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Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea



Melogmo Dongmo Yanickkevin, Lunga Paul Keilah, Toghueo Kouipou Rufin Marie, Djague Fred, Dize Darline and Fekam Boyom Fabrice
 
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ABSTRACT

Background and Objective: Diarrhea is a bacterial disease classified as the third leading cause of death for children under 5. Due to the frequent setbacks that surround the use of first line antibacterials, this thematic outline was designed with the aim of finding future alternative means of treatment on the basis of Cassia alata, Garcinia lucida and Alstonia boonei, medicinal plants used traditionally for the treatment of diarrhea. Materials and Methods: The various plant extracts were obtained by methanol and hydroethanol maceration and their inhibitory potential was evaluated on 5 diarrheal-causing enterobacteria by the broth microdilution method. Phytochemical screening was performed by colorimetric methods. Cytotoxicity was assessed on Vero cell line by the spectrophotometric method. The effects of the extracts on bacterial (S. flexneri) membrane destabilization and nucleotide leakage was evaluated spectrophotometrically, while the effect on loss of salt tolerance and time kill kinetics was done by enumerating colonies after treatment. Results: MIC values ranged from 500-1000 μg mL1. Phytochemical screening revealed the presence of flavonoids, phenols, anthocyanins, glycosides, quinones and steroids. The CC50 values ranged from 357.9±10.818 to 161.7±65.195 μg mL1. Methanolic extract of C. alata leaves showed its ability to destabilize the S. flexneri membrane as well as foster loss of salt tolerance. The time kill kinetics of S. flexneri showed a bacteriostatic effect up to 8h with the methanolic extracts of leaves of C. alata. Conclusion: The overall results support the traditional use of these plants and show that they could serve as potent sources of non-cytotoxic antidiarrheal phytomedicines.

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Melogmo Dongmo Yanickkevin, Lunga Paul Keilah, Toghueo Kouipou Rufin Marie, Djague Fred, Dize Darline and Fekam Boyom Fabrice, 2020. Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea. Research Journal of Medicinal Plants, 14: 53-63.

DOI: 10.3923/rjmp.2020.53.63

URL: https://scialert.net/abstract/?doi=rjmp.2020.53.63
 
Received: January 03, 2020; Accepted: January 19, 2020; Published: March 15, 2020



INTRODUCTION

Infectious diarrhea is a microbial pathology caused by several germs, which together with their produced endotoxins have the ability to destabilize the membranes of the epithelial cells of the intestines. The direct consequence is the non-adsorption of water, nutrients and solutes, leading to diarrhea1. This is the second leading cause of death for children under five, especially in developing countries, with 2.5 million deaths per year2. However, several factors contribute to the development of this pathology, namely non-compliance with hygiene rules, difficulties in accessing uncontaminated drinking water, immunodeficiency and certain chronic diseases3. The management of this disease is based on prophylaxis and curative treatment. The latter is based on the use of rehydration solutions and chemotherapy. However, these different strategies have several limitations such as resistance phenomena and undesirable side effects. These limitations raise the need for new therapeutic substances to address this problem4. Traditional medicine through the use of medicinal plants is one of the promising alternatives.

Cassia alata (Caesalpiniaceae) is a shrub with a wide range of uses. The different parts of C. alata are used in mouthwash and lotions of eczema and scab. Decoctions of wood are used for the treatment of liver infections, rhinitis and loss of appetite due to gastrointestinal problems5. In Cameroon, C. alata is used for the treatment of many infections including typhoid fever and diarrhea6. Garcinia lucida (Clusiaceae) is a small tree used in food and traditional medicine. The barks are used against gastroenteritis infections, in gynecology and also as an aphrodisiac stimulant. In the South Region of Cameroon it is considered antidote (against venom of the snake). In the South West Region it is used in combination with some plants for the treatment of typhoid6. Alstonia boonei (Apocynaceae) is a tree used in Ivory Coast for the treatment of malaria, typhoid fever, gonorrhea, asthma, dysentery. It is also applied to ulcers, snake bites, rheumatic pains and toothaches7. Due to limited and at times shallow scientific works on the titled species, this study was designed to evaluate the antibacterial and cytotoxic properties of C. alata, G. lucida and A. boonei.

MATERIALS AND METHODS

This research was carried out in the Laboratory for Phytobiochemistry and Medicinal Plants Studies of the Antimicrobial and Biocontrol Agents Unit (AmBcAU), Department of Biochemistry, University of Yaoundé I, Cameroon, from January, 2018-April, 2019.

Plant material: The samples of different parts of C. alata, G. lucida and A. bonei plants were harvested from Kumba (South West Region, Cameroon) in January, 2018. Botanical identification was done by Mr. Nana Victorat the National Herbarium of Cameroon by comparing the specimens (roots, leaves, bark) to those previously registered under reference numbers 62835HNC, 45146HNC and 43368HNC, respectively.

Extraction: The extracts were obtained by macerating each plant powder in methanol (1:10, w/v) for 72 h at room temperature according to Tchakam et al.8, with slight modifications. The mixtures were stirred twice a day and the macerates obtained were filtered using a hydrophilic cotton wool and then evaporated using a rotary evaporator (Buchi, 011) at 60°C. The process was repeated 3 times in order to deplete the plant material and the crude extracts were obtained.

Bacterial species: The in vitro antibacterial activity of the extracts was evaluated on 5 bacterial isolates including Shigella flexneri, Salmonella typhimurium, Salmonella enteretidis, Staphylococcus aureus from the Pasteur center of Cameroon (CPC) and Salmonella typhi from the ‘Centre Hospitalier Universitaire’ (CHU) of Yaoundé-Cameroon. These isolates were stored in the laboratory in tubes containing Muller Hinton agar by slant culture at 4°C.

Preparation of bacterial Inocula: The different bacterial inocula were prepared according to the standard 0.5 McFarland. For this purpose, a mother suspension was prepared at 0.5 McFarland turbidity (corresponding to a concentration of approximately 1.5×108 CFU mL1) from 24 h young cultures and then diluted to 1.5×106 CFU mL1 for the tests9.

Preparation of stock solutions of extracts and reference antibacterial: The stock solutions of extracts were prepared at 100 mg mL1 by dissolving 100 mg of extract in 1 mL of absolute DMSO. As for ciprofloxacin, it was prepared under the same conditions, at 2 mg mL1 in sterile distilled water and served as a positive control during the tests.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC): The microdilution method on Muller Hinton broth was used to test the susceptibility of bacteria. The tests were carried out on 96-well microplates according to the M07 A9 protocol described by CLSI9. For this, two-folds serial dilutions of extracts were carried out in the Muller Hinton broth to obtain volumes of 100 μL per well. One hundred microliters of a bacterial suspension (1.5×106 CFU mL1) were added into each well containing the test substances to obtain final concentration range of 1000-62.5 μg mL1. The percentage of DMSO in the first wells was 1% and showed no effect on bacterial growth. Ciprofloxacin was used as a positive control and the plates were covered and incubated at 37°C for 24 h. MICs were determined by the addition of 50 μL of INT (0.2 mg mL1) to the wells and re-incubated at 37°C for 30 min. Membrane dehydrogenases from viable cells reduce the yellow-colored dye (INT) to formazan pink. The MIC was determined as the lowest concentration of test substance which hindered bacterial growth, marked by no change in color of the medium.

MBCs were determined by sub-culturing 50 μL aliquots of the inhibitory cups (not having received INT) into the wells of sterile plates containing 150 μL of Muller Hinton broth. The plates were then covered and incubated at 37°C for 48 h. Cell viability was determined by the INT colorimetric method as above. The lowest concentration of the test substances showing no color change was considered MBC. The tests were performed in triplicate at two different occasions.

Evaluation of the phytochemical profile of the most active extracts: The extracts of the various plants were freshly prepared and subjected to a qualitative evaluation of the presence of phytochemical groups of secondary metabolites. This was done by colorimetric and/or complexation reactions as described by Bruneton10 and Harborne11.

Evaluation of the cytotoxic activities of the extracts: Vero ATCC CRL 1586 cells from the ‘Centre Pasteur’ of Cameroon (CPC) were used to evaluate the safety of extracts.

Determination of median cytotoxic concentrations (CC50): The determination of the median cytotoxic concentrations was evaluated by the MTT colorimetric method as described by Mosmann12. The mitochondrial enzymes of viable cells reduce the yellow MTT to formazan violet. Accordingly, 100 μL cell suspension titrated at 5×103 cells/well was introduced into the wells of a microplate 4 h before exposure to the extracts for adhesion and cell confluence. Subsequently the extracts (100 μL) prepared at different concentrations were brought into contact with these adherent cells and incubated at 37°C, 5% CO2 for 48 h. DMSO at 0.2-10% were used as negative and positive controls respectively, while sterility control wells contained only culture medium. At the end of the incubation time, 20 μL of MTT were introduced into all the wells and re-incubated at 37°C for 5 h. The formed formazan was dissolved in absolute DMSO and the optical densities (ODs) were measured at 570 nm using the TECAN Infinite M200 plate reader. These ODs were used to calculate cell viability percentages:

Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea

Non-linear regression curves of percentage viability against sample concentrations led to the determination of the CC50 values. The tests were carried out in triplicate.

Evaluation of the effect of extraction the destabilization of the outer membrane: The evaluation of the potential effect of the extracts on the destabilization of the bacterial membrane of S. flexneri was carried out according to the protocol previously used by Oliveira et al.13 with some modifications. Cells from 24 h cultures titrated at 1.5×106 CFU mL1 were incubated with extracts at MIC, 2MIC and 4MIC in 96-well microplates at 37°C for 24 h. The optical densities were measured at 405 nm (wavelength at which the complex form between Lipopolysaccharides and membrane stabilizing divalent cations absorbs) via a Tecan Infinite M200 plate reader. These ODs made it possible to calculate the percentage of membrane destabilization. The tests were performed in triplicate:

Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea

Evaluation of the effect of extract on nucleotide leakage: The test was carried out according to the protocol previously used by Oliveira et al.13. Briefly, an overnight culture of S. flexneri was washed in sterile physiologic water (2 mL of 0.9% NaCl) and the resulting solution centrifuged at 10 000 rpm for 10 min. After this time, the supernatant was discarded and the resulting pellet re-suspended in 10 mM PBS (pH 7.4) and the turbidity adjusted to 0.5 McFarland (1.5x108 CFU mL1). The inoculum (100 μL) was introduced into 100 μL of MHB containing each extract at varying concentrations (MIC, 2MIC, 4MIC) and incubated at different times intervals (0, 2, 4, 6, 8 and 12 h). 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.

Evaluation of the effect of extraction the loss of salt tolerance: The ability of S. flexneri to form colonies in the presence of extracts on NaCl-supplemented MHA was evaluated according to the protocol previously used by Etame et al.14. In effect, a preliminary test was carried out by culturing the bacterium at 37°C for 24 h on MHA supplemented with NaCl at different concentrations (10-100 mg mL1). At the end of this incubation period, the number of colonies was counted and NaCl concentrations that did not affect the growth of the microbe were selected. Subsequently the cells (0.5 McFarland) were mixed with the C. alata methanol leaf extract at different concentrations (MIC, 2CMI and 4MIC) followed by incubation for 1 h at 37°C. The content of each well was subcultured on MHA supplemented with NaCl at the selected concentrations (60, 70 and 80 mg mL1). The petri dishes were incubated at 37°C for 24 h and the number of colony forming units (CFU) was plotted against the extract and NaCl concentrations. The tests were carried out in triplicate.

Evaluation of the time kill kinetics of C. alata methanol leaf extract on S. flexneri: The time killing kinetic of the C. alata methanol leaf extract was performed according to the method described by Klepser et al.15 with slight modifications. 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. flexneri suspension (1.5×106 CFU mL1) 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. Wells containing the bacteria incubated with MHB were used as growth controls. The test was performed in triplicate and results were presented as Mean±SD.

Statistical analysis: Data were analyzed by the One-way analysis of variance (ANOVA) using the Statistical package for social science (SPSS) software version 16.0. The GraphPard Prism.7 software was used to calculate the CC50 using the nonlinear regression curve. The results were expressed, where appropriate, as Mean±SD. The differences between the means were compared by the Waller Duncan test at 95% confidence (p<0.05).

RESULTS

Extraction yields: The extraction yields of the extracts according to the plant part and the solvent are grouped in Table 1. The leaves of C. alata (34.2%) and leaves of A. boonei (49.29%) contain more secondary metabolites extractable by methanol.

Anti-bacterial activity of the methanolic and hydroethanol extracts of C. alata, G. lucida and A. boonei: The Minimum inhibitory concentration values vary from 500-1000 μg mL1, while the minimal bactericidal concentrations was 500 μg mL1 for the methanolic extracts of G. lucida bark on 40% of the pathogens (Table 2). G. lucida extracts were active on all bacterial isolates tested. The best MIC (500 μg mL1) was obtained with the bark methanolic extracts, on S. typhi, S. aureus, S. typhimurum and S. enteritidis while S. flexneri was the least sensitive with a MIC of 1000 μg mL1. Extracts of C. alata were less active than those of G. lucida, with best activities on S. typhi (MIC of 500 μg mL1). However, MBCs of the methanolic and hydroethanolic extracts of the leaves, barks and stems of C. alata were higher than the tested concentrations (1000 μg mL1). The activity of the various extracts tested was less important than that of ciprofloxacin (reference antibiotic) with MICs between 0.039-0.078 μg mL1 depending on the isolates.

Phytochemical profiles of active extracts: The results of the phytochemical screening of the various extracts show the presence of three classes of secondary metabolites, phenolic compounds, terpenes and glycosides (Table 3). Flavonoids and phenols were present in all extracts. In addition to the above two groups of metabolites, G. lucida extracts contained quinones and tannins, on the other hand, the hydroethanolic extracts of C. alata were rich in anthocyanins, glycosides and tannins. In addition, anthocyanins, glycosides, quinones and steroids were present in the methanol extracts of the leaves of C. alata unlike those of the stems that contained quinones and steroids.

Table 1:
Extraction yield as a function of plant part and solvent
Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea

Table 2:
Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) in (μg mL1) of selected extracts
Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea
*Not determined (MBC>1000 μg mL1)

Table 3:Qualitative phytochemical constituents of extracts of G. lucida and C. alata
Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea
+: Present, -: Absent

Table 4:
Median cytotoxic concentrations of extracts
Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea
Values carrying the same letter superscripts are not significantly different (p>0.05), Waller Duncan

A critical observation of these results showed that either a combination of quinones and tannins is vital to the antibacterial activities of these extracts or specific active principles are found in the G. lucida extracts.

Cytotoxic activity of active extracts: The results of the cytotoxic activity showed that the median cytotoxic concentrations (CC50) range from 357.9±10.81 to 161.7±65.19 μg mL1 (Table 4). The methanol extracts from C. alata twigs showed lower CC50 compared to methanolic and hydroethanolic extracts of leaves and stems of C. alata. The methanolic extract of G. lucida stem bark had significantly higher (p<0.05) CC50 and therefore less cytotoxic compared to the rest of the extracts.

Effect of extracts on the destabilization of the outer membrane of S. flexneri: Figure 1 shows the variation of destabilization percentages of the outer membrane of the bacterium as a function of the concentrations of different extracts. It can be noted that only the methanolic extracts of C. alata leaves showed a mechanism of membrane destabilization by the chelation of divalent cations. The degree of destabilization of the outer membrane of the bacterium was concentration-dependent and was significantly higher (p<0.05) than that of polymyxin at the corresponding highest concentrations (4 MIC).

Effect of C. alata methanol leaf extract on membrane lysis: The period variation of the optical densities of supernatants collected after treatment of S. flexneri isolates with different concentrations of the methanolic extract of the leaves of C. alata is presented in Fig. 2.

Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea
Fig. 1:
Curve of the destabilization (%) of the outer membrane of S. flexneri by the active extracts
 
GLB (MeOH): Methanolic extract of G. lucida bark, CAB (EtOH/H2O): hydroethanolic extract of C. alata bark, CAL (MeOH): Methanolic extract of leaves of C. alata, CAS (MeOH): Methanolic extract of C. alata stems, Polymyxin: Positive control, MIC: Minimum Inhibitory concentration

Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea
Fig. 2:Variation of the optical density as a function of the concentration of the methanolic extract of the leaves of C. alata with time
  MIC: Minimum inhibitory concentration, CN: Negative control, CP: Positive control (Ciprofloxacin)

It shows that the extract did not cause any membrane damage materialized by the absence of the release of the nucleic acids (DNA) in the extracellular medium. Indeed, an increase in the optical density at 260 nm would translate a release of nucleic acids (DNA) in the medium. Compared with growth control, no variation was observed, meaning that this extract would not exert its antibacterial activity by this mechanism.

Effect of extract on loss of salt tolerance: The potential of salt tolerance of S. flexneri in the presence of C. alata leaf extract at different concentrations is shown in Fig. 3. These results showed that the number of colony forming units (CFU) of the bacterium simultaneously depends on the extract as well as the salt concentrations. For a given salt concentration, this number CFU decreases significantly (p<0.05) with increase in extract concentration. Likewise, for a given extract concentration the CFU decrease significantly (p<0.05) with the salt concentration. Thus, the C. alata leaf extract at 2MIC and 80% salt concentration reveals the best inhibitory activity on the S. flexneri isolate. This shows the inability of S. flexneri to tolerate the presence of NaCl in its growth medium.

Time kill kinetic effect of methanolic extract of leaves of C. alata on S. flexneri: Due to the fact that ethanolic extract of leaves of C. alata was the only extract with mechanism of action on the destabilization of the outer membrane of S. flexneri, its effect on the kinetics of mortality was evaluated on this isolate. For different extract concentrations, the number of colonies was evaluated at different times and presented in Fig. 4 to determine the time when the destabilization of the membrane by the extract was maximal.

Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea
Fig. 3:
Variation of the number of S. flexneri colonies as a function of the concentration of extract and NaCl
 
For the same salt concentration, bars with same letters are not significantly different (p 0.05), for the same extract concentration, bars with same Greek alphabet are not significantly different (p 0.05), Waller Duncan test, MIC: Minimum inhibitory concentration, NC: Negative control

Image for - Antibacterial and Cytotoxic Activities of Three Medicinal Plants from Cameroon (Alstonia boonei, Cassia alata and Garcinia lucida) Against Diarrhea
Fig. 4:
Time kill kinetics of S. flexneri as a function of the concentration of methanolic extract of the leaves of C. alata
 
MIC: 1000 μg mL1, CP: Positive control (ciprofloxacin 0.078 μg mL1), NC: Negative control

With regards to this figure, it was found that the methanol extract of the leaves of C. alata at concentrations of MIC, 2MIC, 4MIC and 8MIC affected bacterial growth during first 8 h following initial contact with the microbe whereas the effect of ciprofloxacin (0.039 μg mL1) was felt throughout the experimental period (Fig. 4). The membrane destabilization effect of the extract was concentration-dependent and was maximum at 8 h, after which there was resurgence of the bacterial growth. After this resurgent time, the activity of the extract was no longer concentration-dependent, as there was no significant difference in the rate of increase of the colonies. This suggested that this resurgent time could be considered as the time interval for the re-administration of the extract in a subsequent in vivo evaluation of its therapeutic efficacy.

DISCUSSION

The antibacterial parameters obtained show the inhibitory potential of the methanolic extract of the bark of G. lucida and the methanolic and hydroethanolic extracts of the leaves, stems and barks of C. alata. According to the classification scale defined, all the tested pathogens were susceptible to the different methanolic extracts of G. lucida barks as well as ethanolic and hydroethanolic bark, leaves and stems of C. alata. Ethanolic extracts of the barks of G. lucida as as well as extracts from different parts of C. alata presented MIC values varying from 500-1000 μg mL1. The antimicrobial activity of a plant extract is considered to be highly active if the MIC<100 μg mL1, significantly active when 100<MIC <512 μg mL1, moderately active when 512<MIC<2048 μg mL1, weakly active if MIC> 2048 μg mL1 and not active when MIC16>10 000 μg mL1. Therefore, the extracts of G. lucida bark exhibited significant activities on all the tested microbes, while those of C. alata showed significant to moderate activities as a function of the bacterial species. These results are in agreement with those of Momo et al.17 who obtained a MIC range of 128-256 μg mL1 respectively, on S. typhi and S. aureus strains with methanolic extracts of G. lucida bark. In contrast, Dzoyem et al.18 showed that the methanolic extracts of leaves of G. lucida have a MIC of 500 μg mL1 on a strain of S. aureus. Pissang et al.19 obtained a MIC of 1250 μg mL1 with alcohol extracts from C. alata leaves on S. aureus and Promgool et al.20 obtained substantially the same results on S. typhimurum strain with MICs ranging from 620-1280 μg mL1 depending on the parts.

The presence of total phenols, flavonoids, tannins, quinones, glycosides and terpenoids in the methanolic extracts of G. lucida bark and all the methanolic and hydroethanolic extracts of C. alata may explain the source of antibacterial activities of these extracts. The variability of these groups of secondary metabolites which may be influenced by environmental factors21 as well as the modes of action could justify the differences in activities recorded with the extracts. The richness of these extracts in secondary metabolites having diversified modes of action justifies the activities obtained. Indeed, phenolic compounds such as tannins, flavonoids, simple phenols, quinones have been shown to possess inhibitory effects on the alteration of membrane structures22. Quinones have the ability to irreversibly complex with nucleophilic amino acids and proteins, leading to the loss of their functions23. Furthermore, phenols have chemical and biological effects known to be due to the redox system, phenol/semiquinone/quinone and the semiquinone intermediate is therefore the basic molecule responsible for chemical and biological effects24. The antimicrobial mechanisms of several flavonoids could be attributed to the inhibition of nucleic acid synthesis25-27. Tannins have the ability to induce chelation with proteins4. Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, membrane enzymes and membrane transport proteins28. These results are in agreement with those obtained by Karthika et al.29 who showed the presence of phenolic compounds in the methanolic extracts of leaves of C. alata. Sylvie et al.30 demonstrated the presence of phenols and flavonoids in the methanolic extracts of fruits and barks of G. lucida, while Owoyale et al.31 showed that anthocyanins were absent in extracts of C. alata. Sylvie et al.30 and Ramaraj et al.32 showed the presence of steroids in leaf extracts of C. alata and methanolic extracts of bark and fruit of G. lucida, respectively.

The search for new drugs for the treatment of pathologies is increasingly critical because of undesirable side effects such as the cytotoxicity of isolated, semi-synthetic or synthetic substances. Thus, the methanolic extracts of G. lucida bark and all the methanolic and hydroethanolic extracts of the bark, leaves and stems of C. alata were evaluated for cytotoxicity on Vero cell line and theirCC50 values varied from 357.95±10.81 to 161.7±65.19 μg mL1. According to the American National Cancer Institute33, all the extracts tested were not cytotoxic since their CC50 values were greater than 30 μg mL1. To our knowledge, no scientific research has been done on the cytotoxicity of these plants with the Vero cell line. Nevertheless, Arthanari et al.34 showed that the CC50 of the methanolic extract of C. grandiflora flowers was greater than 20 μg mL1.

The resistance of most Gram-negative bacteria is due in part to the outer membrane which is a semi-permeable barrier to different antibiotics35. The measurement of membrane permeability of bacteria is therefore essential in the study of the mode of action of antibiotics36. The literature shows that the most lipophilic flavonoids can disrupt bacterial membranes by destabilizing them23. In addition, phenolic compounds are known as chelators of bivalent cations for bridging LPS. This chelation induces the release of LPS thus destabilizing the outer membrane of bacteria35,37. Thus, the presence of flavonoids in the leaves of C. alata could explain the destabilization of the outer membrane of S. flexneri as presented above. These results are in agreement with those obtained by Puupponen-Pimia et al.38, which showed that gallic acid isolated from Caesalpiniamimosoides has the ability to permeabilize the outer membrane of the Salmonella typhimurium strain by chelating the divalent cations. Although, the other extracts were rich in flavonoids, tannins, quinones and phenols capable of complexing with membrane proteins and divalent cations28, they did not show destabilization of the bacterial membrane, probably due to low concentrations and/or presence of non-destabilizing specific metabolites of the above classes. They would therefore certainly act by other mechanism(s) such as intracellular efflux of K+, chelation with nucleic acids, lysis of cellular proteins.

As food is the main source of diarrhea-causing bacteria, some food industries use saline solutions for food preservation. However, several studies have shown a development of salt tolerance by these bacteria. The loss of salt tolerance by the bacterium S. flexneri after treatment with methanolic extracts of leaves of C. alata may explain the ability of the extracts to disrupt the expulsion of salts from the cell, which is associated with the alteration bacterial membrane14,39. This alteration could induce the permeability of the membrane; affect cellular exchanges and the inadequate regulation of cell osmosis as well as the exclusion of toxic elements. The consequence of loss of salt tolerance and other toxic molecules can be used to demonstrate damage to the bacterial membrane40. This induction of loss of salt tolerance by C. alata leaf extract can be beneficial for the treatment of ailments caused by bacteria ingested from salt-preserved foods.

Membrane damage can induce permeability of the membrane, thereby promoting the release of cellular content such nucleic acids. The release of nucleic acids thus makes it possible to evaluate the degree of damage of the membrane. From the results, no continuous increase of optical density was recorded with concentration and time, indicating that the extract does not act by membrane lysis.

Time-kill assays allow antibacterial agents to be classified as bacteriostatic or bactericidal and characterize the relationship between agent concentration and activity over time. Measurement of bacterial kinetics showed a concentration-dependent retardation in cell growth with extract. The extract’s effect lasted during the first 8 h, after which a resurgence of the microbe was observed indicating its bacteriostatic effect, which in turn gives the immune system of the host the time needed to clear the microbes from the system41. Keeping aside toxicological studies, a phytomedicinal preparation of this plant could be administered at 8 h intervals.

This study shows that these extracts are not toxic to human cells, provides scientific data in support of the traditional use of these plants in the treatment of bacterial infections and suggests that doses should be repeated at 8 h intervals (3 times daily). However, more work needs to be carried out to demonstrate their in vivo therapeutic efficacies as well as their toxicological profiles.

CONCLUSION

The study revealed that the extracts of G. lucida and C. alata possess phytochemicals exhibiting significant (MICs ranging from 500-1000 μg mL1) bacteriostatic activities within the first 8 h following administration and are non- cytotoxic. In addition, the mode of action of the C. alata methanol leaf extract was achieved through the destabilization of the outer membrane of bacteria and/or prevention of salt expulsion from the bacterial cells. These results showed that the title species could offer great perspective in the development of antibacterial phytomedicines.

SIGNIFICANCE STATEMENT

This study discovers the outer membrane destabilization effect of C. alata methanol leaf extract on human pathogenic bacteria that could be very beneficial in the elucidation of the mechanisms of action of its phytochemicals. It also demonstrates the inability of S. flexneri to tolerate the presence of NaCl in its growth medium when supplemented with the extract that many researchers were not able to explore. This study will help researches to unveil new strategies in the incorporation of salt in phytomedicinal formulations. Thus a more efficient way of preparing phytomedicines may be arrived at.

ACKNOWLEDGMENT

Authors are very grateful to the Seeding Labs’ Instrumental Access Grant (SL2012-2) to Prof. Fekam Boyom Fabrice. 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 author would like to thanks the Research Journal of Medicinal Plants for publishing this article FREE of cost and to Karim Foundation for bearing the cost of article production, hosting as well as liaison with abstracting & indexing services, and customer services.

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