http://orcid.org/0000-0003-3642-377X
Figures
Abstract
Drug resistant Salmonella species and shortcomings related to current drugs stress the urgent need to search for new antimicrobial agents to control salmonellosis. This study investigated the antisalmonellal and antioxidant potentials of methanolic and hydro-ethanolic extracts of Garcinia kola and Alchornea cordifolia as potential sources of drugs to control Salmonella species and to reduce related oxidative stress. The antisalmonellal activity was assessed using the broth microdilution, membrane destabilization and time-kill kinetic assays. While, the DPPH, ABTS and FRAP assays were used for the determination of the antioxidant activities. The minimum inhibitory concentrations ranged from 125 to 1000 μg/mL, with the methanolic root extract of G. kola being the most active. The time kill kinetic assay revealed a concentration-dependent bacteriostatic activity for promising extracts. Potent extracts from G. kola showed the ability to destabilize S. typhi outer membrane, with the methanolic root extract presenting the highest activity; two-fold higher than those of polymyxin B tested as reference. In addition, this methanolic root extract of G. kola also provoked nucleotide leakage in a concentration-dependent manner. From the antioxidant assays, the hydro-ethanolic extract from the stem bark of A. cordifolia presented significant activities comparable to that of Vitamin C. The methanolic root extract of G. kola also presented appreciable antioxidant activities, though less than that of A. cordifolia. Overall, the phytochemical screening of active extracts revealed the presence of anthocyanins, flavonoids, glycosides, phenols, tannins, triterpenoids and steroids. These results provide evidence of the antibacterial potential of G. kola and offer great perspectives in a possible standardisation of an antisalmonellal phytomedicine.
Citation: Djague F, Lunga PK, Toghueo KRM, Melogmo DYK, Fekam BF (2020) Garcinia kola (Heckel) and Alchornea cordifolia (Schumach. & Thonn.) Müll. Arg. from Cameroon possess potential antisalmonellal and antioxidant properties. PLoS ONE 15(8): e0237076. https://doi.org/10.1371/journal.pone.0237076
Editor: Christophe Hano, Universite d'Orleans, FRANCE
Received: May 14, 2020; Accepted: July 19, 2020; Published: August 4, 2020
Copyright: © 2020 Djague et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: Authors are very grateful to the Seeding Labs’ Instrumental Access Grant (SL2012-2) to Prof. Boyom. This work also received materials and equipment support from the Yaoundé- Bielefeld Bilateral Graduate School for Natural Products with Antiparasite and Antibacterial Activity (YaBiNaPA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Salmonellosis is an infectious disease caused by S. enterica, and S. bongori [1]. Historically, it has been clinically categorized as typhoidal and non-typhoidal [2]. The former category is due to the human host-restricted S. enterica serovars typhi, paratyphi A, paratyphi B and certain strains of S. paratyphi C that cause typhoid and paratyphoid fevers, referred collectively as enteric fevers. The latter is due to ubiquitous non-typhoidal Salmonellae (NTS) such as S. enterica serovar typhimurium and S. enterica serovar enteritidis having broader host range and predominantly causing self-limiting gastroenteritis in animals and humans [3]. Salmonella bacteria are widely distributed in domestic and wild animals.
Transmission is generally via the faecal-oral route, by either the consumption of contaminated food or water, person-to-person contact, or from direct contact with infected animals [4]. Salmonellosis is manifested in several different ways and both sex are affected equally [5]. The predominant symptom for typhoidal salmonellosis is fever. The temperature rises gradually during the first week of the illness and reaches a plateau of 39 to 40°C the following week. Other common symptoms include: abdominal pain, diarrhea, constipation, loss of appetite, headache etc. Some patients experience distinctive rash on their chest and abdomen, which is rose-coloured and flat, while others develop hepatosplenomegaly [6]. Acute gastroenteritis is the most common presentation of NTS infection. Typical symptoms include nausea, and/or vomiting. Fever, abdominal cramps and bloody diarrhea may also be reported [7].
Salmonellosis remains a major public health problem worldwide [1,8]. It is the second most commonly reported foodborne infection in the European Union and in the United States [9]. In Africa, recent investigation showed that the relative proportion of bacteremia due to invasive Salmonella infections has increased dramatically [10]. Typhoidal salmonellosis continue to be a significant contributor to global morbidity and mortality, with an estimated 17 million illnesses worldwide and approximately 178,000 deaths each year as of 2015 [8]. Besides enteric fever, non-typhoidal salmonellosis also represent a considerable burden in both developing and developed countries [11]. Each year, an estimated 94 million cases and approximately 155 000 deaths occur due to gastroenteritis worldwide [12]. There are several drugs (Amoxicillin, Ampicillin, Ciprofloxacin, Cefotaxime, Ceftriaxone, etc.) used to fight this infection [13]. However, due to many reasons including the extensive and inappropriate use of the same antimicrobials, delay in diagnosis or poor hygiene conditions, increase in multidrug resistant Salmonella species has been reported, restricting the therapeutic options [14].
Apart from causing salmonellosis, Rastaldo et al. [15] reported that the entrance of Salmonella equally causes the production of superoxide and nitric oxide which react together to form peroxynitrite a strong biological oxidant, leading to increase levels of oxidative species (hydrogen peroxide, and hydroxyl radical) and oxidative stress [16]. Stress is at the origin of other human illnesses like sickle cell diseases, artherosclerosis, Parkinson’s disease, heart failure, myocardial infarction, Alzheimer’s disease etc. [17]. Oxidative stress can be prevented or delayed by using antioxidant agents. However, beside their unavailability, high cost and side effects, frequently used synthetic antioxidants such as Butylated Hydroxyl Anizole (BHA) and Butylated Hydroxyl Toluene (BHT) have been proven to be neurotoxic, hepatotoxic, and carcinogenic [18]. Consequently, there is need to search for complementary and alternative sources of medicines.
Garcinia kola Heckel is a dicotyledonous plant belonging to the family of Clusiaceae [19]. It is mostly found in lowland forests of India, Indochina, Indonesia, and Brazil [20]. It has been recognized as an indigenous medicinal plant found in the tropical rain forest of Central and Western Africa, especially Benin, Sierra Leone, Democratic Republic of Congo, Ivory Coast, Gabon, Ghana, Liberia, Nigeria, Senegal and Cameroon, where it is one of the most valued tree [21]. Known as bitter cola, the fruits of G. kola, is a well-recognized aphrodisiac, and a decoction of the leaves and stem bark is traditionally used in the treatment of typhoid fever in Cameroon. Alchornea cordifolia Schum. & Thonn. on the other hand, belongs to the family of Euphorbiaceae and occurs mainly in west to central Africa in countries as Congo, Ivory Coast, Nigeria, Ghana and Cameroon. [22]. It is called Bambami in Hausa (Nigeria), Mabounji in the southwest region of Cameroon, Arbre de djeman in French and commonly Christmas bush or dove wood in English. It is commonly used throughout its areas of distribution as a remedy for skin diseases, abdominal pains and vomiting.
Previous studies [23–25] reported the antibacterial activity of G. kola. Despite the few works done on the antisalmonellal activity of G. kola, investigations related to the possible mechanisms of action of G. kola extracts are still not very much explored. In addition, most of the biological activities have been performed on seeds; and reports of these activities on the roots, stem barks and leaves of G. kola are lacking. More so, most of the activities are evaluated by using preliminary and inconclusive tests such as the agar disc or well diffusion methods. For A. cordifolia, despite the works done on this plant, its hydro-ethanolic extract has not been used. Thus, the aim of the present study was to evaluate the antisalmonellal and antioxidant properties of the leaves, roots and stem barks of G. kola and A. cordifolia and their possible antimicrobial modes of action.
Materials and methods
Plant materials and extraction
Fresh parts of a specimen of both G. kola and A. cordifolia were harvested from Kumba, Southwest Region of Cameroon in March 2018. These parts included the leaves, roots and stem bark of G. kola and the leaves, stem bark and twigs of A. cordifolia. Botanical identification was done by Mr Nana Victor at the Cameroon National Herbarium in Yaoundé, where a voucher specimen was deposited under the reference number 41604HNC and 29705HNC respectively for G. kola and A. cordifolia. No permission was necessary for sample collection. The fresh plant materials were later washed separately with fresh water to remove dirt and other contaminants, shade-dried and ground to fine powders. The powders were macerated in hydro-ethanol (1:4 v/v) and absolute methanol for 72 hours accompanied by occasional shaking and stirring. The solutions were sieved using a hydrophilic cotton and filtered using Whatman filter paper N0 1 after which the filtrates were concentrated using a rotary evaporator (BÜCHI 461) at 65ºC (for hydro-ethanol) and at 60ºC (for methanol) under reduced pressure till dryness to obtain the crude extract. The extracts were preserved in sterile bottles and conserved at 4 ºC for further experiments.
In vitro antisalmonellal assay
Microbial species and culture media.
Three Salmonella species were used in this study, one strain (S. typhi) from the ‘‘Centre Hospitalier Universitaire” (CHU) of Yaounde-Cameroon and two clinical isolates (S. typhimurium and S. enteritidis) from ‘‘Centre Pasteur” of Cameroon (CPC). Mueller Hinton Agar (MHA) was used to revive the bacteria while Mueller Hinton Broth was used for the antisalmonellal assay. Salmonella-Shigella Agar (SS agar) was used for the time kill kinetic assay.
Determination of MIC and MBC.
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth micro-dilution method, using the ρ-iodonitrotetrazolium-Chloride (INT) based assay as previously reported by Lunga et al. [26]. Briefly, 196 μL of Muller Hinton broth (MHB) were introduced into the wells of the first line followed by 4 μL of each extract (initially prepared at 100 mg/ml in 100% DMSO). Serial two-fold dilutions were performed and 100 μL of the bacterial suspension (1.5 x106 CFU/mL) was added into each well. The final concentration ranged from 1000 to 15.625 μg/mL for the extracts and from 5 to 0.0195 μg/mL for the positive control (Ciprofloxacin and Polymyxin B).The plates were incubated at 37°C for 24 hours after which 50 μL of 0.2 mg/mL INT solution was added and further incubated at 37°C for 30 min. MICs were determined as the lowest sample concentration at which no visible colour change was observed. Wells containing MHB and bacteria constituted the negative control while the sterility control contained MHB alone. The final concentration of DMSO was at most 1% and preliminary test did not inhibit bacterial growth
The MBC was determined by transferring 50 μL of INT-free aliquots from the wells with no colour change into 150 μL of freshly prepared MHB. These preparations were further incubated at 37°C for 48 hours and revealed as above to obtain the MBC. All tests were carried out in triplicates.
Phytochemical screening
The presence of major phytochemical classes of compounds such as Alkaloids, Anthocyanins, Flavonoids, Glycosides, Phenols, Quinones, Saponins, Sterols, Tannins and Triterpenoids were qualitatively evaluated in the plant extracts according to previously described methods [27].
Evaluation of possible mode of action
The outer membrane permeability (OMP) assay.
This was determined according to the method described by Hao et al. [28], with some modifications. In a 96 well micro-plate, concentrations of MIC, 2MIC, and 4MIC of each extract in triplicate were prepared by two-fold serial dilution. One hundred microliters (100μL) of S. typhi suspension (1.5 x 106 CFU/mL) were added and the plate incubated at 37°C for 24h. The optical densities were read at 405 nm (wavelength at which the complex form between Lipopolysaccharides and membrane stabilizing divalent cations absorbs) and the percentage destabilization was calculated using the formula below.
Where %D: Percentage Destabilization; Ao: Absorbance of negative control; As: absorbance of test samples.
Polymyxin B was used as the positive control and was equally prepared at MIC (0.0781 μg/mL), 2MIC (0.1562 μg/mL), and 4MIC (0.2124 μg/mL). MHB and the bacteria constituted the negative control while the sterility control contained MHB alone. Wells containing MHB and the extracts alone constituted the blank. A graph of %D against extract concentration was plotted in order to determine the concentration which causes maximum destabilization.
Nucleotide leakage.
This was determined according to the method described by Tang et al. [29] with slight modifications. Briefly, an overnight culture of S. typhi was washed in sterile physiologic water (2 mL of 0.9% NaCl) and the resulting solution centrifuged at 10 000 rpm for 10 min. The supernatant was discarded, and the resulting pellet was re-suspended in 10 mM PBS (pH 7.4) and the turbidity adjusted to 0.5 McFarland (1.5x108 CFU/mL). In a 96 well micro-plate, 100 μL of the bacterial suspension were added to 100 μL of MHB containing the methanolic root extract of G. kola (GKRM) at MIC, 2MIC, 4MIC (initially prepared in 10 mM PBS (pH 7.4)). The plate was incubated at 37°C for different times (0, 2, 4, 6, 8, 12 and 24h). Following each incubation period, the cell suspension was centrifuged at 10 000 rpm for 10 min, the supernatant appropriately diluted and the optical densities were recorded at 260 nm. The test was performed in triplicate and simultaneously for positive (ciprofloxacin), negative (PBS + cell suspension), sterility control (MHB alone) and blank (MHB + extract). The optical densities were plotted against the different times to determine the time dependent degree of leakage of the different extract concentrations.
Time-kill kinetics test.
The time kill kinetic of the G. kola root methanol (GKRM) extract was performed according to the method described by Tsuji et al. [30]. Here, extract concentrations of MIC, 2MIC, 4MIC and 8MIC were prepared by serial two-fold dilution in a 96 well micro-plate. One hundred microliters (100 μL) of S. typhi suspension (1.5 x 106 CFU/mL) were added and the plate incubated at 37°C for different time intervals (0, 1, 2, 4, 6, 8, 12, and 24 h). Following each incubation period, the cell suspensions were appropriately diluted (in NaCl 0.9%) and the resulting solution sub-cultured on SS agar plates for further 24 h at 37°C. Ciprofloxacin was used as positive control while the bacteria incubated with MHB was used as growth control. The test was performed in triplicate and results were presented as Mean ±SD.
In vitro antioxidant activity
DPPH radical scavenging assay.
This was performed according to the method described by Scherer and Godoy [31] with slight modifications. Briefly, the extracts were first diluted to obtain final extract concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg/mL in a 96 well micro-plate. Twenty-five microliters (25 μL) of each dilution was introduced into a new micro-plate and 75 μL of 0.02% DPPH added to obtain final concentrations of 500, 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL. The reaction mixtures were kept in the dark at room temperature for 30 min after which the absorbance was measured at 517 nm against the blank (DPPH in methanol). L-ascorbic acid was used as positive control and was treated in the same conditions as the extracts with final concentrations of 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391 and 0.195 μg/mL. The assay was performed in triplicate.
The percentage (%) radical scavenging activities of the plant extracts were calculated using the following formula; from which other parameters like the radical scavenging activity 50 (RSA50), effective concentration 50 (EC50), and antiradical power (ARP) were deduced.
Where, RSA: Radical Scavenging Activity; Ao: Absorbance of the blank (DPPH + methanol); As: Absorbance of DPPH radical + plant extract.
ABTS radical scavenging assay.
It was performed according to the method described by Re et al. [32] with slight modifications. In a 96 well micro-plate, the assay was performed at the same extract concentrations as above, using the same positive control. The absorbance of ABTS radical after 30 min incubation was read at 734 nm. The assay was performed in triplicate.
The percentage (%) radical scavenging activities of the plant extracts were calculated and from it, the same parameters were deduced as for the DPPH assay.
Ferric ion reducing antioxidant power (FRAP) assay.
The assay was performed according to the method described by Benzie et al. [33] with slight modifications. Briefly, the extracts were first dissolved as for the DPPH and ABTS assays. 25 μL from each dilution were added to 25 μL of 1.2 mg/mL Fe3+ solution in a new micro-plate. The plates were pre-incubated for 15 min at ambient temperature. Fifty microlitres (50 μL) of 0.2% ortho-phenanthroline solution was added to obtain final extract concentrations of 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 3.90625 μg/mL. The reaction mixtures were further incubated for 20 min at ambient temperature after which the absorbance was measured at 505 nm using a 96 well micro-plate reader (Infinite M200 (TECAN) against the blank (25 μL methanol + 25 μL Fe3+ + 50 μL ortho-phenanthroline). Hydroxylamine was used as positive control and was treated in the same way as the extracts with final concentrations of 6.6, 3.3, 1.65, 0.825, 0.413, 0.206, 0.103 and 0 μg/mL. The assay was performed in triplicate.
From a concentration-activity curve of NH2OH used as standard, the optical densities of the test wells were projected, and the results expressed quantitatively as μg equivalent NH2OH/g of extracts.
Statistical analysis
Where appropriate, the data were subjected to one-way analysis of variance (ANOVA) and results were presented as the Mean ± SD of the replicated values. Significance differences for multiple comparisons were determined by Waller-Duncan Post Hoc test at p ≤ 0.05 using the Statistical Package for the Social Sciences (SPSS, version 16.0) program.
Results
Antisalmonellal activities of the extracts
The microbial growth inhibition capacities of the extracts were assessed based on their minimum inhibitory concentrations (MICs) and bactericidal concentrations (MBCs) (Table 1).
The MIC values ranged from 125 to 500 μg/mL. G. kola extracts were generally more active (MIC ranging from 125 to 500 μg/mL) than those of A. cordifolia (MIC 500 μg/mL). Of all G. kola extracts, both the hydro-ethanolic stem bark and its methanolic leaf extracts were not active against S. typhimurium while the methanolic leaf extract alone was inactive against S. enteritidis at the tested concentrations. The results also showed that S. typhi was the most susceptible of the three serovars (MIC of 125μg/mL with the methanolic root and hydro-ethanolic stem bark extracts of G. kola and 250 μg/ml with other G. kola extracts).
Phytochemical composition of extracts
Phytochemical screening revealed the presence of Anthocyanins, Flavonoids, Glycosides, Phenols, Tannins, Triterpenoids and Steroids (Table 2). The methanolic leaf extract of G. kola and the Hydro-ethanolic stem extract of A. cordifolia were the most complex in phytochemical groups, though being the least antisalmonellal. The methanolic root extract of G. kola, with the best antibacterial activity, was the least complex in phytochemical groups of compounds, differing from its hydro-ethanolic extract by the presence of glycosides and terpenoids on one hand, and the absence of flavonoids on the other hand in the latter. This may suggest the central role played by flavonoids in attributing activity to this plant.
Effect of extracts on outer membrane permeability of S. typhi
The ability of the extracts to permeabilize the outer membrane of S. typhi was evaluated over a period of 24 h at three different concentrations (MIC, 2MIC and 4MIC). Fig 1 shows that, apart from the hydro-ethanolic stem bark extract of A. cordifolia, all the other extracts from G. kola presented the same trend as that of Polymyxin B, on the destabilization of S. typhi outer membrane. The results showed that the percentage permeabilization was concentration dependent. Interestingly, the destabilization percentage of the extracts of G. kola were generally significantly greater (p≤0.05) than those of Polymyxin B at their corresponding concentrations. The root methanolic extract showed the highest effect, closely followed by its hydro-ethanolic extract, then the stem and leaf methanolic extracts. The hydro-ethanolic stem bark extract of G. kola presented sky-rocketing activities at high concentrations; showing the highest effect at 4 MIC.
Pol B: Polymyxin B; GKRM: G. kola roots methanol; GKRH: G. kola roots hydro-ethanol; GKSM: G. kola stem bark methanol; GKSH: G. kola stem bark hydro-ethanol; GKLM: G. kola leaf methanol; ACSH: A. cordifolia stem bark hydro-ethanol.
Effect of G. kola methanolic root extract concentration on S. typhi nucleotide leakage with time
Being the most active antisalmonellal extract, the methanolic root extract of G. kola (GKRM) was further evaluated for its ability to permeabilize the cytoplasmic membrane. The results showed that GKRM extract caused leakage of nucleotides (DNA and RNA), as revealed by the increase in absorbance of the supernatant at 260 nm with time (Fig 2). This indicated that nucleotides were released outside the cell. The extract presented high nucleotide leakage effect at 2MIC, not significantly different (p≤0.05) from that of 4MIC; both of which were significantly different from the effect at MIC, the latter being similar to the effect of ciprofloxacin, the reference mechanistic antibiotic.
CIP: Ciprofloxacin; ST: Salmonella typhi.
The time-Kill kinetics effect of G. kola methanolic root extract against S. typhi
All concentrations of the methanolic root extract of G. kola inhibited the growth cycle curve when compared to the negative control (Fig 3). There was an initial increase in the number of viable cells over the first 4 hours, followed by a reduction up to the 8th h and a gradual rise to the 24th h. The activities of the extract at 2MIC and 4MIC were not significantly different (p˃0.05), while at the MIC value, the extract concentration was insufficient to significantly reduce bacterial growth. The inhibitory activity (through membrane destabilization) was pronounced during the first 8 hours. After 8 hours (resurface or re-emergent time), the bacterial regained its normal growth cycle, indicating that the extract exhibited a concentration-dependent bacteriostatic effect on S. typhi as opposed to the bactericidal effect exhibited by ciprofloxacin (continuous declining trend of the growth curve).
CIP: Ciprofloxacin; NC: negative control; MIC: minimum inhibitory concentration.
In vitro antioxidant potentials of the extract
The DPPH and ABTS assays revealed that the extracts exhibited good free radical scavenging activities (Tables 3 and 4). The hydro-ethanolic stem bark extract of A. cordifolia exhibited the highest radical scavenging activity (12.606 μg/mL and 1.330 μg/mL for DPPH and ABTS respectively), very close to that of Vitamin C (8.926 μg/mL and 2.710 ± 0.080 μg/mL). The methanolic root extract of G. kola presented appreciable antioxidant activities though less than that of A. cordifolia, the most antioxidant. The EC50 and ARP showed the same trend as the RSA50.
FRAP assay equally showed that, there was a significant correlation between the concentration of the extracts and their reducing power. By projecting the optical densities of the extracts on a concentration-activity curve of NH2OH used as standard, the results showed that ACSH generally exhibited the highest reducing power per gram of extract, followed by the hydro-ethanolic stem extract of G. kola (Table 5). At low concentrations (≤62.5 μg/mL), the activity of hydro-ethanolic stem bark extract of A. cordifolia was significantly (P≤0.05) higher than those of the other extracts, while at high concentrations (≥125 μg/mL), its activity was comparable to, at least, one of the extracts. At the maximum concentration (500 μg/mL), the ferric ion reducing capacities of all the extracts, except the methanol leave extract of G. kola, were not significantly (P>0.05) different.
Generally, the results of the antioxidant assays showed that, the hydro-ethanolic extract presented greater antioxidant potentials than the methanolic extracts. The high antioxidant effect of the hydro-ethanolic extracts could be due to the diversity of compounds extracted by the conjugate solvent system (water/ethanol).
Discussion
Antibacterial activities and phytochemical compositions of extracts
The results of antimicrobial tests showed that the different parts of G. kola and the stem bark of A. cordifolia contained substances with antisalmonellal activities. The existence of these substances was confirmed by phytochemical screening which revealed the presence of certain classes of compounds (flavonoids, phenols, tannins, etc.), whose members have already been known to exhibit antimicrobial activities. According to Tamokou et al. [34], a plant extract is considered to be highly active if the MIC < 100 μg/mL; significantly active when 100 ≤MIC ≤512 μg/mL; moderately active when 512 <MIC ≤2048 μg/mL; weakly active if MIC > 2048 μg/mL and not active when MIC >10 000 μg/mL. Consequently, all G. kola extracts presented significant antibacterial activities (100 ≤ MIC ≤ 512 μg/mL) while moderate activities (MIC of 1000 μg/mL) were observed with the hydro-ethanolic stem bark extract of A. cordifolia (on S. typhimurium and S. typhi). It is likely that the amount of active ingredients in this plant extract does not occur in quantities large enough to produce significant activity. Such extract upon fractionation, purification and concentration of the active ingredients, may still lead to the isolation of therapeutically useful compounds against salmonellosis. However, among G. kola extracts, some of them acted on two or three Salmonellal species while others acted narrowly on one. Critical analysis of the composition and antisalmonellal activities of the extracts revealed that flavonoids, phenols and tannins may be responsible for the observed differences, as their mechanisms of action and/or concentration may differ. This probably explains why the methanolic root extract of G. kola, though presenting the best antisalmonellal activity, was the least complex in phytochemical groups of compounds, differing from its hydro-ethanolic extract by the presence of glycosides and terpenoids, and the absence of flavonoids in the latter, suggesting that flavonoids may play a central role in attributing activity to this plant. Contrarily, the methanolic leaf extract of G. kola and the stem bark hydro-ethanolic extract of A. cordifolia with the highest phytochemical groups of compounds showed the least antisalmonellal activity. This suggests that the presence of anthocyanins, glycosides, steroids in their extracts may have contributed to the low activity recorded, given that these were the major phytochemical groups of compounds that differentiated them from the most active. The findings of this study were in conformity with that of Flora et al. [35] which showed that the methanolic root and stem extracts of G. kola contained flavonoids, phenols and tanins; as well as that of Gatsing et al. [25] which revealed that the methanolic leaf extract did not contain alkaloids, quinones and saponins.
Mode of action of extracts and growth kinetics
Due to the variety of bacterial targets, several models for studying the mode of antibacterial action of drugs have been described in the literature [36]. As a result, the outer membrane permeability and nucleotide leakage assays were chosen to evaluate the mode of action of the extracts against the most sensitive Salmonella serova (S. typhi). Apart from the hydro-ethanolic stem bark extract of A. cordifolia, all the other extracts from G. kola presented the same trend as that of Polymyxin B, indicating that they have similar modes of action, on the destabilization of the bacterial outer membrane. Looking at the percentage destabilization as a function of concentration, putting aside toxicity, the hydro-ethanolic stem extracts of G. kola could be administered at 2MIC, and those of the other G. kola extracts at MIC given that at these concentrations, their respective destabilization capacities were greater than that of Polymyxin B. The effect of these extracts on the outer membrane permeability of S. typhi could be attributed to their phytochemical contents. In effect, certain small terpenoids and phenolic compounds have been reported to disorganize and weaken the interactions between lipopolysaccharide molecules by chelating outer membrane-stabilizing divalent cations [37,38]. This antibacterial action can result in membrane expansion, increase membrane fluidity and permeability, disturbance of membrane embedded proteins, inhibition of respiration, and alterations in the ionic homeostasis between intracellular and extracellular compartments of Gram-negative bacteria [39]. This explains the significantly high destabilization effect of the terpenoid- and phenolic-rich extracts of G. kola.
Nucleotides leakage results from the damage of the cell membrane or a change in permeability of the cell membrane [40]. Various vital intracellular materials including small ions such as K+ and (PO4)3- tend to leak out, followed by the leakage of large molecules such as DNA, RNA and other materials [41]. Phenolic compounds have been reported to disturb the cytoplasmic membrane, disrupt the proton motive force, electron flow and active transport [42]. Hence, they could be responsible for the observed leakage, probably triggered by the phenol-rich methanolic root extract of G. kola. Moreover, the nucleotide leakage exhibited by the extract may be associated with the presence of flavonoids. Flora et al. [43] attributed the antimicrobial activities of flavonoids to the disruption of microbial cells’ membranes due to their ability to chelate membrane proteins. Thus, the antibacterial flavonoids, phenols and tannins in this extract plausibly acted through the destabilization of the cell membrane.
Time-kill assays allow antibacterial agents to be classified as bacteriostatic or bactericidal and characterize the relationship between agent concentration and activity over time. The time kill profile of G. kola methanolic root extract showed that its nucleotide leakage and outer membrane permeability effects on S. typhi were bacteriostatic and concentration dependent. Bacteriostatic antimicrobial agents only inhibit the growth and multiplication of the microbes giving the immune system of the host the time to clear the microbes from the system [44]. A concentration dependent bacteriostatic effect takes place when an antimicrobial agent has a high concentration at the binding site in order to eliminate the microorganism [45]. This might explain why there was an initial increase in the number of viable cells at all concentrations over the first few hours, as the extract had to attain its binding sites before exerting its effect. This time-kill kinetics reveals that if an antisalmonellal phytomedicine is prepared from this plant, it should be administered at intervals of 8 hours (re-emergence time) following its contact with the pathogen.
Antioxidant activities of extracts and salmonellosis related stress conditions
In addition to causing salmonellosis, it has been demonstrated that the entrance of Salmonella causes the production of superoxide and nitric oxide which react together to form peroxynitrite, a strong biological oxidant. This consequently increases the levels of reactive oxygen species [15]. Oxidative stress can be prevented or delayed by using antioxidant agents. Thus, having unraveled the possible antibacterial mode of action of the extracts and characterizing the relationship between their concentrations and activities over time, their antioxidant potentials were equally determined to see if in addition to their antibacterial properties, they could fight against a possible oxidative stress caused by the pathogenic invasion.
The hydro-ethanolic stem extract of A. cordifolia showed the best antioxidant potential. However, this extract, though having the best antioxidant activity, did not present significant antisalmonellal effect. This indicates that, its Salmonella inhibiting capacity may not be directly linked to its antioxidant activity. The extracts of G. kola presented dual (antisalmonellal and antioxidant) activities and this lends credit to their possible exploitation in the formulation of an anti-typhoid phytomedicine. The extracts could scavenge reactive oxygen species which is implicated in the pathogenesis of typhoidal and non-typhoidal Salmonellae [15] and therefore can fight against oxidative stress that may result from Salmonella infection. From the phytochemical screening and antioxidant results, the antioxidant potential of the extracts may not only be attributed to the diversity of the phytochemical groups identified, but also to the concentration of the specific group, as the methanolic leaf extract of G. kola, though being the riches in terms of phytochemical groups did not show the highest antioxidant effects. Even though most Euphorbiaceae are known to be toxic, A. cordifolia has been reported to be safe in sub-acute toxicity studies at doses ranging from 250–2000 mg/kg bw. [46]. However, the frequently used aqueous decoction of the stem barks of G. kola was shown to be toxic, with an LD50 value in the “very toxic” classification range (50–500mg/kg), whereas its methanol extract did not show noticeable toxicity effect; instead, the tendency of increasing erythrocyte number over time [47]. View the significant activities obtained herein, there is therefore need to do profound in vivo studies including toxicological analysis in the perspective of the development of a potential antityphoid phytomedicine.
Conclusion
The results obtained in this study may thus serve as preliminary scientific data on the title species in the treatment of Salmonella infections in Cameroon, as they might provide good sources of active principles that could either be used as lead molecules for the synthesis of novel drugs or for a possible standardization of a phytomedicine against salmonellosis/typhoid fever. However, this could only be possible after profound in vivo therapeutic and toxicological studies, as well chemical profiling of the potent extract.
Acknowledgments
Authors are thankful to the Cameroon National Herbarium (Yaounde) for the plant identification. The authors also acknowledge the ‘Centre Hospitalier Universitaire’ of Yaounde-Cameroon and ‘Centre Pasteur’ of Cameroon for providing the bacteria species.
References
- 1. Saravanan S, Venketaraman P, Thippichettypalayam RGKM, Kuppannan S, Palani S, Vasudevan G, et al. Molecular Epidemiology of Nontyphoidal Salmonella in Poultry and Poultry Products in India: Implications for Human Health. Indian J Microbiol. 2015;1. pmid:26843692
- 2. Okoro CK, Kingsley RA, Connor TR, Harris SR, Parry CM, Al-Mashhadani MN, et al. Intra-continental spread of human invasive Salmonella typhimurium pathovariants in sub-Saharan Africa. Nat. Genet. 2012; 44(11): 1215–21. pmid:23023330
- 3. Gal-Mor O, Boyle EC, Grassl GA. Same species, different diseases: how and why typhoidal and non typhoidal Salmonella enterica serovars differ. Front Microbiol. 2014; 5: 391. pmid:25136336
- 4.
Jay LS, Davos D, Dundas M, Frankish E, Lightfoot D. Salmonella. In: Hocking AD, editor. Foodborne microorganisms of public health significance. Sydney: Australian Institute of Food Science and Technology (NSW Branch); 2003. p. 207–266.
- 5.
Huckstep RL. Typhoid fever and other Salmonella infections. E & S Livingstone: Edinburgh, Scotland; 1962.
- 6. Gasem MH, Smits HL, Goris MG, Dolmans WM. Evaluation of a simple and rapid dipstick assay for the diagnosis of typhoid fever in Indonesia. Journal of Medicinal Microbiology. 2002; 51: 173–77.
- 7. Crum-Cianflone NF. Salmonellosis and the GI tract: More than just peanut butter. Curr Gastroenterol Rep. 2008; 10(4): 424–31. pmid:18627657
- 8. Gordon MA, Ao TT, Feasey NA, Keddy KH, Angulo FJ, Crump JA. Global burden of invasive non typhoidal Salmonella disease. Emerg Infect Dis. 2015; 21: 941–49.
- 9. Aaydha CV, Tien AN, Krishna K, Pia E, Vivek PD, Mohammad-Ali S, et al. Rapid detection of Salmonella enterica in food samples by a novel approach with combination of sample concentration and direct PCR. Biosens. Bioelectron. 2019; 129: 224–230. pmid:30318404
- 10. Global Burden of Disease. In: Global Burden of Disease Results Tool. GBD Results Tool j GHDx. 2018. http://ghdx. Healthdata.org/gbd-results-tool. Accessed 10 May 2017.
- 11.
World Health Organisation. In: Salmonella (non-typhoidal) [WWW Document]. 2018. URL http://www.who.int/en/newsroom/factsheets/detail/salmonella-(non-typhoidal).
- 12. Majowicz SE, Musto J, Scallan E, Angulo FJ, O'Brein SJ, Jones TFet al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis. 2010; 50: 882–9. pmid:20158401
- 13. Sharma P, Dahiya S1, Balaji V, Kanga A, Panda P, Das R, et al. Typhoidal Salmonellae: Use of Multi-Locus Sequence Typing to Determine Population Structure. PLoS ONE. 2016; 11(9): 0162530.
- 14. Guimarães DO, Momesso LS, Pupo MT. Antibióticos: importância terapêutica e perspectivas para a descoberta e desenvolvimento de novos agentes. Quim Nova. 2010; 33(3): 667–79.
- 15. Rastaldo R, Pagliaro P, Cappello S, Penna C, Mancardi D, Westerhof N, et al Nitric oxide and cardiac function. Life Sci. 2007; 81: 779–93. pmid:17707439
- 16. Moukette BM, Anatole PC, Biapa CPN, Njimou JR, Ngogang JY. Free radicals quenching potential, protective properties against oxidative mediated ion toxicity and HPLC phenolic profile of a Cameroonian spice: Piper guineensis. Toxicology Reports. 2015; 2: 792–805. pmid:28962415
- 17. De Diego-Otero Y, Romero-Zerbo Y, El Bekay R, Decara J, Sanchez L, Rodriguezde FF, et al. Alpha tocopherol protects against oxidative stress in the fragile X knockout mouse: an experimental therapeutic approach for the Fmr1 deficiency. Neuropsychopharmacol. 2009; 34(4): 1011–26.
- 18. Farombi EO, Britton G, Emerole GO. Evaluation of the antioxidant and partial characterisation of extracts from browned yam flour. Food Res Int. 2000; 33:493–499.
- 19. Dah-Nouvlessounon D, Adoukonou-Sagbadja H, Diarrassouba N, Sin H, Noumavo PA, Baba-Moussa F, et al. Nutritional and Anti-Nutrient Composition of Three Kola Nuts (Cola nitida, Cola acuminata and Garcinia kola) Produced in Benin. Food Nutri Sci. 2015; 6: 1395–1407.
- 20. Gustafson MHG, Stevens P, Bittrich V. Phylogeny of Clusiaceae based on rbc L sequences. Int. J. Plant Sci. 2002; 163(6): 1045–1054.
- 21. Esiegwu AC, Okoli IC, Emenalom OO, Esonu BO, Udedibie ABI. The Emerging Nutricitical Benefits of the African Wonder Nut (Garcinia Kola Heckel): A Review. GJASR. 2014; 2(2): 170–83.
- 22. Adeshina GO, Kunle OF, Onaolapo JA, Ehinmidu JO, Odama LE. Antibacterial Activity of Aqueous and Ethyl Acetate sub-fractions of Alchornea cordifolia leaf. European J Med Plants. 2012; 2(1): 31–41.
- 23. Odeyemi OA, Oluwajoba SO. Antimicrobial activity of Garcinia kola against human upper respiratory tract pathogens. Int J Res Biomedicine Biotechnol. 2011; 1(2): 5–8.
- 24. Gatsing D, Moudji ST, Kuiate J-R, Nji-Nkah BF, Fodouop SPC, Njateng GSS, et al. In vitro Antibacterial Activity of Alchornea cordifolia Bark against Salmonella Species Causing Typhoid Fevers. Ethiopian Pharm J. 2008; 26: 83–94.
- 25. Gatsing D, Christiane FNN, Nji-Nkah BF, Kuiate J-R, Tchuanguep FM. Antibacterial Activity, Bioavailability and Acute Toxicity Evaluation of the Leaf Extract of Alchornea cordifolia (Euphorbiaceae). Int J Pharmacol. 2010; 6(3):173–82.
- 26. Lunga PK, Tamokou J-D, Fodouop PCS, Kuiate J-R, Tchoumboue J, Gatsing D. Antityphoid and radical scavenging properties of the methanol extracts and compounds from the aerial part of Paullinia pinnata. SpringerPlus. 2014; 3: 302. pmid:25279277
- 27.
Harborne JB. Phytochemical Methods: A Guide of modern techniques of plants analysis. Chapman and Hall. London. p. 150, 1976.
- 28. Hao G, Shi Y-H, Tang Y-L, Le G-W. The membrane action mechanism of analogs of the antimicrobial peptide Buforin 2. Peptides. 2009; 30: 1421–27. pmid:19467281
- 29. Tang YL, Shi YH, Zhao W, Hao G, Le GW. Insertion mode of a novel anionic antimicrobial peptide MDpep5 (Val-Glu-Ser-Trp-Val) from Chinese traditional edible larvae of housefly and its effect on surface potential of bacterial membrane. J Pharm Biomed Anal. 2008; 48: 1187–94. pmid:18926657
- 30. Tsuji BT, Yang JC, Forrest A, Kelchlin PA, Smith PF. In vitro pharmacodynamics of novel rifamycin ABI-0043 against Staphylococcus aureus. J Antimicrob Chemoth. 2008; 62(1): 156–160.
- 31. Scherer R, Godoy HT. Antioxidant activity index (IAA) by the 2, 2-diphenyl-1-picrylhydrazyl method. Food Chem. 2009; 112: 654–58.
- 32. Re R, Pellegrini N, Proteggent A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999; 26(10): 1231–37.
- 33. Benzie IF, Strain J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 1996; 239(1): 70–6. pmid:8660627
- 34.
Tamokou JD, Mbaveng TA, Kuete V. Antimicrobial activities of African medicinal spices and vegetables. In: Medicinal spices and vegetables from Africa: therapeutic potential against metabolic inflammatory infectious and systemic diseases, 1st edition. Elsevier; 2017. p. 207–237,
- 35. Flora TM, Igor KV, Veronique PB, Victor K. Antibacterial activities of methanol extracts from Alchornea cordifolia and four other Cameroonian plants against MDR phenotypes. J. Taibah Univ. Med. Sci. 2016; 11(2): 121–127.
- 36. Pinto NCC, Silva JB, Menegati LM, Guedes MCMR, Marques LB, Da Silva TP, et al. Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts. An Acad Bras Ciênc. 2017; 89 (3): 2053–73.
- 37. Frirdich E, Whitfield C. Lipopolysaccharide inner core oligosaccharide structure and outer membrane stability in human pathogens belonging to the Enterobacteriaceae. J Endotoxin Res. 2005; 11: 133–44. pmid:15949142
- 38. Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev. 1999; 56: 395–411.
- 39. Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristiani M, Daniele C, et al. Antimicrob Agents Ch. 2005; 49: 2474–2478.
- 40. Naufalin R, Herastuti SR. Antibacterial activity of Nicolaia speciosa fruit extract. Int Food Res J. 2017; 24(1): 379–85.
- 41. Xing K, Chen XG, Kong M, Liu CS, Cha DS, Park HJ. Effect of oleoyl-chitosan nanoparticles as a novel antibacterial dispersion system on viability, membrane permeability and cell morphology of Escherichia coli and Staphylococcus aureus. Carbohydr. Polym. 2009; 79: 17–22.
- 42. Naufalin R, Jenie BSL, Herastuti SR. Antibacterial activity of kecombrang (Nicolaia speciosa) flower extract toward pathogenic and spoilage bacteria. Journal Teknologi dan Industri Pangan. 2005; 16(2): 119–25.
- 43. Flora O, Folasade D. Antimicrobial Effect of Phyllantus amarus and Parquetina nigrenscens on Salmonella typhi. Afr. J. Biochem. Res. 2008; 11: 215–219.
- 44. Pankey GA, Sabath LD. Clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram-positive bacterial infections. Clin Infect Dis. 2004; 38: 864–70. pmid:14999632
- 45. Anantharaman A, Rizvi MS, Sahal D. Synergy with rifampin and kanamycin enhances potency, kill kinetics, and selectivity of de novo-designed antimicrobial peptides. Antimicrob Agents Ch. 2010; 54(5): 1693–9.
- 46. Ezeokeke EE, Ene AC, Igwe CU. Sub-acute toxicity studies of Alchornea cordifolia leaf extract in Swiss albino rats. J Anal Bioanal Tech. 2017; 8: 353.
- 47. Kagbo HD, Ejebe DE. Phytochemistry and preliminary toxicity studies of the methanol extract of the stem bark of Garcinia kola (Heckel). Internet J Toxicol. 2010; 7(2).