ISSN: 2320-5407 Int. J. Adv. Res.

12(12), 409-418

Journal Homepage: -www.journalijar.com

Article DOI:10.21474/IJAR01/20038
DOI URL: http://dx.doi.org/10.21474/IJAR01/20038

RESEARCH ARTICLE
COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF ROOTS, BARKS AND LEAVES
OF JATROPHA MULTIFIDA, JATROPHA CURCAS AND ZANTHOXYLUM ZANTHOXYLOIDES

Djaboutou Kafuyemon Ismanth1,2, Lauris Fah1,2, Phénix Assogba1, Eric Agbodjento1, Esther Deguenon1,
Edna Hounsa1, Hans Ohouko1, Hornel Koudokpon1, Boris Legba1, Arnaud Soha1, Jean Robert Klotoe1 and
Victorien Dougnon1
1. Research Unit in Applied Microbiology and Pharmacology of Natural Substances, Laboratory Research in
Applied Biology, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, Benin.
2. Department of Continuing Education, National Medical and Health Institute, University of Abomey-Calavi,
Benin.
……………………………………………………………………………………………………....
Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History Antimicrobial resistance is a major public health issue. This study
Received: 10 October 2024 compares the antibacterial activity of leaves, bark, and roots of
Final Accepted: 14 November 2024 Jatropha curcas, Jatropha multifida, and Zanthoxylum zanthoxyloides.
Published: December 2024 Extracts (water, ethanolic, and hydro-ethanolic) were tested against
Staphylococcus aureus and Klebsiella pneumoniae, and their
Key words:-
Jatropha Multifida, Jatropha Curcas, antioxidant activity was assessed using the DPPH test. Polyphenol and
Zanthoxylum Zanthoxyloides, Anti- flavonoid levels were quantified spectrophotometrically.Hydro-
Bacterial Activity ethanolic extracts of J. multifida bark and Z. zanthoxyloides leaves
showed strong activity against K. pneumoniae (MIC, MBC: 6.25mg for
J. curcas, J. multifida; 3.12mg for Z. zanthoxyloides). Against S.
aureus, the best were J. curcas, J.multifida (3.12mg), and Z.
zanthoxyloides(1.5mg). Leaves and bark were richer in flavonoids and
polyphenols (p=0.001) and exhibited superior antioxidant activity.
Using leaves and bark over roots enhances sustainability.

Copyright, IJAR, 2024,. All rights reserved.

……………………………………………………………………………………………………....
Introduction:-
Bacterial resistance to antibiotics is one of the most important challenges for anti-infective treatments for healthcare
professionals around the world and for the pharmaceutical industry(Onzo et al., 2016). The inappropriate and often
exaggerated use of antibiotics, particularly in human and animal health, is at the origin of an evolutionary selection
pressure on microbial populations (Bihari et al., 2011). The latter results in the growth and spread of resistant and
multidrug-resistant strains, leading to increasingly common treatment failure (Bihari et al., 2011).In order to
mitigate this phenomenon, a lot of scientific research is directed towards the identification of new antimicrobial
agents of natural origin such as bioactive molecules of plants (Monciardini et al., 2014). African flora is known for
its richness, which is the basis for frequent exploitation with the aim of developing therapeutic recipes(Gurib-Fakim
et al., 2010; Veilleux et al., 1996). Akoègninou et al.(2006) estimated the Beninese flora at more than 2807 plant
species. This flora consists of several medicinal plants with antimicrobial properties that are used in the treatment of
infections (Djego et al., 2012; Dougnon et al., 2017; Koudokpon et al., 2017). The roots of some medicinal essences
are widely used in recipes to treat various infections (Arekemase et al., 2011).Jatropha curcas, Zanthoxylum
Zanthoxyloidesand Jatropha multifida, are among these plants whose roots are commonly used in Benin in the
treatment of several microbial infections(Dougnon et al., 2017). These plants have the potential to act on several

Corresponding Author:-Victorien Dougnon 409

Address:-Research Unit in Applied Microbiology and Pharmacology of Natural Substances,
Laboratory Research in Applied Biology, Polytechnic School of Abomey-Calavi, University
of Abomey-Calavi, Benin.
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

bacterial strains such as Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus
epidermidis, Staphylococcus saprophyticus, Escherichia coli, Klebsiella pneumoniae, Shigella dysenteriae,
Salmonella Typhimurium and Proteus mirabilis(Aiyelaagbe et al., 2007; Arekemase et al., 2011; Ynalvez et al.,
2012). The biological properties of these plants are much more attributed to the roots than to the other organs (leaves
and bark) of these plant species, which then proves the frequent use of these roots in traditional medicine(Agbulu et
al., 2015; Aiyelaagbe et al., 2008; Arekemase et al., 2011). However, the frequent use of the roots of these plants
can contribute to their rarity or even the total extinction of these species. In addition, other plant organs (leaves and
roots) can have biological properties similar to those of their roots. But very few studies have been done on the
antibacterial activity of extracts from the renewable organs (leaves and bark) of these plants(Agban et al., 2020;
Sharma et al., 2012; Tine et al., 2020). In addition, the mechanism of action of the antibacterial effect of extracts of
these three plants has not been specified in these studies. The same applies to the resistance profile of the bacterial
strains used. It is therefore wise to determine the biological properties as well as the mechanism of action of the
renewable organs of these plants in order to limit the use of roots in traditional medicinal recipes. This is why our
study aims to promote the use of the renewable organs of these three plants of the Beninese flora.

Methods:-
Collection of plant material and preparation of extracts
The organs (leaves, roots and fresh bark) were dried in the laboratory at a temperature varying between 16°C and
22°C. After drying, they were crushed in the mill. The powders were then used for aqueous and ethanolic
extractions of each plant species by the methodology described by Fah et al.(2015).Fifty (50g) grams of powder
from each plant part were macerated in 500 ml of solvent (water and ethanol). The mixtures were left for 72 hours
under continuous stirring at room temperature. The homogenate obtained was filtered three times on hydrophilic
cotton and once on Wattman No. 1 paper. The filtrate obtained was evaporated at a temperature of 40°C in an oven
to a dry mass which is the extract. The extract obtained was stored in the refrigerator at 4°C. The hydro-ethanolic
extraction was carried out based on the methodology used by Klotoé et al.(2020). Briefly modified, 50g of powder
was macerated in 500 ml of the 50% v/v ethanol and water mixture. After 72 hours of stirring at room temperature,
the homogenate obtained was filtered three times on cotton wool and once on Wattman paper.

Determination of total polyphenols and flavonoids

The amount of polyphenols and flavonoids contained in the extracts was determined by quantitative phytochemical
analysis following the methodology applied byKlotoé et al.(2020).

Total phenols were determined using Folin Ciocalteu reagent (FCR). 50 μL of the extract was mixed with 250 μL of
the RCF (10 times diluted in distilled water) and 750 μL of an aqueous solution of sodium carbonate Na 2CO3
(7.5%). After 8 min of incubation, 950 μL of distilled water was added and mixed with the vortex and incubated for
2 hours. Optical densities (ODs) were read at 760 nm using a spectrophotometer. The reading was taken against a
blank composed of a mixture of 250 μL of FCR, 750 μL of Na2CO3 and 1 ml of distilled water. The total
polyphenol content in the different extracts was calculated from a linear calibration curve (y = ax + b), established
with precise gallic acid concentrations as the reference standard (0-200 μg/ml). Total phenolic content was
determined as mg gallic acid equivalent/g extract (mg GAE/g) using the formula below:
TFC = (X xV) / m

With: TFC is a total phenolic content, X is the concentration of gallic acid in mg/ml; V is the extraction volume used
in ml and m is the weight of the extract in grams.

Flavonoid content was measured using aluminum trichloride (AlCl3) as a reagent. 500 μl AlCl3 (2%), 500 μl of the
extract and 3 ml of methanol were mixed. The white was composed of 500 μl of AlCl3 and 3.5 ml of methanol. The
reading was made with a spectrophotometer at 415 nm after 10 min of incubation. Samples were prepared three
times for each assay and average values were taken. The amounts of flavonoids in the extracts were calculated from
the calibration curve of a standard flavonoid (Rutin) as the reference molecule (0-1 mg/ml). The total flavonoid
content was determined using the formula below:
TFC = (X xV) / m

With: TFC is the content of total flavonoids, X is the concentration of rutin in mg/ml; V is the extraction volume
used in ml and m is the weight of the extract in grams.

410
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

Antioxidant activity of plant extracts studied by DPPH test

The method adopted in this study is that of Klotoé et al.(2020)that 100 μL volume of different concentrations of
each extract was added to 1900 μL of the ethanolic solution of DPPH (0.4 mg/mL). The white was prepared by
mixing 100 μl of the extraction solvent with 1900 μl of the DPPH solution. After incubation in the dark for 1 hour at
room temperature, absorbance readings were performed at 517 nm using a spectrophotometer. These measured
absorbances were used to calculate the percentage of DPPH radical scavenging that is proportional to the antioxidant
potency of the sample. Vitamin C was used as the gold standard. Antioxidant activity is expressed as the percentage
of trapping determined by the formula:
P = (Ab- Ae) / Ab x 100
With P: Percentage of trapping; Ab: absorption of white; Ae: Absorption of the sample.

Determination of the antimicrobial activity of Jatropha curcas, Jatropha multifidaand Zanthoxylum

zanthoxyloides extracts
Characteristics of the selected bacterial strains
For the evaluation of antibacterial properties, the bacterial strains used came from the strain collection of the
Research Unit in Applied Microbiology and Pharmacology of Natural Substances (U.R.M.A.Pha). The criterion for
choosing Strains was based on species. The bacterial species chosen are the most predominant in cases of clinical
infection(Agbulu et al., 2015; Arekemase et al., 2011; Igbinosa et al., 2009).
1. Staphylococcus aureus: Golden-yellow colonies, characteristic of Staphylococcus aureus, from the
transplanting of isolates on Chapman medium were selected. Then the Gram staining of these colonies was
carried out followed by their identification using catalase, freestaphylocoagulase and D-nase.
2. Klebsiella pneumoniae: the brownish colonies, mucous membranes characteristic of the Klebsiella from the
transplanting of isolates on EMB medium were selected. Then the Gram staining of these colonies was carried
out followed by their identifications thanks to the Leminor gallery.

Control of the resistance profile of selected strains

The control of the resistance profile of the selected strains was done according to the method described by the
Antibiogram Committee of the French Society of Microbiology, (2020) (Table 1).

Determination of antimicrobial activity in liquid and solid media

It was performed in microplates from 96 wells following the method described by Agbankpe et al.(2016). A stock
solution sample was prepared at a concentration of 100mg/ml in distilled water. In fact, 100 μl of MH broth and 100
μl of the stock solution of each extract concentrated at 100 mg/ml were deposited in the 1st well. After
homogenization, 200 μl of a 50mg/ml extract solution was obtained. A volume of 100 μl of this new solution was
then taken and mixed with the MH broth contained in the 2nd well and this series of dilutions was carried out up to
the 10th well. Then 100 μL of the bacterial suspension was added to each well. The positive (100μL Mueller Hinton
Broth + 100μL bacterial suspension) and negative (100μL Mueller Hinton Broth + 100μL test extract stock solution)
controls were taken in the 11th and 12th wells, respectively. The microplates were covered with parafilm and then
placed for 18 hours in the oven at 37°C. Bacterial growth was revealed by tetrazolium.

Determination of the Minimum Bactericidal Concentration

To determine the Minimum Bactericidal Concentration, all IJC wells with higher concentrations were inoculated on
HD agar and incubated at 37°C for 18 to 24 hours. This made it possible to assess the minimum bactericidal
concentration, which corresponded to the lowest concentration of extract that did not reveal bacterial colonies. The
antibiotic potency (Pa) of each extract was then calculated with the MBC/MIC formula. The antibacterial effect or
potency is judged to be bactericidal or bacteriostatic on the basis of Pa = MBC/MIC. If 1 ≤ Pa ≤ 2, the effect is
bactericidal and if 4 ≤ Pa ≥ 16, the effect is bacteriostatic.

Mode of action of the antibacterial effect plant extracts

The mode of action of the antibacterial effect was evaluated for the extracts that were Improved antibacterial activity
on the bacterial strains tested. Thus, the external membrane permeability test was performed. It was determined
according to the method described by Dougnon et al. (2021), with some modifications. In a 96-well microplate,
concentrations of MIC and 2MIC of each extract were prepared as a triplicate by dilution in series of four. One
hundred microliters (100 μl) of suspension of each strain were added and the plate was incubated at 37 C ̊ for 24
hours. The optical densities were read at 405 nm and the percent destabilization was calculated using the formula
below:

411
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

%D = [(AB – AE)  Ao] x 100

With %D: Percentage of destabilization; AB: Absorbance of White; AE: absorbance of the samples tested.

Imipenem was used as a positive control and was prepared equally at the MIC (0.0781 μg/mL) and 2MIC (0.1562
μg/mL). The HD broth and bacterial suspension constituted the negative control while the sterility control contained
the HD broth alone. The wells containing the MH broth and the extracts alone made up the white. A graph of the
%D in relation to the concentration of the extract was plotted in order to determine the concentration that causes
maximum destabilization of the bacterial membrane.

Data processing and analysis

Data were collected and saved in the Excel 2019 spreadsheet and graphs were made with Graph Pad Prism 7
software. Descriptive statistics were produced using SPSS 20 software. The ANOVA test was used for the
comparison of the means. Comparisons were made at the 5% significance level.

Results and Discussion:-

Results:-
Determination of total polyphenols and flavonoids
Total polyphenol content
The comparison of the different types of extracts according to the organs of Jatropha curcas is presented in Table 2.
This table shows that for the aqueous extract, the leaves and bark are significantly richer in polyphenols than the
roots (p < 0.05). For the ethanolic extract, the leaves and bark are significantly richer in polyphenols than the roots
(p < 0.05). In addition, for the hydro-ethanolic extract, the leaves and bark are significantly richer in polyphenols
than the roots (p < 0.05). Therefore, the extracts obtained from the leaves and bark showed a better polyphenol
content compared to the roots.

Table 3 shows the comparison of the different types of extracts according to the organs of Jatropha multifida. From
this table it appears that the aqueous extract of the bark showed a richness in polyphenols compared to the leaves (p
< 0.05). Also, with regard to the hydro-ethanolic extract, the bark exhibited a significantly better content than the
leaves (p < 0.05). While there is no significant difference between the polyphenol content of the ethanolic extract of
the different organs. Hence the best contents were obtained for the bark extracts while the low contents were noted
for the leaves.

Table 4 shows the comparison of the different types of extracts according to the organs of Zanthoxylum
zanthoxyloides. From the analysis of this table it can be seen that there is no significant difference between the
polyphenol content of the aqueous extract of the different organs. For the bark, the ethanolic extract was rich in
polyphenols compared to the roots. While for the hydro-ethanolic extract, the leaves and bark showed a significantly
better polyphenol content compared to the roots (p < 0.05). Generally speaking, all extracts of extracts from roots
are less rich in polyphenols than those of other organs.

Total flavonoid content

The comparison of the different types of extracts according to the organs of Jatropha curcas is presented in Table 5.

This table shows that for the aqueous extract, the leaves were significantly richer in flavonoids compared to the bark
(p< 0.05) and the roots (p< 0.05). Similarly,to the hydro-ethanolic extract, the leaves were richer in flavonoid than
the bark and roots. While for the ethanolic extract, the bark was richer in flavonoids compared to the leaves (p <
0.05). In general, the extracts obtained from the leaves and bark were richer in flavonoids than those from the roots.

Table 6 shows the comparison of the different types of extracts according to the organs of Jatropha multifida. From
this table it can be seen that for the aqueous extracts, the bark followed by those of the leaves showed a significantly
high flavonoid richness compared to the roots (p < 0.05). While for the ethanolic extract, the leaves are significantly
richer in flavonoids than the bark and roots (p < 0.05).

The same is true for the hydro-ethanol extract. In short, for this plant, the extracts of the bark followed by those of
the leaves are richer in flavonoids than those of the roots.

412
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

Table 7 illustrates the comparison of the different types of extracts according to the organs of Zanthoxylum
zanthoxyloides. From the analysis of this table it appears that for the aqueous extract, the leaves are richer in
flavonoids than the other plant organs (p < 0.05). Similarly, for the ethanolic extract, the leaves are richer in
flavonoids than the other plant organs (p < 0.05). In addition, for the hydro-ethanolic extract, the leaves showed a
significantly better flavonoid richness than the bark (p < 0.05) and the roots (p < 0.05). Thus, extracts from the
leaves of this plant are particularly richer in flavonoids than other organs.

Antioxidant activity
All the extracts tested reduced the DPPH radical to varying proportions. This inhibition of the DPPH radical showed
that the antioxidant activity of the different extracts was proportional to the increase in the concentration of the
extracts. The comparison of the different types of extracts according to the organs of Jatropha curcas is presented in
Table 8. This table shows that for the aqueous extract, the roots significantly inhibited the DPPH radical compared
to the leaves and bark (p < 0.05). On the other hand, for the ethanolic extract, the bark significantly inhibited the
DPPH radical compared to the leaves (p < 0.05) and roots (p < 0.05). Concerning the hydro-ethanolic extract, the
leaves significantly inhibited the DPPH radical compared to the roots (p < 0.05). In short, with the exception of the
aqueous extract, all the other extracts (hydro-ethanolic and ethanolic) of the leaves and bark were more antioxidant
than those of the roots.

Table 9 provides information on the comparison of the different types of extracts according to the organs of
Jatropha multifida. This table shows that for the aqueous extract, the bark significantly inhibited the DPPH radical
compared to the leaves (p < 0.05) and the roots (p < 0.05). Similarly, for the ethanolic extract, the bark inhibited the
DPPH radical compared to the leaves (p < 0.05). The same is true for the hydro-ethanol extract. From all of the
above, it can be said that the extracts of the bark exhibited the best inhibitory powers of the DPPH radical.

The comparison of the different types of extracts according to the organs of Zanthoxylum zanthoxyloides is
presented in Table 10. From the analysis in this table, we can see that for the aqueous extract, the roots showed a
better inhibitory potential of the DPPH radical compared to the leaves (p < 0.05) and the bark (p < 0.05). On the
other hand, for the ethanolic extract, the leaves showed a better inhibitory potential of the DPPH radical compared to
the roots (p < 0.05). The same observation is made for the hydro-ethanolic extract. Finally, apart from the aqueous
extract, the leaves followed by the bark showed a better inhibitory power of the DPPH radical for the other extracts
(ethanolic and hydro-ethanolic)

Effect of extracts from different plant organs on bacterial strains

Determination of the antibiotic power of plant extracts
Table 11 shows the results of the MIC, MBC and antibiotic potency of extracts of J. curcas, J. multifidaand Z.
zanthoxyloidestested on S. aureus strain ATCC 25923. From this table it appears that the aqueous extract of the
leaves of J. curcashad a lower Minimum Inhibitory Concentration (MIC) than that of the bark at the level of the S.
aureus strain ATCC 25923. In contrast, the ethanolic extract of the roots showed a smaller MIC than that of the bark
and leaves. Similarly, the hydro-ethanolic extract of the roots showed a smaller MIC than that of the leaves and
bark. The aqueous extracts of the leaves and bark showed the same bacteriostatic powers on S. aureus strain ATCC
25923. On the other hand, the hydro-ethanolic extract of the roots showed a lower bactericidal power than that of the
bark on the same strain. As for J. multifida, the aqueous extract of the bark showed a lower Minimum Inhibitory
Concentration than that of the roots and leaves for S. aureus strain ATCC 25923. Similarly, the hydro-ethanolic
extract of the bark showed a lower Minimum Inhibitory Concentration than that of the roots and leaves at the level
of S. aureus strain ATCC 25923. Ethanolic extracts from the bark and roots showed a lower Minimum Inhibitory
Concentration than that of the leaf at the level of the S. aureus strain. The extracts (aqueous, ethanolic and
hydroethanolic) of the bark and roots showed the same bacteriostatic powers on S. aureus strain ATCC 25923. On
the other hand, only the aqueous extract of the leaves showed bactericidal potential (MBC/MIC ratio equal to 2) on
this same strain. With regard to Z. zanthoxyloides, the aqueous extract of the leaves showed a lower Minimum
Inhibitory Concentration than that of the roots on S. aureus ATCC 25923. Similarly, hydro-ethanolic extracts from
the leaves and bark showed smaller MICs than those from the roots. In contrast, the ethanolic extract of the roots
had a smaller MIC than that of the bark at the level of S. aureus strain ATCC 25923. Ethanolic extracts from the
bark and roots showed the same bacteriostatic properties on S. aureus strain ATCC 25923.On the other hand, only
the aqueous extract of the leaves showed bactericidal potential (MBC/MIC ratio equal to 1) on this same strain.

413
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

Table 12 shows the results of the MIC, MBC and antibiotic potency of Jatropha curcas, J. multifidaand
Zanthoxylum zanthoxyloidesextractstested on the K. pneumoniae strain. For J. curcas, the strain of K. pneumoniae,
the aqueous extract of the bark had a MIC of 25. The ethanolic extract of the leaves showed a smaller MIC than that
of the bark. Hydro-ethanolic extracts from the bark and roots, on the other hand, showed lower MICs than those of
the leaves.

All the extracts (aqueous, ethanolic and hydro-ethanolic) from the bark as well as the ethanolic and hydro-ethanolic
extracts from the leaves showed the same bacteriostatic powers; the same for the hydro-ethanolic extract of the
roots. Tan disk for J. multifida, the aqueous extract of the leaves showed a smaller MIC than that of the bark and
roots on K. pneumoniae. Hydro-ethanolic extracts of the leaves and bark showed a smaller MIC than that of the root.
In contrast, the ethanolic extract of the bark had a smaller MIC than that of the leaf. All extracts (aqueous, ethanolic
and hydroethanolic) from the bark and leaf showed the same bacteriostatic properties, while only the hydro-
ethanolic extract of the roots showed bactericidal potential (MBC/MIC ratio equal to 1). For Z. zanthoxyloides, the
aqueous extract of the roots showed a smaller MIC than that of the bark on K. pneumoniae. In contrast, the ethanolic
extract of the bark showed a smaller MIC than that of the roots and leaves. The hydro-ethanolic extract of the leaves
showed a smaller MIC than that of the bark and roots. All extracts (ethanolic and hydroethanolic) from the leaves,
bark and roots showed the same bacteriostatic properties (MBC/MIC ratio equal to 1).

Determination of the percentage of destabilization of extracts on the membrane of the stumps

The ability of plant extracts to penetrate the membrane of resistant bacterial strains was evaluated for 24 hours at
two different concentrations (MIC and 2MIC). It appears from the figures below that the extracts showed variable
capacities of variable destabilization depending on the strain tested. Figures 1 and 2 illustrate the destabilization
rates of the active plant extracts on the membrane of the different bacterial strains used.

Figure 1 shows that the destabilization varies with the concentration of extracts (aqueous and ethanolic) from the
leaves and roots on the membrane the S. aureus strain ATCC 25923. The percentage of destabilization of aqueous
and ethanolic extracts of leaves and roots at the IJC was the same with the reference molecule (imipenem). For
2MIC, membrane destabilization of S. aureus ATCC 25923 by leaf and root extracts was better compared to
imipenem. On the other hand, hydro-ethanolic extracts from the leaves and roots showed a percentage of
destabilization higher than the reference molecule (imipenem) at the MIC. The best percentages of destabilization
were noted with aqueous extracts from the leaves (MIC: 64.11 ± 0.11%; 2MIC: 81.11 ± 0.02%); Ethanol (MIC:
64.11 ± 0.08%; 2MIC: 81.11 ± 0.57%) and hydro-ethanolic (MIC: 91.22 ± 0.12%; 2MIC: 88.62 ± 0.12%) of the root
of J. multifidaon the membrane of S. aureus ATCC

Figure 2 illustrates the membrane destabilization capabilities of extracts (aqueous, ethanolic and hydroethanolic)
from leaves and roots on the K. pneumoniae strain. It varies according to the concentrations of extracts used. The
percentage of destabilization of the ethanolic and hydro-ethanolic extracts of the J. multifida leaf at the IJC is higher
than that of the root and reference molecule (imipenem) extracts. As for 2MIC, the destabilization of the K.
pneumoniae membrane by leaf and root extracts was better compared to imipenem. The best percentages of
destabilization at MIC and 2MIC concentrations were noted with aqueous extracts (MIC: 50.02 ± 0.07%; 2MIC:
62.20 ± 3.25%); ethanolic (MIC: 50.02 ± 0.03%; 2MIC: 62.20 ± 0.04%) and hydro-ethanolic (MIC: 71.88 ± 0.04%;
2MIC: 79.57 ± 1.35%) extracts from J. multifidaleaveson the membrane of the K. pneumoniae strain.

Discussion:-
The present study aimed to compare the antibacterial activity of aqueous, ethanolic and hydro-ethanolic extracts
from the organs (roots, leaves and bark) of Jatropha curcas, J. multifidaand Zanthoxylum zanthoxyloides. These
plants are species whose roots are traditionally used in Benin in the treatment of infections. Antibacterial activity
was assessed on S. aureus strains ATCC 25923 and K. pneumoniae. The present study shows that aqueous, ethanolic
and hydro-ethanolic extracts from plant organs showed variable yields at extraction. This variation in yields can be
explained by the solvents used and the extraction capacity of each solvent (Akinmoladun et al., 2022).

Determination of the content of polyphenols and total flavonoids on plant extracts. Indeed, the extracts (aqueous,
ethanolic and hydro-ethanolic) obtained from the leaves and bark of Jatropha curcas are richer in flavonoids than
those from the roots. Extracts from the bark of Jatropha multifida followed by those from the leaves are
significantly richer in flavonoids than those from the roots. As for Zanthoxylum zanthoxyloides, the extracts of the
leaves are particularly richer in flavonoids than the other organs. The polyphenol contents of the aqueous, ethanolic

414
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

and hydro-ethanolic extracts, leaves and bark of J. curcasare better. For J. multifidathe best concentrations were
obtained for the bark extracts while the low concentrations were noted for the leaves. Concerning Z. zanthoxyloides,
all extracts from the roots are less rich in polyphenols than those of the other organs. The presence of
phytochemicals such as saponin, steroids, tannins, glycosides, alkaloids, polyphenols and flavonoids in extracts from
the leaves and bark of J. curcas, J. multifida and Z. zanthoxyloideshas been reported in several studies (Carvalho et
al., 2018; El Diwani et al., 2009; Igbinosa et al., 2009; Kosh-Komba et al., 2017). Based on the work of Igbinosa et
al.(2011) and El Diwani et al.(2009), the determination of the total polyphenol and flavonoid content of J.
curcasbark extracts showed lower polyphenol and flavonoid values compared to those found in the present study.
This difference in content between the extracts obtained and other previous studies could be equated with the
extraction conditions, the extraction process, and the quality of the solvents used (Akinmoladun et al., 2022). The
presence of these chemical compounds in these plants is the basis of their antimicrobial powers. Indeed, the
antimicrobial and antioxidant properties of these chemical compounds are well known (Agban et al., 2020; El
Diwani et al., 2009; Hirota et al., 2012).

In terms of antioxidant activity, ethanolic and hydro-ethanolic extracts from the leaves and bark of J. curcas and
Z.zanthoxyloidesinhibited the DPPH radical at better concentrations compared to extracts from their roots. Also, all
extracts of the bark of J. multifidahave shown the best inhibitory powers of the DPPH radical. The results obtained
are lower than those found by Hirota et al.(2012) in a study carried out on J. multifida. The difference observed in
the results of antioxidant activity obtained and other previous studies could be due to the level of maturity of the
leaves, bark and roots collected, the time of harvest and the drying conditions (Assefa et al., 2008; Chen et al.,
2022). The antioxidant power of medicinal plants could be attributed to the presence of phenolic compounds and
flavonoids(Carvalho et al., 2018; Garde-cerdán et al., 2017; Igbinosa et al., 2011).

For the antibacterial activity of the extracts of the different plant organs with regard to the bacterial strains, it
appears that all the extracts showed a variable activity depending on the strains tested. All extracts from the leaves,
bark and roots of J. curcaswere active on S. aureus ATCC 25923with the exception of the aqueous extract of the
roots. The hydro-ethanolic extract of the roots showed better activity compared to the other extracts. As for the
bacterial strain of K. pneumoniae, only bark extracts and ethanolic and hydroethanolic extracts from the leaves as
well as hydroethanolic extracts from the roots were active.

Hydro-ethanolic extracts from the bark and roots presented the best activities. According toIgbinosa et al.(2009),
aqueous and ethanol extracts have antibacterial activities on Staphylococcus aureus ATCC 25923and Klebsiella
pneumoniae. This study, consistent with the present study, demonstrated the ability of J. curcasextracts to inhibit
bacterial strains.

All extracts from the leaves, bark and roots of J. multifida were active on S. aureus ATCC 25923but only the
ethanolic extract from the bark was the most active. For the strain of K. pneumoniae, all extracts showed activity
except the ethanolic extract of the roots; and only the hydro-ethanolic extracts of the leaves and bark showed better
bactericidal activities. According to Fitria, (2018), extracts of Jatropha multifidabark showed antibacterial and
antibiotic activity on methicillin-resistant Staphylococcus aureus and Staphylococcus aureus. This antimicrobial
potential of extracts against Staphylococcus aureus NCTC6571 has been shown with all aqueous and ethanolic
extracts of leaves, bark and roots (Aiyelaagbe et al., 2008). These studies are in line with the results obtained in the
present study by confirming the antibacterial properties of the extracts of the different organs of J. multifidaon
bacteria.

For Z. zanthoxyloides, all extracts of the leaves, bark and roots were active on S. aureus ATCC 25923 with the
exception of ethanolic leaf extracts and aqueous bark extract. Only the ethanolic extract of the roots showed good
bactericidal activity. For K. pneumoniae, all extracts from the leaves, bark and roots were active with the exception
of the aqueous extract from the leaves. Only the hydro-ethanolic extract of the leaves showed the best bactericidal
activity. According to Kosh-Komba et al. (2017), hydro-ethanolic extracts from the leaves, bark and roots of Z.
zanthoxyloides showed an antibacterial effect on Klebsiella pneumonia and Staphylococcus aureus.

The work carried out by Agbulu et al.(2015) showed the antibacterial potential of Z. zanthoxyloides extracts on
Staphylococcus aureus ATCC 25923 and Klebsiella pneumoniae. The results obtained in this work are consistent
with those of Agbulu et al.(2015)andKosh-Komba et al.(2017), who demonstrated that extracts from the leaves and
bark of Z. zanthoxyloideshave activity on bacterial strains. Only ethanolic and hydro-ethanolic extracts from plant

415
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

organs (leaves, bark and roots) (J. curcas, J. multifidaand Z. zanthoxyloides) showed the best antibacterial activities
depending on the plants, organs and strains used compared to aqueous extracts. This is due to the strong polarity of
ethanol (Akinmoladun et al., 2022). Several bacterial strains have been used to evaluate the mode of action of the
active extracts.

In the present study, the bacterial external membrane permeability test was chosen to assess the mode of action of
aqueous, ethanolic and hydro-ethanolic extracts of the leaves and roots of J. multifida on K. pneumoniae and
S.aureus ATCC 25923. The results obtained revealed the membrane-destabilizing power of the bacterial strains of
the tested extracts. These extracts showed a superior destabilizing power compared to the Imipenem used here as a
reference molecule. These results show that the extracts have a consequent mode of action on the destabilization of
the outer membrane of the bacterial strains tested. In addition, regardless of the concentration of leaf extracts used,
the destabilization of the membrane of S. aureus ATCC 25923 strainsdoes not change. However, destabilization is
dependent on the concentration on the membrane of K. pneumoniae strains. In the case of S. aureus ATCC 25923,
destabilization increased as the concentration of leaf extracts increased. While it is independent on the membrane of
the K. pneumoniae strains. Therefore, the administration of leaf extracts could vary between MIC and 2 MIC on S.
aureus ATCC 25923strains.But, it could be administered at the IJC on strains of K. pneumoniae. The phytochemical
composition of plant extracts could be at the origin of their high potential for membrane destabilization (Frirdich and
Whitfield, 2005). In addition, it has been reported that the destabilization and dysregulation of interactions between
lipopolysaccharide molecules are caused by phenolic compounds (flavonoids, tannins, etc.) and terpenoids (Frirdich
and Whitfield, 2005; Vaara, 1992). The explosion of the cytoplasmic membrane and the dysregulation of ionic
homeostasis between the intracellular and extracellular compartments of Gram-negative bacteria are responsible for
the antibacterial effect of plant extracts (Kumar et al., 2013; Yala et al., 2016). The results of this study therefore
confirm the previous results.

Conclusion:-
The present study was carried out as part of a comparison of the antibacterial activity of renewable organs (leaves,
bark) of 03 medicinal plants of the Beninese flora whose roots are used in the treatment of infections. The present
study showed that hydro-ethanolic extracts from the bark and leaves of J. curcas and Z. zanthoxyloides, respectively,
showed good antibacterial activities. Similarly, hydro-ethanolic extracts from the leaves and bark of J. multifidahave
shown strong antibacterial potential. The other plant organs expressed great therapeutic potential in the present study
and should be advocated in the treatment of infections instead of roots in order to preserve biodiversity. The results
obtained on the mechanism of action of plant extracts give them potential to destabilize the membrane of bacteria. It
is therefore very important to carry out research on the toxicity of these plant organs in order to minimize health
risks and propose them as candidates for ATMs.

Acknowledgements:-
The auhors congrats the members of Research Unit in Applied Microbiology and Pharmacology of natural
substances

Competing Interests
The authorsdeclarethat they have no competinginterestconcerningthis article.

Authors’ Contributions
All authorscontributed to the realization of the work and to the manuscript preparation.

References:-
1. Agban A, Hoekou Y, Pissang P, Tchacondo T, Batawila K. 2020. Evaluation du potentielantimicrobien et de la
toxicité des extraits de Jatropha multifida Linn (Euphorbiaceae). J. Appl. Biosci,151: 15550–15558.
https://doi.org/10.35759/JABs.151.4
2. Agbankpe AJ, Dougnon TV, Bankole SH, Houngbegnon O, Dah-Nouvlessounon D, Baba-Moussa L. 2016. In
vitro antibacterial effects of Cratevaadansonii, Vernonia amygdalina and Sesamum radiatum used for the
treatment of infectious diarrhoeas in Benin. J. Infect. Dis. Ther,4: 2332–0877.
3. Agbulu CO, Aboje EE, Akande T. 2015. Antimicrobial Activity of Zanthoxylum zanthoxyloides root used as
chewing stick (in Nigeria) on oral pathogens. Inter Sci Res,1: 75–81.

416
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

4. Aiyelaagbe OO, Adeniyi BA, Fatunsin OF, Arimah BD. 2007. In vitro antimicrobial activity and phytochemical
analysis of Jatropha curcas roots.
5. Aiyelaagbe OO, Oguntuase BJ, Arimah BD, Adeniyi BA. 2008. The antimicrobial activity of Jatropha multifida
extracts and chromatographic fractions against sexually transmitted infections. J Med Sci, 8: 143–147.
6. Akinmoladun AC, Falaiye OE, Ojo OB, Adeoti A, Amoo ZA, Olaleye MT. 2022. Effect of extraction
technique, solvent polarity, and plant matrix on the antioxidant properties of Chrysophyllum albidum G. Don
(African Star Apple). Bull. Natl. Res. Cent,46: 40. https://doi.org/10.1186/s42269-022-00718-y
7. Akoègninou A, Van der Burg W, Van der Maesen LJG. 2006. Flore analytique du Bénin. Backhuys Publishers.
8. Akouétévi GT, Bouraïma D, Simplice DK, Ségla T, Yaovi A, Comlan de S. 2017. Prévalence des
souchesd’entérobactériesproductrices de bêta-lactamases à spectreélargiisolées au Togo et de leursensibilité aux
antibiotiques.Int. J. Biol. Chem. Sci. https://www.ajol.info/index.php/ijbcs/article/view/161889
9. Arekemase MO, Kayode RMO, Ajiboye AE. 2011. Antimicrobial activity and phytochemical analysis of
Jatropha curcas plant against some selected microorganisms. Int. J. Biol,3: 52.
10. Assefa G, Sonder K, Wink M, Kijora C, Steinmueller N, Peters KJ. 2008. Effect of variety and harvesting
management on the concentration of tannins and alkaloids in tagasaste (Chamaecytisuspalmensis). Anim. Feed
Sci. Technol,144: 242–256.
11. Bihari CG, Behera Manaswini BM, Kumar JP, Kumar TS. 2011. Pharmacognostical and phytochemical
investigation of various tulsi plants available in south eastern Odisha.
12. Carvalho C, Mariano LV, Negrão VS, Gonçalves P, Marcucci C. 2018. Phenols, flavonoids and antioxidant
activity of Jatropha multifida L. collected in Pindamonhangaba, Sao Paulo State, Brazil. J Anal Pharm Res7:
581–584.
13. Chen C, Mokhtar RAM, SaniMSA, Noor NQ. 2022. The effect of maturity and extraction solvents on bioactive
compounds and antioxidant activity of mulberry (Morus alba) fruits and leaves. Molecules27: 2406.
14. Djego J, Djego-Djossou S, Cakpo Y, Agnani P, Sinsin B. 2012. Evaluation du potentielethnobotanique des
populations rurales au Sud et au centre du Bénin. Int. J. Biol. Chem. Sci, 5: 1432–1447.
https://doi.org/10.4314/ijbcs.v5i4.10
15. Dougnon TV, Attakpa E, BankoléH, Hounmanou YM, Dèhou R, Agbankpè J, Souza M, Fabiyi K, Gbaguidi F,
Baba-Moussa L. 2017. Etude ethnobotanique des plantes
médicinalesutiliséescontreunemaladiecutanéecontagieuse : La gale humaine au Sud-Bénin.
PharmacopéeMédecineTradit. Afr, 18: 16–22.
16. Dougnon V, Hounsa E, Agbodjento E, KeilahL, Sintondji K, Afaton A, Klotoe J, Baba-Moussa L, Bankole H.
2021. Percentage Destabilization Effect of Some West African Medicinal Plants on the Outer Membrane of
Various Bacteria Involved in Infectious Diarrhea. PubMed, 1: 16–22
17. El Diwani G, El Rafie S, Hawash S. 2009. Antioxidant activity of extracts obtained from residues of nodes
leaves stem and root of Egyptian Jatropha curcas. Afr J Pharm Pharmacol, 3: 521–530.
18. Fah L, Koudokpon H, Dougnon T, Klotoé J, Fanou B, Dougnon V, LokoF. 2015. Evaluation des
propriétésantihyperglycémiantes de Schwenckia americana L. :uneplanteutilisée dans le traitement du diabète au
Bénin. Ethnopharmacologia,4: 78–83.
19. Fitria A. 2018. The Bactericidal and Antibiofilm Activity of Stem Bark of Jatropha multifida L. Against
Staphylococcus aureus and MRSA. EKSAKTA J. Sci. Data Anal,3: 42–55.
https://doi.org/10.20885/eksakta.vol18.iss1.art5
20. Frirdich E, WhitfieldC. 2005. Lipopolysaccharide inner core oligosaccharide structure and outer membrane
stability in human pathogens belonging to the Enterobacteriaceae. J. Endotoxin Res, 11: 133–144.
https://doi.org/10.1179/096805105X46592
21. Garde-cerdán T, Gonzalo-diago A, Pérez-álvarez EP. 2017. Phenolic Compounds: Types, Effects and Research.
Nova Science Pub Inc,3: 521–530.
22. Gurib-Fakim A, Brendler T, Phillips L, Eloff L. 2010. Green gold—success stories using southern African plant
species. Assoc. Afr. Med. Plants Stand, 7: 525–530.
23. Hao G, Shi YH, Tang YL, Le GW. 2009. The membrane action mechanism of analogs of the antimicrobial
peptide Buforin 2. Peptides30: 1421–1427. https://doi.org/10.1016/j.peptides.2009.05.016
24. Hirota BC, Miyazaki CM, Mercali CA, Verdan MC, Kalegari M, Gemin C, Lordello AL, Miguel MD, Miguel
OG. 2012. C-glycosyl flavones and a comparative study of the antioxidant, hemolytic and toxic potential of
Jatropha multifida leaves and bark. Int J Phytomedicine, 4: 1–5.
25. Igbinosa OO, Igbinosa EO, Aiyegoro OA. 2009. Antimicrobial activity and phytochemical screening of stem
bark extracts from Jatropha curcas (Linn). Afr. J. Pharm. Pharmacol,3: 058–062.

417
ISSN: 2320-5407 Int. J. Adv. Res. 12(12), 409-418

26. Igbinosa OO, Igbinosa IH, Chigor VN, Uzunuigbe OE, Oyedemi SO, Odjadjare EE, Okoh AI, Igbinosa EO.
2011. Polyphenolic Contents and Antioxidant Potential of Stem Bark Extracts from Jatropha curcas (Linn). Int.
J. Mol. Sci,12: 2958–2971. https://doi.org/10.3390/ijms12052958
27. Klotoé JR, Agbodjento E, Dougnon VT, Yovo M, Sacramento TI, Déguénon E, DougnonJT, Atègbo JM. 2020.
Exploration of the chemical potential and antioxidant activity of some plants used in the treatment of male
infertility in southern Benin. J. Pharm. Res. Int, 32: 1–12.
28. Kosh-Komba E, Toumnou LA, Zinga I, Touckia I, Lembo PNZ, Mukeina G, Semballa S, Yongo OD, Syssa-
Magale JL. 2017. Phytochemical screening, antifungal and antibacterial effect of Zanthoxylum zanthoxyloides
and Zanthoxylum macrophylum used in traditional medicine in Yamboro (Central African Republic). Medicine
(Baltimore)1: 3-12.
29. Koudokpon H, Dougnon T, Bankolé H, FahL. Hounmanou Y, Baba-Moussa L.2017. Ethnobotanical survey of
plants used in the treatment of infections in southern Benin. Health Sci Dis,18: 92–9.
30. Kumar K, AhmadT, Ali S, Rizvi IA, Agarwal A, Kumar R, Chaubey AK. 2013. Influence of projectile breakup
on the 16 O + 115 In reaction at energies ≈ 4–7 MeV/nucleon. Phys. Rev, 88: 064613.
https://doi.org/10.1103/PhysRevC.88.064613
31. Monciardini P, Iorio M, Maffioli S, Sosio M, Donadio S. 2014. Discovering new bioactive molecules from
microbial sources. Microb. Biotechnol,7: 209–220. https://doi.org/10.1111/1751-7915.12123
32. Onzo CF, AzokpotaP, Dah-Nouvlessounon D, Lehmane TH, Adjatin A, Baba-Moussa L. 2016. Évaluation de
l’activitéantimicrobienne de quatre feuillesutiliséescommeemballages dans l’artisanatagroalimentaire au Bénin.
J. Appl. Biosci,95: 9015. https://doi.org/10.4314/jab.v95i1.11
33. Sharma AK, Gangwar M, Tilak R, Nath G, Sinha AS, Tripathi YB, Kumar D. 2012. Comparative in vitro
antimicrobial and phytochemical evaluation of methanolic extract of root, stem and leaf of Jatropha curcas
Linn. Pharmacogn. J, 4: 34–40.
34. TineY, Diop M, Mahieux S, Ndoye I, Diallo A, Costa J, Neut C, WéléA. 2020. Antibacterial Activity of
Methanolic Extracts of Zanthoxylum zanthoxyloides (Lam.) B. Zepernich and Timler (Rutaceae) Justifying its
Use in Traditional Medicine to Fight against Infection.
35. Vaara M. 1992. Agents that increase the permeability of the outer membrane. Microbiol. Rev, 56: 395–411.
https://doi.org/10.1128/mr.56.3.395-411.1992
36. Veilleux C, King SR, Morganstein L. 1996. An introduction to ethnobotany. Linda Morgenstem Ed.
37. Yala JF, Ntsameso-Mve-MbaV, Issembe YA, Alexis N, Souza A. 2016. Évaluation in vitro de
l’activitéantimicrobienne de l’extraitaqueuxd’Eryngiumfoetidiumrécolté dans la ville de Franceville.
38. Ynalvez RA, Cardenas C, Addo JK, Adukpo GE, Dadson BA, Addo-Mensa A, 2012. Evaluation of the
Antimicrobial Activity of Zanthoxylum za nthoxyloides Root Bark Extracts. Res. J. MedPlant,6: 149–159.

418