In developing countries such as Benin, infections are serious public health problems that threaten populations because of their frequency and severity associated with low access to health care .
The use of synthetic antibiotics is one of the most effective means available for humanity to face these microbial invasions. Unfortunately, today there is an increasingly growing resistance of germs against these antibiotics . It is important to notice that the most affected by these infections have almost no access to primary health care. They usually use traditional medicine whose effectiveness is most of the time devoid of scientific evidence . Thus, it is urgent to screen traditional medicines to assess their effectiveness and serve as the start point for the discovery of new molecules that can enrich the current armamentarium.
Desmodium ramosissimum is consumed as food but it is also used in traditional medicine against various infections including salmonellosis, candidiasis, diarrhoea, abdominal pain, wounds, diphtheria, dysentery, haemophilia, hepatitis and pneumonia . This study aims to verify if beyond its food use, this plant could help support the above pathologies. It therefore proposes to evaluate the antibacterial and antifungal activity of aqueous, methanol and ethanol extracts of the leaves and stems of Desmodium ramosissimum. These microbiological tests covered several bacteria such as staphylococci, Candida albicans, Pseudomonas aeruginosa, Proteus mirabilis, Micrococus luteus, Streptococcus oralis, Proteus vulgais, Enterococcus foecalis and, Escherichia coli involved in several diseases that this plant is assumed to treat. The knowledge of the chemical composition of this plant is also part of this study’s objectives.
2. Material and Methods
2.1. Collection of Plant Material
The leaves and stems of D. ramossisimum were collected in March 2018 in the town of Porto-Novo. Once in the laboratory, the leaves and stems were washed with water then dried at a temperature of 25˚C ± 3˚C for 16 days. They were then powdered using a Retsch mill type SM 2000/1430/Upm/Smfet. The powder obtained was used for the identification of bioactive chemical groups and for the preparation of different extracts.
2.2. Microbial Strains
The microorganisms used include both bacteria and yeast
- Ten reference strains including five Gram + bacteria (Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis T22695, Micrococcus luteus ATCC 10240, Streptococcus oralis NCTC 8029 and Enterococcus foecalis ATCC 29212), four bacteria Gram-(Pseudomonas aeruginosa ATCC 27853, Proteus miriabilis A24974, Proteus vulgaris A25015, Escherichia coli ATCC 25922) and one yeast (Candida albicans MHMR).
- Four strains of Staphylococcus genus (S. hominis, S. simulans, S. aureus, S. xylosus) Isolated from meat products of food origin .
- Three clinical strains of the multi-resistant Staphylococcus genus isolated from urine (S. hemolyticus) and blood (S. hemolyticus and S. lungdunensis) of patients at National Hospital and University Center of Cotonou .
All these tested strains belong to the collection of Biology and Molecular Typing Microbiology Laboratory.
2.3. Obtaining Extracts
- Methanol extract and Water-ethanol extract
The extraction method described by  was used. A mass of 50 g of powder of the leaf and steam was macerated in 500 ml of solvent: methanol and water ethanol mixture in the proportions 30:70 (v/v) under stirring for 48 hours. The macerated was then filtered thrice with cotton wool and once on the filter paper Whatman # 1. The extract recovered by filtration was subjected to evaporation in a rotary evaporator (RE 300). The resulting residues were dried at 40˚C in an oven and the collected powders constitute the methanol extract and water-ethanol extract ready for use.
- Successive extraction with hexane, ethanol, and ethyl acetate
The used extraction technique is an adaptation of the protocol used in the work of  and . A mass of 100 g of Desmodium ramosissimum powder was macerated in a litter of ethanol 96˚ under continued stirring for 72 hours. The obtained mixture was filtered thrice. To the filtrate, 50 ml of distilled water and 50 ml of hexane were added. The obtained mixture was then poured in a separator funnel where the two phases of the formation are observed: a lower aqueous phase and an upper organic phase. The organic layer was collected then dried to obtain the hexane extract. The first half of the filtrate of the lower layer was dried at 40˚C to obtain the ethanol extract. As for the second half 200 ml of distilled water and 100 ml of ethyl acetate were added. The resulting mixture was left decanted to obtain two phases. The upper layer was recovered and dried to obtain the extract of ethyl acetate. The lower phase was dried to obtain the residual ethanol extract. The extracts obtained were stored in sterile bottles for future use.
2.4. Preparation of Standard Inoculum
The bacterial suspension in physiological water was carried out by emulsifying a few colonies of bacteria taken from the Mueller Hinton agar to achieve a turbidity equivalent to that of the standard range 0.5 McFarland. To do this, after stirring vigorously the turbidity standard on a vortex, a comparison of the turbidity of the bacterial suspension to that of the standard range 0.5 McFarland was made. Bacterial density (0.5 McFarland), was adjusted with physiological water. Each bacterial inoculum was used in 15 min to 30 min following its preparation.
2.5. Preparation of the Control
Three Controls were used. The first consisted only of the culture medium (2 ml) to check the sterility of the workplace; the second contained the culture medium and the extract (1:1) to verify the sterility of extracts and have the reference extract-culture medium system; the third consisted of erythromycin as a reference antibacterial substance to assess the behaviour of an active substance in the medium and verify the effectiveness of the test.
2.6. Microorganisms Sensibility Tests
Seeding was carried out using a sterile cotton swab introduced into the bacterial suspension with removal of the excess. The entire agar surface was swabbed in three directions.
Sterile disks of blotting paper were firmly placed on the surface of the inoculated and dried agar. The disks placed on the boxes were carefully impregnated with 30 μl plant extracts with a concentration of 20 mg/ml using a micro pipette. Incubations took place ideally within 15 minutes (without exceeding 30 minutes) following the deposit of disks at 37˚C ± 1˚C.
2.7. Determination of the Minimum Inhibitory Concentration (MIC)
The method of macro-dilution in liquid medium described by  was used for MIC determination. It’s consisted in interacting germs and extracts (at various dilutions) with visual assessment of the growth of microorganisms after 24 hours of incubation . The minimum inhibitory concentration is the lowest concentration at which the extract prevented visible growth of bacteria incubation from 18 to 24 hours. For its implementation, a known charge of bacterial inoculum (106 CFU/ml) was put in contact with a dilution range of plant extracts. The procedure is summarized as follows: In a series of 10 test tubes numbered from T1 to T10, 1 ml of sterile distilled water was introduced except in tube T1. One millilitre of the extract at the starting concentration (sensitivity test concentration) was then introduced into the tubes T1 and T2. From the tube T2, successive dilutions of 1/2 were performed up to tube T9. To all the tubes, it was added 1 ml of inoculum at 106 CFU/ml of MH nutrient broth. At final all the tubes contained a total volume of 2 ml and, were incubated at 37˚C. After 24 h of incubation, the tubes turbidity was examined and compared to the control tube T10.
2.8. Determination of Minimal Bactericidal Concentration (MBC) or CMF
It was made in conjunction with the MIC determination. After identifying the MIC, all tubes starting from the MIC to the highest concentrations, were inoculated with a platinum loop on Petri dishes containing agar MH medium. These plates were incubated at 37˚C for 24 h. Upon observation, the concentration of the extract that reveals no microbial growth is the Minimum Bactericidal Concentration (MBC). Microbial growth was visually checked. Consistent with  and , the MBC is the lowest concentration at which the extract has prevented visible bacterial growth after transplanting and the CMF, the lowest concentration at which the extract prevented visible growth of fungi after transplanting.
2.9. Effect of the Extracts and Categorization
The effect of the extracts was determined by taking the ratio CMB (or CMF)/MIC. If the ratio is less than or equal to 4, the extract is said bactericidal (or fungicidal); if, on the other hand, it is greater than 4 the extract is bacteriostatic or fungi static as appropriate .
2.10. Preliminary Chemical Screening
The chemical screening used the standard reactions in solution based on colouration, precipitation or the formation of foam. It was consisted in finding bioactive groups with antimicrobial potential notably alkaloids, anthocyanins, flavonoids, quinones, saponins, steroids, tannins, terpenoids, mucilage, coumarins, reducing compounds, anthracene, and glycosides. Furthermore, the research of cyanogenic glycosides will give information about possible toxicity to cyanide. Alkaloids were detected by using the reagents of Dragendorff according to the method described by Hager and Wagner . Flavonoids have been identified with the reagent of Shinoda when the mixture of aqueous acid extract and magnesium shavings revealed a pink-red or red-purple colouration; as for anthocyanins, their presence is revealed by a red colouration in the absence of magnesium shavings in the above mixture . Cyanogenic glycosides have been underlined by the reaction of picric acid with an aqueous decoction’s vapour when picrosoda paper changed to orange or red  ; Quinones were found by reacting ether extracts with 1% KOH. The appearance of a characteristic colour ranging from orange-red to violet-purple indicated their presence; Saponins were detected by their ability to form a foam (height: >10 mm) after the stirring of an aqueous solution . Steroids have been researched by the reaction of Lieberman-Burchard. It consisted in putting the ethereal organic extract in the presence of anhydrous acetic acid and concentrated sulphuric acid and observe a purple or green colouring; Tannins have been investigated by reacting an aqueous infused with a ferric chloride solution at 1%. The reaction was considered positive with the formation of blue-green precipitates, dark blue or green  ; the Mixing of the aqueous infused with Stiasny reagent helped found catechol tannins while adding sodium acetate and ferric chloride to the filtrate resulting in the formation of precipitates, helped found Gallic tannins . Tannins have been investigated by reacting an aqueous infused with a ferric chloride solution at 1%. The reaction was considered positive with the formation of blue-green precipitates, dark blue or green  ; Mixing the brewed with the reagent Stiasny helped found catechol tannins while adding sodium acetate filtrate and ferric chloride resulting in the formation of precipitates, helped found Gallic tannins .
2.11. Statistical Analysis
The standard errors of means of experiments were analyzed and analysis of data was performed using GraphPad Prism 8. Differences were taken as statistically significant when p < 0.05.
3. Results and Discussion
The yields of the various extractions are summarized in Table 1. They are expressed as a percentage relative to the mass of the initial powder. The analysis of this table shows that the extraction yields vary according to the extract type. And that the best yield is that of the ethanol extract (16%) while the hexane extract has the lowest yield (2.24%).
Sensitivity of reference’s strains to different extract
The inhibition zones’ results of the ten (10) reference strains over time (24 h and 48 h), are recorded on the Figures 1-3. The analysis of these figures reveals that the larger zones of inhibition of methanol extracts, ethanol and residual ethanol extracts were observed respectively on Micrococcus luteus strains (14.5 ± 0.6 mm), E. coli (18.6 ± 0.02 mm) and Streptococcus oralis (15.6 ± 0.02 mm). It should also be noted that the lowest measured inhibition diameter is that of Staphylococcus epidermidis’ strains (9 ± 0.5 mm), and therefore considered to be the least sensitive microorganism. Analysis of variance shows that there is no difference (p > 0.05) over time (between 24 and 48) between the inhibition’s diameters per strains. However, it shows a very highly significant difference (p < 0.001) between treatments. Furthermore, no zone of inhibition was observed around the disks impregnated with water-ethanol extracts, hexane and ethyl acetate extracts.
Sensitivity of Foodborne Strains
Figure 4 shows the inhibitions’ diameters of ethanol extracts, acetate, water-ethanol, methanol and hexane extract of D. ramosissimum on the in vitro growth of foodborne strains. The analysis of this figure shows that the largest diameters of inhibition were observed with the ethanol extract on the S. xylosus strain (13.5 ± 0.07 mm), followed by hydro-ethanol extract on S. hominis strain (13.2 ± 0.8 mm). No significant differences (p > 0.05) were observed in the inhibition’s diameter after 48 hours.
Table 1. Yield of extraction.
Figure 1. Inhibitions’ diameters of the methanol extract on reference strains as over time (24 h and 48 h).
Figure 2. Inhibitions’ diameters of residual ethanol extract on reference strains over time (24 h and 48 h).
Figure 3. Inhibitions’ diameters of ethanol extract on the reference strain over time (24 h and 48 h).
Figure 4. Inhibition diameters of ethanol, acetate, water-ethanol, methanol and hexane of D. ramosissimum on the in vitro growth of food’s strains.
Sensitivity of Multi-Resistant Clinical Strains of Staphylococcus
The sensitivity of clinical strains of Staphylococcus to our extract is shown in Figure 5. We note that the largest diameter of inhibition (20 ± 0.08 mm) was observed on Staphylococcus lungdunensis strain with the methanol extract. Apart from methanol and ethanol residual extracts residual that inhibited each 33.33% (1/3) of strains, hexane, acetate and water-ethanol extracts showed no inhibitory effect on multi-resistant strains of Staphylococcus.
Minimum Inhibitory and Bactericidal Concentrations of D. ramosissimum extract on the different reference strains used
From the analysis of results relating to MIC (Tables 2-4), the methanol extract inhibited the growth of all tested germs, with a significant effect on E. coli and S. aureus strains where its MIC values are equal to 1.25 mg/ml. However, this extract has a low activity on P. vulgaris strain with an MIC of 10 mg/ml.
Figure 5. Inhibition diameters of ethanol, residual ethanol and methanol extracts of D. ramosissimum on in vitro growth of clinical strains.
Table 2. Minimum Inhibitory Concentration (MIC) and bactericidal (MBC) in mg/ml of D. ramosissimum’s methanol extracts and MBC/MIC ratio.
Table 3. Minimum Inhibitory Concentration (MIC) and bactericidal (MBC) in mg/ml of D. ramosissimum’s ethanol extracts and MBC/MIC ratio.
Table 4. Minimum Inhibitory Concentration (MIC) and bactericidal (MBC) in mg/ml of D. ramosissimum’s ethanol residual extracts and MBC/MIC ratio.
The ethanol extract also inhibited the growth of all the tested strains, but with a more intense activity than the methanol extract. C. albicans and S. oralis were more sensitive to this extract with an MIC of 0.625 mg/ml, while S. aureus and P. vulgaris were less sensitive (MIC = 5 mg/ml).
The residual ethanol extract exerted inhibition on all microorganisms at a concentration of 20 mg/ml except for the E. foecalis strain that was resistant to this extract.
Analysis of results for the Minimum Bactericidal Concentrations varies depending on the strain and types of extracts. The most remarkable CMB of methanol, residual ethanol and ethanol extracts are 5 mg/ml (E. coli and P. mirabilus), 5 mg/ml (S. epidermidis, E. coli) and 5 mg/ml (S. aureus) respectively. Furthermore, these methanol, ethanol and residual ethanol extracts did not have bactericidal effect on some strains. This is the case of S. aureus, P. aeruginosa and P. vulgaris for the methanol extract; S. aureus and P. vulgaris for ethanol extract and finally E. coli and P. vulgaris strains for the residual ethanol extract.
Preliminary chemical screening:
The preliminary phytochemical screening (Table 5) performed on the D. ramosissimum reveals the presence of several pharmacological groups with penitential interest. All parts of this plant contain saponins, flavonoids, anthocyanins and, tannins. It was reported that this plant is used in traditional Beninese medicine in form of decoction and since glycosides are absent, this implies that there is no risk of cyanide poisoning due to the way traditional recipes are prepared. During this study, the phenolic compounds and derivatives, like anthocyanins, flavonoids and quinones identified in D.ramosissimum could justify its strong antimicrobial activity observed on the studied strains according to Ebana et al., 1991  and Nguyi, 1988 . This confirms the use of Desmodium ramosissimum in the wounds and ulcers’ treatment (Bade et al., 2018) .
These results could help to understand its use in traditional medicine against respiratory and epidermal diseases . The literature does not seem to present in a singular way the composition of this plant; nevertheless, a study carried out by Muanda in 2010  on a similar plant D. adscendens revealed the presence of flavonoids, steroids, tannins and saponins to which biological properties are attributed. These include, the antimicrobial activity on Trichophyton rubrum and Candida albicans due to phenols and saponins according to Nwachukwu et al., 2008  as well as in vivo antidiarrheal activity attributed to flavonoids, tannins and terpenoids (Sule et al., 2001) . It should be noted that this study confirms those earlier findings, by identifying flavonoids, steroids and saponins and shows the antimicrobial activity on C. albicans of this plant. In the flowers of Cucurbita maxima, Attarde et al., 2010  reported the presence of steroids from which spinastrérol was isolated by Consolacio & Kathlun in 2005  as well as flavonoids, saponins and other phenols (Saha & Haldar, 2011) . These compounds are responsible for several properties of the flower including the antimicrobial activity on Salmonella thyphi, E. coli, Escherichia faecalis, Bacillus cereus, Candida albicans and Candida lunata (Muruganantham et al., 2016) . The activity on Candida albicans was confirmed by this study and its antibacterial spectrum was extended notably to multi-resistant Staphylococcus’s stains used in this study. The results obtained in this study are therefore for this species the first scientific report. Indeed, the present study reports the presence of alkaloids, anthocyanins, flavonoids, tannins, saponins and reducing compounds of this plant. Besides, the presence of quinones and terpenoids was noted in D. ramosisimum. From antimicrobial’s activity point of view, this plant is very active.
Table 5. Qualitative phytochemical screening of the D. ramosissimum powder.
This study shows that some plants consumed as food in Benin, like the D. ramosissimum are also endowed with great antibacterial and antifungal activities. It suggests that this aspect of the plant should be sufficiently exploited and that a thorough phytochemical screening and toxicology studies should be carried out to characterize the antimicrobial compounds contained in this plant and determine their limits of use.
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