Vol. 10(25), pp. 360-366, 3 July, 2016
DOI: 10.5897/JMPR2016.6122
Article Number: 17DDCEC59276
ISSN 1996-0875
Copyright © 2016
Author(s) retain the copyright of this article
http://www.academicjournals.org/JMPR
Journal of Medicinal Plants Research
Full Length Research Paper
Modulatory effects of Hilleria latifolia and Laportea
ovalifolia on activity of selected antibiotics
Susana Oteng Dapaah, Christian Agyare*, Yaw Duah Boakye and Theresa Appiah
Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of
Science and Technology, Kumasi, Ghana.
Received 13 April, 2016; Accepted 9 June, 2016
Microorganisms are becoming resistant to almost all existing and newly discovered antimicrobial
agents. This has led to ineffective treatment of infectious diseases with increased risk of complications.
Few medicinal plants have been found to exhibit the ability of reversing the resistance mechanisms of
microbes to antibiotics. The study investigates the influence of leaf methanol extracts of Hilleria latifolia
and Laportea ovalifolia on some commonly used antibiotics. Micro-dilution technique was used to
determine the antimicrobial activity and minimum inhibitory concentration (MIC) of the leaf extracts and
the selected antibiotics. MICs of the antibiotics in presence of sub-inhibitory concentration of the
extracts were determined. MIC of H. latifolia and L. ovalifolia extracts ranged from 50 to 100 mg/ml. In
the presence of sub-inhibitory concentration (5 mg/ml) of the extracts, the activity of the antibiotics was
modified with enhanced or reduced activity. The activity of amoxicillin was potentiated by 8-folds, 4folds, 2-folds, 8-folds, and 2-folds against Escherichia coli, Bacillus subtilis, Salmonella typhi,
Staphylococcus aureus and Pseudomonas aeruginosa, respectively in the presence of leaf extract of H.
latifolia. Activity of ampicillin was potentiated by 2- and 4-folds against E. coli and S. typhi, respectively,
as well as tetracycline, 4-folds, against Klebsiella pneumonia in the presence of leaf extract of H.
latifolia. Sub-inhibitory concentrations of H. latifolia and L. ovalifolia extracts reduced the activities of
erythromycin and ciprofloxacin against all the test organisms. Sub-inhibitory concentrations of H.
latifolia and L. ovalifolia extracts modified the activities of the selected antibiotics.
Key words: Minimum inhibitory concentration, antibiotics, microbial resistance, antibiotic-resistance modifying
agents.
INTRODUCTION
Pathogenic organisms which were previously known to
have been killed or inhibited by antibiotics are now
resistant to these same antimicrobial agents (Levy and
Marshall, 2004). This has forced a number of
pharmaceutical companies to leave the field of antibiotic
discovery and production to rather produce more
profitable medications for treating other diseases
especially non-communicable diseases (Projan, 2003).
The problem of resistance is now posing great threat on
public health more than ever before, due to increasing
*Corresponding author. E-mail: cagyare.pharm@knust.edu.gh or chrisagyare@yahoo.com. Tel: +233246369803.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
Dapaah et al.
multi-drug resistance in a single organism drastically
limiting therapeutic options (Levy, 2005).
Naturally, bacteria have the ability to genetically
develop resistance to antibacterial agents (Nascimento et
al., 2000). They use different ways and strategies to
acquire or develop resistance to antibiotics, which
include, active efflux of drugs, alteration of target sites
and inactivation of antibiotics by producing enzymes that
degrade them (Sibanda and Okoh, 2007). Also,
inappropriate diagnosis, drug counterfeit, use of
antibiotics in food production and animal rearing, noncompliance and under dosing and sometimes
uncontrolled use of antimicrobial agents are factors that
contribute greatly to the overwhelming increase in the
microbial resistant menace in this generation (Adu et al.,
2014).
Microbial resistance to antimicrobial agents has limited
the use of known cheap but effective antibiotics (Ranjan
et al., 2012). This has necessitated the search for new
potent antimicrobial agents to combat the threat posed by
resistant microbes. In search of antimicrobial agents,
various sources such as the synthetic compounds as well
as bioactive agents from natural products (aquatic
microorganisms and medicinal plants) are taken into
consideration (Agyare et al., 2012). Medicinal plants are
great sources of antimicrobial agents and the idea that
plants have been used as effective drugs for treatment of
infectious diseases was well accepted even before the
discovery of microbes (Rios and Recio, 2005).
Coates et al. (2002) reported the occurrence of cross
resistance to newly identified antibiotics and other
antimicrobial agents suggesting that newly discovered
antimicrobial agents may be rendered ineffective in the
near future. Even though the appropriate use of these
antimicrobial agents can reduce the rate of resistance
development, it cannot eliminate the emergence of
resistant strains (Sibanda and Okoh, 2007). There is
therefore the need to discover and develop new
compounds that will target and block resistance
mechanisms to help treat infections from these resistant
strains (Oluwatuyi et al., 2004).
Medicinal plants have been found to contain
compounds with or without antimicrobial property that
can cause resistant microorganisms to be susceptible to
a previously impotent antibiotic (Aiyegoro et al., 2009). In
most developing countries, where majority of the people
rely on medicinal plants and natural products, they
combine their orthodox medications with these plants
in the treatment of various diseases (Adu et al., 2014;
Agyare et al., 2009). This study, therefore, investigates
the influence of leaf methanol extracts of Hilleria latifolia
and Laportea ovalifolia on some commonly used
antibiotics. H. latifolia (Lam.) H. Walt belongs to the
family Phytolaccaceae and is locally known by the Ewes
as ‘avegboma’ and ‘anafranaku’ by the Asantes in
Ghana. It is a perennial herb of 30 to 120 cm high, with
ovate-elliptic leaves and numerous short hair-like
361
structures on lower surface. The leaves are used in
Ghana for the management of rheumatism, boils and
wounds (Agyare et al., 2009). The leaves are used to
treat general oedema, asthma and some skin diseases
(Mshana, 2000; Dokosi, 1998). It is also used to treat
cough with blood (Schmelzer and Gurib-Fakim, 2008).
L. ovalifolia (Schumach.) Chew belongs to the family
Urticaceae. It is known by the Asantes in Ghana as
‘akyekyenwonsa’, ‘abrewa nom taa’ or ‘Kumasi otuo’. It is
a herbaceous weed more often creeping than erect and
densely covered with stinging hairs (Chew, 1969). L.
ovalifolia is of two varieties, that is, male and female
(Essiett et al., 2011). Leaves are used to treat wounds
(Agyare et al., 2009) and the fruits are used as a poison
antidote (Bouch, 2004). The root extract is used to
prevent or reduce excessive menstrual bleeding
(Sofowora, 1996)..
MATERIALS AND METHODS
Plant collection
Leaves of H. latifolia and L. ovalifolia were collected from Aburi in
the Eastern region of Ghana on February, 2014. The plants were
authenticated by Dr. Alex Asase of the Department of Botany,
University of Ghana, and voucher specimen AA 63 and AA 71,
respectively deposited in the Ghana Herbarium, Department of
Botany, University of Ghana, Legon, Accra, Ghana.
Plant extraction
The fresh leaves collected were washed thoroughly under running
tap-water and dried under shade between 25 and 28°C for two
weeks, after which they were pulverized into coarse powder using
the laboratory milling machine (Christy and Norris, Chelmsford,
England). 800 g each of the powdered plant materials were soaked
in 2.5 L of 70% v/v methanol. They were extracted with the aid of
ultra-turrax (T 25 Janke and Kunkel, Labortenik, Germany) under
ice-cooling at a speed of 24000 rpm for 3 to 5 min, and then filtered
using a laboratory sieve (Retsch, Haan, Germany) of mesh number
200 with aperture of 75 μm and Whatmann filter paper No.1. The
filtrates were concentrated with the rotary evaporator (Rotavapor
BÜCHI R-200 with heating bath B-490, Büchi, Konstanz) at 40°C
under reduced pressure and lyophilized and then stored in air tight
containers at 4 to 8°C. The yields of the extracts of H. latifolia and
L. ovalifolia were 17.4 and 11.29% w/w, related to the dried
material, respectively.
Preliminary phytochemical screening
Methanol leaf extracts of H. latifolia (HLLE) and L. ovalifolia (LOLE)
and their respective powdered dried plant materials were subjected
to qualitative phytochemical analysis to identify various secondary
metabolites such as tannins, glycosides, saponins, alkaloids,
flavonoids, steroids and terpenoids present using standard methods
described by Usman et al. (2014), Trease and Evans (2002) and
Sofowora (1993).
High performance liquid chromatography (HPLC) profile of
extracts
HPLC analysis was performed to identify the profile or finger-prints
362
J. Med. Plants Res.
Table 1. Phytochemical screening of methanol leaf extracts of H. latifolia and L. ovalifolia, and their dried powdered plant materials.
Secondary metabolites
Tannins
Flavonoids
Glycosides
Saponin
Alkaloids
Sterols
Terpenoids
HLLE
+
+
+
+
+
+
+
H. latifolia leaf
Powdered plant material
+
+
+
+
+
+
+
LOLE
+
+
+
+
L. ovalifolia leaf
Powdered plant material
+
+
+
+
+
(+) = Presence of secondary metabolites; (-) = absence of secondary metabolites. HLLE: Methanol leaf extract of H. latifolia; LOLE: methanol
leaf extract of L. ovalifolia.
of the crude extracts with a UV-detector set at a wavelength of 254
nm. The running conditions included injection volume of 10 μl,
mobile phase of methanol:water (20:80 v/v, isocratic condition), flow
rate of 1 ml/min and pressure of 15 MPa. The chromatographic
data were determined using Chrom Quest® software.
Test organisms
Test organisms were obtained from the microbiology laboratory of
the Department of Pharmaceutics, Faculty of Pharmacy and
Pharmaceutical Sciences, Kwame Nkrumah University of Science
and Technology (KNUST), Kumasi, Ghana. They include
Pseudomonas aeruginosa ATCC 4853, Escherichia coli ATCC
25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis
NCTC 10073, Enterococcus faecalis ATCC 29212 and clinical
strains of Streptococcus pyogenes, Salmonella typhi and Klebsiella
pneumonia. They were stored in 30% glycerol broth at -4°C in a
frost free freezer (Mistral, UK) until needed, whereby 100 μl of the
stock suspension was transferred into 10 ml nutrient broth (Oxoid
Limited, United Kingdom) and incubated at 37°C for 24 h (subcultured) before use.
Determination of antibacterial activity and minimum inhibitory
concentration (MIC) of extracts
Micro-dilution method described by Eloff (1998) and modified by
Agyare et al. (2012) was used to determine the antibacterial activity
and the MIC of the extracts. Each well of micro-titre plate (96 wells)
was filled with 100 μl of double strength nutrient broth, 20 μl of 106
cfu/ml of the test organisms and 80 μl of different concentrations of
HLLE and LOLE ranging from 1.56 to 100 mg/ml. Ciprofloxacin
(Sigma-Aldrich, Michigan, USA) at concentration range of 1.0 to
128 μg/ml was used as reference antibiotic drug. Control wells were
filled with broth only and broth and test organisms only. After 24 h
of incubation at 37°C, 20 µl of 1.25 mg/ml of 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich,
Taufkirchen, Germany) was added to each well and observed for a
purple colouration after incubation at 37°C for 30 min which
indicated microbial growth. The minimum concentrations of HLLE,
LOLE and reference drug that did not show any colour change in
the wells were recorded as the MIC. The method was replicated
three times to validate the results.
concentration of the extracts on the activity of some selected
antibiotics including amoxicillin, erythromycin, ciprofloxacin,
tetracycline and ampicillin (Sigma-Aldrich, Michigan, USA). The
micro-dilution technique with some modifications as described by
Adu et al. (2014) was employed. The MICs of the antibiotics were
first determined using concentrations ranging from 1 to 1024 μg/ml.
Each of the 96 wells of the micro-titre plate was filled with 100 μl of
double strength nutrient broth, appropriate volume of different
concentrations of the antibiotics and 20 μl of 106 cfu/ml of the test
organisms. The plate was incubated for 24 h at 37°C, after which
20 μl MTT was added to the wells and MIC determined as the
lowest concentration at which no growth was observed (that is, no
colour change from yellow to purple). MICs of the antibiotics were
re-determined in the presence of sub-inhibitory concentration (5
mg/ml) of the extracts (HLLE and LOLE).
RESULTS
Various phytochemical tests were performed on HLLE
and LOLE and their dried powdered plant materials to
identify their phytochemical composition. Phytochemical
screening of HLLE and dried powdered leaf material of H.
latifolia revealed the presence of tannins, flavonoids,
glycosides, saponins, alkaloids, sterols and terpenoids.
LOLE and the pulverized leaf material of L. ovalifolia also
showed the presence of tannins, glycosides, sterols and
terpenoids. However, saponins was found in the
powdered leaf material of L. ovalifolia but was absent in
its extract (Table 1).
HPLC profiles of extracts
Chemical profiles of the extracts indicate the present of
metabolites/compounds at the wavelength used and
these will serve as a guide in identifying the plants
(Figures 1 and 2).
Antibacterial activity and MIC of extracts
Microbial resistance modifying activity of the extracts
This study was done to determine the effect of a sub-inhibitory
MICs of HLLE and LOLE against typed and clinical
strains of organisms, consisting of Gram-positive bacteria
Dapaah et al.
363
Table 2. MIC of methanol leaf extracts of H. latifolia and L. ovalifolia against test organisms.
Test organisms
B. subtilis
S. aureus
E. feacalis
S. pyogenes
E. coli
S. typhi
K. pneumoniae
P. aeruginosa
MIC of extracts and reference drug
HLLE (mg/ml)
LOLE (mg/ml)
Cip (μg/ml)
50.0
50.0
2.0
50.0
100.0
4.0
50.0
100.0
4.0
50.0
100.0
4.0
50.0
100.0
2.0
50.0
50.0
4.0
50.0
50.0
4.0
50.0
50.0
4.0
HLLE: Methanol leaf extract of H. latifolia; LOLE: methanol leaf extract of L. ovalifolia; Cip:
Ciprofloxacin.
(B. subtilis, S. aureus, E. feacalis, S. pyogenes) and
Gram-negative bacteria (E. coli, S. typhi, K. pneumonia,
P. aeruginosa) were between 50 and 100 mg/ml (Table
2).
Antibiotic modulatory activity of methanol leaf
extracts of H. latifolia (HLLE) and L. ovalifolia (LOLE)
Activities of the selected antibiotics (amoxicillin,
erythromycin, ciprofloxacin, tetracycline and ampicillin)
against the test microorganisms were modified in the
presence of sub-inhibitory concentration (5 mg/ml) of the
extracts, either by enhanced or reduced activity. For
instance, the activity of amoxicillin against E. coli, B.
subtilis, S. typhi, S. aureus and P. aeruginosa potentiated
by 8-, 4-, 2-, 8- and 2-folds, respectively, in the presence
of the sub-inhibitory concentration of HLLE. Sub-inhibitory
concentration of HLLE again enhanced the activity of
ampicillin 2- and 4-folds against E. coli and S. typhi,
respectively, as well as tetracycline, 4 folds, against K.
pneumonia. The activity of amoxicillin against S. aureus
and E. coli was enhanced (2-folds) in the presence of
LOLE. However, both HLLE and LOLE sub-inhibitory
concentrations reduced the activities of erythromycin and
ciprofloxacin against all test organisms (Table 3).
DISCUSSION
Secondary metabolites such as tannins, flavonoids,
glycosides, alkaloids, terpenoids and steroids present in
the plants may be responsible for their pharmacological
and biological properties (Maganha et al., 2010; BarbosaFilho et al., 2006; Mbagwu et al., 2007; Sofowora, 1993).
These secondary metabolites act alone or in synergy
leading to the healing potentials of plants (Jenke-Kodama
et al., 2008; Gurib-Fakim, 2006). Phytochemical
screening of plant materials revealed the presence of
tannins, glycosides, saponins, flavonoids, alkaloids,
sterols and terpenoids in the leaves of H. latifolia, which
is similar to reports on the same plant by Abotsi et al.
(2012) and Schmelzer and Gurib-Fakim (2008). Tannins,
glycosides, sterols and terpenoids were also present in
the leaves of L. ovalifolia, however, flavonoids and
alkaloids were absent (Table 1). Essiett et al. (2011)
reported the presence of phytochemicals such as tannins,
glycosides, saponins, flavonoids and alkaloids in the
leaves of L. ovalifolia. The absence of flavonoids and
alkaloids in the leaves of L. ovalifolia as observed in this
study may be due to the different geographical location of
the plant, the season and time of collection which are all
contributing factors leading to variations in the
phytochemical constituents of plants of the same species
(Stackpole et al., 2011; González‐Martínez et al., 2006).
In addition to the phytochemical screening, HLPC
profile of the 70% methanol extracts (HLLE, and LOLE)
were developed for identification purposes. HPLC
profiling is more specific and helps in easy identification
and confirmation of plant on the basis of specific
phytochemicals present. The HPLC profiles of the
extracts (Figures 1 and 2) showed that the peaks
representing compounds present in the extracts
appeared in the early part (early elution) of the
chromatogram. This observation may be due to the polar
solvent (70% methanol) used for the extraction. The
profiles indicate the complex chemical composition of the
extracts and provide identification parameters to figure
out alterations in crude extracts (Tistaert et al., 2012).
HLLE and LOLE exhibited a broad spectrum
antibacterial activity against B. subtilis, S. aureus, E.
feacalis, S. pyogenes, E. coli, S. typhi, K. pneumonia and
P. aeruginosa with MIC ranging from 50 to 100 mg/ml
(Table 2). The antibacterial activity observed may be
attributed to the phytochemical constituents present in
the extracts, since these phyto-constituents have been
reported to exhibit antimicrobial properties (Edeoga et al.,
2005; Nweze et al., 2004).
The low antibacterial activity of HLLE and LOLE may
be as a result of low amount of the active constituents
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J. Med. Plants Res.
UV1000-254nm
Retention Time
7.227
20
4.395
1.017
20
mAU
40
1.235
1.362
1.408
1.662
2.0681.855
2.722
3.382
40
0
0
0
5
10
15
20
25
30
Minutes
Figure 1. HPLC profile of methanol leaf extract of H. latifolia (HLLE) at λ 254 nm.
UV1000-254nm
Retention Time
1.997
7.5
5.0
2.5
2.5
0.0
mAU
5.0
1.063
1.475
2.342
3.111
7.5
0.0
0
5
10
15
20
25
30
Minutes
Figure 2. HPLC profile of methanol leaf extract of L. ovalifolia (LOLE) at λ 254 nm.
in the extracts. Similar to this observation, Okwulehie and
Akanwa (2013) reported a low antimicrobial activity of L.
ovalifolia. At MIC of 50 mg/ml, L. ovalifolia did not inhibit
the growth of the test organisms. Also, Assob et al.
(2011) reported on the antimicrobial activity of H. latifolia
with MIC of 0.6 to 2.5 mg/ml. The high MIC (50 to 100
mg/ml) observed for H. latifolia in this study may be as a
result of different extraction procedures used and
different locality of the plant materials used which may
lead to different composition in terms of primary and
secondary metabolites.
Even though, HLLE and LOLE may not be potential
source of antimicrobial agents as reported by Navarro
and Delgado (1999) and Fabry et al. (1998) because of
their relatively high MICs, it is also well noted that
plant extracts with low antimicrobial activity may have
some phyto-constituents that can modify the antimicrobial
activity of some existing antimicrobial agents, especially
against resistant bacteria (Adu et al., 2009). Subinhibitory concentrations (5 mg/ml) of HLLE and LOLE
modified the activity of amoxicillin, erythromycin,
ciprofloxacin, tetracycline and ampicillin by either
potentiating or reducing their activity against the test
organisms.
The increased activity of these antibiotics in the
presence of the sub-inhibitory concentration of the
extracts may be attributed to the phytochemicals present
in the extracts. For instance, flavonoids have been
reported to have the ability to reverse the resistance of S.
aureus to some antibiotics (Aiyegoro et al., 2009). The
Dapaah et al.
365
Table 3. Effect of sub-inhibitory concentration (5mg/mL) of HLLE and LOLE on activity of selected antibiotics
against test organisms.
Antibiotic
only/Antibiotic + HLLE/
LOLE
BS
SA
Test organisms
EF
SP
EC
ST
Number of folds increase in activity
1
>0.25
8
2
1
>0.25
2
0.5
KP
PA
1
0.5
2
1
Amx
HLLE
LOLE
4
1
8
2
Amp
HLLE
LOLE
1
0.25
1
1
1
1
>0.0625
>0.0625
2
1
4
0.25
1
0.5
1
>0.0625
Tet
HLLE
LOLE
1
0.125
1
0.25
1
0.25
1
0.25
0.5
0.125
1
0.25
4
0.5
2
0.5
Ery
HLLE
LOLE
0.25
0.5
0.0625
0.125
0.125
0.25
0.125
0.0625
0.0625
0.0625
0.0625
0.125
0.125
0.125
0.25
0.125
Cip
HLLE
LOLE
0.125
0.0625
0.5
0.125
0.5
0.25
0.5
0.25
0.125
0.125
0.25
0.125
0.5
0.125
0.25
0.125
EC: E. coli, BS: B. subtilis, ST: S. typhi, KP: K. pneumonia, SA: S. aureus, EF: E. feacalis, SP: S. pyogenes, PA: P.
aeruginosa. Amx: amoxicillin, Amp: ampicillin, Tet: tetracycline, Ery: erythromycin, Cip: ciprofloxacin, HLLE: methanol leaf
extract of Hilleria latifolia, LOLE: methanol leaf extract of Laportea ovalifolia.
activity of the test antibiotics was enhanced mainly by
HLLE as compared to LOLE. This may be as a result of
the presence of flavonoids in HLLE and its absence in
LOLE Antimicrobials from plants, at sub-inhibitory
concen-trations, have been reported to be efficient in
synergism with antibiotics by enhancing their
antimicrobial actions (Kamatou et al., 2006). The
phytochemicals act by reversing the resistance
mechanisms of some microorganisms, thereby rendering
them susceptible to previously inactive antibiotics
(Tenover, 2006). Plants have also been known to
produce multi-drug resistance inhibitors (MDRIs) to
enhance the antimicrobial activities of compounds
(Stermitz et al., 2000).
The reduced or nullified activity of the antibiotics may
be as a result of interactions between the phytochemicals
in the extract and the antibiotics or the microorganisms.
The phyto-constituents may react chemically with the
antibiotics leading to loss of activity (Adu et al., 2009,
2014). It has been established that certain substances
including plant constituents can shield microorganisms
from the lethal effects of some antimicrobial agents
(Keweloh et al., 1989). For example, some
phytochemicals can bind to the surface structures of
microorganisms thereby reducing their permeability to
antibiotics (Adu et al., 2014). Furthermore, some of the
phyto-contituents may act as protein activators or coenzymes which bind to and activate enzymes responsible
for resistance in an organism, making them resistant to a
previously potent antibiotic (Lambert, 2002). There is
need to isolate the bioactive agents or compounds
responsible for the antibiotic resistance modifying
properties especially those that potentiated the activity of
the extracts against resistant bacterial strains.
Conclusion
The sub-inhibitory concentrations of the methanol leaf
extracts of H. latifolia and L. ovalifolia modified the
activity of some antibiotics by either potentiating or
reducing their antibacterial activities.
Conflict of interests
The authors have declare no conflict of interests.
ACKNOWLEDGEMENTS
The authors gratitude goes to Dr. Alex Asase and Mr.
John Yaw Amponsah of Ghana Herbarium and
Department of Botany, University of Ghana, Legon,
Accra, Ghana for the identification and collection of the
plant material. Also to Mr. Francis Amankwah,
Department of Pharmaceutics, Faculty of Pharmacy and
Pharmaceutical Sciences, Kwame Nkrumah University of
Science and Technology, Kumasi, Ghana, for his
assistance during the study.
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