Antioxidant and Antimicrobial Capacities of Two Medicinal Plants Used against Urinary Infections in Burkina Faso ()
1. Introduction
Urinary tract infections (UTIs) are one of the most common bacterial infections [1]. Indeed, UTIs affect approximately 250 million people annually worldwide, and are the main cause of medical visits which impose a substantial financial burden on society [2]. Antibiotic therapies remain the most effective approach for the management of UTIs [3].
Unfortunately, many therapeutic failures are observed because of the abusive and inappropriate uses of antibiotics [4]. Recently there is a resurgence of interest in phytochemicals, to search for new safe potential inhibitors, due to the emergence of multi-antibiotic-resistant pathogens [5]. Phenolic compounds (PCs) are among the most phytochemicals explored because they have enormous scope of biological effects [6].
PCs include classes of molecules known as tannins, lignins, and flavonoids. That exhibited various physiological activities, including anti-inflammatory, antioxidant, anti-carcinogenic, antihypertensive, anti-arthritic, and antimicrobial [7]. PCs could be also a good supplement during antibiotic therapy that may accelerate the antibacterial action, as well as inhibiting oxidative damages generated by the action of antibacterial agents [5].
Numerous ethnobotanical surveys carried out worldwide have revealed that various medicinal plants are commonly used to treat UTIs [8] [9] [10]. Euphorbia hirta L. (E. hirta) and Terminalia avicennioides GUILL & PERR (T. avicennioides) are two plants frequently used in the management of UTIs in the Hauts-Bassins areas of Burkina Faso. Those plants are widely applied as hypoglycemic, antimicrobial and they are used for treating inflammatory diseases, parasitosis, cough and rheumatism [11]. The aim of this study was to investigate the antioxidant and antibacterial effects of extracts and fraction from E. hirta and T. avicennioides.
2. Materials and Methods
2.1. Standards and Reagents
The Folin-Ciocalteu reagent, NaH2PO4, Na2HPO4, sodium carbonate, aluminum trichloride, gallic acid, tannic acid and quercetin were purchased from Sigmaaldrich chemie, Germany. 2,2-diphenylpicrylhydrazyl (DPPH), trichloroacetic acid, and solvents used were from Fluka Chemie, Switzerland. Potassium hexacyanoferrate [K3Fe(CN)6] was from Prolabo and ascorbic acid was from Labosi, France. All chemicals and solvents used were of analytical grade.
2.2. Plant Collection
The leaves stem barks, roots of T. avicennioides and the whole plant of E. hirta were collected during the month of May 2022 from the classified forest of Dinderesso, a village located about 15 km west of Bobo-Dioulassso/Burkina Faso. The plants were identified by Dr Ouaba Yempabou Hermann a botanist-cytoecologist from the University NAZI Boni of Bobo-Dioulasso. The organs of plants collected were washed, dried in the shade and then powdered for further analysis.
2.3. Microbial Strains
Standard reference strains, including Escherichia coli (ATCC 25922), Escherichia coli (ATCC 35218), Klebsiella pneumoniae (ATCC 700603) and Staphylococcus aureus (ATCC 29213). Clinical strains from suspected infected with UTIs patients, including Staphylococcus aureus, Klebsiella pneumoniae (n = 4), Escherichia coli (n = 11), Enterobacter aeroginosa, Acinetobacter baumannii (n = 2), Klebsiella oxytoca, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus haemolyticus (n = 2), Streptococcus agalactiae and Candida albicans (n = 3). All the strains were obtained from the bacteriology research laboratory of the Muraz center/Burkina Faso. A previous antibiogram identified bacterial isolates of ESBL (4 K. pneumoniae et 2 E. coli) and methicillin-resistant (2 Staphylococcus haemolyticus) phenotypes.
2.4. Preparation of Extracts
Decoction: 25 g of each plant powder were extracted with 250 ml of distilled water. The mixture was heated and boiled under reflux at 100˚C for 30 min. After filtration, the extracts were frozen and lyophilized for obtaining the aqueous crude extract.
Maceration: 25 g of each powdered plant samples were extracted with aqueous acetone (80%). After 24 hours of mechanical stirring at room temperature, the acetone was removed using a rotary evaporator at 60˚C, and the rest was lyophilized for corresponding to hydro-acetonic crude extract.
Fractionation: crude extracts was made by successive liquid-liquid fractionation with an equal volume of n-Hexane, Dichloromethane, Ethyl acetate and n-Butanol. The extracts were concentrated to dryness and stored at 4˚C until being used.
2.5. Phenolic Compounds Content
Total Phenolic and total flavonoids: Total phenolic and total flavonoids were assessed using the Folin-Ciocalteu and aluminum chloride reagents, respectively as described previously [12]. The results were expressed in mgEAG/100mg and mgEQ/100mg extract, respectively.
Total Tannins: Tannin estimation was done by treating extracts with vanillin acid [13]. The results were expressed in mgTAE/100mg extract.
2.6. Evaluation of Antioxidant Properties
The free radical (DPPH) scavenging capacity and the ferric reducing power (FRAP) of extracts and fractions of both plants were evaluated as described previously by other author [12].
2.7. Antimicrobial Testing
Antimicrobial activities of plant extracts were carried out by applying the disc diffusion method [14]. The evaluation of the efficacy of the extracts was made according to the criteria from a previous study [15].
2.7.1. Determination of Antibacterial Parameters
Minimum Inhibitory Concentration (MIC): The MIC values were determined using a broth microdilution assay as described earlier [16]. The lowest concentration of extract required for inhibiting the growth of bacteria was recorded as the MIC.
Minimum Bactericidal/Fungicidal Concentration (MBC/MFC): The MBC/ MBF was determined from the positive MIC well, according to other study [17]. The MBC/MBF value was estimated as the lowest concentration of extract that inhibits 98% - 99.9% of microbes.
The MBC/MIC ratio defines the mode of action of the substance. The effect is bactericidal if the ratio is below 4, bacteriostatic if it is over 4 or tolerant if it exceeds 32 [18].
2.7.2. Synergy Assay
The double disc method was used to explore the synergistic action between antibiotic discs with an extract. Antibiotic discs uniquely and the disc combination with 20 µl of extract were put apart on a Mueller-Hinton agar plate which was inoculated with pathogens of 0.5 McFarland turbidity. After 24 h incubation at 37˚C, a positive interaction is suggested by the enlargement of the size of inhibition zones [19].
2.8. Statistical Analysis
The analysis was performed in triplicate and the results were expressed as mean value ± standard error. The direction and the magnitude of the correlation between the variables were calculated using analysis of variance (ANOVA test) and Pearson’s correlation test (r2). The criterion for statistical significance was p ≤ 0.05.
3. Results
3.1. Polyphenols Content
Variable contents in phenolics, flavonoids and total tannins from crude extracts and fractions of E. hirta and T. avicennioides are shown in Table 1. The n-Butanol
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Table 1. Phenolics contents of E. hirta and T. avicennioides extracts.
Values with superscript letters a mean highest content.
fraction (145.14 ± 1.37 GAE/100mg extracts) from E. hirta and the Aqueous fraction of stem barks from T. avicennioides (81.41 ± 0.91 mg GAE/100mg extracts) had high levels of phenolic. For total flavonoids, the best contents (23.56 ± 0.68 and 18.32 ± 0.17 mg QE/100mg extracts) were obtained with ethyl acetate and n-Butanol fractions of E. hirta, respectively. Total tannins were high in Aqueous acetone extract (11.18 ± 0.31 mg TAE/100mg extracts) as well as Aqueous fraction (11.12 ± 0.28 mg TAE/100mg extracts) and n-Butanol fraction (10.60 ± 0.45 mg TAE/100mg extracts) of T. avicennioides stem barks.
3.2. Antioxidant Capacity
Free radical scavenging (DPPH) and Ferric Reducing Antioxidant Power (FRAP) methods have been used to measure the antioxidant activity of E. hirta and T. avicennioides extracts (Table 2). In DPPH assay the results ranged from 39.14 ± 8.95 µmol AAE/g extract (n-Hexane fraction of E. hirta) to 920.06 ± 41.28 µmol AAE/g extract (Ethyl Acetate fraction of T. avicennioides roots). Concerning the ferric reducing capacity of extracts, the values varied between 373.37 ± 12.29 µmol AAE/g extract (Dichloromethane fraction of T. avicennioides leaves) and 12399.21 ± 196.75 µmol AAE/g extract (n-Butanol fraction of E. hirta).
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Table 2. Antioxidant activity of E. hirta and T. avicennioides extracts.
Values with superscript letters a mean greatest activity.
3.3. Antimicrobial Activity
3.3.1. Inhibition Zone Diameters of Crude Extracts
The effectiveness of crude aqueous and aqueous acetone extracts of E. hirta and T. avicennioides has been evaluated against 32 causative pathogens of urinary tract infections. It was found an insignificant antimicrobial activity with E. hirta extracts (Table 3). The extracts of T. avicennioides were effective against 16 strains including 13 bacteria (8 Gram-negative and 5 Gram-positive) and 3 yeasts. The diameters of inhibition zones varied between 7 mm and 16 mm according to the type of plant, organs, extract and microbial strains tested. Aqueous extracts of different parts of T. avicennioides and aqueous acetone extracts of leaves and roots of the same species were demonstrated the best results (diameters ≥ 15) against methicillin-resistant Staphylococcus haemolyticus and Candida albicans strains. Of all these extracts, the aqueous extract of T. avicennioides leaves was identified as the most effective.
3.3.2. Efficiency of Fractions of Aqueous Extract of T. avicennioides Leaves
The fractions at 10 mg/ml of leaves extract of T. avicennioides exhibited variable degrees of antimicrobial activity against certain strains (Table 4). The aqueous fraction of leave was presented the best diameters of inhibition (from 13 mm to 16 mm) against S. aureus ATCC 29213, S. aureus and the two methicillin-resistant S. haemolyticus strains.
3.3.3. Antibacterial Parameters
The values of MIC and MBC/MFC have been determined for the strains which were most susceptible to the aqueous fractions of T. avicennioides leaves. The
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Table 3. Antimicrobial activity of crude aqueous and hydro-acetonic extracts at 50 mg/ml.
AqAc = Aqueous acetone; Aq = Aqueous; A = k. pneumoniae strain1; B = k. pneumoniae strain2 C = E. coli strin3; D = Enterobacter aeroginosa; E = Acinetobacter baumannii strain1 F = Acinetobacter baumannii strain2; G = Klebsiella oxytoca; H = Pseudomonas aeruginosa; I = Proteus mirabilis; J = S.aureus ATCC 29213; K = S. aureus; L = methicillin-resistant Staphylococcus haemolyticus strain1; M = methicillin-resistantStaphylococcus haemolyticus strain2; N = Streptococcus agalactiae; O = Candida albicans strain1; P = Candida albicans strain2; Q = Candida albicans strain 3.
aqueous fraction of leaves has shown a bactericidal effect with a concentration of MIC (5 mg/ml) and of MBC (10 mg/ml) against strains recorded in (Table 5).
3.3.4. Synergy Assay
Table 6 indicate the interaction between 9 antibiotic discs and aqueous fraction of T. avicennioides leaves against methicillin-resistant S. haemolyticus 1 and 2; S. aureus and S. aureus ATCC 29213. The extract has exhibited synergistic effects
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Table 4. Antimicrobial activity of fractions from leaves aqueous crude extracts.
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Table 5. Antibacterial parameters of aqueous fraction of T. avicennioides leaves.
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Table 6. Mean zone of inhibition (mm) of different antibiotics (without and with extract) against bacteria.
A = Inhibition zone of disc without extract; B = Inhibition zone of disc associated to extract; zones of inhibition highlighted mean a synergetic effect.
with some antibiotic. The best improvement of the diameter was obtained with Penicillin G (10 µg) and Tetracycline (30 µg) followed by Cefoxitin (30 µg).
4. Discussion
The quantitative analysis of total phenolic, flavonoids and tannins contents ofE. hirta and T. avicennioides indicated variable contents with the vegetable species, parts and solvent used. This result could be linked to extraction solvent polarity, environmental conditions and genetic factors. The greatest polyphenol content was found in n-Butanol fraction from E. hirta. Ethyl acetate fraction of E. hirta yielded the best amount in total flavonoids. Aqueous acetone extract from T. avicennioides stem barks was most riche in total tannins. Many beneficial effects derived from phenolics have been attributed to their antioxidant activity [20]. Several analytical methods have been carry out to explore the antioxidant properties of plant extracts [21].
In this study free radicals scavenging (DPPH) and ferric reducing antioxidant power (FRAP) methods have been used to assess the antioxidant activities of crude extracts and fractions of E. hirta and T. avicennioides. All the extracts demonstrated antioxidant activities. The best antioxidant capacities were obtained with Ethyl Acetate fraction from T. avicennioides roots in DPPH assay; and n-Butanol fraction from E. hirta in FRAP testing. These activities within the extracts could be due to high correlations between polyphenols contents and antioxidant capacity [22] [23]. Thus, the direction and the magnitude of the correlation were calculated in order to estimate the correlation between the phenolics contents and free radicals scavenging activity or ferric reducing power.
In this work, a high correlation was demonstrated between the total polyphenol content and DPPH scavenging activity (p = 3.089e−12, r2 = 0.7281619) or ferric reducing power (p < 2.2e−16, r2 = 0.9508803). There was a moderate correlation of total flavonoids levels with antioxidant capacity in DDPH method (p = 7.405e−07, r2 = 0.4257956) and FRAP assay (p = 7.339e−10, r2 = 0.5629441). A weak correlation of total tannins was shown with Both DPPH (p = 0.1035, r2 = 0.1468837) and FRAP (p = 0.02893, r2 = 0.2163953) antioxidant tests. This variation could be explaining by some phenolic intrinsic factors such as: structure-activity relationships, polymerization grade and possible synergy or antagonism among different classes of compounds [24]. Antioxidant properties of phenolic compounds could be an advantage for further anti-bacterial compounds investigation.
Regarding antibacterial activity, crude extracts (at 50 mg/ml) and a fraction (at 10 mg/ml) from E. hirta and T. avicennioides have been evaluated against 32 causative pathogens of urinary tract infections. The extracts of T. avicennioides were effective against 16 strains. This difference in results between the two species could be linked to the environmental conditions of both plants and the polarity of the extraction solvents. Of all extracts from T. avicennioides, aqueous fraction of T. avicennioides leaves allowed the best results (diameter from 13 mm to 16 mm) against S. aureus ATCC 29213, S. aureus and 2 methicillin-resistant Staphylococcus haemolyticus strains. This result could be explained by a concentration of the active molecules in this fraction.
A further antibacterial parameters study revealed the bactericidal effect (CMB/ CMI < 4) of this fraction. Other studies realized in Nigeria have also proven anti-methicillin resistant activity of certain extracts from T. avicennioides [25] [26]. These results prove anti-methicillin resistant property of T. avicennioides and its potency to be efficient in inhibiting the UTI causing pathogens. The antimicrobial activity of this plant could be attributed to the presence of tannins, flavonoids and phenols.
Aqueous fraction of T. avicennioides leaves was then used to screen synergistic action with antibiotic discs. It was found in this study the increasing of the size of some inhibition zones, involving the synergic effect. Several studies have reported the potentiation of antibiotic effect with phytochemicals [27] [28]. This could be due to the mechanism of action of drugs against organisms used and proper selection of natural compounds [29]. There is a need for more studies concerning the molecular basis of synergistic interactions for the development of novel therapies against methicillin-resistant and multi-resistant strains in general.
5. Conclusions
The present study revealed that E. hirta and T. avicennioides exhibited variable degrees of antioxidant potentials in DPPH and FRAP assays.
Great to weak correlation were demonstrated between this activity and total phenolics, flavonoids and tannins contents. The antimicrobial activity of these plants could be attributed to the presence of phenolics.
The extracts of T. avicennioides were effective against 16 causative pathogens of urinary tract infections; and its leaves allowed bactericidal effect against S. aureus ATCC 29213, S. aureus and 2 methicillin-resistant Staphylococcus haemolyticus strains. Further studies must be done in order to identify the bioactive molecules. T. avicennioides leaves could be recommended as an herbal drug formulation for treating UTI.
Acknowledgements
The authors are grateful to the authorities of Université Nazi Boni and Institutes of Research in Sciences of health (IRSS, Centre MURAZ).