Antimicrobial, antioxidant activities and toxicity on Cavia porcellus of Dialium angolense Welw. Ex Oliv, a traditional medicinal plant from Bagira in Eastern of DR Congo

Dialium angolense is used in Bagira for its various medicinal properties particularly in the management of infectious diseases. In this study, the methanol and aqueous extracts of leaves and fruits were evaluated for their in vitro antioxidant and antimicrobial properties and their in vivo toxicity on Cavia porcellus. The major phytochemical classes of extracts were screened using standard in-tube reactions. The antimicrobial study was tested on Candida albicans, Escherichia coli, Salmonella typhi, Staphylococcus aureus and Streptococcus pneumoniae using agar well diffusion and dilution methods, while the antioxidant activity was evaluated by a DPPH assay. For the acute toxicity study, animals (6/group) were orally given in a single dose 5000, 1000 or 15000 mg of extract/kg body weight (BW) then observed for 14 days. In sub-acute toxicity assays, 150 or 300 mg/kg BW/day were orally given, and animals observed for 28 days. Total phenolics and total flavonoids contents ranged 1.19 to 1.61 mg GAE.g-1 and 0.45 to 1.01 mg QEg-1, respectively. The extracts presented antioxidant activity with IC50 ranging 4.9 to 6.9 μg/mL. The minimal inhibitory concentration (MIC) on tested strains ranged from 1.9 to 500 μg/mL with the aqueous extract of fruits as a most active extract: MIC=1.9 μg/mL on E. coli and C. albicans. No signs of toxicity were noted during the acute and sub-acute toxicity assessments, suggesting a maximal tolerate doses (MDT) and LD50 > 15000 mg/kg BW. This study highlights the antioxidant and antimicrobial activities of Dialium angolense and suggests that further studies be directed towards the isolation of active compounds.


Introduction
Microbial infections are a major health concern not only because of their high prevalence but also of their high mortality in most African countries like the DR Congo [1][2][3][4]. Despite the existence of a varied therapeutic arsenal [5], the emergence of antimicrobial resistances reduces its impact and justifies the need for the research of new active therapeutic substances with various mechanisms of action [6][7][8][9]. Rich in secondary metabolites, plants used in traditional medicine have advantages that could offer humanity such expected antimicrobials [10,11]. Several screening studies, carried out in different African regions, have resulted in the isolation and characterization of antimicrobial compounds [12][13][14]. Several antimicrobial compounds have antioxidant potential [15][16][17]. That is why, there is currently a tendency to investigate antimicrobial and antioxidant activities as shown by several recent studies carried out on African medicinal plants [18][19][20]. Despite their reported medicinal properties, indicated by ethnobotanical studies, the pharmacology of the many plants used in traditional African medicine is quite often insufficiently investigated and their safety is mostly unknown. [21,22].
Dialium angolense Welw. ex Oliv., also called Dialium evrardii (Steyaert) belongs to the Fabaceae family, with many local names like Kizimya (Shi) or Cituzo (Havu); is one of those medicinal plants that are not well known. It is a tree of 3 to 12 meters high, endemic in tropical Africa. In DR Congo, the leaves and the fruits are used in the treatment of headaches, fever, gastritis, conjunctivitis, urethritis, amebiasis, malaria, digestive and respiratory diseases. Both organs are edible, the fruits are consumed by humans and the leaves by primates [23,24]. A recent study reported antioxidant and antiplasmodial activities in vitro of aqueous and methanolic extracts of Dialium angolense leaves [25]. Unfortunately, no information has been reported on the antimicrobial potential or less on the toxicity and phytochemical composition of the leaves and fruits of this plant. This study aims to evaluate the antimicrobial activity of aqueous and methanolic extracts of leaves and fruits of Dialium angolense on germs responsible for some digestive, respiratory and nosocomial infections. It also intends to establish its phytochemical profile in secondary metabolites and assess the acute and subacute toxicity of its two organs, on Cavia porcellus.

Plant material and experimental animals
Leaves and fruits of Dialium angolense were collected on June 2016 from Bagira (2°28'12.9''S; 28 ° 49'18''E; 2,883.1 m) and was identified at the herbarium of Meise in Belgium (voucher number: BR0000018879285). Healthy Cavia porcellus (275.5 ± 5.2 g) were obtained from animals holding unit of the zoo-technology Department of the Faculty of Agronomic Sciences, University of Lubumbashi. They were allowed unrestricted access to food for rodents (MIDEMA-DRC) and water ad libitum.

Preparation of extracts
Methanolic extracts (ME) were obtained by macerating 350 g of coarsely powdered dried vegetal material in 1.5 L of methanol. After 72 h, the extract was filtered on paper (Whatman, USA) and the residue was macerated twice in a similar manner. The filtrates were combined, concentrated, and dried using a rotary evaporator (Büchi R-210, Switzerland) at 40 °C under reduced pressure :130-180 mbar (Yield, 12.8 %, W/W). Aqueous extracts (AE) were prepared according to the protocol used in traditional medicine by decocting 320 g of the sample in 2 L of local tap water, boiling for 1 h in a close recipient and filtration on paper. The extract was lyophilized (Yield, 11.9 %, m/m). For all tests, each extract was dissolved in its extraction solvent before mixed with the vehicle.

Screening for secondary metabolites
The plant extract was analyzed for the presence of some secondary metabolite including alkaloids, coumarins, flavonoids, saponins, steroids, tannins, terpenoids and phenols, using standard in-tube reactions [26,27].

Determination of total phenolics, flavonoids and tannins contents
The total phenolics content of each sample was measured by a Folin-Ciocalteu method [28] and expressed as milligrams gallic acid equivalents per gram of dry plant extract (mg GAE/g DE) through a calibration curve gallic acid (y = 0.011x + 0.001, R 2 = 0.998 ; linearity range, 1 -200 mg. L -1 ). The total flavonoids content was determined using an aluminum trichloride assay [29] and expressed as milligrams quercetin equivalents per gram of dry plant extract (mg QE.g -1 DE) through the calibration curve of quercetin (y = 0.008 x + 0.001, R 2 = 0.996; linearity range, 0.1 to 150 mg/mL). The total tannins were determined by a vanillin method [30] and expressed as milligrams gallic acid equivalents per gram of dry plant extract (mg GAE.g -1 DE) through the calibration curve established for gallic acid (y = 0.006 x + 0.0011, R 2 = 0.997; linearity range, 1 -100 mg. L -1 ).

Antimicrobial Activity Assay
Candida albicans ATCC 10231, Escherichia coli ATCC 25922, Salmonella typhi ATCC 14028, Staphylococcus aureus ATCC 6538, and Streptococcus pneumoniae ATCC 49619 were supplied by the Provincial Laboratory of Lubumbashi where the tests were carried out using an agar diffusion method [31]. A broth dilution method was used in triplicates to determine the Minimum Inhibitory Concentrations (MIC) and Minimum Microbicidal Concentrations (MMC) of the extract [32]. The germs used during this study were chosen according to the anti-infectious uses of D. angolense in Traditional Congolese Medicine (TCM): E. coli, S. typhi, (for digestive infections), S. aureus (for nosocomial infections) and S. pneumonia (for respiratory infections). The measurement of the diameter of inhibition (ID) made it possible to determine the sensitivity of different germs by the diffusion method and the dilution test made it possible to determine the MIC and the MMC. These various parameters made it possible to determine and characterize the antimicrobial activities of the extracts.
For the determination of the sensitivity, in sterile Petri dishes, 20 mL of Muller-Hinton agar were poured and then left to stand for 20 minutes. After solidification, on each culture medium, 1 mL of bacterial suspension of 10 8 CFU /mL was seeded over the entire surface. Blotting paper discs (ø = 6 mm) were impregnated with a volume of 5 μL of extracts (50 and 100 μg/mL) and placed on the surface of the solidified and infected medium. The Petri dishes were then incubated at 37 °C for 48 h in the oven. The sensitivity of the germs to the extracts was estimated by measuring the diameter (mm) of the zone of inhibition induced by the different concentrations around the discs and each experiment was repeated three times [33].
Regarding the determination of the MIC, 100 µL of each extract at 1 mg/mL (dissolved in methanol or water depending on the extract) was mixed with 1900 µL of the culture medium. Eight successive dilutions of order 2 (from 50 μg / mL to 1.9 μg / mL) were then carried out for each extract and placed in different aseptic tubes of 5 mL; 1000 µL of the standard inoculum was then added to each tube and the mixture was incubated for 24 h at 37 °C. The growth of the microorganisms was observed visually. The MIC, considered to be the lowest concentration at which the extract prevented the visible growth of bacteria was determined [26].
Regarding the determination of the minimum microbicidal concentration (MCM), the sampling was carried out in tubes used for the determination of the MIC. The inoculation was carried out in the Petri dishes on Salmonella-shigella agar (bacteria) or Sabouraud agar (fungi) medium and the incubation was carried out at 37 °C (bacteria) or 28 °C (fungi) for 24 h. Microbial growth was checked visually and MMC, defined as the smallest concentration at which the extract prevented the visible growth of microbes (fungi or bacteria) after sub culturing was determined [32].

DPPH assay
Antioxidant activity was evaluated using DPPH method [25,34]. Briefly, 50 μL of extract (or positive control) prepared at different dilutions of order 2 in methanol from a 100 μg/mL solution were interacted with 1950 μL of 0.002% DPPH in a plate 96 wells (Nunc WVR, Germany). After mixing and incubating in the dark for 30 minutes, the solution was read at 492 nm (Thermo Fisher Scientific Inc., Waltham, USA). The tests were carried out in triplicate and the 0.002% DPPH solution was used as a negative control. The percentage of antioxidant activity was calculated by the formula: Where, Ab = absorbance measured in the presence of the negative control; Ae = absorbance measured in the presence of the extract, AAO (%) = Percentage of inhibition.

Toxicological Study
Acute toxicity was carried out as previously described [35] using, 0 (i.e. vehicle = control), 5000, 10000, 15000 mg/kg BW in single dose (oral administration; 6 animals per group, followed over14 days). For the subacute toxicity study, Cavia porcellus (6 animals per group) orally received for 28 days, 0 (i.e. vehicle = control), 150, and 300 mg/kg BW/day. During blood collection and serum preparation for biochemical analysis, validated procedures were followed [35]. The activities of alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), and the levels of urea and creatinine were determined by colorimetric assays with Labtest® kits (Minas Gerais, Brazil). When evaluating subacute toxicity, we used the dose of 150 mg/kg, corresponding to the dose used in traditional medicine, and double of this dose (300 mg/kg); As for the doses used for the assessment of acute toxicity, we took into account the doses from the preliminary tests following the Organization for Economic Cooperation and Development OECD procedure for which up to 3000 mg/kg no sign of toxicity was observed.

Statistical Analysis
Values were analyzed using GraphPad Prism 6 (GraphPad Software, La Jolla, USA). Comparisons between different groups were carried out by analysis of variance, ANOVA; a probability level p < 0.05 was considered significant.

Ethical Approval
The principles governing the use of laboratory animals as laid out by OECD, Minna Committee on Ethics for Medical and Scientific Research and also existing internationally accepted principles for laboratory animal use and care as contained in the Canadian Council on Animal Care Guidelines and Protocol Review [36] were duly observed. The project proposal and procedures were reviewed and approved by the Department of Pharmacology in the faculty of Pharmaceutical Sciences from the University of Lubumbashi, DR Congo (UNILU/FSP/DPCOL/PT/00/2014).

Chemical screening of Dialium angolense
The phytochemical screening of Dialium angolense revealed the presence of polyphenols, tannins, terpenoids, flavonoids, coumarins, anthraquinones but the absence of alkaloids and saponins ( Table 1).
The highest values in total phenols (1.6118 ± 0.006 mg GAEg -1 ), total flavonoids (1.0112 ± 0.006 mg QE. g -1 ) and total tannins (0.2830 ± 0.001 GAE.g -1 ) were observed in the methanolic extract of fruits (MEF). Overall, the total flavonoids levels are more than 3 times higher than the total of tannins and the fruit contents are higher (p < 0.01) than leaves ( Table 2).

Antioxidant activity
Depending on their IC50 values, extracts were classified as following: (i) very active if IC50 ≤ 5 µg/ mL, (ii) active if 5 µg/mL ≤ IC50 ≤ 15 µg/mL, (iii) moderately active if 15 µg/mL <IC50 <50 µg/mL, (iv) weakly active if IC50 ≥ 50 µg/mL [25]. The scavenging ability of tested samples showed a concentration-dependent activity. The anti-free radical activity, expressed in the form of IC50, varied between 1.62 and 6.87 μg/mL suggesting that all extract were very actives. In line with their higher content in phenols, flavonoids and tannins, the fruits have an anti-radical power superior to that of the leaves (p < 0.01) and particularly AEF, although less than that of ascorbic acid, used as positive control (Figure 1).

Figure 1
Dialium angolense DPPH radical scavenging activities expressed as percent reduction (a) and as IC50 -1 (b). The results are expressed as the mean ± SD, n = 5

Sensitivity of tested bacteria and fungus
The microbial sensibility was assessed by the measurement of inhibition diameter (ID) on agar. A microbial strain was considered "sensitive" for ID ≥ 10 mm [37]. The highest dose-dependent sensitivity was observed by E. coli (ID of AEL is 24.0 ± 0.2 mm). In this study, S. pneumoniae and S. typhi are the most sensitive microbes in the study with 100% and 80% of positive tests, respectively (Table 3).

MIC (Minimum Inhibitory Concentration) and Minimum Microbicidal Concentration (MMC)
The MIC allowed to categorize the activity of extracts in one of 4 following groups: i) very active extract if MIC ≤ 5 µg/mL, ii) active extract if 5 µg/mL ≤ MIC ≤ 50 µg/mL, iii) moderately active extract if 50 µg/mL ≤ MIC ≤ 325 µg/mL and iv) low activity extract if MIC > 325 µg/mL [32]. All extracts were found to be at least active on S. typhi and S. pneumoniae and AEF was the most active (MIC = 3.9 µg/mL and 7.8 µg/mL, respectively) extract; overall the AEF extract was the most active on other strains, E. coli and C. albicans (Table 4).

Clinical signs, weight variation, MTD (maximal tolerate dose) and 50 % lethal dose (LD50)
As for the control group, no sign of acute or sub-acute toxicity was observed in animals following administration of D. angolense extracts; there were no significant variations in weight, either for body ( Figure 2) or heart, liver, spleen and kidney (Figure 3a-b).

Correlation between antioxidant activity and different content in phenols
The contents in total phenolics and flavonoids showed a correlation with the measured antioxidant (hydrogendonating) activities; this was particularly the case for flavonoids (Table 5).

Correlation between Antimicrobial activity and content in different phenols quantified
A positive correlation was observed between the content of phenols, especially flavonoids, and antimicrobial activity. This correlation was very significant in particular on the strains S. typhi (RTPC = 0.998; RTFC = 0.997) and S. pneumonia (RTPC = 0.845; RTFC = 0.838), suggesting that the polyphenols in particular the flavonoids contribute very significantly in the expression of the antimicrobial activity of the different extracts (table 6).

Correlation between antioxidant and antimicrobial activity
Very significant correlation (R> 0.87) between antioxidant and antibacterial activity was observed in particular with S. typhi, S. pneumoniae, S. aureus and Candida albicans and the relationship between antioxidant activity and antimicrobial (AOA / AMA) for these 4 germs ranged between 0.014 and 2.65 (Table 7).

Discussion
Given the paucity of information on Dialium angolense, the present study was devoted to the phytochemical screening and evaluation of antioxidant and antibacterial activities as well as to the acute and sub-acute toxicities of leaves and fruits aqueous and methanolic extracts.
Very few studies have reported the phytochemical composition of secondary metabolites of species of the genus Dialium. We show in the leaves of D. angolense the presence of polyphenols, particularly flavonoids and tannins, as previously reported for D. guineense Willd and D. indum L. [38,39] and in the fruits flavonoids, saponins, and steroids as reported in D. guineense [40,41]. The fruits of D. angolense have higher flavonoid and tannin contents than D. indum [41,42] but lower than D. guineense [43,44]. There is a need to conduct an extensive phytochemical study of the thirty or so species accepted in this genus [45] to determine whether the flavonoids and/or tannins could constitute chemotaxonomic markers.
According to the correlation between antioxidant activity and different content in phenols. In this study, there are significant differences between extracts, indicating that the quality of polyphenols/flavonoids in the extract is certainly more important than their content [55]. This is confirmed by Vasco et al. [56] who states that the correlation depends on the extraction solvent, the hydrophilicity of the compounds, the sample, and the type of phenolic compounds. By evidence, not all antioxidant characteristics are assessed by the test performed here; notably, the ability to quench in vivo oxidative damage and lipid peroxidation largely depends on the lipophilicity of the compounds (phenols, tocopherols, carotenoids, and flavonoid aglycones) and the chelation of metals: ascorbic acid, tannins, flavonoid aglycones and glycosides [57].
The antibacterial activity of D. angolense fruits is higher compared D. guineense aqueous extract on E. coli (MIC = 225 mg/mL,), S. typhi (150 mg/mL), S. pneumoniae (mg/mL) and S. aureus (225 mg/mL) [58]. It is more active than Dialium corbisieri Staner, aqueous extract on E. coli (ID = 0 and 11 mm), S. aureus (ID = 0 and 10 mm), C. albicans (ID = 0 and 10 mm) [48]. It is likely that the polyphenolic compounds, and particularly flavonoids [59][60][61], would be both responsible for antioxidant and antibacterial activities of leaves and fruits of D. angolense. The antimicrobial activity of flavonoids has previously been demonstrated [62][63][64] and several antimicrobial mechanisms of compounds in these groups have been demonstrated [59]. It has been established that flavonoids aggravate microorganisms in various ways including to form a complex with the cell wall components and consequently inhibit further adhesions and the microbial growth [65], adhesion to and invasion of hosts by Gram-positive bacteria [66], interaction with liposomes [67], eradication of biofilm bacteria [68] or inhibition of the DNA, RNA and proteins bacterial synthesis [69].
All studied extracts showed a particularly interesting antibacterial activity on S. typhi, justifying the use of D. angolense against a condition identified as "typhoid fever" in traditional medicine [23]. This activity is particularly interesting since co-infections Plasmodium falciparum -S. typhi are commonly reported in Bukavu city, as well as elsewhere [70]. As, in the region, the plant is also used as an antimalarial [23] its use may have various benefits.
In this study, it have been established a correlation between antioxidant and antimicrobial activity (Table 7). These results agree with the previous literature where it has been established a correlation between the antioxidant and antimicrobial activity of some plant extracts [71][72][73]. This correlation would probably be linked to the presence of polyphenols quantified in the plant during this study, as suggested by previous work [74,75].

Conclusion
This study highlights for the first time the antioxidant and antibacterial activities as well as an absence of sub-acute toxicity of the leaves and fruits of D. angolense, while pointing to the role of its polyphenols, especially flavonoids. It thus supports the traditional use of this medicinal plant and opens the way to a bio-guided-isolation of these antioxidant and antimicrobial compounds.