Antimicrobial, Antiparasitic and Antioxidant Activities of Medicinal Plants from Sudan

Medicinal plants continue to play a vital role as therapeutic agents in primary health care in developing countries [1]. Sudan is located in tropical Africa and has high plant diversity and a multinational population. In Sudan and other developing countries, traditional medicine plays a major role particularly in rural regions due to both economic and cultural reasons [2]. Comprehensive ethnobotanical investigations on Sudanese folk medicine was reported previously [3-6]. (The ethnomedicinal data of selected plant materials are provided in (Table S1) in the Supporting information).


Introduction
Medicinal plants continue to play a vital role as therapeutic agents in primary health care in developing countries [1]. Sudan is located in tropical Africa and has high plant diversity and a multinational population. In Sudan and other developing countries, traditional medicine plays a major role particularly in rural regions due to both economic and cultural reasons [2]. Comprehensive ethnobotanical investigations on Sudanese folk medicine was reported previously [3][4][5][6]. (The ethnomedicinal data of selected plant materials are provided in (Table S1) in the Supporting information).
The frequent use of medicinal plants for treatment of different diseases has encouraged a number of researchers to study their biological activities [7,8]. Additionally, natural products can contribute to the discovery of novel antimicrobial [9], and antioxidant components [10]. A number of pharmaceutical studies have demonstrated the antibacterial, antimalarial, antitrypanosomal, and antioxidant activities of Sudanese medicinal plants [11,12,2,[13][14][15][16][17][18]. It is well-known that plants, which are rich in a diversity of secondary metabolites such as polyphenols, tannins, terpenoids and alkaloids are usually interesting for their antiparasitic, antimicrobial, and antioxidant activities [19][20][21][22][23][24]. The antioxidant hypothesis is however under discussion, and recent evidence suggested that its role has heavily been overestimated [25]. Therefore, the aim of the present study was to evaluate the antibacterial, antiplasmodial, antitrypanosomal, and antioxidant activities of some Sudanese medicinal plants. The antimicrobial effect of some phenolic compounds extracted from edible and medicinal plants from Europe was also assessed. We have also isolated thirteen bioactive phenolic constituents from Z. spina-christi, S. oleraceus and H. sabdariffa by preparative-HPLC (PHPLC).

Evaluation of antiplasmodial and antitrypanosomal activities
The antiplasmodial and antitrypanosomal activities were evaluated in vitro against a chloroquine sensitive strain of Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense STI900 (African strain), respectively. Both assays were carried out at two different concentrations (2 and 10 µg/ mL). These experiments were conducted in collaboration with Swiss Tropical and Public Health Institute (Swiss TPH), Basel, Switzerland as previously described [38].

Evaluation of antioxidant activities
The scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals of the crude plant extracts was measured according to a method previously described [39]. Assays were carried out in 3mL reaction mixtures containing 2mL of 0.1mM DPPH-ethanol solution, 0.9ml of 50mM Tris-HCl buffer (pH 7.4) and 0.mL of plant extracts at two different concentrations (10 and 100µg/mL). The reaction mixture was vortexed and left in the dark at room temperature (27 °C) for 30min. The absorbance was measured spectrophotometrically at 517nm. Gallic acid was used as a positive control, while ethanol was used as a blank sample. The inhibitory effect of DPPH was calculated using the standard equation [40]. The experiments were conducted in triplicates, and the data are given as mean values ± standard deviation (SD).

HPLC & HPLC-MS n
The HPLC separation and HPLC-MSn analysis was achieved as previously described [41,42].

Preparative-HPLC isolation
Preparative-HPLC isolation of compound 1-13 was carried out as in our previous study [37]. NMR 1 H NMR and 13 C NMR spectra were acquired on a JEOL ECX-400 spectrometer operating at 400MHz for 1 H NMR and 100 MHz for 13 C NMR in CD 3 OD using a 5mm probe. The chemical shifts (δ) are reported in parts per million (ppm) and were referenced to the residual solvent peak. The coupling constants (J) are quoted in hertz (Hz).

Results and Discussion
Phytochemical profiling and characterization of the isolated constituents  (Table 1) was determined as in our previous study [37] by total ion chromatograms in negative ion mode and UV chromatograms at 280nm. (Table 2) Retention times, high resolution MS data and amounts of the isolated constituents.

Evaluation of antibacterial activities
The antibacterial activities of the crude plant extracts and some selected phenolic compounds isolated by preparative HPLC from the plant material analysed in this study were evaluated for their efficacies against Gram-positive and Gram-negative bacteria. As suitable model organisms B. subtilis S168, B. aquimaris MB-2011, and C. michiganensis GSPB 390 were chosen for Gram-

Journal of Complementary Medicine & Alternative Healthcare
positive and E. coli DH5α, E. amylovora 1189, and P. syringae pv tomato DC300 for Gram-negative. The organisms chosen can be viewed as suitable model organisms for both pathogenic Grampositive and Gram-negative bacteria. Ampicillin and DMSO were used as positive and negative controls, respectively. The MIC values of the plant extracts obtained using the micro-dilution methods are presented in (Table 3). Interestingly, the plant extracts tested herein showed antibacterial activity only against Gram-positive bacteria, with MIC values varying from 195 to 1562μg/mL, while Gram-negative strains were not affected at all. The higher sensitivity of the Gram-positive bacteria compared to Gram-negative bacteria could be attributed to their differences in cell envelope components. Gram-positive bacteria have an external peptidoglycan layer, which only is a permeable and thus ineffective barrier against toxic compounds Mallik [51]. Extracts of Acacia nilotica and Punica granatum (pomegranate) peals showed the highest activities against B. aquimaris MB 2011 with a MIC value of 195μg/mL. The antibacterial activities of these two plants against Streptococcus viridans, S. aureus, E. coli, B. subtilis, Shigella sonnei and Salmonella typhimurium were reported in previous studies [52,53] However, the reference antibiotic as a positive control showed variable inhibitory activity on the all strains of bacteria with MIC values ranging from 3.9 to 250μg/ mL (Table 3). No inhibition zone was detected for the negative control (DMSO).   Phenolic compounds found in medicinal plants have been extensively studied against a wide range of microorganisms, and among them chlorogenic acids, flavanols and tannins received more interest due to their broad spectrum and the fact that most of them process antimicrobial properties [54][55][56]. Consequently, we have evaluated the in vitro antibacterial activities of some phenolic compounds against the selected bacterial strains (Table  4). Generally, the antibacterial activities the pure compounds were found to be comparatively higher than that of crude extracts. Additionally, Gram-positive bacteria were found to be more susceptible to the phenolic compounds than Gram-negative bacteria. Among the tested bioactive compounds, phloretin and resveratrol showed the strongest inhibitory activities against the all Gram-positive bacteria with MICs ranged from 9 to 125μg/mL (Table 4), followed by luteolin 7-O-glucoside and then epigallocatechin gallate (EGCG) (MICs 62 to 625μg/mL), whereas the MIC values of chlorogenic acids ranged from 260 to 540μg/mL, and therefore showed comparably low inhibitory activity. Nevertheless, the antibacterial activity of chlorogenic acids was already documented in previous studies [55,57]. The hydroxyl groups in the polyphenols are believed to be play an important role in the antimicrobial activity [58] because these groups can inactivate the microbial enzymes and interact with the cell membrane of bacteria to disrupt membrane structures and causing leakage of cellular components [59,60] (Figure 3).  Table 5 shows the antiplasmodial and antitrypanosomal activities of selected Sudanese medicinal plants against a chloroquine sensitive strain P. falciparum NF54 and T. brucei rhodesiense STI900 (African strain), respectively. For most of these plants, no specific studies of antitrypanosomal and antitrypanosomal activities exist in the literature. Most of the plant extracts exhibited dose dependent antiparasitic activities. These plants (Table 4) are traditionally used for the treatment of many ailments, parasitic and microbial infections including malaria, virus infections, digestive disorders, weakness, hepatic diseases, obesity, diabetes, skin infections, fever, diarrhea, insomnia, heart problems, colds, toothaches, hypertension, bronchial asthma, spasms, frequent urination, urinary tract infections and elimination of kidney stones [5,17,[26][27][28][29][30][31][32][33][34][35]. This could explain the good observed inhibitory activities of the most of these extracts against the tested parasites. The extracts S. oleraceus and B. aegyptiaca were found to be the most promising ones. The plant extracts showed however weaker antiparasitic activity than that reported for Chrysanthemum cinerariifolium flower extract (86% inhibition against P. falciparum and 99% inhibition against T. brucei rhodesiense at test concentrations of 4.8μg/mL) [

Evaluation of antioxidant activities
It is believed that the antioxidant activity of plant extracts rich phenolic phytoconstituents is due to their ability to be donors of hydrogen atoms or electrons and to capture the free radicals. Scavenging activity for free radicals of 1.1-diphenyl-2picrylhydrazyl (DPPH) has been commonly used to assess the antioxidant activity of medicinal plants and natural products. Plant extracts from Sudan were prepared for investigation of their antioxidant activities. They showed significant free radical scavenging activity at the high concentration (100µg/mL) on DPPH (Figure 4). The extracts of M. pigra stem, M. pigra leaves, O. basilicam arial part and I. coccinea leaves were the most effective radical scavengers with the inhibition of 33.4 ± 3.3%, 29.1±2.4%, 26.3 ± 1.2% and 26.7 ± 1.4%, respectively, as compared to 83.5±1.5% for gallic acid standard. Nevertheless, these crude plant extracts showed better DPPH scavenging activity than that reported for Crataegus monogyna fruits extract (15±1% scavenging activity at a test concentration of 100µg/mL) a flavonoid drug included in most European pharmacopeia [20]. At lower concentration (10 µg/mL) the extracts were not effective as the positive standard (Figure 4), indicating that the activity was concentration dependent. Many studies have demonstrated that the antioxidant activity is significantly affected by the phenolic constituents of the sample [20,21]. Thus, the radical-scavenging activity of the plant extracts may be attributed to their phenolic and flavonoid contents. Furthermore, the antioxidant property of M. pigra was in agreement with those mentioned in the literature [61,63-70] (Figure 4). Percentage inhibition as means±SD of free radical scavenging by the plant extracts.

Conclusion
In conclusion, our findings strongly support the traditional use of the studied Sudanese plants in the treatment of bacterial and parasitic infections. The results revealed that some of these plants, such as A. nilotica, O. basilicam, Z. spina-christi, B. aegyptiaca, S. oleraceus, P. granatum, M. pigra and I. coccinea have the potential to be investigated further to identify the antioxidative, antiplasmodial, antitrypanosomal, and antibacterial metabolites in these plants. The study also reports on the antibacterial activities of some naturally occurring phenolic compounds. Among the tested phytochemicals, phloretin, resveratrol, luteolin 7-O-glucoside and epigallocatechin gallate showed the highest antimicrobial activities. By means of preparative HPLC, HPLC-ESI-TOF, HPLC-ESI-MS n , 1 H-NMR and 13 C-NMR, thirteen phytoconstituents were isolated and identified in the methanolic extracts of Z. spina-christi, S. oleraceus and H. sabdariffa including chlorogenic acids, flavonoid glycosides, coumarins and derivatives. The results of this study highlight the importance of the Sudanese medicinal plants as potential source of plant derived antimicrobial and antiparasitic drugs. However, pharmacological and toxicological studies will be necessary to confirm this hypothesis.