In vitro assessment of the synergism between
extracts of Zanthoxylum zanthoxyloides
and Zanthoxylum leprieurii and some
Marta Gonçalves1,2 Ana M Madureira3, Luís Catarino4, Ana Monteiro1 and Generosa Teixeira4,5*
1LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, 1349-017, Lisboa, Portugal.
2Adv. Inst. Nanotechnology (SAINT), Sungkyunkwan University, Suwon 16419, South Korea.
3iMed, Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisboa, Portugal.
4cE3c, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.
5DCFM, Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisboa, Portugal.
Submission: May 25, 2021; Published: June 22, 2021
*Corresponding author: Generosa Teixeira
How to cite this article:Marta G, Ana M, Madureira, Luís C, Ana M, Generosa T. In vitro Assessment of the Synergism between Extracts of Zanthoxylum
Zanthoxyloides And Zanthoxylum Ieprieurii and Some Standard Antibiotics . Glob J Pharmaceu Sci. 2021; 8(3): 555742. DOI: 10.19080/GJPPS.2021.08.555742.
Purpose: To survive in harsh environments, plants developed functional and metabolic adaptive mechanisms. One of the most relevant defense strategies is the biosynthesis of secondary metabolites, including terpenoids, alkaloids, flavonoids, and phenolics that are accumulated in cellular organelles or secretory structures. Hence, plants are recognized as a valuable source of natural products and for thousands of years very diverse herbal formulations were created to treat several diseases. Zanthoxylum zanthoxyloides and Zanthoxylum leprieurii, two Rutaceae species native to Guinea-Bissau, are well known for their ethnopharmacological relevance.
Methods: In the present study, the in vitro antimicrobial activity of these plants against human pathogens was assessed and the phytochemical profile was screened. The extracts of roots and young leaves were obtained by sequential extraction of increasing polarity (n-hexane, CH2Cl2, EtOAc, MeOH and H2O) and tested against Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined, as well as the evaluation of the synergic potential of the extracts.
Results:Z. leprieurii leaves extracts, the most apolar ones, had the highest antimicrobial activity, being able to inhibit the growth of Enterococcus hirae and all the Staphylococcus strains assayed, including the resistant ones. A synergic effect between the Zanthoxylum species extracts and standard antibiotics was found, reverting the activity of resistant strains. The phytochemical screening revealed the presence of terpenes, flavonoids, and phenolic compounds, known to have antibacterial properties.
Conclusions: The obtained results point to the validation of their use in tradition medicine and emphasize the worthwhile of additional studies of these species to better understand the compounds and mechanisms that may be valuable to restore antibacterial activity.
The use of plants as a medicine has been reported since the beginning of civilization. Diverse plant parts and extracts led to innumerous formulations created to treat a wide variety of diseases [1-3]. Nowadays, plants are still part of traditional medicine but also play a greater role on the source of new compounds that can be active by themselves or be used as lead molecules to developed new drugs.
The discovery of penicillin in the early 1940s, led to the development of several classes of compounds with antibacterial activity which allowed the reduction of mortality and morbidity caused by infectious diseases. However, the irrational and uncontrolled use of antibiotics by health professionals, animal industry and agriculture resulted in the emergence of resistant microbial populations. As a result of this strong selective pressure and increased volume of intercontinental travel, the development
and transmission of multiresistant bacteria is turning into a
serious public health problem [4,5].
According to , the mechanisms of antibiotic resistance can
be summarized in four major classes: I. Modifications of the
antibiotic molecule; II. Decreased antibiotic penetration and
efflux; III. Changes in target sites; IV. Resistance due to global
cell adaptations. The combined effects of these mechanisms
associated with rapid growth rates and the ability to exchange
genes led to the development of methicillin resistant (MRSA),
Vancomycin Intermediate Staphylococcus aureus (VISA) and
Vancomycin Resistant Enterococci (VRE). For instance, in 15
European countries more than 10% Staphylococcus aureus
infections are caused by methicillin-resistant strains (MRSA), with
several countries, namely Portugal presenting resistance rates
closer to 50%. Several highly resistant gram-negative pathogens
like Acinetobacter species, multidrug-resistant, Pseudomonas
aeruginosa, carbapenem-resistant Klebsiella species and
Escherichia coli are also emerging as significant pathogens,
transforming antibiotic resistance in one of the greatest public
health threats of the 21st century .
The occurrence of various infectious diseases and the
increasing prevalence of antibiotic-resistant pathogens have
become a serious threat to human health, and it is necessary to
find new antimicrobial agents capable of reversing antibiotic
resistance. Pharmaceutical companies, in the past few decades,
have shifted their development efforts to chronic diseases and antiviral
compounds instead of the development of new antibiotics
molecules . Several previous studies proved that there is a
huge potential of plant-derived compounds as antibacterial and
as a resistance-modifying of other antibiotics through synergistic
behavior . Therefore, in vitro antibacterial assessment of plants
extracts, their phytochemical profile screening and the evaluation
of the synergic affect when combined with antibiotics reversing
the bacteria resistance may lead to a new approach on antiinfective
The selected species for the present study are autochthonous
plants from Guinea-Bissau, a small African Portuguese speaking
country, where there is a wide range of plants used in herbal
medicine to treat endemic diseases as result of fragile health
services, easily found in local markets and pharmacies .
This country is estimated to have a vascular flora with around
1507 species, 1495 of which are native. Since 1997, to protect
biodiversity, a network of protected areas was established in
Guinea-Bissau, a collaboration between National Institute for
Biodiversity and Protected Areas and International Union for
the Conservation of Nature . Traditionally used and present
in Guinea-Bissau flora, there are two species of Zanthoxylum with
medical properties. Zanthoxylum zanthoxyloides is used to treat
malaria, sick cell anemia, tuberculosis, ulcers, hemorrhoids,
injuries, and syphilitic wounds as well as arthritic pain .
Zanthoxylum leprieurii is traditionally used in the treatment of HIV,malaria, urinary infections, rheumatic pain and used as antiseptic
. The present work aims to evaluate the in vitro antimicrobial
activity of the two Zanthoxylum species, Z. zanthoxyloides and
Z. leprieurii, against a selected panel of microorganisms, to
better understand their application in traditional medicine.
The potential synergistic effect of plant extracts with standard
antibiotics was tested as well as screening of the phytochemical
Samples of Z. zanthoxyloides and Z. leprieurii roots and young
stems were collected during 2016-2017, in Orango island, Bagos
archipelago, Guinea-Bissau and dried at room temperature.
Voucher specimens were deposited and identified at the Lisbon
University Herbarium (LISC).
The plant material was powdered, obtaining 6g of leaves
and 30g of roots of Z. zanthoxyloides and 13g of leaves and 27g
of roots of Z. leprieurii. Each powdered material was subjected
to extractions with five ascending polarity solvents: n-hexane
(n-hex), dichloromethane (CH2Cl2), ethyl acetate (AcOEt),
methanol (MeOH) and water (H2O). Ensuing the usual procedures
 each solvent extraction took about 24h, at room temperature,
with occasional shaking followed by decantation, filtration, drying
and storage at -20ºC, until use.
To assess the in vitro antimicrobial activity of each plant, extract
the model proposed by  was considered. The microorganisms
include a range of Gram-positive and Gram-negative bacteria, all
known to be the cause of several human diseases Table 1.
The minimum inhibitory concentration (MIC) values were
determined by the microplate broth microdilution method
according to CLSI (2019). Briefly, on each well of the microplate,
100 μL of the medium plus 100μL of each extract solution to be
tested were added, obtaining concentrations ranging between
500-7.5μg/mL. An inoculum of each microorganism was also
added (10μL; final concentration 104cfu/mL). Appropriated
antibiotics were used as reference for antibacterial activities.
After incubation at 37ºC for 24 h, the optical density at 630nm was
measured in a Biotek ELX 808 plate spectrophotometer to assess
the bacterial growth and confirmed by macroscopic evaluation.
The samples with MIC value ≤ 100μg/mL were determined to
have antibacterial activity. All assays were performed in triplicate.
The minimum bactericidal concentration (MBC) was evaluated
following the CLSI method  with some modifications. 10μL of
suspension from each well showing no visible growth was spread
out on a Muller-Hinton agar plate. Colony growth was observed
after 24h incubation at 37ºC. MBC corresponds to the lowest
concentration of extract that reduces almost at 100% the viability
of the bacteria.
1ATCC: American Type Culture Collection, Maryland, USA; 2CIP – l’Institut Pasteur Collection, Paris, France; 3MSSA: Methicillin-Sensitive
Staphylococcus aureus; 4MRSA: Methicillin-Resistant S. aureus; 5VISA: Vancomycin-Intermediate S. aureus.
In order to determine the type of interaction between the
extracts and the standard antibiotics (amoxicillin and oxacillin), a
checkerboard assay was performed against the MRSA strain ATCC
43866 and VISA strain CIP 106760 according to . Two-fold
serial dilutions of antibiotic was prepared on the horizontal rows
of microtiter plate and then cross- diluted vertically by two-fold
serial dilutions of the extracts. The concentration of each antibiotic
ranged from 1 to 1/2048 of the MIC and the concentration of the
compounds from 1/2 to 1/64 of the MIC.
The synergic effect was determined based on the fractional
inhibitory concentration index (FICI), calculated according to the
where, FIC(A)= MIC (A in the presence of B)/MIC (A alone)
and FIC(B)= MIC (B in the presence of A)/MIC (B alone) .
When FICI ≤ 0.5 a synergic effect is considered. FICI ranging 0.5-4
are classified as indifference and FICI values > 4 an antagonistic
effect is pondered .
A semi-quantitative phytochemical analysis to detect the
major chemical groups found in each extract was carried out
through thin layer chromatography (TLC) on silica gel plates .
Proper mixtures of eluents were used to develop and the spots
were revealed with appropriated spray-reagents made according
to . being: anisaldehyde-sulfuric acid reagent for terpenoids,Dragendorff reagent for alkaloids, natural products–polyethylene
glycol (NEU) reagent for flavonoids and Fast Blue salt reagent for
phenolic compounds. Results were displayed between absent (-)
and a strong intensity (+++).
A total of 20 extracts of two Zanthoxylum species were
prepared where the bactericidal and bacteriostatic activity
(MBC and MIC) were evaluated against an enlarged panel of
Gram positive and Gram negative sensitive and resistant bacteria
strains. The results are presented in Table 2.
The MSSA growth was inhibited by Z. zanthoxyloides n-hex root extract (MIC 60µg/mL) and by both Z. leprieurii leaves and roots AcOEt extracts (MIC 60µg/mL). Z. zanthoxyloides AcOEt leaves extract and Z. leprieurii n-hex and CH2Cl2 leaves extracts inhibit the development of the MRSA strains (MIC 60 µg/mL). The VISA strain was inhibited by Z. leprieurii CH2Cl2 leaves extract (MIC 60µg/mL). Z. leprieurii CH2Cl2 and AcOEt leaves extracts also presented activity against S. epidermidis (MIC respectively 15 and 60µg/mL). Z. zanthoxyloides leaves n- hex extract, Z. leprieurii leaves CH2Cl2 and AcOEt extracts and Z. leprieurii roots AcOEt extract exhibited inhibitory activity against E. hirae (MIC 60μg/mL). The most active plant part was the Z. leprieurii leaves and the extract presenting major activity was the CH2Cl2 extract which displayed activity against MRSA, VISA, S. epidermidis and E. hirae strains.
In order to evaluate the interactions between the extracts
and two reference antibiotics used to treat S. aureus infections
(amoxicillin and oxacillin), the checkerboard assay was performed
with two S. aureus strains one methicillin-resistant (MRSA, ATCC
43866) and vancomycin-resistant (VISA, CIP 106760). The results
for extracts with fractional inhibitory concentration index (FICI)
1 or lower are displayed in Table 4 and Table 5. FICI values under
0.5 are considered to show a synergic interaction between the
antibiotic and the plant extracts .
Analyzing the results displayed in (Table 2), the bioactive
extracts are the non-polar ones, mainly CH2Cl2 and AcOEt
extracts which are rich in terpenes and flavonoids (Table 3).
Terpenoids have antibacterial properties in nature, acting by
an unclear mechanism. They seem to disrupt the membrane of
bacteria cell . Flavonoid’s bioactivity is markedly related with
the antioxidant action. Nevertheless, several properties have
been reported as anti- inflammatory, antimicrobial, antiviral,
antiallergic and antitumor [21-23].
No antibacterial activity was found for Gram-negative bacteria
and B. subtilis. The presence of a highly hydrophobic outer
membrane that works as a permeability barrier in this group,
can explain those results . Comparing the MIC / MBC values
(Table 2), it is possible to deduce that the extracts that presented
antibacterial activity will act as bacteriostatic agents . MIC is
considered the standard parameter to determine the susceptibility
of bacteria to an external agent like a compound or extract and
corresponds to the lowest concentration of compound/extract
that inhibits the bacterial grow. MBC is considered a relevant
index of the bactericidal activity of antimicrobial agents. MBC
corresponds to the lowest concentration of extract that reduces
almost at 100% the viability of the bacteria. If the MIC and MBC
are in the same range, the extract will probably kill the bacteria.
The antimicrobial activity of Z. zanthoxyloides polar extracts (ethanol and water extracts) against resistant isolates of S. aureus was previously reported by . Similarly, the antibacterial activity of Z. leprieurii and Z. zanthoxyloides essential oils against a MSSA strain was reported by [26,27] published the results of the antibacterial activity of steam bark MeOH/H2O extract of Z. leprieurii against a MSSA strain. Those results sustained the ethnopharmacological properties described for these Zanthoxylum species, once they are mostly used for intestinal disorders and wound care.
Staphylococcus aureus strains are one of the major pathogens
worldwide related with a large range of clinic manifestations
with different levels of severity, triggering lethal infections, by
synthesizing a large variety of toxins and enzymes. S. aureus
strains developed an increasing antibiotic resistance such as a
methicillin resistance (MRSA) and a vancomycin-intermediate
resistance (VISA) . The emergence of multidrug resistant
bacteria strains and the increase of the pathogenicity of the strains
are intimately related . One approach to overcoming bacterial
resistance mechanisms and restoring antibiotic efficacy is the
use of inactive plant extracts when administered individually, in
combination with antibiotics. This combinatory strategy may play
an important role on the management of challenging infectious
In order to establish the potential interaction between the
plants extracts and references antibiotics the checkerboard assay
was performed as previously described. The extract that stood
out was the Z. zanthoxyloides roots MeOH extract, which by itself presents no relevant antibacterial activity against resistant VISA
strain (MIC= 500μg/mL) but was able to restore synergistically
the antibacterial activity of the two antibiotics tested, amoxicillin
from 60 to 4 μg/mL, (FICI = 0.18) and oxacillin from 125 to
7.5μg/mL (FICI = 0.31), corresponding in both cases to a 16-
fold reduction. Z. leprieurii leaves AcOEt was also able to interact
synergistically with oxacillin against the VISA strain, lowering
the antibiotic MIC from 125 mg/mL to 7.5μg/mL (FICI = 0.31),
corresponding to a 16-fold reduction.
For the MRSA strain (ATCC 43866) Z. leprieurii CH2Cl2 extract
was able to revert synergistically the antibacterial activity of
amoxicillin decreasing the MIC value from 60 to 4μg/mL (FICI=
0.46) corresponding to a 16-fold reduction. Z. leprieurii roots
MeOH extract also displayed some synergistic interaction with
amoxicillin against this strain, lowering the antibiotic MIC from
60 to 30μg/mL (FICI =0.5). The water extract of Z. zanthoxyloides
roots when combined with oxacillin, decreased the antibiotic
MIC value from 125μg/mL to 15μg/mL against MRSA strain
Due to the growing problematic of drug-resistant bacteria
it is urging to discover new compounds and new pathways to
control and revert antibiotic resistance, to regulate the increasing
bacterial derived diseases worldwide. The use of natural products
as plant extracts reveal to have a multi target mechanism of action
leading to a reduced prevalence of bacterial resistance. Therefore,
the study of plant compounds and their synergistic interaction is
a fundamental step in overcoming community health problems
arising from current bacterial infections.
Some of the studied extracts were active against Gram positive
strains but the results that stood out were the synergistic activity
that some of the extracts presented, being able to restore the
activity of the tested antibiotics. Overall, these results emphasize
the worthwhile of additional studies of these species to better
understand their efficacy and safety in traditional medicine and
the mechanisms behind the restore of the antibacterial activity.
O’ Neil J. Review on Antibiotic resistance. Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations. Heal Wealth Nations, 2016; December 1-16. Review Paper - Tackling a crisis for the health and wealth of nations_1.pdf.