24.3% terpenoids, 15.18% fatty acids, 3.26% tocopherols, 3.33% phenolic compounds, 1.03% iodo compounds,
0.28% steroids, 0.28% benzene derivatives, 0.09% heteroaromatic molecules, 0.09% volatile organic compounds,
0.07% cyclic secondary amines, 0.15% aminopyridine, 0.05%
alkaloids, and 0.03% oxazoline compounds.
4. Discussion
Traditional plants produce natural products that have been
known to be effective against bacterial infections, with few
side effects compared to commercial antibiotics [61]. Many
plants have been traditionally used to cure bacterial diseases;
however, they lack scientific validation and documentation
on their usage. This study evaluated the in vitro antibacterial
properties of ethyl acetate extract of the Kenyan grown
S. singueana on B. subtilis, S. typhi, E. coli, and S. aureus.
S. singueana extract exhibited antibacterial potentials on
the tested Gram-positive and Gram-negative bacterial
pathogens. The mean zones of inhibition (MZI) recorded
against all the studied bacterial pathogens were dependent
on the extract concentrations (zones decreased with a decrease in extracts concentration). This is in agreement with
Adedoyin et al. [62], who demonstrated that the essential oil
of S. singueana flowers had antibacterial properties in a dosedependent manner. Our findings also agree with a study by
Jambwa et al. [24], which demonstrated that the ethyl acetate
fraction isolated from S. singueana leaves crude extract had
antibacterial effects on both Gram-positive (S. aureus) and
Gram-negative (Salmonella Enteritidis and E. coli) bacterial
pathogens tested.
In this study, S. singueana extract inhibited the bacterial
growth of the tested pathogens, producing MZI ranging
from 07.67 ± 0.33 to 17.67 ± 0.33 mm with higher effects
against Gram-positive bacterial pathogens. This is in consensus with a past report which illustrated that the methanol,
acetone, and chloroform root extracts of S. singueana had
greater activities on Gram-positive (+ve) bacteria (Streptococcus pyogenes, S. aureus, and Streptococcus pneumonia)
than Gram-negative (−ve) pathogens (Pseudomonas aeruginosa, S. typhi, E. coli, and Klebsiella pneumonia) [63]. This
also concurs with a report by Kareru et al. [27], which
showed that the aqueous leaf extracts of S. singueana had
higher effects on S. aureus and B. subtilis than it had on
E. coli. This is also in agreement with Jibril et al. [64] who
demonstrated the broad-spectrum antibacterial effects of
methanol and ethyl acetate leaf extracts of S. singueana.
However, our findings are partly contrary to the reports of
Shawa et al. [20], which suggested that aqueous leaf and root
extracts of S. singueana were inactive on S. aureus and
Pseudomonas aeruginosa.
Gram-positive microbes are more susceptible to antibacterial agents [65], thus making them more sensitive to
crude plant extracts and bioactive constituents. This could be
the possible explanation as to why the studied extract had
higher activities on Gram-positive bacteria pathogens.
The antibacterial potentials of the studied plant extract in
this experiment are ascribable to the presence of various
phytocompounds like terpenoids, alkaloids, fatty acids,
hydrocarbons, phytosterols, and phenolic compounds. This
concurs with a past study that showed the presence of such
compounds in the roots, leaves, and seeds of S. singueana
[21]. This is also in accordance with a study by Kolawole et al.
[31], which demonstrated the presence of phenols, terpenoids, steroids, and alkaloids in ethanolic leaf extract of
S. singueana.
Evidence-Based Complementary and Alternative Medicine
9
Table 6: Quantitative phytochemical compound analysis in ethyl acetate extract of S. singueana.
RT
(mins)
38.78
44.42
47.31
46.71
Compound
β-Sitosterol
c-Cyano-3-methyl-5,10-dihydrobenzo[f]
indolizine
1(3H)-Isobenzofuranone, 6,7– dimethoxy-3[2-(2-methoxyphenyl)-2-Oxoethyl]-
C29H50O
MW (g/
mol)
414.70
Conc (µg/
g)
0.17 ± 0.00
0.09
C14H12N2
208.26
0.06 ± 0.00
0.09
C19H18O6
342.30
0.05 ± 0.00
0.24
C11H13NO3
207.00
0.14 ± 0.00
Volatile
organic
substances
Hydrocarbon
2.53
C11H19NO3
213.27
1.51 ± 0.02
Cyclohexane
0.57
0.95
C25H50
C20H42O
350.70
298.50
0.34 ± 0.00
0.57 ± 0.01
Hydrocarbon
Terpene alcohol
0.72
C17H14O4
282.29
0.43 ± 0.01
Hydrocarbon
0.05
C11H21N
167.29
0.03 ± 0.00
Alkaloid
0.15
C10H13N3S
207.29
0.09 ± 0.00
Aminopyridine
5.57
0.56
C20H40O
C12H26O
296.50
186.33
3.32 ± 0.04
0.33 ± 0.00
Terpene alcohol
Fatty alcohol
0.02
not found
1.78
2.05
4.53
0.28
C10H18O
C19H36O2
C23H46
C11H15NO
154.25
296.50
322.60
177.24
1.06 ± 0.01
1.22 ± 0.02
2.70 ± 0.04
0.17±0.00
%
abundance
0.28
MF
25.51
25.06
28.18
44.35
1,2,3-Propatriol, 1-indol-4-yl (ether)
1,4-Dioxaspiro [4.5]decane-6-carboxylic acid,
dimethylamide
1-Cyclopentyleicosane
1-Hexadecanol, 3,7,11,15-tetramethyl2-(Acetoxymethyl)-3-(methoxycarbonyl)
biphenylene
2-Cyclohexylpiperidine
2-Pyridinamine, N-(4, 5-dihydro-5-methyl-2thiazolyl)-3-methyl3,7,11,15-Tetramethyl-2-hexadecen-1-ol
4-Nonanol, 2,6,8-trimethyl5-(2-Oxo-6-phenyl-1,2-dihydropyrimidinyl-4)
uracil
7-Octenal, 3,7-dimethyl9-Octadecenoic acid, methyl ester, (E)9-Tricosene, (Z)Acetamide, N-methyl-N-(2-phenylethyl)-
03.39
Butane, 2-chloro-2-methyl-
0.33
C5H11Cl
106.59
0.20 ± 0.00
16.95
23.37
25.88
19.40
20.16
19.65
21.64
30.38
29.14
29.89
34.24
24.07
24.33
40.55
23.46
18.88
23.84
26.73
30.72
06.68
28.37
28.65
25.21
26.08
10.55
31.07
32.85
25.72
31.70
21.19
24.21
35.06
Cyclohexadecane, 1,2-diethylDecane, 3,8-dimethylDocosane
Dodecane, 2,6,11-trimethylDodecanoic acid
Dodecanoic acid, methyl ester
Eicosane (C20)
Fumaric acid,4-methyl pent-2-yl tridecyl ester
Hexacosane
Hexadecane (C16)
Hexadecane, 8-hexyl-8-pentylHexadecanoic acid, ethyl ester
Isopropyl hexadecanoate
Lupan-3-ol
Methyl hexadecanoate
Methyleugenol
n-Hexadecanoic acid
Nonadecane (C19)
Octacosane
Oxazolidine, 2-ethyl-2-methylPentacosane
Phenol, 2,4-bis (1-methyl-1-phenylethyl)Phytol
Phytol acetate<E->
Pyrrolidine, 2-decyl-1-methylSqualene
Tetracosane
Tetratetracontane
Tricosane
Tridecane, 1-iodoUndecane, 5,5-dimethylVitamin E
0.52
0.41
3.51
0.68
4.17
0.72
0.95
1.28
3.44
4.50
1.82
2.31
1.01
0.33
1.60
0.52
1.48
2.61
3.41
0.03
3.63
2.81
3.54
3.34
0.07
8.79
8.48
2.40
5.72
1.03
0.83
3.26
C20H40
C12H26
C22H46
C15H32
C12H24O2
C13H26O2
C20H42
C23H42O4
C26H54
C16H34
C27H56
C18H36O2
C19H38O2
C30H52O
C17H34O2
C11H14O2
C16H32O2
C19H40
C28H58
C6H13NO
C25H52
C24H26O
C20H40O
C22H42O2
C17H35N
C30H50
C24H50
C44H90
C23H48
C13H27I
C13H28
C29H50O2
280.50
170.33
310.60
212.41
200.32
214.34
282.50
382.58
366.70
226.44
380.70
284.50
298.50
428.40
270.50
178.23
256.42
268.50
394.80
115.17
352.70
330.50
296.50
338.60
253.50
410.70
338.70
619.20
324.60
310.26
184.36
430.70
0.31 ± 0.00
0.24 ± 0.00
2.09 ± 0.03
0.40 ± 0.01
2.48 ± 0.03
0.43 ± 0.01
0.57 ± 0.01
0.76 ± 0.01
2.05 ± 0.03
2.68 ± 0.04
1.08 ± 0.01
1.38 ± 0.02
0.60 ± 0.01
0.20 ± 0.00
0.95 ± 0.01
0.31 ± 0.00
0.88 ± 0.01
1.50 ± 0.02
2.03 ± 0.03
0.02 ± 0.00
2.16 ± 0.03
1.67 ± 0.02
2.11 ± 0.03
1.99 ± 0.03
0.04 ± 0.00
5.24 ± .07
5.05 ± 0.03
1.43 ± 0.02
3.41 ± 0.05
0.61 ± 0.01
0.50 ± 0.01
1.94 ± 0.03
27.34
21.79
27.06
44.18
13.72
46.38
29.69
21.51
49.10
Chemical Class
Steroid
Heteroaromatic molecule
0.01 ± 0.00
Conc, concentration; Mins, minutes; MF, molecular formula; RT, retention time; MW, molecular weight.
Monoterpenoid
Fatty acid methyl ester
Hydrocarbon
Benzene derivative
Alkyl chloride /chlorinated
hydrocarbon
Hydrocarbon
Hydrocarbon
Hydrocarbon
Aliphatic alkane
Fatty acid
Fatty acid methyl ester
Hydrocarbon
Fatty acid ester
Hydrocarbon
Hydrocarbon
Hydrocarbon
Fatty acid ester
Fatty acid ester
Tritepenoid
Fatty acid methyl ester
Phenylpropanoid
Fatty acid dervivative
Hydrocarbon
Hydrocarbon
Oxazoline compound
Hydrocarbon
Phenolic compound
Diterpenoid
Diterpenoid
Cyclic secondary amine
Triterpenoid
Hydrocarbon
Hydrocarbon
Hydrocarbon
Iodo compound
Hydrocarbon
Tocopherol
10
Compounds like terpenoids have known antibacterial
potentials [66]. They interfere with bacterial oxygen uptake
and oxidative phosphorylation which are two important
essential processes in bacteria [67]. Squalene, a triterpenoid,
which had the highest concentration in the studied plant
extract, has been shown to exhibit antibacterial effects [31].
Phytol, a diterpenoid, also present in the extract has known
antibacterial activity [68]. Similarly, phytol acetate<E->,
another diterpenoids, has also been shown to have antibacterial effects [69].
Majority of the hydrocarbons found in this extract were
alkanes. Alkanes act by interfering with bacterial cell
membrane integrity and function leading to bacterial cell
death [70]. Octacosane, a straight-chain alkane present in
the studied extract has known antibacterial properties [71].
Additionally, tetracosane and tricosane, other straight-chain
alkanes, have also demonstrated antibacterial effects [72, 73].
Many plants utilize fatty acids in defense against pathogenic bacteria. Their prime target is disrupting the electron
transport chain of bacterial cell membranes. Fatty acids can
also act by inhibiting bacterial enzyme activity, impairment
of nutrient uptake, and direct bacterial cell lysis [74]. Fatty
acid like Hexadecanoic acid ethyl ester, present in this extract, has been found to possess antimicrobial capabilities
[66]. Similarly, lauric acid, another fatty acid, has also
previously been demonstrated to have antibacterial potential
[75].
Alkaloids, a structurally diverse group of plant secondary
metabolites, exert their antibacterial activity by inhibiting
bacterial enzyme activity as well as causing disruption of the
bacterial membrane thus killing the bacteria [76]. An alkaloid 2-cyclohexylpiperidine which was present in the
studied extract has been known to have antimicrobial activities [77].
Phenolic compounds, large heterogeneous secondary
plant metabolites, are known for their cell lysis in addition to
membrane-disturbing capabilities as their mode of antibacterial activity [78]. Methyleugenol, a phenolic compound, present in the extract, has been shown to have
antibacterial activities [79].
Phytosterols stabilize plant cell phospholipid bilayers
just like cholesterols in animal cell membranes [80]. They
have a resemblance to sterols which are found in the bacterial cells; thus, the phytosterols replace the normal sterols
in the bacterial cell membrane, thus disrupting the bacterial
cell membrane hence killing the bacteria [81]. β-Sitosterol,
which was present in the extract, has been found to exhibit
antimicrobial activities [82].
Vitamin E (α tocopherol) confers its antibacterial potential by acting as an antibacterial adjuvant in combination with other antibacterial agents [83]. Vitamin E, which
was present in S. singueana extract, has been shown to
exhibit antimicrobial activities [84]. Other compounds like
fatty alcohols previously demonstrated antibacterial potentials [85]. A previous experiment by Malarvizhi et al.
[86] confirmed that 3,7,11,15-tetramethyl-2-hexadecen-1ol, a terpene alcohol, has been demonstrated to have antibacterial effects.
Evidence-Based Complementary and Alternative Medicine
The high availability of free radicals in excess of antioxidants results in oxidative stress [87], which leads to
cellular impairment and oxidative stress (OS)-related diseases [88]. To reduce the effects of oxidative stress in cells,
antioxidants are produced, which counteracts the upshot of
unstable free radicals by either reacting with them or
neutralizing them by donating electrons to stabilize them
[57]. Curative plants have for long been utilized to manage
illnesses as a result of oxidative stress. The plant extract
studied in this study showed potent in vitro antioxidant
capabilities.
In this study, plant extract’s and standards’ antioxidant
capacities in all the assays were in a dose-dependent trend.
As the extract’s/standards’ concentration decreased, the
antioxidant capacity also decreased. These findings concur
with a previous study that demonstrated dose-dependent
antioxidant potentials of ethyl acetate, petroleum ether, and
methanol root bark extracts of S. singueana [89].
DPPH radical scavenging method is the most popular in
vitro antioxidant method because it is easy, accurate, more
sensitive, and more economical, whose outcome is highly
reproducible as well as easily comparable with other free
radical scavenging assays. In this method, when antioxidants
in the tested extract react with DPPH, the DPPH accepts
hydrogen atoms from the antioxidant making it lose its color
from purple to yellow in a concentration-dependent manner
measured at 517 nm [90].
In this experiment, the reference, ascorbic acid, had a
greater scavenging capacity of DPPH radicals with IC50
value of 20.54 ± 2.24 µg/ml in comparison to the plant extract, which had an IC50 value of 47.97 ± 0.69 µg/ml. This is
in agreement with the findings of Jambwa et al. [24], which
demonstrated that the ethyl acetate fraction isolated from
S. singueana leaves crude extract exhibited DPPH radical
scavenging potential but with a lower IC50 in comparison to
the standard ascorbic.
Our findings also concur with Hilawea et al. [89], who
demonstrated that DPPH radical scavenging ability of ethyl
acetate, petroleum ether, and methanol root bark extracts of
S. singueana and ascorbic acid followed a similar dose-dependent trend. The researchers also found out that ascorbic
acid had a greater DPPH radical scavenging activity than
ethyl acetate, petroleum ether, and methanol root bark
extracts of S. singueana.
The ferric reducing power of a substance depends on its
ability to convert Fe3+ (ferric) to Fe2+ (ferrous) complex
forming a Prussian blue-colored solution, with a directly
comparative intensity to the substance’s concentration. A
greater absorbance read at 700 nm indicates a higher reducing
capability of the substance [57]. Ferric reducing power results
in this study demonstrated that vitamin C had significantly
higher reducing power than the studied plant extract. Our
findings concur with Hilawea et al. [89], who confirmed that
the reducing capacity of ethyl acetate, petroleum ether, and
methanol root bark extracts of S. singueana decreased with a
decrease in extracts concentration. Additionally, they also
noted that ascorbic acid had a greater reducing activity than
the S. singueana extracts studied.
Evidence-Based Complementary and Alternative Medicine
The extract’s potential to scavenge hydroxyl radicals
utilizes the principle that the extract will hinder •OH radicalmediated deoxyribose deterioration via Fenton’s reaction
using Fe3+ + EDTA + ascorbic acid + hydrogen peroxide reaction blend [57]. Among the free radicals, hydroxyl radicals
are considered extremely reactive and the most harmful free
radical as its interaction with the cell membranes can
damage sugar groups and the DNA base pairs leading to cell
death and eventually mutation, which might also cause
cancer, aging, and other chronic-related diseases [91].
Findings of this study confirm that the plant extract
demonstrated significant hydroxyl radical scavenging activities. However, these findings showed that the standard
had greater hydroxyl radical scavenging capacity in comparison to the studied extract. This is in agreement with a
previous study by Gerezgher et al. [25], who found that the
standard had a higher hydroxyl radical scavenging activity
than the ethanolic leaf extract of S. singueana.
Antioxidant activities exhibited by S. singueana extract
could be attributed to the availability of various phytocompounds that work synergistically to overcome free
radicals [92]. Several biological compounds including
phenolic compounds; lipids like fatty acids, phytosterols,
and fatty acid esters; terpenoids like monoterpene, diterpenes, and triterpenes; alkaloids; and hydrocarbons like
alkanes and alkenes were detected. They have been shown to
exert their antioxidant capability through multi-step processes that involve initiating propagating and eventually
terminating free radicals [92].
Terpenoids are known to have antioxidant activities.
Terpenoids act as antioxidants by scavenging free radicals
[93], through the donation of hydrogen to free radicals to
stabilize them [94]. They also act as chelating agents [95]. A
7-octenal,3,7-dimethyl-monoterpenoid, present in S. singueana extract has been known to have antioxidant effects
[96]. Squalene, a triterpenoid, has also been shown to possess
antioxidant effects [31].
Fatty acids exert antioxidant activity by scavenging free
radicals [97]. Dodecanoic acid, a fatty acid, present in the
extract, has previously been demonstrated to have antioxidant properties [98]. Ethyl hexadecanoate acid ethyl ester,
another fatty acid, which was also present in the studied
extract, has been shown in previous studies to have antioxidant activities [99].
Phenolic substances protect against free radicals by
donating hydrogen atoms or electrons to unstable radicals
[100] and chelating metal cations [101]. Methyleugenol, a
phenolic compound, present in S. singueana extract is
known to have antioxidant properties [102].
Phytosterols exert their antioxidant properties through
the donation of electrons to unstable free radicals to make
them stable [103]. β-Sitosterol, which was present in
S. singueana extract, has been found to have antioxidant
effects [104].
Tetratetracontane, a hydrocarbon present in S. singueana
extract, has been shown to have antioxidant activities [105].
Tetracosane, another hydrocarbon also present in the
studied extract, has also been shown to exhibit antioxidant
potentials [106]. Other compounds like vitamin E, which
11
was present in the studied extract, has been confirmed to
possess antioxidant potentials [84].
The purpose of this study was to validate the traditional
use of the leaves of S. singueana medicinal plant against
common bacteria that cause several human infections, such
as E. coli, Bacillus subtilis, Salmonella typhi, and Staphylococcus aureus, by evaluating its in vitro antibacterial and
antioxidant properties and the presence of phytochemicals
with such activities.
5. Conclusions and Recommendations
The findings of this study give a basis for the utilization of
S. singueana in the treatment of bacterial infections and
oxidative stress related infections. The extract also showed
the presence of several phytocompounds that could be used
in developing new antibacterial and antioxidant agents. The
fatty acids, terpenoids, phenols, and others in this extract
justify the obtained results.
Data Availability
The data utilized to support the findings in this study are
included in this article.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Authors’ Contributions
Mathew Ngugi Piero and George Isanda Omwenga supervised Paul Ochieng Nyalo as he conducted the study. Prior to
submission of this manuscript, all the authors approved its
final draft after reading it.
Acknowledgments
The authors express their gratitude to Kenyatta University’s
(KU) Department of Biochemistry, Microbiology, and
Biotechnology (DBMB) for having allowed them to conduct
the research in their laboratories. They also acknowledge Mr.
Kimani James, Mr. Daniel Gitonga, Ms. Spora Mavanza, and
Mr. Ibrahim Waweru of the same department for their
technical support.
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