Original Article
Antibacterial Activity of Ritchiea albersii Gilg and Cynoglossum
amplifolium Leaves Extracts against Selected Bacteria
Hizkel Engiso1, Teshale Worku2, Dejen Nureye3, Mohammed Salahaddin3,4, Workineh Woldeselassie3,
Solomon Hambisa3, Nymathullah Sharief5
Department of Clinical Pharmacy, School of Pharmacy, College of Medicine and Health Sciences, Hawassa University, Hawassa,
Department of Medical Laboratory, College of Medicine and Health Sciences, Mizan‑Tepi University (Aman Campus), Mizan‑Aman,
3
Department of Pharmacy, College of Medicine and Health Sciences, Mizan‑Tepi University (Aman Campus), Mizan‑Aman, Ethiopia,
4
Department of Biomolecular Sciences, Pharmacology Division, University of Mississippi, Oxford, Mississippi, United States of America,
5
Department of Biochemistry and Biotechnology, Minerva Degree College, East Godavari District, Andhra Pradesh, India
1
2
Abstract
Background: The increase in antimicrobial resistance worldwide has necessitated the search for alternative
therapeutic agents. The leaf extracts of Ritchiea albersii and Cynoglossum amplifolium have been used as
traditional medicine for the management of eye, ear and wound infections in Ethiopia.
Objective: The objective of the study was to evaluate the antibacterial activity of R. albersii and C. amplifolium
against three common bacteria.
Materials and Methods: In this experimental study, the antimicrobial properties of 80% methanol,
chloroform and acetone extracts of R. albersii and C. amplifolium were evaluated against two Gram-positive
bacteria (Staphylococcus aureus ATCC 25923 and Streptococcus pneumoniae ATCC 49619) and one Gram-negative
bacterium (Escherichia coli ATCC 25922) using the agar-well diffusion method. Ciprofloxacin 0.05 mg/disc
was used as a positive control. Furthermore, a preliminary phytochemical study was carried out.
Results: The zones of inhibition shown by all extracts of the two plants against the tested bacteria were
significantly lesser (P < 0.05) than the standard drug. E. coli and S. aureus were the most susceptible strains for
most extracts studied. The acetone extract of R. albersii exhibited a higher inhibitory effect (P < 0.05) against
S. pneumoniae (16 mm) and E. coli (19 mm) compared with its methanol extract. The chloroform extract of R. albersii
was more effective than its methanol extract (P < 0.05) against all tested bacteria. The acetone extract of C.
amplifolium displayed a higher inhibitory effect (20 mm) against E. coli than its methanol and chloroform extracts.
Conclusions: The leaf extracts of R. albersii and C. amplifolium exhibited broad-spectrum antimicrobial activity,
highlighting their potential as phytotherapeutic drugs in preventing and treating infections caused by
S. aureus, S. pneumoniae and E. coli. Further investigations for isolating specific compounds and elucidating
mechanisms are required to address the need for novel antibacterial drugs.
Keywords: Antibacterial activity, Cynoglossum amplifolium, Escherichia coli, Ritchiea albersii, Staphylococcus aureus,
Streptococcus pneumoniae
Address for correspondence: Mr. Dejen Nureye, Department of Pharmacy, College of Medicine and Health Sciences, Mizan‑Tepi University (Aman Campus),
P.O. Box 260, Mizan‑Aman, Ethiopia.
E‑mail: dejenureye@gmail.com
Submitted: 23‑Jul‑2019 Revised: 03‑Nov‑2019 Accepted: 12‑Mar‑2020 Published: 20‑Aug‑2020
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Website:
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DOI:
10.4103/sjmms.sjmms_276_19
For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com
How to cite this article: Engiso H, Worku T, Nureye D, Salahaddin M,
Woldeselassie W, Hambisa S, et al. Antibacterial Activity of Ritchiea albersii
Gilg and Cynoglossum amplifolium Leaves Extracts against Selected
Bacteria. Saudi J Med Med Sci 2020;8:201‑7.
© 2020 Saudi Journal of Medicine & Medical Sciences | Published by Wolters Kluwer - Medknow
201
Engiso, et al.: Bioactivity of R. albersii and C. amplifolium
INTRODUCTION
Infectious diseases are a leading cause of mortality
worldwide, particularly in low‑income countries. [1]
According to the World Health Organization, in 2016, lower
respiratory infections, diarrheal diseases and tuberculosis
accounted for 3 million, 1.4 million and 1.3 million deaths,
respectively, making them three of the top ten causes of
deaths worldwide.[2]
Antimicrobials remain valuable resources for treating
and preventing infectious disease despite the global
increase in antimicrobial resistance (AMR).[3] However,
AMR is a key issue in public health and has increased the
rates of morbidity, mortality and socioeconomic costs.[4]
The increase in multi‑drug resistant (MDR) pathogenic
bacteria is limiting the choices of effective antibacterial
treatment, as this phenomenon has not been paralleled by
the development of new antibiotics.[5] Consequently, by the
year 2050, an increase in AMR is estimated to annually put
10,000,000 lives at risk.[6] Hence, there is an urgent need
for newer antibacterial agents with novel mechanisms of
action.[7,8] Some areas for discovering such antibacterials
are natural products of plant origin and antimicrobial
peptides.[9‑11]
In Ethiopia, about 90% of the population is reliant
on traditional remedies for the management of
diseases.[12] Studies conducted on numerous traditionally
used ethnomedical plants of Ethiopia have shown
antibacterial activities including Nuxia congesta,[13] Zehneria
scabra, Ricinus communis,[14] Rhamnus prinoides,[15] Justicia
schimpriana, [16] Jasminium abyssinicum, Myrsine africana,
Foeniculum vulgare,[17] Verbasicum sinaticum, Calpurnia aurea,
Salvia schimperi, Hypericum revolutum, Pterolobium stellatum,[18]
Datura stramonium, Croton macrostachyus and Acokanthera
schimperi.[19]
Ritchiea albersii Gilg (Capparidaceae) is a small tree with a short
thick trunk (11 m high). In Ethiopia, its various parts are
used in traditional medicine for the treatment of meningitis,
wound, cataract, respiratory tract problems and tonsillitis.
Cynoglossum amplifolium (Boraginaceae) is a perennial herb
or subshrub that is 0.3–1.8 m tall and has a thick tuberous
root of up to 45 cm and large leaves and tall stems. In
Ethiopia, C. amplifolium is prescribed by traditional medicine
practitioners to treat ear, eye and wound infections.[20‑22]
Despite their use as traditional medicines, no antibacterial
studies of R. albersii and C. amplifolium have been conducted
to date. Therefore, the aim of this study was to screen
the antibacterial activities of 80% methanol, chloroform
and acetone crude extracts of R. albersii and C. amplifolium
202
leaves against Streptococcus pneumoniae, Staphylococcus aureus
and Escherichia coli, which are MDR and common causes
of ear and wound infections.
MATERIALS AND METHODS
Collection of plant material
For this experimental study, fresh leaves of R. albersii and
C. amplifolium were collected in December 2017 from its
natural habitat in Bench district, namely Temenja Yaxi
and Andekel Kebele, Southwest Ethiopia, about 574 km
from Addis Ababa. The leaves were covered in plastic
sheet during transportation. The collected plants were
then identified and authenticated as R. albersii and C.
amplifolium by a taxonomist at the National Herbarium,
College of Natural and Computational Sciences, Addis
Ababa University, where voucher specimens (no. HE
001 and HE 002, respectively) were deposited for future
reference.
Preparation of plant extract
Fresh leaves of the plants were thoroughly washed by
tap water and cleaned with gauze to remove dirt and
soil. These samples were then air‑dried under the shade
and crushed into coarse powder using sterile pestle and
mortar. Subsequently, 231 g powder of R. albersii and
210 g powder of C. amplifolium were divided into three
portions and extracted by cold maceration technique
with 80% methanol (800 mL), acetone (1000 mL) and
chloroform (1000 mL) solution in an Erlenmeyer flask
for 3 consecutive days at room temperature to get the
crude hydroalcoholic, acetone and chloroform extract,
respectively. The same volume of solvent was used for
the successive extraction of the residues. The extraction
process was facilitated using a mechanical shaker (Bibby
Scientific Limited, Stone Staffordshire, UK) at 120
revolutions per minute. The resulting crude extracts were
separated from the marc with gauze and then filtered by
Whatman filter paper Grade‑1 using suction twice by the
addition of fresh solvent to acquire the maximum yield.
The filtrates were combined and concentrated by Rotary
evaporator (Buchi Rotavapor R‑200, Flawil, Switzerland)
under reduced pressure. All extracts were then dried
and further concentrated using a dry oven (Leaders
Engineering, Hastings, UK). Finally, the extracts were
transferred into an amber glass bottle and kept at −20°C
until use. The respective percentage yield of 80% methanol,
acetone and chloroform of R. albersii was 15.9%, 16.3%
and 14.8% and of C. amplifolium was 16%, 14.9% and
14.6%, respectively. All the extracts were reconstituted
with dimethylsulfoxide (DMSO) to obtain 100 mg/mL
concentrations.
Saudi Journal of Medicine & Medical Sciences | Volume 8 | Issue 3 | September-December 2020
Engiso, et al.: Bioactivity of R. albersii and C. amplifolium
Phytochemical screening
Statistical analysis
Each crude extract obtained by different solvent extractions
was separately tested using standard procedures for the
presence of various phytoconstituents, namely alkaloids,
flavonoids, saponins, terpenoids, tannins and phenolic
compounds.[23‑25]
Data were organized, edited and analyzed using SPSS
version 22 for Windows (IBM Corp., Armonk, NY, USA).
The results of the antibacterial activity were expressed as
mean ± standard error of mean. Statistical significance
was determined by a one‑way analysis of variance followed
by the Tukey post hoc test to compare the inhibition zone
against the selected bacteria between control and treatment
groups. P < 0.05 was considered statistically significant at
a 95% confidence interval.
Test organisms
Standard bacterial strains of two Gram‑positive
bacteria (S. pneumoniae ATCC 49619 and S. aureus ATCC
25923) and one Gram‑negative bacterium (E. coli ATCC
25922) were obtained from Ethiopian Public Health
Institute and used in this study. They were preserved
at −20°C until the preparation of inoculums. Each
bacterial strain was activated by streaking on culture
media aseptically. For S. aureus ATCC 25923 and E. coli
ATCC 25922, nutrient agar was used, and for S. pneumoniae
ATCC 49619, 5% sheep blood agar was used. The culture
media inoculated with S. pneumoniae was enclosed in a
candle jar to supply 5%–10% of carbon dioxide. All
the inoculated strains were incubated for 24 h at 37°C
in an incubator. Then, the inoculum of each bacterium
was prepared by taking 3–5 colonies and transferring
them to tubes containing 5 ml of normal saline. The
immersed colonies of bacteria were mixed gently to
form a homogeneous suspension until the turbidity
of the suspension became attuned to 0.5 McFarland
standards (1.5 × 108 CFU/mL).[26]
Antibacterial activity assay
A sterile cotton swab was implemented to remove
surplus suspension by gentle rotation of the swab
against the surface of the tube. It was then used to
dispense the bacteria evenly over the whole surface of the
Mueller–Hinton Agar (MHA). For S. pneumoniae, MHA
supplemented with sheep blood (5%) was used. The agar
well diffusion method, which is equivalent to Kirby–Bauer
disc‑diffusion method, was used to assess the antibacterial
effects of all extracts extracted from the study plants, as
described previously.[27] Wells of 6‑mm diameter were
formed on the inoculated agar media with a sterile cork
borer. Around 100 μL of each extract solution (100 mg/
ml) was added into each well. Ciprofloxacin 0.05 mg/disc
was used as a positive control. The solvent (DMSO) used
for the reconstitution of each extract was used as a negative
control. The plates were then incubated at 37°C for 18
h. Antibacterial activity was interpreted by measuring the
diameter of clear inhibition zones surrounding the wells
according to the standards of Clinical and Laboratory
Standards Institute, 2015.[28] Each extract was examined
in triplicate to ensure the quality and the mean value was
calculated.
RESULTS
Phytochemical screening
The qualitative phytochemical screening of the crude
methanol and chloroform leave extracts of R. albersii revealed
the presence of all the tested phytoconstituents except
saponins [Table 1]. However, only alkaloids and phenols were
detected in the acetone extract. Similarly, the methanol extract
of C. amplifolium comprised all of the tested constituents
except terpenoids, while its chloroform extract contained
flavonoids, tannins, phenols and terpenoids and the acetone
extract comprised flavonoids, tannins and saponins.
Screening of antibacterial activity
The antibacterial activities of three solvent extracts
from R. albersii and C. amplifolium against each bacterium
are tabulated in Table 2. The inhibition zones shown
by all extracts of both the plants against each bacterial
species were significantly lesser (P ≤ 0.001) than the
positive control [Table 2]. From R. albersii test groups, the
chloroform extract demonstrated a greater inhibition zone
against E. coli (21.67 mm) and S. aureus (19.67 mm). For
the acetone extract, E. coli was the most susceptible strain
with a growth inhibition of 19 mm followed by S. aureus (18
mm). There were no significant differences between
the inhibition sizes by chloroform and acetone extracts.
Overall, the methanol extract of R. albersii exhibited a
significantly lower inhibitory effect than acetone against
S. pneumoniae and E. coli (P ≤ 0.001) and than chloroform
against S. aureus (P = 0.002), S. pneumoniae (P = 0.014) and
E. coli (P ≤ 0.001).
From C. amplifolium extract groups, the largest area of
inhibition was attained by the acetone extract against
E. coli (20 mm) followed by methanol extract against S.
aureus (19.33 mm). All of its extracts showed an inhibitory
effect against S. aureus and S. pneumoniae, with no significant
differences between them. However, the methanol and
chloroform extracts had a significantly smaller zone of
growth inhibition (P ≤ 0.001) against E. coli as compared
with the acetone extract [Table 2].
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Engiso, et al.: Bioactivity of R. albersii and C. amplifolium
Table 1: Phytochemical constituent of Ritchiea albersii and Cynoglossum amplifolium
Constituents
Methanol extract
Ritchiea albersii
Chloroform extract
Acetone extract
+
+
+
−
+
−
+
+
++
−
+
+
++
−
−
−
+
−
Alkaloids
Flavonoids
Tannins
Saponins
Phenols
Terpenoids
Cynoglossum amplifolium
Methanol extract Chloroform extract Acetone extract
+
+
+
+
+
−
−
+
+
−
+
+
−
+
+
+
−
−
+: Trace amount; −: Absent; ++: High amount
Table 2: Inhibition zone diameter of extracts from leaves of Ritchiea albersii and Cynoglossum amplifolium against three
pathogenic bacteria
Test groups
Staphylococcus aureus (ATCC 25923)
MRA
CRA
ARA
MCA
CCA
ACA
PC
NC
Staphylococcus pneumoniae (ATCC 49619)
a,c
a,c,d
16.33±1.86
19.67±0.88a
18.00±1.16a
19.33±0.88a
17.33±0.88a
18.67±1.33a
30.00±0.00
−
11.33±1.67
14.00±1.00a
16.00±0.00a
14.00±1.00a
16.67±1.67a
16.33±0.88a
26.00±0.00
−
Escherichia coli (ATCC 25922)
15.00±0.58a,c,d
21.67±1.76a
19.00±0.58a
15.00±0.00a,g
14.00±0.58a,g
20.00±1.00a
32.00±0.00
−
Data are expressed as mean±SEM (n=3). aAs compared to PC; bAs compared to MRA; cAs compared to CRA; dAs compared to ARA; eAs compared
to MCA; fAs compared to CCA; gAs compared to ACA; P<0.05. The NC has shown no antibacterial activity. MRA – Methanol extract of Ritichea
albersii; CRA – Chloroform extract of Ritichea albersii; ARA – Acetone extract of Ritichea Albersii; MCA – Methanolic extract of Cynoglossum
amplifolium; CCA – Chloroform extract of Cynoglossum amplifolium; ACA – Acetone extract of Cynoglossum amplifolium; PC – Positive control;
NC – Negative control; SEM – Standard error of mean
In terms of comparison across the various extracts, it was
found that all the tested extracts were lesser effective against
S. pneumoniae than S. aureus and E. coli, except the chloroform
extract of C. amplifolium. The methanol extracts of both
the plants exhibited the same inhibition diameter (15 mm)
against E. coli, but the inhibition diameter of C. amplifolium
methanol extract was greater against S. aureus compared with
that of R. albersii (19.33 mm vs. 16.33 mm, respectively).
Against S. pneumoniae, the chloroform extract of R. albersii
and the methanol extract of C. amplifolium had the same
inhibition diameter (14 mm). Of all the extracts, the
chloroform extract of R. albersii had the highest inhibition
diameter (21.67 mm against E. coli, followed by 19.67 mm
against S. aureus). In terms of the acetone extracts, the zones
of inhibitions of both the extracts against the Gram‑positive
bacteria were almost identical but differed by about 1 mm
against the Gram‑negative bacteria.
DISCUSSION
An increase in AMR has resulted in medicinal plants gaining
importance for their therapeutic potential in producing
bioactive substances that inhibit the growth of microbes.
The preliminary results of this study, therefore, justify the
use of such plants in the complementary and alternative
medicine system against some common microbes of public
health importance as well as highlight potential sources for
developing effective antimicrobial agents in the future.[29]
204
Organic solvents were used in this study for extraction, as
these have been reported to result in higher antibacterial
activity compared with aqueous extract.[30] Acetone has been
reported to be highly effective for extraction, as it dissolves
a wide range of active compounds from plants including
both hydrophilic and hydrophobic components.[31,32] In
addition, the use of organic solvent as an extractant does
not confer any negative effect on the bioactivity against
the bacteria tested.[33]
To the best of the authors’ knowledge, this is the first
study on the antibacterial activity and phytoconstituents of
R. albersii and C. amplifolium extracts. Here, the qualitative
phytochemical analysis of the extracts of both plants
verified the existence of different secondary metabolites
[Table 1], which are well known to produce antimicrobial
effects in other plants.[34,35] Thus, the antibacterial activity
of both the plants in this study may be associated with
the availability of these chemicals that act synergistically
or individually.
Despite showing a zone of inhibition, the extracts of these
plants did not produce a significant growth inhibition as
compared with the standard control. The use of crude
extracts of plants can limit their antibacterial potency.[36]
In the current study, a single dose of the crude extract was
used, which may have resulted in a lower concentration of
the active components. Therefore, future studies should
be conducted with multiple doses of the extracts with
Saudi Journal of Medicine & Medical Sciences | Volume 8 | Issue 3 | September-December 2020
Engiso, et al.: Bioactivity of R. albersii and C. amplifolium
increasing concentrations to determine its effectiveness
compared with the standard control.
Among the extracts of R. albersii leaves, a maximum zone
of inhibition against E. coli and S. aureus was observed with
the chloroform extract [Table 2]. This may be because the
chloroform extract had a greater amount of the active
component(s) such as flavonoids and, particularly, tannins
than that in the 80% methanol extract; these components
were absent or present in an undetectable amount within
acetone extract [Table 1]. Tannins enhance the therapeutic
efficacy, as they are able to (a) bind proteins and thus
inhibit cell protein synthesis, (b) form a complex with
the microorganism membrane because of its astringent
properties and (c) deprive iron through precipitation
and/or its effect on bacterial metabolism through inhibition
of oxidative phosphorylation.[37,38] Moreover, the presence
of terpenoids in chloroform extract, which was absent
in the methanol and acetone extracts, may have directly/
indirectly enhanced its growth inhibitory effects against
the studied bacteria.
The acetone extract of R. albersii had only slightly lower
inhibition against E. coli and S. aureus strains as compared
with the chloroform extract. In the qualitative test, this
acetone extract was found to have high alkaloid contents
and phenols, which have antimicrobial properties[39] and
thus may have contributed to the effectiveness against
these strains. The comparable antibacterial effects of the
acetone and chloroform extracts against all tested bacteria
suggest that medium polar and nonpolar compounds of
R. albersii are likely responsible for its bioactivity, which
is similar to the findings of Teka et al.[40] The methanol
extract had lesser activity on most tested bacteria compared
with that of acetone and chloroform extracts, indicating
that the active components that inhibit the growth of the
studied bacteria might be dissolved better in acetone and
chloroform than in 80% methanol. Nonetheless, the zone
of inhibition by the 80% methanol extracts of both plants
against E. coli is similar to that of the same solvent extract
of P. stellatum,[18], Ceterach officinarum DC and Echinophora
tenuifolia L. subsp. sibthorpiana (Guss) Tutin.[41]
In terms of the extracts of C. amplifolium, the acetone
extract had significantly higher efficacy against E. coli than
the 80% methanol and chloroform extracts [Table 2]. The
acetone extract consisted of saponins, which was absent
in the chloroform extract and may be the differentiating
factor for this higher efficacy. This also justifies the
localization of active compounds in acetone extract, as 80%
methanol and chloroform extract exhibit lesser effect on
this microorganism. Against S. aureus and S. pneumoniae, all
crude extracts of C. amplifolium showed comparable effects.
This may describe the relativity of active phytoconstituent
composition among those extracts and/or may be because
these are Gram‑positive bacteria.
All the extracts of this study were found to inhibit growth in
all three studied bacteria. This indicates that these extracts
contain compounds with broad‑spectrum antibacterial
activity, highlighting their potential as alternatives to
antibiotics. In terms of the mechanism, the active ingredients
in the extracts may affect the overall impermeability and
integrity of the bacterial cell wall.[35,42] Flavonoids, which
are a diverse group of secondary metabolites that often
present in relatively high concentrations in plants,[43,44] have
been shown to have effective antimicrobial phytochemicals
against various disease‑causing organisms (i.e., have a wide
range of bioactivities). This biological activity is because
of their ability to form a complex with the bacterial cell
wall and with extracellular and soluble proteins. Similarly,
alkaloids and phenols, which occurred in most extracts
in this study, have been recognized to have a growth
suppression tendency against various Gram‑positive and
Gram‑negative bacteria.[45,46]
The bioactivity of all extracts against all studied bacteria
varied except for the 80% methanol extracts of both the
plants against E. coli and the chloroform extract of R.
albersii and 80% methanol extract of C. amplifolium against
S. pneumoniae [Table 2]. These dissimilarities in activity could
be linked to the disparity in solvent used for extraction
purpose,[13] in addition to phytochemical variations in
composition and/or concentration in the respective
plant extracts. The sensitivity of microorganisms to
chemotherapeutic compounds can change even in different
strains of a single bacterial species. Like this study, the
extract of various plants inhibited the growth of selected
microorganisms at different ratios in another study. The
phytochemicals and their concentrations differ across plants,
which explains the difference in antimicrobial effect.[41] On
the other hand, the extract from the same plant species has
shown variable activity against different bacterial species,
presumably because of the difference in sensitivity of the
microorganisms to specific active ingredients in a plant.[27]
It is interesting to note that S. pneumoniae is less susceptible
than S. aureus to different extracts of the respective plants
despite both microbes being Gram‑positive, thereby
suggesting that genetic variations between the two
bacteria[47] could be a factor making S. pneumoniae more
resistant to the extracts. Furthermore, this might be because
of the more complex nature of cell wall of S. pneumoniae
compared with that of S. aureus.
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Engiso, et al.: Bioactivity of R. albersii and C. amplifolium
Although the in vitro finding of this study suggests
that the extracts from R. albersii and C. amplifolium are
effective against the selected bacterial species, this may
not necessarily be the same in in vivo studies, as seen in
previous studies.[27,36] Therefore, more detailed in vitro
studies, including determination of minimum inhibitory
concentration and minimum bactericidal concentration,
and in vivo investigation of these medicinal plants should
be carried out.
6.
7.
8.
9.
10.
11.
CONCLUSIONS
The preliminary findings of this study revealed that because
the extracts of R. albersii and C. amplifolium have a wide
spectrum of activity against selected bacteria, they may
have potential beyond their current use in ethnomedicine.
However, further detailed investigation and isolation of
compounds from the extracts should be done so enable
more precise testing for the development of newer and
safer antibacterial agents. This work can be a basis for
elucidation of the actual mechanism of action of these
plants.
12.
13.
14.
15.
Peer review
This article was peer‑reviewed by five independent and
anonymous reviewers.
16.
Acknowledgments
17.
The financial support of Mizan–Tepi University is
gratefully acknowledged.
18.
Financial support and sponsorship
The research was funded by the Department of Pharmacy,
College of Medicine and Health Sciences, Mizan–Tepi
University, Research Grant no. HSR/079/06.
19.
20.
Conflicts of interest
There are no conflicts of interest.
21.
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