PHARMACEUTICAL AND BIOLOGICAL EVALUATIONS
February 2016; vol. 3 (Issue 1): xxx-xxx.
www.onlinepbe.com
ISSN 2394-0859
Research Article
Comparative study of phytochemical and synergistic antibacterial activity of Tribulus terrestris (L.) and Pandiaka
heudelotii (Moq.) Hien on some clinical bacterial isolates
S. Abubakar1,2*, B. O. Akanbi3, V. A. Etim1, O. Segun2, J. C. Ogbu3
1
Biotechnology and Genetic Engineering Laboratory, Sheda Science and Technology Complex, P.M.B. 186, Garki,
Abuja, Nigeria
2
Department of Biological Sciences, University of Abuja, P.M.B 117, Nigeria
3
Department of Microbiology, University of Abuja, P.M.B. 117, Abuja, Nigeria
ABSTRACT
*For correspondence
Dr. S. Abubakar,
Biotechnology and Genetic
Engineering Laboratory, Sheda
Science and Technology
Complex (SHESTCO), P.M.B.
186, Garki, Abuja, Nigeria.
Email:
salisuabubakar99
@yahoo.com
Objective: This research work focus on synergistic effect of
ethnomedicinal plants namely, Tribulus terrestris and Pandiaka heudelotii
methanolic and aqueous leaves extracts on two species of clinical bacterial
isolates namely, Staphylococcus aureus and Escherichia coli, using disc
diffusion method.
Methods: The leaves were collected, air dried, pulverized and extracted by
maceration. The extracts were subjected to preliminary phytochemical
screening using standard procedures. The susceptibility test of individual
plant as well as the synergistic effect of combinations of the plants extract
was carried out using disc diffusion method.
Results: Collectively the plant extracts revealed the presence of alkaloids,
flavonoids, steroids, saponins, glycosides, amino acids, reducing sugars
and tannins. The in-vitro antimicrobial activity of the crude extracts was
examined against both the plants leaves extract showed antibacterial
activity against the tested microorganisms. However, some of the eluents
collected from methanolic extract fractions of column chromatography
were also showed activity on tested organisms. The minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC) of
the leaves extract were determined within the ranges of (125 to 250
mg/ml) and (250 to 500 mg/ml) respectively, the highest synergistic
activity was attained against E. coli by combined both methanolic P.
heudelotii and T. terrestris extract which showed a zone of (27.54±00).
Conclusions: The result indicates antibacterial activity of the leaves
extract of P. heudelotii in combination with that of T. terrestris against the
clinical bacterial isolates tested and laid credence to the advantage of the
combination of the two plants over the two other individual forms.
Statistically using, Microsoft excel version 2010.
Received: 19 January 2016
Accepted: 08 February 2016
Keywords: Antibacterial, Phytochemical screening, Synergistic effect, Tribulus
terrestris, Pandiaka heudelotii
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Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx.
‘maasun
kadangaree’
(lizard’s
spears).
Traditionally in Burkina Faso P. heudelottii has
being used as a tonic drink for parturient, as a
spasmolytic, in the treatment of, blennorrhoea,
annexite salpingitis, in the treatment of women
genital apparatus inflammation, in the treatment
of malaria 13. The plant is highly esteemed by
traditional healers and used in treatment of
asthma, bleeding in facilitating delivery, boils,
bronchitis, cold, cough, colic, debility, dropsy,
dog bite, dysentery, ear complications,
headache, leucoderma, pneumonia, renal
complications, scorpion bite, snake bite, and
skin disease. Traditional healers claim that
addition of this plant would enhance the efficacy
of any drug of plant origin.
Introduction
Herbal medicine represents one of the most
important fields of traditional medicine all over
the world. To promote the proper use of herbal
medicine and determine their potential as source
of new drugs, it is essential to study medicinal
plants which have folklore reputation in more
intensified way. However, over 80% of the
World’s population uses plant as their primary
source of medication.1
T. terrestris is a flowering plant in the family
Zygophyllaceae, blooms with small yellow
flowers containing only 5 petals. It grows with
multiple stems that spring out from one crown. It
has pinnate leaves made of two rows of little
leaflets which are arranged opposite each other
along the stem. The fruit appears a week after
blooming; its seeds are firm and stiff and have
two sharp spines. The common name for T.
terrestris is devil’s eyelashes, puncture vine or
cat’s head.2
The aim of this present work therefore, is to
evaluate the implication of the individual and
synergistic antibacterial activity of two
ethnomedicinal plant of Nigeria namely: - T.
terrestris and P. heudelotii towards achieving
greater potency at significantly well-defined
concentration ratios. Based on our knowledge,
little or nothing is known about the combined
activity of the Nigerian T. terrestris and P.
heudelotii or has not been documented.
T. terrestris is used in folk medicine as tonic,
aphrodisiac, analgesic, astringent, stomachic,
anti-hypertensive, diuretic, lithontriptic and
urinary anti-infective.3 The antimicrobial effects
of T. terrestris from other countries have been
reported. Turkish and Iranian spp. showed
activity against all test bacteria oral pathogens4
while the Yemeni species showed no detectable
antimicrobial activity against any of the
reference bacteria.2 The T. terrestris extract is
also used for urinary dysfunction, asthma and
opthalmia and different,5,6 and has been shown
to have antihypertensive and vasodilatory
properties.7,8 It may also protect against
oxidative stress and exhibits antitumor,
cytotoxic,
antifungal
and
antihelmentic
properties.9,10 T. terrestris is also used in folk
medicine as tonic, aphrodisiac, analgesic,
astringent,
stomachic,
anti-hypertensive,
diuretic, lithon-triptic, and urinary antiinfective.11
Materials and Methods
This research work was carried out in the Sheda
Science and Technology Complex/Microbiology
laboratory of the University of Abuja Main
Campus, Gwagwalada, Abuja, FCT, North Central Zone of Nigeria. The FCT is located at
Longitude 9 ºN and Latitude 7 ºE with a land
mass of 7, 315 sq km of which Gwagwalada
occupies 1, 043 sq km. It is situated within the
Savannah region with moderate climatic
conditions.
Collection/identification of plant materials
Fresh leaves of P. heudelotii and T. terrestris
were collected in the month of August, 2013.
The plants were further identified and
authenticated by a trained taxonomist Mr. O.
Segun of biological sciences department,
University of Abuja, Gwagwalada, Abuja,
Nigeria. The authenticated specimens were
deposited at the biological garden, University of
Abuja, Nigeria.
P. heudelotii is a long branched herb, 2-3 ft,
high; tips of calyx and bracteole often reddish.12
Belongs to the Amaranthaceae family, occurring
from Senegal to South Nigeria and across central
Africa to Sudan. Its common name in Hausa is
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Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx.
and finally allowed to stand for two weeks with
constant shaking at regular intervals at room
temperature (30±5oC). Thereafter, the percolates
were filtered using Whatman No. 1 filter paper
and solvent (methanol) were evaporated at room
temperature to obtain the methanolic extract of
the leaves, which was subsequently stored in
sterile bottles until required for use. The same
procedures was used to obtained the aqueous
extract.
Sterilizations
This was carried out by use of autoclave
(Serado, Model YX-241D), which was done at a
temperature of 1210C, pressure of 15psi for
fifteen minutes. It was used to sterilize media,
glass wares before use.
Collection and confirmatory test of the isolates
The test organisms (Staph. and E. coli) were
clinical isolates obtained from the diagnostic
laboratory of University of Abuja Teaching
Hospital, Gwagwalada, Abuja, Nigeria.The
identity and purity of the test organisms were
confirmed as follows:
Extracts sterilization and concentration
The stock solutions of the P. heudelotii and T.
terrestris extracts (both aqueous and methanolic)
were prepared in screw capped bijoux bottles.
0.5 g of each of the extract powder was weighed
on an analytical weighing balance and dissolved
in 1 ml of methanol to arrive at 500 mg/ml
concentration of stock solution. The stock
solutions were filter-sterilized using a membrane
filter (pore size 0.45 µm) (Sterlitech). Three
varied extract concentrations 500 mg/ml, 250
mg/ml and 125 mg/ml, were prepared from the
stock solutions using double dilution. The sterile
extracts were stored aseptically under
refrigeration temperature at 40C for further use.17
Staphylococcus aureus
An inoculum of the test organism was cultured
on mannitol salt agar. The growth produced
yellowish colonies. This is due to their ability to
use mannitol as food source leading to the
production of acidic byproducts of fermentation
that will lower the pH of the medium and caused
the pH indicator phenol red to turn yellow. The
colonies were further subjected to Gram staining
and were gram positive. The test organism was
inoculated on a nutrient agar slant in a bijoux
bottle and refrigerated at 4oC.14
Phytochemical screening of the extracts
The aqueous and methanolic extracts of P.
heudelotii and T. terrestris were analysed for the
presence of alkaloids, flavonoids, tannins,
steroids, saponins, glycosides, amino acids and
reducing sugars using method adopted from.18-20
Escherichia coli
The isolate obtained was plated on Eosin
methylene blue agar (EMB) by streaking from
18-24 hours. Colonies with distinctive metallic
green sheen were observed which indicate a
positive result for E. coli. The colonies were
further subjected to Gram staining and was gram
negative. The test organism was inoculated on a
nutrient agar slant in a bijoux bottle and
refrigerated at 4oC.15
Antibacterial testing of the extracts
Agar disc diffusion method modified by Khan
and Saeed was employed.21 The agar-disc
diffusion method was employed to determine the
growth inhibition abilities of the test organisms
by the extracts. Discs of about 6mm were made
from Whatman’s No. 1 filter paper using paper
puncher. The discs were transferred into bijou
bottles and sterilized in the oven at 121oC for
15mins.The sterilized discs were aseptically
placed into each of the labeled bijou bottles to
obtained a disc potency of 500 mg/disc, 250
mg/disc, and 125 mg/disc respectively of each
extracts. Mueller-Hinton agar was prepared
according to manufacturer’s guideline and 25 ml
Sample preparation and extractions
The fresh leaves were allowed to dry under
shade to constant weight and pulverized using
clean wooden mortar and pestle. The contents
were stored in air-tight containers until required
for further analysis.16 50 g of the fine powder of
the leaves of P. heudelotii and T. terrestris was
weighed and suspended in a bottle after which it
was percolated with 500 ml of 95% methanol
©Pharmaceutical and Biological Evaluations
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Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx.
each was poured into sterile Petri dishes. The
agar was allowed to solidify and dry. The agar
was aseptically inoculated uniformly with the
test organism using streak method, it was
allowed undisturbed for 30 minutes. With the
aid of sterile forceps, impregnated paper discs
containing P. heudelotii and T. terrestris extracts
at different concentrations were arranged
radially and pressed firmly onto the inoculated
agar surface to ensure even contact. Each disc
was sufficiently spaced out and kept at least
15mm from the edge of the plate and 25 mm
from disc to disc to prevent overlapping of zones
and the plates were incubated at 37oC for 24
hours. With the aid of a meter rule, the zone
diameters of inhibition of growths were
measured and recorded to the nearest
millimeter.17,22 The same procedure where
applied for the synergy and for the different
eluents collected from column chromatography.
Synergistic activity
The synergistic activity was achieved by
combining the different extracts at 1:1,T.
terrestris methanolic + P. heudelotii aqueous, P.
heudelotii aqueous + T. terrestris aqueous, P.
heudelotii methanolic + T. terrestris aqueous
and T. terrestris methanolic + P. heudelotii
methanolic, to obtained disc potency of
500mg/disc of each combinations.
Thin layer chromatography
The crude methanolic and aqueous extracts of P.
heudelotii and T .terrestris were subjected to
thin layer chromatographic analysis using
different solvent systems. The spots were
visualized using UV-lamp.
Column chromatography
The crude methanolic extract of P. heudelotii
and T. terrestris as observed in TLC plate
indicated
some
components
at
70/30
ethylacetate/n-hexane, 50/50 ethylacetate/nhexane, completely soluble methanol; hence the
need for proper separation with column
chromatography. The glass column was washed,
dried, and clamped vertically onto a retort stand;
the column was packed using the dry pack
method. A piece of cotton wool was inserted
into a clean dry glass column followed by 20 g
adsorbent silica gel of mesh size 120-150 mm
was slowly poured into the column. The column
was tapped gently to give a uniform packing. 5 g
of the crude extract was pre-absorbed with silica
gel which was allowed to dry and then loaded
into the glass column. Silica gel was again added
on top of the sample layer before addition of
solvent. The elution started with 100% nhexane, n-hexane / ethyl acetate 90:10, ethyl
acetate / n-hexane 80:20, ethyl acetate (100%),
methanol (100%). Methanol washed out the
extract completely due to the high polarity of the
sample26.
Minimum inhibitory concentration (MIC)
MIC is defined as the lowest concentration
where no visible turbidity is observed in the test
tube (bacteriostatic concentration). The broth
dilution technique was utilized where the plant
extract was prepared to the highest concentration
of 500 mg/ml (stock concentration) in sterile
distilled water and serially diluted to 250 mg/ml,
125 mg/ml, 62.5 mg/ml and 31.25 mg/ml.0.2 ml
suspension of the test organisms were
transferred into 20 ml Mueller Hinton broth and
0.2 ml of each extracts concentrations were
added and the test tubes were incubated for 24
hours at 37 ºC, the test tubes were observed for
turbidity with the aid of a spectrophotometer.
The least absorbance value was determined and
noted as the MIC value.23
Minimum bactericidal concentration (MBC)
The MBC is the lowest concentration of
antibacterial substance required to produce a
sterile culture.24 This was determined from the
broth dilution resulting from the MIC tubes by
streaking the contents of the tubes and incubated
at 37ºC for 18 hours. The lowest concentration
of the extract which showed no bacterial growth
was noted and recorded as the MBC.25
©Pharmaceutical and Biological Evaluations
Statistical analysis
Statistical analysis was carried out using
Microsoft excel version 2010. An ANOVA
(Analysis of Variance) was conducted to find the
4
Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx.
significant differences of the extracts at (P
<0.05).
Table 1 showed the physical characteristics of
the extracts. These includes, the solvent of
extraction used, initial weight of the extracts,
final weight of the extracts, percentage yield of
the extracts, colour and the texture of the
extracts observed.
Results and Discussion
Physical characteristics and percentage yield of
P. heudelotii and T. terrestris extracts.
Table 1: Physical characteristics of the extracts.
Plants
Solvents
T. terrestris
P. heudelotii
T. terrestris
P. heudelotii
Methanol
Methanol
Aqueous
Aqueous
I.W- Initial weight
I.W (G)
F.W (G)
25.0
25.0
25.0
25.0
2.7
2.7
4.8
4.2
%Yield
9.3
8.0
10.0
12.2
Colour
Texture
Dark green
Dark green
Light brown
Dark brown
gluey
gluey
Powdery
gluey
FW- Final weight
Table 2: Phytochemical constituents of T. terrestris and P. heudelotii extracts.
Phytochemical
Alkaloids
Flavonoids
Steroids
Amino acids
Saponins
Glycosides
Tannins
Reducing sugars
T. terrestris
Methanol
+
+
+
+
+
+
P. heudelotii
Methanol
+
+
+
+
+
+
Aqueous
+
+
+
+
+
Aqueous
+
+
+
+
+
-
+ = Presents; - = Absents
Table 3: Average zone of inhibition in (mm) of T. terrestris and P. heudelotii extracts.
Test organism
Extract
Staphylococcus
aureus
PA
PM
TA
TM
PA
PM
TA
TM
Escherichia coli
Extract concentrations in (mg/disc)
500mg/disc
250mg/disc
125mg/disc
10.13±01
14.51±00
14.04±04
19.23±00
14.14±00
15.53±00
14.60±01
19.88±00
8.42±00
11.23±05
12.83±01
11.41±00
10.79±00
12.85±01
10.58±03
17.94±00
NA= No Activity
©Pharmaceutical and Biological Evaluations
5
NA
9.27±03
8.27±00
NA
7.18±04
9.72±00
NA
9.72±00
Positive control
(streptomycin)
10µg/disc
22.12±00
22.12±00
22.12±00
22.12±01
21.23±00
21.23±00
21.23±00
21.23±00
Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx.
Table 2 showed the results of qualitative
phytochemical screening of both the methanolic
and aqueous extracts of T. terrestris and P.
heudelotii extracts, each solvents divulged the
present of various phytochemical constituents
while Alkaloids and steroids were present in
both the solvents used for the extractions.
zones of inhibition in millimeter using disk
diffusion method (mean±SD) of the triplicate.
Table 6: Activity of the different fractions of
methanolic T. terrestris on S. aureus and
E. coli.
Table 4: Synergistic average zone of
inhibition in (mm) of T. terrestris and
P. heudelotii extracts.
Test
organis
ms
E. coli
S.
aureus
Negativ
e
control
(DMSO
)
Concentration (500 mg/disc)
TM/PA PA/TA PM/TM PM/TA
19.53±
00
20.55±
00
NA
19.65±
01
18.00±
01
NA
27.54±
00
25.50±
03
NA
7:3 hex : ethyl
8:2 ethyl : hex
9:1 ethyl : hex
100% methanol
+
_
+
_
+
+
+
+
Table 1 showed the total percentage (%) yield of
the two solvent used for the extraction namely;
methanol and aqueous, both the aqueous and
methanolic extract yield (10.0 g and 9.3 g) and
(12.2 g and 8.0 g) for T. terrestris and P.
heudelotii respectively, from the original weight
of 25 g. The qualitative phytochemical analysis
of the T. terrestris and P. heudelotii leaf extracts
in Table 2. The result showed that the aqueous
and methanolic leaf extracts of T. terrestris
contains alkaloids, steroids, saponins and
reducing sugars with the addition of flavonoids
and amino acid in methanolic extract while
glycosides was only present in aqueous extract.
The methanolic and aqueous leaf extract of P.
heudelotii contains alkaloids, steroids, amino
acids, and reducing sugars in addition flavonoids
and tannins were present in methanolic extract
while glycoside was present only on aqueous
extract.
E. coli.
_
_
_
+
+
hex: n-hexane; ethyl: ethylacetate
Table 3 and 4 showed the susceptibility test of
individual extract of P. heudelotii and T.
terrestris as well as the combinations (synergy)
of the two plants extract on the two clinical
isolates used S. aureus and E. coli at various
concentrations. The extract showed broad
spectrum action on both gram negative and gram
positive
bacteria,
based
on
different
concentrations of the extracts employed with
©Pharmaceutical and Biological Evaluations
E. coli
_
Table 5 and 6 showed the susceptibility of the
tested clinical isolates on different fractions of
methanolic extracts for both P. heudelotii and T.
terrestris.
Table 5: Activity of the different fractions of
methanolic P. heudelotii on S. aureus and
E. coli.
S. aureus
_
+
_
+
_
S. aureus
_
hex: n-hexane; ethyl: ethylacetate
18.21±
03
20.54±
00
NA
PA: P. heudelotii (aqueous). PM: P. heudelotii (methanol).
TA: T. terrestris (aqueous). TM: T. terrestris (methanol);
DMSO- Dimethylsulfoxide ; NA- No Activity
Fractions
100% hex
7:3 hex : ethyl
8:2 ethyl : hex
9:1 ethyl : hex
100%
methanol
Fractions
100% hex
The two extracts showed the presence of most of
the secondary metabolites such as: - Tannins,
steroids, saponins, alkaloids, amino acid,
flavonoids and reducing sugar. Alkaloid
contributes to a plant species fitness of
survival.27 They often have pharmacological
effects and are used as medication and
recreational drugs.28 Similarly, tannins are well
6
Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx.
known for their antioxidant and antimicrobial
properties as well as skin regeneration, antiinflammatory and diuretic properties.29,30
Flavonoids are widely recognized for exerting
antioxidant, antimicrobial, anti-carcinogenic and
antitumor properties.29,30 Many pharmacological
activities such as antibiotic, antifungal, antiviral,
hepato protective, and anti-inflammatory and
ant-ulcer activities have been reported for
saponins31 and steroids have been reported to
exert analgesic properties.32
extract on both the test isolates were unable to
determine within the prepared concentrations.
The antimicrobial activity demonstrated by the
combination of the two extracts is probably due
to synergizing effect of the phytochemical result
obtained. The bioactivity was prominent against
E. coli at 500 mg/disc by PM/TM combination
with 27.54±00 mm zone of inhibition. This
clearly exhibits the advantage of the
combination of the two plants over the two other
individual forms coupled with enhanced
synergistic activity.
The antimicrobial susceptibility pattern of the
crude extracts showed in table 3 the results
shows that the methanolic and aqueous extracts
of both T. terrestris and P. heudelotii shows
activity on both the test organisms, which
signified the broad action of the extracts. The
methanolic extracts of both the samples shows
high zone of inhibition ranges between
(19.23±0, 14.51±00) on S. aureus and
(19.88±00, 15.53±00) at 500 mg/disc each
respectively. The aqueous extracts also illustrate
zone within the range of (10.13±01 to14.04±04)
on both the test isolates at 500 mg/disc.
In similar study that involves the combination of
two different extracts A. comosus and Allium
sativum against S. typhi, the activity of each
extract was significantly different from their
combination and this shows an interaction
between the two plants extracts.32 Soleimanpour4
evaluated the antimicrobial activities of an
ethanol extract of T. terrestris alone and in
combination with Capsella bursa-pastoris and
Glycyrrhiza glabra against six pathogens
namely Streptococcus mutans, Streptococcus
sanguis, Actinomyces viscosus, Enterococcus
faecalis S. aureus, and E. coli and the results
shown that mixed extracts were more effective
against all bacteria than any of the cases alone
that indicates the synergistic effect between
these three extracts.
The MIC and MBC on table 7 indicates that both
the methanolic and aqueous extract the MIC
where determined within the ranges of 125
mg/ml to 250 mg/ml while the MBC where
determined within the ranges of 250 mg/ml to
500 mg/ml. The MBC of P. Heudelotii aqueous
Table 7: Minimum inhibitory/Bactericidal Concentration of P. heudelotii and T. terrestris extracts
on S. aureus and E. coli.
Test organism
Extracts 500 mg/ml
250 mg/ml
125 mg/ml
62.5 mg/ml
Staphylococcus
aureus
PA
PM
TA
TM
PA
PM
TA
TM
MIC
MBC
MIC
MIC
MIC
MBC
+
MIC
+
MIC
MIC
+
+
MIC
++
++
++
++
++
+
++
+
Escherichia coli
ND
MBC
MBC
ND
MBC
MBC
-
ND= MBC Not determined at prepared Concentrations, += Mild turbidity, ++=Strong turbidity
©Pharmaceutical and Biological Evaluations
7
31.25
mg/ml
++
++
++
++
++
++
++
++
Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx.
part of Nara desert, Pakistan. Pak J Bot.
2010;42(2):839-51.
7. Phillips, S.J., R.P. Anderson and R.E.
Schapire. Maximum entropy modeling of
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8. Sharifi, A.M., R. Darabi and N. Akbarloo.
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Tribulus terrestris in 2K1C hypertensive
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iaka+heudelotii
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pratiques medicinales du Burkina Faso: cas
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Conclusions
Based on the result of this research, there is clear
evidence that the two plants can be a good
source of bioactive substances that could possess
broad spectrum activity. Therefore, there is an
increasing need for researchers to investigate the
synergistic capacity of plants. The results of the
present research seem to be promising and may
enhance the natural product uses, showing the
potentiality of P. heudelotii and T. terrestris in
the treatment of various infectious diseases
caused by bacteria. It also indicates the need for
understanding of synergism mechanism is
fundamental to development of pharmacological
agents to treat diseases causes by various
bacteria using medicinal plants.
Funding:
No funding sources
Conflict of interest: None declared
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