Sickle cell anemia (SCA) or Drepanocytosis is a genetic disease characterized by the presence of haemoglobin S in the blood      . All over the world, the number of individuals suffering from SCA is estimated at approximately 50 million of the people  and according to the World Health Organization (WHO), 300,000 children are born each year with a major anomaly from the hemoglobin of which most frequent is the SCA  . In Africa, SCA is the first genetic disease by the number of the patients  . In Democratic Republic of the Congo (DRC), 2% of the populations are sicklers and the majority of these patients die before the age of five years when they do not receive health care and those which survive however present an attack at the level of certain vital organs, which reduces considerably their life expectancy  .
It was reported that in DRC, 12% of the children hospitalized in the hospital are sicklers and it is estimated that the annual cost of the treatment is higher than 1000 USD per patient  , a cost hard to bear for the majority of the population whose average income is lower than 2 USD per day and who for the needs for primary health care turns mainly to traditional medicine and in particular the medicinal plants    .
The plant species Uvariodendron molundense (Diels) R.E.Fr. belongs to the Annonaceae family. It leaves are consumed as traditional tea or used as spice by local communities of Nord Ubangi province in DRC and is effective against bacterial infections. The plant harvested in Cameroon was reported to possess antifungal, antioxidant and antiplasmodial activities   .
Recent findings revealed that the consumption of some tropical plants as foods by the non-human primates (great apes) protect them against hemolytic disease like malaria by inhibiting the Plasmodium falciparum inducing hemolysis of infected erythrocytes   . Like for great apes, we hypothesized that the consumption of traditional foods/tea could also inhibit the erythrocytes hemolysis in human including those suffering from SCD. Indeed, great apes are reported as a good model for the understanding of eukaryotic cell hemolysis because they share with human a common gut anatomy   . Thus, the traditional tea Uvariodendron molundense is expected to contain phytochemicals with anti-sickle cell anemia and antibacterial properties, which would act either individually, or in synergy. Indeed, the majority of the SCA children generally die by bacterial infections.
The aim of this study was to evaluate the chemical composition and bioactivity of U. molundense against Sickle cell disease and associated pathogenic bacteria agents in order to promote this dietary supplement as putative nutraceuticals. Indeed, patients with SCA usually suffer from intermittent clinical or hematologic crises. However, infection, and not crisis, is the most common cause of death, particularly in children. The antimicrobial resistance is therefore one of the most serious public health problems and constitutes one of the major causes of failure in the SCA infection treatment   . Thus, direct or indirect antimicrobial effect U. molundense could warrant further studies as to the nature of active compounds and to determine the new strategy for prophylaxis in SCA infections.
2. Materials and Methods
2.1. Plant Material Collection and Identification
The tested plant material (leaves and bark) used in the present study was collected in Nord Ubangi Province (Democratic Republic of the Congo) during a field work in 2015 and was authenticated by Mr. Justin A. Asimonyio of the CSB (Centre de surveillance de la Biodiversité, University of Kisangani). Voucher specimen N0 JAA02NU is on deposit at the Laboratory of Molecular bio-pros- pection (Department of Biology, Faculty of Science, University of Kinshasa).
2.2. Extraction, Chemical Screening and Preparation of Increasing Polarity Extracts
The dried and powdered plant material (10 g) was repeatedly extracted by cold percolation with 95% ethanol (EtOH) and water (100 mL × 2) for 48 hours.
Chemical screening was performed on the aqueous and organic extracts to investigate the presence of alkaloids, saponins, total polyphenols, flavonoids, tannins, anthocyanis, leuco-anthocyanins, quinones, terpenes and steroids according to standard protocol  . Fractions were filtered and concentrated to dryness under reduced pressure using a rotary evaporator. Organic/triterpenic acids were extracted as follow: the powdered material (40 g) were macerated with 100 mL of dichloromethane-methanol-NH4OH (100:1:1; v/v/v) and then percolated with 300 mL of the same solvent mixture at room temperature. The extract was concentrated under reduced pressure until 100 mL (pH 10). The resulting solution was then mixed with 5% citric acid (v/v) to precipitate organic/triterpenic acids  . To obtain the increasing polarity extracts, the powdered plant material was exhaustively extracted with n-hexane (1:10, p/v), ethyl acetate, and methanol and acidified methanol (HCl 1%). The resulting fractions were evaporated to dryness on an evaporator apparatus. All extracts were stored at +4˚C.
2.3. Quantification of Secondary Metabolites
2.3.1. Total Phenolic
Total phenolic contents were determined according to the Folin-Ciocalteu method with slight modifications  . The extract (200 μL) was mixed with 1.5 mL of Folin-Ciocalteu reagent (previously diluted 10 times with double distilled water) and allowed to stand at room temperature for 5 min. 1.5 mL of sodium bicarbonate solution (60 g/L) was added to the mixture and after incubation for 90 min at room temperature, the absorbance was measured at 725 nm using a UV- Visible spectrophotometer (GENESYS 10S). Total phenolic were quantified by calibration curve obtained from measuring the absorbance of the known concentrations of gallic acid standard solutions (10 - 150 μg/mL in 80% methanol). The results were calculated as gallic acid equivalent (GAE) per one gram dry powder and reported as mean value ± standard deviation (SD) (the standard curve equation: Y = 0.006x − 0..002; R2 = 0.997).
Total flavonoid content was measured by the aluminum chloride colorimetric method  . An aliquot (1 mL) of each extract was added to 10 mL volumetric flask containing 4 mL of double distilled water. Then 0.3 mL NaNO2 5% was added to the flask and after 5 min, 0.3 mL AlCl3 (10%) was also added. At 6th min, 2 mL NaOH (1 M) was added and the total volume was made up to 10 mL with double distilled water.
The solution was mixed completely and the absorbance was measured versus prepared reagent blank at 510 nm. Total flavonoid content was expressed as mg quecertin equivalents (QE) per one gram dry powder. One mL of standard solution (quecertin: 5 - 100 µg/mL) was used to construct calibration curve (the standard curve equation: Y = 0.009x + 0.006; R2 = 0.999).
The samples were diluted with the mixture ethanol/water/HCl conc. (70:30:1; v/v/v) and the absorbance was measured at the wavelength of 540 nm. The anthocyanins content (expressed as malvidin-3-glucoside equivalent, M-3-GE) was calculated using the following relation: Anthocyanins = A540 *(10/0.6)*d (with A540 = maximum of absorption at 540 nm; d = dilution factor; 0.6 maximum of absorption of 10 mg/L of M-3-GE standard solution)  .
To 1 ml of the extract was added 7.5 ml of distilled water and 0.5 ml of Folin- Ciocalteu reagent and 1 ml of sodium carbonate (Na2CO3 35%). The absorbance was measured at the wavelength of 725 nm. The tannins content (expressed as tannic acid equivalent, TAE) was calculated using the following relation: Y = 0.443x − 0.264; R2 = 0.720  .
2.4. Biological Experiments
2.4.1. In Vitro Antisickling Bioassay
Blood samples used to assess the antisickling activity of the selected plant extracts were taken from known SCD patients attending the “Centre de Médecine Mixte et d’Anémie SS” located in Kinshasa, Democratic Republic of the Congo. None of the patients had been transfused recently with Hb AA blood and all antisickling experiments were carried out with freshly collected blood. In order to confirm their SS nature, the above-mentioned blood samples were first characterized by Hemoglobin electrophoresis on cellulose acetate gel to confirm their status and were then stored at +4˚C in a refrigerator. An informed consent was obtained from all the patients participating in the study and all the research procedures have received the approval of Department of Biology Ethics Committee.
An aliquot of Hb S-blood was diluted with 150 mM phosphate buffered saline (NaH2PO4 30 mM, Na2HPO4 120 mM, NaCl 150 mM) and mixed with an equivalent volume of 2% sodium metabisulfite. A drop from the mixture was spotted on a microscope slide in the presence or absence of plant extracts and covered with a cover slip.
Paraffin was applied to seal the edges of the cover completely to exclude air (Hypoxia). Duplicate analyses were run for each extract. The red blood cells (RBCs) were analyzed by a computer assisted image analysis software (Motic Images 2000, version 1.3; Motic China Group Co LTD) and statistical data analysis were processed using Microcal Origin 8.5 Pro package software as previously reported      .
2.4.2. Determination of Antibacterial Activity
・ Microbial strains
The activity of the plant samples was tested toward Staphylococcus aureus (S. aureus ATCC 33591) and Escherichia coli (E. coli ATCC 27195) strains. The tested strains were obtained from the American Type Culture Collection (ATCC, Rockville MD, USA).
・ Determination of Minimum inhibitory concentration (MIC)
The Minimum inhibitory concentration (MIC) was determined by broth micro-dilution method as reported in our previously research work   . The inocula of used microorganisms were prepared from 24 hours old broth cultures. The absorbance was read at 600 nm and adjusted with sterile physiological solution (0.9% NaCl) to match that of a 0.5 McFarland standard solution (108 cells/mL). The prepared microbial suspension was diluted (1/100) to achieve 106 CFU/mL. Stock solutions of the plant extracts were prepared in Tween 80 (Fisher chemicals) (3 mg/300µL) and diluted to 2.7 ml with Mueller Hinton Broth (MHB) (Conda, Madrid, Spain) to achieve a Tween 80 final concentration of 0.1%. This solution was transferred in 96-wells plates (200 µL/well) and two- fold serially diluted with MHB to give final concentrations ranging from 1000 to 3906 µg/mL.
An aliquot (10 µL) of 106 CFU/mL overnight culture was added to wells of a sterile 96-well micro-plate titer. The positive control wells contained MHB+ bacteria suspension without plant extract while negative control wells contained MHB only. The MIC was determined as the lowest plant extract concentration at which no growth were observed after 24 hours. Resasurin (30 μL) in aqueous solution (0.01%) was used to evaluate the micro-organism viability.
Three independent experiments were run for the optimization of each in vitro bioassay.
3. Results and Discussion
3.1. Phytochemical Study
The chemical screening performed on the aqueous and alcoholic extracts of both leaves and bark of Uvariodendron molundense revealed the presence of alkalo- ids, saponins, total polyphenols, flavonoids, tannins, anthocyanis, leuco-antho- cyanins, quinones, terpenes and steroids. Phenolic compounds such as antho- cyanins               , rosmarinic acid  and lunularic acid  and triterpenes like betulinic, maslinic, olea- nolic  and ursolic acid  were reported to display antisickling activity in vitro in our previous research works.
Table 1 gives the extraction yield and chemical composition of stem bark extracts from Uvariodendron molundense.
It is deduced from this table that the high yield (weight of the extract divided by weight of the plant powder multiplied by 100) was obtained with methanol following respectively by ethanol and ethyl acetate. However, n-hexane and aci- dified methanol displayed a poor yield. These results indicate that the abundant secondary metabolites/compounds in this plant species are those which pass eas- ily through the polar solvents (methanol, ethanol and ethyl acetate).
This is the case of phytomarkers like flavonoids and tannins (which are most concentrated in methanol) and les anthocyanins which are concentrated in ethyl acetate. The presence of anthocyanins in all examined extracts suggests that they
Table 1. Extraction yield and chemical composition of leaves and bark of U. molundense (data from three independent experiments in triplicate).
could possess antisickling properties as reported elsewhere               . The bioactive anthocyanins could be those which are acylated by organic acids  . In the present study, the extraction yield of organic/triterpenoic acids (betulinic acid rich extract) is 1.70%. This value is greater than that of Uvariopsis congensis  . All of these extracts were tested for their antisickling and antibacterial effects.
3.2. Antisickling Activity
Figure 1 gives the morphology of untreated sickle erythrocytes and treated with Uvariodendron molundense extracts.
Figure 1. Morphology of untreated sickle erythrocytes (a) or SS RBCs treated with 50 µg/mL of Uvariodendron molundense extracts (b)-(g) (X500), [NaCl 0.9%; Na2S2O5 2%].
Figure 1(a) reveals that the control sample contains in majority sickle-shaped erythrocytes, confirming the SS homozygous status of the used blood. Mixed together with different plant extracts (Figures 1(b)-(g)), the majority of erythrocytes are reversed normal-shape.
This indicates that Uvariodendron molundense have antisickling properties. The bioactivity displayed could be due to secondary metabolites like anthocyanins, phenolic or triterpenoic acids as previously reported                   . The morphology/ phenotype of treated sickle erythrocytes was remarkably similar to normal red blood cells (Figures 1(b)-(g)).
Figure 2 shows the dose dependent antisickling activity of both anthocyanins and triterpenic acids rich/organic acids extract from Uvariodendron molundense.
It can deduce from this figure that the normalization rate of sickled cells in hypoxic conditions increases with the extract concentration. At 100 µg/mL, the rates of normalization were 89% for organic extract (ED50 ie dose of extract for which 50% of the sickled erythrocytes are reversed equal to 0.391 µg/ml) and 82% for anthocyanins extract (ED50 = 0.659 µg/mL). Thus, the antisickling activity of the tested plant is dose dependent (Figure 2). It can be noticed also that the area under the curve was greater for organic acids extract than that of anthocyanins extract. This means that organic acids extract displays great bioacti- vity than anthocyanins extract one.
Figure 2. Dose-dependent normalization rate of sickled erythrocytes with bioactive compounds from Uvariodendron molundense (NaCl 0.9%; Na2S2O5 2%) (The curve was fitted with the help of Origin Pro 8.5 package software using B-spline: Fill Area Under the Curve and Inclusive broken by missing values).
In this study, we demonstrated that all tested extracts are biologically active confirming the hypothesis that Uvariodendron molundense contains phytocon- stituents which would act either individually, or in synergy, in order to confer him the antisickling properties. It is therefore suggested that bioactive extracts could exert their pharmacological effect by various mechanisms including the inhibition of free radicals formation, the inhibition of hemoglobin polymerization, and the inhibition of erythrocyte hemolysis as reported for others plants in our previous findings   . Thus, oral supplementation of aqueous Uvariodendron molundense leaf extracts could abate oxidative stress, hemolysis and bacterial infections associated with clinical manifestation of SCA.
3.3. Antibacterial Activity
Due to the high cost of modern therapy for SCA, plant extracts displaying at the same time antibacterial and antisickling activities could be useful in the management of this hereditary blood disorder. The antibacterial activity of Uvariodendron molundense extracts against E. coli and S. aureus two bacterial strains associated to this disease was evaluated and results are shown in Table 2.
It is deduced from this table that S. aureus is more sensitive to Uvariodendron molundense (all extracts have a CMI ≤ 31.25 µg/mL) than E. coli (for which alone organic extract exhibit strong activity: CMI = 31.25 µg/mL). The difference in the bioactivity of tested extracts would be due to the nature of the bacterial wall structure   . Indeed, contrary to E coli, S. aureus is a positive gram bacterium.
Its wall is thick (several layers) and would constitute the pharmacological target of the biologically active compounds present in Uvariodendron molundense whereas for E coli, the external membrane would prevent some of secondary metabolites present in this medicinal plant species from penetrating in the bacterial cell. These results confirm the antimicrobial properties of the natural products of plant origin   . It is well established that prokaryotic parasites like S. aureus and E coli are the principal cause of septicemia and osteomyelitis
Table 2. Antibacterial activity (expressed as minimal inhibitory concentration, MIC) of Uvariodendron molundense extracts.
Legend: ATCC: American Type Cell Collection. The values in the two last columns bearing different superscripts are significantly different (p < 0.05), (n = 3 independent experiments).
in sickler patients  . So, plant extract displaying both antibacterial and antisickling properties is a suitable candidate for the development and standardization of medicine of pharmacological relevance for the management of SCA in endemic regions.
In the present research study, the bioactivity of organic acids extract is particularly interesting because of it inhibitory effect on E. coli (CMI = 31.25 µg/ml), a bacterial strain generally no sensitive to plant extracts   . The isolation, purification and structure characterization of bioactive organic acids would be used as starting point for pharmaco-modulation, galenic formulation or models/precursors for a chemical synthesis on an industrial scale. To our knowledge, it is for the first time that the anti-sickle cell anemia properties of Uvariodendron molundense is reported in the literature. The leaves of this plant species is consumed as traditional tea or used as spice local communities of Nord Ubangi province. Consequently, dietary supplements with proven antisickling and bacterial inhibitory potentialities could be of great interest to promote for the management of SCD in this area as putative nutraceuticals.
4. Conclusions and Suggestions
The present study evaluated the chemical composition and the antisickling and antibacterial activities of Uvariodendron molundense. The results revealed that:
- The leaves and bark of U. molundense contain various secondary metabolites such as total phenols, anthocyanins, flavonoids, tannins, quinones, saponins, alkaloids, steroids, terpenoids and leuco-anthocyanins;
- All tested extracts displayed interesting antisickling activity. At 100 µg/mL, the rates of normalization were 89% for organic extract (ED50 = 0.391 µg/ml) and 82% for anthocyanins extract (ED50 = 0.659 µg/mL).
- The antibacterial activity of the plant extracts was very good toward Staphyloccocus aureus (CMI ≤ 31.25 µg/mL) while, for Escherichia coli, only the organic extract exhibit interesting activity (CMI = 31.25 µg/mL).
This study provided experimental evidence that supports further development of U. molundense extracts as a medicine for the management of SCA in endemic areas. This plant species as dietary supplement with proven antisickling and bacterial inhibitory potentialities could be of great interest to promote for the management of SCA in this area as nutraceuticals. Further work is in progress to identify the compounds responsible for these interesting activities. It would be therefore necessary to evaluate the interaction of such phytochemicals with β- lactam based antibiotics against SCA pathogenic bacteria agents in order to increase the chances of success of prophylaxis with the combination of plant extracts/phytochemicals with oral penicillin as a suitable mean to prevent infections caused by such pathogens in children with SCA.
The authors are indebted to The World Academy of Sciences (TWAS) (Grant No. 15-156 RG/CHE/AF/AC_G?FR3240287018) for providing financial assistance.