Hindawi
Biochemistry Research International
Volume 2021, Article ID 5599129, 8 pages
https://doi.org/10.1155/2021/5599129
Research Article
Ozoroa insignis reticulata (Baker f.) R. Fern. & A. Fern. Root
Extract Inhibits the Production of Extracellular Proteases by
Staphylococcus aureus
Jonathan Katsukunya , Rumbidzai Makurira , and Stanley Mukanganyama
Department of Biotechnology and Biochemistry, University of Zimbabwe, P.O. Box MP 167, Mt. Pleasant, Harare, Zimbabwe
Correspondence should be addressed to Stanley Mukanganyama; smukanganyama@medic.uz.ac.zw
Received 14 February 2021; Revised 14 July 2021; Accepted 20 October 2021; Published 29 October 2021
Academic Editor: Jayanta Kumar Patra
Copyright © 2021 Jonathan Katsukunya 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.
Treatment of infections caused by S. aureus has become a challenge due to the emergency of resistant strains. Ozoroa reticulata
root extracts have been used in traditional medicine to treat throat and chest pains in Zimbabwe. The objective of the study was to
determine the effects of O. reticulata root bark extracts on the production of extracellular proteases by S. aureus. The root barks
were collected, dried, and crushed into powder. To obtain different phytoconstituents, plant extractions were performed. Extractions were carried out using two solvent mixtures: ethanol : water (50 : 50 v/v) and dichloromethane : methanol (50 : 50 v/v).
Serial exhaustive extractions were also performed using methanol, ethanol, dichloromethane, acetone, ethyl acetate, hexane, and
water. The broth microdilution assays were used to assess the antibacterial effects of the Ozoroa reticulata root bark extracts
against S. aureus. Ciprofloxacin was used as a positive control. Qualitative screening for extracellular protease production by
S. aureus on BCG-skim milk agar plates using the most potent extract was carried out. The proteolytic zones were measured and
expressed as the ratio of the diameter of the colony to the total diameter of the colony plus the zone of hydrolysis (Pz values). The
ethyl acetate extract was found to be the most potent inhibitor of the growth of S. aureus with 99% inhibition and a minimum
inhibitory concentration (MIC) of 100 µg/mL. Inhibition of extracellular protease production was directly proportional to the
concentration of the extract. At 100 µg/mL, the ethyl acetate extract had a Pz value of 0.84, indicative of mild proteolytic activity. A
Pz value of 0.94 was observed at a concentration of 200 µg/mL and signified weak proteolytic activity. In conclusion, the extract
inhibited the production of extracellular proteases in S. aureus. Further work on the isolation and purification of bioactive
compounds responsible for inhibiting the production of extracellular proteases is of importance in the discovery of agents with
antivirulent effects on S. aureus.
1. Introduction
Bacterial infections have emerged to be among the biggest
threats in the 21st century due to the emergence of antibioticresistant strains of bacteria. This has led to difficulties in
treating common bacterial infections [1]. Among the most
serious threats are infections that are caused by drug-resistant
Campylobacter, Enterobacteriaceae, Enterococcus, Pseudomonas aeruginosa, Shigella, Salmonella typhi, and Staphylococcus aureus [2]. S. aureus is a pathogen of medical concern
because of its intrinsic virulence [3]. The pathogenesis of
S. aureus is mediated by several virulence factors. The factors
include protein A, α-haemolysin, extracellular adherence
protein, chemotaxis inhibitory protein, enterotoxins, toxic
shock syndrome toxins, and the production of extracellular
enzymes [4, 5]. Extracellular enzymes are a different type of
enzymes that are used by bacteria for invasion. These include
coagulases, hyaluronidases, lipases, staphylokinases, phospholipases, deoxyribonucleases, and proteases [6].
S. aureus can cause a variety of severe infections and
adjust to diverse ecological settings. In Zimbabwe, S. aureus
has been associated with a number of outbreaks, including
food poisoning [7]. In many developing countries, the
occurrence of food-borne diseases is often coupled to
2
resistant bacteria [8]. Methicillin-resistant Staphylococcus
aureus (MRSA) has shown resistance to drugs commonly
used in Zimbabwe [9, 10]. This is mainly pertinent to
Critical Care Units (CCUs) and theatres. From patients
admitted to CCUs in one of the referral hospitals in
Zimbabwe, 94% of the MRSA isolates showed multidrug
resistance [11]. Bacteria that show increased resistance
against antimicrobial agents and form biofilms are of great
medical concern. MRSA strains form more biofilm than
Methicillin-susceptible Staphylococcus aureus (MSSA)
strains [12]. Biofilm-mediated infections produce a poor
prognosis for patients. Therefore, there is a need to search
for new strategies to prevent and treat biofilm-mediated
infections as well as reducing the invasiveness of the
bacteria in vivo.
Plants possess defense mechanisms against invading
pathogens. These include the production of biologically
active compounds known as phytochemicals. Phytochemical
extracts have been reported to have a biological activity such
as anticancer, antimicrobial, antioxidant, antidiarrhoeal,
analgesic, and wound healing activities [13]. This makes
them attractive as alternative therapeutic options against
several disorders [13]. Several plants have been used traditionally to treat several bacterial infections and this
knowledge has provided the need for studies into the antimicrobial activities of different plant extracts [14]. One of
the plants that are widely used traditionally as a remedy for
different ailments and infections is Ozoroa insignis reticulata
[15]. Most traditional healers in Zimbabwe use the leaf and
bark macerate or infusion of this plant to treat diarrhoea,
kidney and liver complaints, ulcers, throat infections, chest
pains, and schistosomiasis. In some instances, a paste of
leaves and bark is usually applied to the skin to treat several
skin diseases and infections. Infusions of the root extracts are
usually taken orally by women after childbirth to increase
lactation [16]. The current study aimed to determine the
effects of the root bark extract on the growth and extracellular protease production in S. aureus.
2. Materials and Methods
2.1. Reagents and Materials. All chemical reagents used in
this study were purchased from Sigma-Aldrich (Darmstadt,
Germany). Hexane, DCM, acetone, ethyl acetate, methanol,
and ethanol used for extractions were of reagent grade and
were distilled before use. Nutrient agar media (1 g/L meat
extract, 2 g/L yeast extract, 5 g/L peptone, 5 g/L sodium
chloride, 15 g/L agar, pH 7.4 ± 0.2 at 25° C) and nutrient
broth media (15 g/L peptone, 3 g/L yeast extract, 6 g/L sodium chloride, 1 g/L D-glucose, pH 7.5 ± 0.2 at 25° C) were
used as growth and enrichment media, respectively.
Ciprofloxacin and 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) were also purchased
from Sigma-Aldrich (Darmstadt, Germany).
2.2. Plant Collection and Preparation. The root barks of
Ozoroa reticulata voucher number BR1 F1 used in this study
were obtained from an urban woodland in Richmond,
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Bulawayo, Zimbabwe (20° 5′6″ S; 28° 33′7″ E) in November
2018. Ethnobotanical surveys were carried out by Ms.
R. Makurira (Department of Biotechnology and Biochemistry, University of Zimbabwe) who interviewed 9 traditional
healers in Bulawayo Province (Zimbabwe) leading to the
identification Ozoroa reticulata (Baker f.) R. Fern. & A. Fern.
Taxonomical authentication of the identity of the plant was
performed by a botanist at the National Botanical Gardens of
Zimbabwe in Harare. The root barks were washed, dried in
an oven at 45° C, and ground to a fine powder using a
mechanical grinder. For total extraction, the powdered
sample was soaked in two mixture solvents: 50 : 50 (v/v)
DCM: methanol and 50 : 50 (v/v) ethanol: water. For serial
exhaustive extraction, seven solvents with different polarities
were used to macerate the powdered sample. The solvents
were hexane, dichloromethane, ethyl acetate, acetone, ethanol, methanol, and water. After the maceration period, all
mixtures were clarified by filtration, using a Whatman filter
paper No. 1 (Sigma-Aldrich, Darmstadt, Germany). The
filtrates were allowed to air dry under a fan at room temperature. The dried filtrates were then scraped, weighed, and
stored for later use.
2.3. Antibacterial Test. The antibacterial activity of the extracts was assessed using the broth microdilution method
[17]. The root bark extracts were tested against Staphylococcus aureus NCTC 6571 (Culture Collections, Public
Health England, Salisbury, UK). Stock solutions were prepared by dissolving the extracts in dimethyl sulfoxide
(DMSO). Two-fold dilutions of the extracts in nutrient broth
were dispensed in a 96-well microplate. Overnight liquid
cultures of S. aureus cells were adjusted to 0.5 McFarland’s
standard to make a final concentration of 2 × 106 CFU/mL.
The inoculum was placed in each well and incubated at 37° C
for 24 hours in an incubator (Lab DoctorTM, MIDSCI Co.,
Valley Park, USA). Ciprofloxacin was used as a positive
control while a mixture of nutrient broth and DMSO was
used as a negative control. The bacterial suspension without
extracts was used as the growth control. Preincubation
readings were recorded at 590 nm using a Genios Pro
microplate reader (Tecan Group Ltd. Zurich, Switzerland).
Postincubation readings were recorded after 24 h and the cell
viability test was carried out by the MTT assay. The assay
involved the addition of the MTT dye to each well, incubation for at least 2 h, and then observation of colour
changes.
2.4. Determination of Minimum Inhibitory Concentration and
Minimum Bactericidal Concentration. To determine the
MIC of the most potent extract, a stock solution of 400 µg/
mL of the extract was prepared. Two-fold serial dilutions
were done in a 96-well microplate using nutrient broth to
obtain the following concentrations: 200, 100, 50, 25, 12.5,
6.25, 3.125, 1.562, and 0.781 µg/mL. Cells exposed to extracts
were incubated at 37° C for 24 h and MIC was determined.
The minimum inhibitory concentration (MIC) was determined as the lowest concentration of the extract at which no
visible growth of S. aureus was observed. To determine the
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Table 1: Percentage yields and inhibitions of O. reticulata root bark extracts.
Extraction solvent
Ethanol : water
DCM : methanol
Hexane
DCM
Acetone
Ethyl acetate
Methanol
Ethanol
Water
Colour and consistency after drying
Dark brown, solid
Dark brown, solid
Pale yellow, viscous
Brown, solid
Brown, solid
Dark brown, viscous
Dark brown, solid
Brown, solid
Brown, solid
Weight of extract (g)
2.906
1.336
1.908
0.861
1.250
0.390
1.502
0.905
0.195
MBC of the most potent extract, inoculations from the wells
with half MIC, MIC, and twice MIC were inoculated on a
nutrient agar plate. The plate was incubated at 37° C and
observed after 24 h. The concentration at which no visible
growth of S. aureus was observed was noted as the MBC.
2.5. Screening for the Production of Extracellular Proteases.
Screening for the production of extracellular proteases was
done by a method for the detection of protease activity on
agar plates described by Vijayaraghavan and Vincent [18]
with some modifications. Overnight cultures of S. aureus
cells were standardised to make a final concentration of
1 × 106 CFU/mL. A nutrient agar stock mixture of 200 µg/mL
of the most potent extract was prepared along with 1% (w/v)
skimmed milk and 0.0015% (w/v) bromocresol green (BCG).
A Two-fold serial dilution of the nutrient agar stock mixture
was performed to obtain a 100 µg/mL nutrient agar mixture
of the same extract. These were poured onto two separate
Petri dishes and allowed to set. The third Petri dish contained the BCG-skim milk agar prepared without the extract.
For protease activity screening, 5 µL of the standardised
S. aureus cells were placed on all the BCG-skim milk agar
plates prepared and incubated at 37° C in an incubator. The
plates were observed after 24 hours for any zones of proteolysis. The proteolytic zones were measured, recorded, and
expressed as Pz values according to the following equation:
Pz �
(colony diameter)
.
(colony diameter + zone of clearance)
(1)
2.6. Statistical Analysis. Analysis was performed using
GraphPad Prism for Windows, version 8.4.3 (GraphPad
Software, San Diego, California, USA). Statistical analysis of
the results was done using one-way ANOVA. This was
followed by Bonferroni’s multiple comparison post tests and
values of P < 0.05 or less were considered significant.
3. Results
3.1. Plant Extractions. The percentage yields for total and
serial exhaustive extractions performed on root barks from
O. reticulata were determined as presented in Table 1. The
percentage yields are expressed as the ratio of the dry weight
of plant extract to the weight of the plant material used for
the extraction process. The highest percentage yield was
Percentage of yield
15
7
10
5
7
3
10
7
2
Percentage inhibition
32
29
62
93
54
99
67
50
56
obtained from the ethanol: water extract (15%) followed by
the methanol extract (10%). The lowest percentage yield was
obtained from the water extract (2%). The colour and
consistencies of the root bark extracts after drying are shown
in Table 1. All root bark extracts had a brown colour except
for the hexane extract which was pale yellow. The ethyl
acetate and hexane extracts were viscous solids while all the
other extracts were solid.
3.2. Antibacterial Activity of O. reticulata Root Bark Extracts.
The percentage inhibition of growth for the 9 extracts of
O. reticulata root barks against S. aureus (NCTC 6571) is
shown in Table 1. Typical results for the determination of the
percentage inhibition of growth are shown in Figure 1. A
decrease in optical density (OD) at 590 nm was observed as
the concentration of the extract increased. The most potent
extract was the ethyl acetate extract with a percentage inhibition of 99%. This was followed by the DCM extract with
93%. The least potent extract was the DCM: methanol extract
with a percentage inhibition of 29%.
3.3. Determination of the Minimum Inhibitory Concentration
and Minimum Bactericidal Concentration. The antibacterial
effect of the most potent ethyl acetate extract was investigated at a broader concentration range of 0 to 200 µg/mL.
The lowest concentration of the extract that exhibited
complete inhibition of growth of S. aureus was 100 µg/mL as
shown in Figure 2(a). The MIC value of the ethyl acetate
extract was greater than that of the standard drug, ciprofloxacin. Ciprofloxacin had an MIC of 0.125 µg/mL as shown
in Figure 2(b). The investigation into the bactericidal effects
of the ethyl acetate extract against S. aureus (NCTC 6571)
showed that the extract was bacteriostatic at tested concentrations and, therefore, the MBC was greater than 200 µg/
mL.
3.4. Screening for the Production of Extracellular Proteases.
The production of extracellular proteases by S. aureus was
detected by the presence of zones of clearance around the
colonies on BCG-skim milk agar plates as shown in Figure 3.
The sizes of the haloes are proportional to the number of
proteases produced. In the presence of the ethyl acetate
extract, the sizes of the haloes were smaller as compared to
the positive control (no extract). The size of the haloes
became less distinct as the concentration of the ethyl acetate
4
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0.6
Cell density at 590 nm
Cell density at 590 nm
1.5
****
1.0
****
****
0.5
****
****
****
0.4
****
0.2
****
99 %
0.0
93 %
-0.2
0.0
0
12.5
25.0
50.0
100
Concentration of extract (µg/mL)
0
media
12.5
25.0
50.0
100
Concentration of extract (µg/mL)
(a)
media
(b)
Figure 1: Antibacterial effects of O. reticulata root bark extracts against S. aureus. The data are obtained by incubating S. aureus cells in the
presence of (a) DCM extract and (b) ethyl acetate extract. All values are expressed as the mean ± SD of four replicates. Statistical one-way
ANOVA and Bonferroni’s multiple comparison test (where ∗ < 0.05, ∗∗ < 0.01, ∗∗∗ < 0.001, ns � no significant difference vs. media) show
that there is no significant difference between media and cells exposed to 100 µg/mL of ethyl acetate extract. The percentage inhibitions of
growth of S. aureus by the DCM and ethyl acetate extracts were 93 and 99%, respectively.
0.6
****
ns
0.0
****
0.4
0.2
ns ns ns ns
Concentration of extract (µg/mL)
(a)
0.063
0.031
0.016
0.008
0
0.004
media
200
50
100
25
6.3
12.5
3.2
1.6
0.8
0
0.0
1.00
**** ****
MIC = 0.125 µg/mL
**** ****
media
****
0.5
****
0.50
**** **** ****
**** ****
0.25
MIC = 100 µg/mL
0.125
****
Cell density at 590 nm
Cell density at 590 nm
1.0
Concentration of ciprofloxacin (µg/mL)
(b)
Figure 2: The minimum inhibitory concentration of (a) ethyl acetate extract and (b) ciprofloxacin. All values are expressed as the mean ± SD
of four replicates. Statistical one-way ANOVA and Bonferroni’s multiple comparison test (where ∗ < 0.05, ∗∗ < 0.01, ∗∗∗ < 0.001, ns � no
significant difference vs. media) show that the lowest concentration where there is no significant difference between media and cells is
100 µg/ml. Therefore, the MIC for the extract is 100 µg/mL.
extract increased from 100 to 200 µg/mL. The assay is based
on the ability of the proteases to hydrolyse the protein in the
agar. The protease activity may, however, be expressed in
terms of proteolytic activity (Pz ) values as shown in Table 2.
A strong proteolytic activity is observed for the positive
control (Pz � 0.62) while moderate (Pz � 0.84) and weak
(Pz � 0.94) proteolytic activities are observed in the presence of 100 µg/mL and 200 µg/mL, respectively, of the ethyl
acetate extract. There was no growth, haloes, or proteolytic
activity observed for the negative control.
4. Discussion
Different percentage yields of extracts were observed for
total extractions and serial exhaustive extractions. These
differences may be attributed to the different polarities of the
solvents used [19] and the solubility of the phytochemicals in
the respective solvents. Differences in the structure of
phytochemicals determine their solubility in solvents of
different polarities [20]. Higher percentage recovery of extractable compounds is observed mostly from polar solvents
such as methanol and ethanol: water (50 : 50 v/v) for both
total and serial exhaustive extractions. This is in contrast to
nonpolar solvents and solvents of intermediate polarity such
as hexane, DCM, acetone, and ethyl acetate. The higher
yields obtained from polar solvents may be due to higher
solubility of extractable bioactive components such as carbohydrates and protein [20]. Therefore, these observations
may suggest that the O. reticulata root barks are rich in polar
phytochemical compounds.
All the extracts demonstrated anti-S. aureus activity. The
difference in antimicrobial activities observed from the
extracts is likely due to the differences in the concentrations
of antimicrobial compounds within the extracts. The higher
the concentration of the antimicrobial compounds in the
extracts, the greater the antimicrobial activity of the extract.
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(a) Positive Control
(c) 200 µg/mL extract
(b) 100 µg/mL extract
(d) Negative control
Figure 3: The effects of the O. reticulata ethyl acetate extract on the production of extracellular proteases by S. aureus. A distinct halo is
produced for the positive control (a), less distinct haloes are produced on plates containing 100 µg/mL (b) and 200 µg/mL (c) of the ethyl
acetate extract while no growth or haloes are obtained for the negative control (d) after 24 h of incubation.
Table 2: The proteolytic activities of the extracellular proteases produced from S. aureus on BCG-skim milk agar plates: A, B, C, and D.
Plate
A
B
C
D
Colony diameter (mm)
Colony diameter + halo (mm)
Pz value
Reference Pz value
Proteolytic activity
10
11
8
—
16
13
8.5
—
0.62 < 0.69
0.84
0.94
—
Very strong
0.80–0.89
0.90–0.99
—
Mild
Weak
—
Pz value is the ratio of the diameter of the colony to the total diameter of the colony plus the zone of hydrolysis.
The most potent extract was the ethyl acetate extract with an
MIC of 100 µg/mL. This means that, at a concentration of
100 µg/ml of the ethyl acetate extract, there was complete
inhibition of the growth of S. aureus. This observation
suggests that the ethyl acetate extract had a higher concentration of antimicrobial compounds as compared to the
other extracts. Ethyl acetate is a solvent of intermediate
polarity and is capable of extracting slightly polar and
nonpolar compounds. Phytochemical analysis of
O. reticulata has revealed that the plant contains compounds
such as essential oils, anacardic acids, ginkgolic acids, tannins, and flavonoids [21]. These compounds were shown to
be extractable using ethyl acetate as an extraction solvent
and have antimicrobial properties [21]. Therefore, these
compounds may also be present in our sample and were
responsible for the antibacterial activity that was observed.
Essential oils consist of terpenes, terpenoids, and aromatic compounds [22]. Some parts of these compounds are
hydrophobic and some may be hydrophilic depending on
their structure. The essential oils found in O. reticulata
include tirucallane triterpenes and a-elemolic acid esters
[21]. Essential oils have been shown to have an inhibitory
activity against Gram-positive cocci such as MRSA. The
peptidoglycan layer of Gram-positive bacteria allows hydrophobic substances such as essential oils to pass and reach
the internal environment [23]. The mechanism of action of
essential oils against Gram-positive bacteria is suggested to
be through disruption of the cell wall and cytoplasmic
membranes, cytoplasm coagulation, and alteration of the
permeability and functions of the lipid bilayer [24].
Anacardic acids inhibit the growth of S. aureus through
several mechanisms. Firstly, anacardic acids disrupt the
membranes of S. aureus by acting as surfactants [25]. Secondly, amphipathic anacardic acids are capable of inhibiting
bacterial respiration. This is through interaction with the
electron transport chain system and ATPases. Thirdly,
anacardic acids have been reported to have a chelating effect
on metal ions such as Fe2+ and Cu2+ [26]. This reduces the
availability of the ions to the bacteria. Fourthly, anacardic
acids have an inhibitory activity towards ß-lactamase which
is responsible for ß-lactam antibiotic resistance [27]. Lastly,
anacardic acids have been reported to inhibit the synthesis of
lipids in bacterial cells. The mechanism is through inhibition
of glycerol-3-phosphate dehydrogenase enzyme [28].
Ginkgolic acids also affect the growth of S. aureus. Studies on
the antibacterial mechanisms of ginkgolic acids revealed that
they could affect the activity of numerous enzymes responsible for the survival of the bacteria such as protein
phosphatases, lipoxygenases, and histone acetyltransferases
[29, 30]. Furthermore, in another study, it was revealed that
6
ginkgolic acids could inhibit DNA replication, RNA transcription, and protein synthesis in vivo in gram-positive
bacteria [31]. Tannins have been reported to be bacteriostatic
or bactericidal against S. aureus. The astringent properties of
tannins may induce complexation with enzymes or substrates [32]. Many bacterial enzymes are inhibited when
mixed with tannins. Furthermore, tannins affect bacterial
cell membranes. Flavonoids have also been reported to
possess interesting antibacterial mechanisms against
S. aureus. These may also account for the effect of the ethyl
acetate extract on the growth of S. aureus observed. Flavonoids affect the growth of S. aureus by inhibiting DNA,
RNA, and protein synthesis. The proposed mechanism of
this action was through the intercalation of flavonoids with
nucleic acids [33]. The inhibition of S. aureus growth by
flavonoids is through membrane disruption as they have
been reported to cause potassium leakage in S. aureus cells
thereby reducing their viability [34]. Flavonoids also disrupt
S. aureus cell membranes through the disordering of
membrane lipids and through altering the fluidity in hydrophilic and hydrophobic regions of the cell membranes
[35].
The ethyl acetate extract was used to screen for the
production of extracellular proteases by S. aureus. The zones
of clearance are directly proportional to the number of
extracellular proteases produced by S. aureus. A larger zone
of clearance observed for the positive control is because
there was no extract supplemented into the media. Therefore, there was no inhibition of the production of extracellular proteases by S. aureus. The zone of clearance
significantly decreased in the presence of the ethyl acetate
extract. The sizes of the zones of clearance are directly
proportional to the concentration of the ethyl acetate extract.
The zone of clearance observed at 100 µg/mL of the ethyl
acetate extract is larger in size compared to that at 200 µg/mL
of the ethyl acetate extract. This indicates the effectiveness of
the ethyl acetate extract in inhibiting the production of
extracellular proteases by S. aureus as its concentration
increases.
The effectiveness of the ethyl acetate extract to inhibit the
production of extracellular proteases can also be explained in
terms of proteolytic activity values. This parameter is used to
quantify the extent to which the extracellular proteases
produced hydrolyse the protein within the agar media. The
greater the Pz value, the weaker the proteolytic activity of the
extracellular proteases. The proteolytic activity gets weaker
as the concentration of the ethyl acetate extract increases
from 0 µg/mL (positive control) to 200 µg/mL. This shows
that, at higher concentrations of the ethyl acetate extract, the
amount of extracellular proteases produced by S. aureus is
low. Fewer proteases are present to hydrolyse the protein in
the agar media. As a result, this demonstrates the effectiveness of the ethyl acetate extract in inhibiting the production of extracellular proteases.
The BCG-skim milk agar plates allow for the qualitative
determination of protease activity. The BCG reagent binds to
the unhydrolysed protein in the skim milk agar plates. The
reagent is pH-dependent and shows a greenish-blue colour
in the presence of unhydrolysed protein [18]. In the presence
Biochemistry Research International
of extracellular proteases from S. aureus, the protein in the
media is hydrolysed to amino acids. This lowers the pH and
liberates the BCG reagent resulting in a distinct clear zone
that is observed.
Inhibition of the production of S. aureus extracellular
proteases by the ethyl acetate extract from O. reticulata root
barks can be attributed to several mechanisms. O. reticulata
has been reported to contain tirucallane triterpenes [21].
These may also be present in the ethyl acetate extract.
Triterpenes and triterpenoids have been associated with the
downregulation of the production of secreted proteins [36].
While the exact mechanism of action of this phenomenon is
still unknown, several suggestions have been put forward.
For example, it is known that triterpenes have the potential
to inhibit protease dimerization [37]. This is due to the
molecular size of triterpenes which allow them to fit into the
hydrophobic interface of the relaxed protease monomer. In
the process, the production of functional proteases may be
inhibited. Furthermore, hydrogen bonding is possible between the proteases and the hydroxyl/carboxyl groups in the
triterpenes scaffold [38].
The production of bacterial virulence factors is regulated
by the quorum sensing system. Quorum sensing (QS) refers
to the way microorganisms regulate their behaviour through
sending and receiving chemical signals (autoinducers).
S. aureus uses QS to regulate the production of virulence
factors including the extracellular proteases. Some phytochemicals have been shown to have an inhibitory effect on
the QS system of S. aureus. Inhibitors that target virulence
factor production by the QS system affect the transcription
and translation of the virulence factors [39]. Therefore, this
suggests that the ethyl acetate extract from O. reticulata root
barks may contain phytochemicals that inhibit or quench the
QS system, thereby, preventing extracellular protease
production.
5. Conclusion
The root bark extracts from O. reticulata inhibited the
growth of S. aureus. The most potent inhibitor of the
growth of S. aureus was the ethyl acetate extract and it was
found to be bacteriostatic. The ethyl acetate extract
inhibited extracellular protease production by S. aureus.
Root bark extracts from O. reticulata, therefore, contain
promising phytochemical compounds that may be able to
combat infections caused by S. aureus. This may minimize
the severity of S. aureus infections and the development of
antibiotic-resistant strains of S. aureus.
Abbreviations
DCM:
MIC:
MBC:
MTT:
Dichloromethane
Minimum inhibitory concentration
Minimum bactericidal concentration
3-(4, 5-Dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide
DMSO: Dimethyl sulfoxide
NCTC: National Collection of Type Cultures
MRSA: Methicillin-resistant Staphylococcus aureus
Biochemistry Research International
MSSA:
BCG:
OD:
QS:
CCU:
Methicillin-susceptible Staphylococcus aureus
Bromocresol green
Optical density
Quorum sensing
Critical care unit.
Data Availability
7
[8]
[9]
The datasets used and/or analyzed during the current study
are available from the corresponding author on reasonable
request.
[10]
Conflicts of Interest
[11]
The authors declare that they have no competing interests.
Acknowledgments
The authors acknowledge the assistance of Mr. Christopher
Chapano, a taxonomist with the National Herbarium and
Botanical Gardens, Harare, Zimbabwe, in the authentication
of the plant sample name. Support from the Swedish International Development Agency (SIDA) through the International Science Programmes (ISP) (International
Program in the Chemical Sciences (Uppsala University,
Uppsala, Sweden, ISP IPICS:ZIM01) and the International
Foundation in Sciences (IFS F/3413-03F, Stockholm, Sweden) is acknowledged. F/3413-03F supported research under
the title “Screening natural plant products from selected
plants from Zimbabwe as a source of anti-infective compounds for phytomedicines development.” ISP IPICS :
ZIM01 supported the research under the title “Biomolecular
interactions analyses.” Support from the Alliance for Global
Health and Science (University of California, Berkeley) is
acknowledged.
[12]
[13]
[14]
[15]
[16]
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