In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease Benjamin Z. Gbolo1,2*, Amandine Nachtergael2, Damien S. T. Tshibangu3, Nicole M. Misengabu4, Nsabatien Victoire1, Patrick B. Memvanga5, Dorothée D. Tshilanda2, K. N. Ngbolua1, Pius T. Mpiana3, Pierre Duez2 1Department of Biology, Faculty of Sciences, University of Kinshasa, Kinshasa, Democratic Republic of the Congo 2Unit of Therapeutic Chemistry and Pharmacognosy, Faculty of Medicine and Pharmacy, University of Mons (UMONS), 7000 Mons, Belgium 3Department of Chemistry, Faculty of Sciences, University of Kinshasa, Kinshasa, Democratic Republic of the Congo 4Department of Biology, Institut de Recherche en Sciences de la Santé (IRSS), Kinshasa, Democratic Republic of the Congo 5Faculty of Pharmaceutical Sciences, University of Kinshasa, Democratic Republic of the Congo Abstract Sickle cell disease (SCD) is an autosomal recessive blood disorder characterized by red blood cells that assume an abnormal, rigid sickle shape under low-oxygen conditions. These sickle-shaped erythrocytes tend to lyse, aggregate, and obstruct small blood vessels, leading to major complications. The present study aims to investigate properties that may underlie the activity of Drepanoalphaâ, an antisickling herbal formulation developed in the Democratic Republic of Congo (DRC) for the prevention and symptomatic treatment of sickle cell disease crises. The Drepanoalpha® Ethanolic Extract (DEE) is a dry extract (drug-extract ratio, DER, 100/11) prepared from ethanol (96 %, v/v) percolation of a 1:1 mixture of 2 food plants, Justicia secunda Vahl and Moringa oleifera Lam. Sickling was classically measured by light microscopy on diluted washed erythrocytes obtained from homozygote patients; erythrocytes were treated with 2 % Na2S2O5 in the presence of DEE (suspension in 9 ‰ NaCl), 9 ‰ NaCl (negative control) or disodium cromoglycate (DSCG, positive control). For all tested conditions, the sickle hemoglobin polymerization, the Fe2+/Fe3+ ratio, and the median corpuscular fragility were measured by spectrophotometry. The DEE reversed sickling by 89.1 %, comparable to DSCG (87.7 %; 60.3 µg/mL), inhibiting sickle cell hemoglobin polymerization of 77.8 % and 74.4 %, respectively. The Fe2+/Fe3+ ratio was improved by 18.0 % for DEE and 15.9 % for DSCG. The median corpuscular fragility values were 0.602, 0.714, and 0.732 for NaCl 9 ‰, DSCG, and DEE, respectively. The measured in vitro parameters validate an effective antisickling effect of DEE and confirm the value of this improved traditional herbal formulation for the management of SCD. Keywords: antisickling activity; drepanoalpha®; erythrocytes; Fe2+/Fe3+ ratio; hemoglobin polymerization; sickle cell disease Data of article Received Reviewed Accepted : : : 27 Aug 2022 30 Dec 2022 23 Feb 2023 DOI 10.18196/jfaps.v2i1.16000 Type of article: Research * Corresponding author, e-mail: benjamin.gbolo@unikin.ac.cd Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 65 INTRODUCTION Sickle cell disease (SCD), or sickle cell anemia or drepanocytosis, is a genetic disease that affects hemoglobin and leads to the synthesis of hemoglobin S (HbS) instead of normal hemoglobin (HbA). In their oxygenated forms, the solubility of HbS decreases 50 times, resulting in its precipitation and intracellular polymerization, which modifies the structure of red blood cells that take a "sickle shape," which tend to lyse but also aggregate and obstruct small blood vessels, leading to major complications.1,2 The disease is not only most prevalent in black people from Africa but is also prevalent around the Mediterranean and in India.3 It is estimated that more than 300,000 babies are born worldwide each year with severe forms of this hemoglobinopathy.1,4,5 Management of the disease is difficult in developing countries, particularly in the Democratic Republic of Congo (DRC), where, with some 25 % AS genotypes, nearly 2 % of the population is affected with SS genotypes.6 Indeed, poverty conjugates to the absence of a welfare system, with huge difficulties in meeting medical costs.5,7 As for many diseases, the relative costs of treatments and their associated adverse effects make the recourse to herbal medicines attractive or even essential, especially for rural populations.4,8 Effectively, an estimated 80 % of the Sub-Saharan population uses traditional medicine for health care; some plants have already proven their effectiveness, and bioactive molecules have been identified.1,4,9,10 The growing interest and use of phytomedicines to treat sickle cell disease are also probably linked to the assumption that medicinal plants are "natural" and "safe". 4,11–13 In DRC, a vast bio-prospecting program has identified a hundred antisickling plants; based on an in vitro antisickling assay, the most active plants were developed in Drepanoalpha®, an improved traditional herbal formulation.1,5,14,15 The Drepanoalpha® powder is a mixture 1:1 (w/w) of 2 edible plant leaves powder, Justicia secunda Vahl (Acanthaceae) and Moringa oleifera Lam. Justicia secunda, a native tropical herbaceous plant originating from South America, is nowadays grown in tropical or subtropical African countries. In the past, this plant was considered ornamental until locals discovered the medicinal properties of its leaves, notably for the treatment of anemia, hypertension16,17 and sickle cell disease.14,18 The phytochemical study of leaves from various J. secunda cultivars revealed the presence of polyphenols, including tannins, leucoanthocyanins, anthocyanins and, mainly, flavonoids.16 The Moringa genus comprises 14 species, among which M. oleifera is sometimes designated as the "tree of life" or "miracle vegetable". This tropical tree, native to the sub-Himalayan mountains, is widely distributed in tropical and sub-tropical areas, both dry and humid.19–21 The leaves used for animal and human feeding, given their alleged richness in proteins, vitamins, b-carotene, and amino acids, are now considered a functional food.14,19,21–25 A wide variety of medicinal uses have been attributed to M. oleifera's various organs for anti-inflammatory, anti-infectious, cardiovascular, gastrointestinal and hematological properties, including the management of sickle cell disease.14,19,21,24 Phytochemical studies reported the highest level of phenolic compounds, mainly flavonoids, in M. oleifera leaves.26 The originality of this study resides in the extraction that allowed to prepare of an Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 66 improved phytomedicine, easier to manage compared to traditional decoctions, and the combination of a series of anti-sickling tests (on cells and hemoglobin) to evaluate, in vitro, the effectiveness of this extract. The present study was carried out on a polar extract of the 2 plants mixture to investigate properties that may underlie the use of Drepanoalpha in the prevention and symptomatic treatment of sickle cell disease crises. METHOD Chemicals and reagents All reagents were of analytical grade. 2- aminoethyl diphenylborinate (97 %) and quercetin hydrate (≥ 95 %) were purchased from Sigma-Aldrich (Merck); polyethyleneglycol 400 (PEG 400) (for laboratory use), methanol (99 %), absolute ethanol (≥ 99.8 %), methyl ethyl ketone (GPR Reactapur), formic acid (98 %) and ethyl acetate (ACS reagent) were obtained from VWR Chemicals. A 10 g/L solution of diphenyl boric acid 2-amino ethyl ester (NP reagent) and a 50 g/L solution of PEG 400 were prepared in methanol to detect polyphenols. Herbal material The Drepanoalpha® powder, a 1:1 (w/w) mixture of the leaves from 2 food plants, Justicia secunda Vahl (Herbarium MNHN- P-P00719831) and Moringa oleifera Lam. (Herbarium MNHN-P-P05401821) has been provided by Research for Sustainable Development (RESUD, Kinshasa, DRC), approved by producer and distributor of the phytomedicine. Extraction The dry extract (drug-extract ratio, DER, 100/11) was obtained by percolating 100 g of the herbal material mixture with 1000 mL ethanol 96 % for 48 h and drying under vacuum at 40°C. The resulting Drepanoalpha® ethanolic extract (DEE) was stored at  4°C for a maximum of 3 months. The leaves of J. secunda and M. oleifera were individually extracted similarly. High-performance thin-layer chromatography (HPTLC) HPTLC was performed according to the procedure of the European Pharmacopeia 10,27 using Automatic TLC Sampler (ATS 4), Automatic Developing Chamber 2 (ADC 2), Derivatizer and TLC Visualizer 2 (Camag, Muttenz, Switzerland). The systems were driven by the software visionCATS version 2.5. The HPTLC was performed on silica gel 60 F254 HPTLC plates (Merck, Germany); 2 µL of quercetin (1 mg/mL MetOH), 10 µL of DEE (30 mg/mL MetOH) and 5 µL of J. secunda and M. oleifera (30 mg/mL MetOH) extracts were applied in 6-mm wide bands, the plates were activated on MgCl2 (~ 33 % RH) and the tank saturated for 20 min; the solvent system was ethyl acetate - methylethylketone - formic acid -water (60:30:5:5, V/V/V/V) and the plate was developed over a path of 60 mm (Fig.1). The plate was heated at 100 °C for 3 min, sprayed with 2 mL of the NP reagent (Derivatizer with green nozzle, level 3) and PEG (Derivatizer with blue nozzle, level 2), and photographed immediately after derivatization under UV365nm, using the Visualizer system. Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 67 Figure 1. HPTLC profile of the ethanolic extracts of Drepanoalpha® and its constituent plants. Stationary phase: HPTLC plate (Silica gel 60 F254) Mobile phase: ethyl acetate - methylethylketone - formic acid - water (60:30:5:5, V/V/V/V); Detection: NP-PEG-400; 1% solution of 2-aminoethyl diphenylborinate in methanol, followed by 5% polyethylene glycol in ethanol; examination under UV365nm. Legend: Tracks: 1-Quercetin, 2-Justicia secunda, 3-Moringa oleifera, 4- Drepanoalpha®. Blood samples Blood samples were left-overs of specimens sampled for the regular monitoring of known sickle cell disease homozygote patients attending the “Centre de Médecine Mixte et d’Anémie SS” (Kalamu district, Kinshasa, DRC) and the “Hôpital Civil Marie Curie” (Charleroi, Belgium). None of these patients had experienced a recent transfusion with Hb AA blood. All antisickling experiments were carried out with blood freshly collected on citrate and stored at ± 4 °C for a maximum of 72 h. Red cell pellets, obtained by centrifugation (1500 g, 10 min) of 0.5 mL of SS blood, were washed thrice with NaCl 9 ‰, in a 1:10 (v/v) ratio, centrifuged (1500g, 10 min) and resuspended in 4 mL NaCl 9 ‰. The study was conducted after receiving the approval of the ethical and scientific committee of the School of Public Health, Faculty of Medicine, University of Kinshasa, Kinshasa-DRC (Approval No.: ESP/CE/237/2019) and of the I.S.P.P.C. OM008 ethical committee of “C.H.U. Charleroi”, Charleroi, Belgium (Approval No P19/55-23/10 CHRAU: UMONS CCB: B325201941714). In vitro antisickling activities Induction of sickling and inhibition of falciformation Samples of 950 µL of blood, obtained from homozygote patients, diluted 1:10 in 9 ‰ NaCl, were added with 10 µL of Drepanoalpha Ethanolic Extract (DEE) (suspension in 9 ‰ NaCl), 9 ‰ NaCl 1 2 3 4 Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 68 (negative control) or disodium cromoglycate (DSCG, positive control) and homogenized. Upon adding 50 µL of 2 % Na2S2O5 and homogenizing, 5 µL of samples were placed on a microscope slide, covered and smeared with clear varnish to isolate from oxygen and induce hypoxia and sickling. The sickled/normal erythrocyte ratios were measured in light microscopy at times 0 and 60 min on images of 5 different microscopic fields acquired with a digital camera (Olympus U-CMAD3): F (%) = SRB TRB 𝑥100 Where F: Sickling Cell Rate; SRB: Sickled Red Blood Cell Count and TRB: Total Red Blood Cell Count. The proportion of sickle cell inhibition SI was calculated as follows (28): 𝑆𝐼 = 𝐹0 − 𝐹𝑛 𝐹0 𝑥 100 Where SI is the percentage of sickling inhibition, 𝐹0 is the % of sickling of the mixture [SS blood + Na2S2O5] (negative control) and 𝐹𝑛 is the % of sickling of the mixture [SS blood + tested extract or compound + Na2S2O5]. For a test to be considered valid, the ratio TRB60 min/TRB0 min should be over 80  5 % to control that only limited hemolysis has been induced by the tested extract or compound. The capacity of extracts to prevent, reverse or protect against falciformation To understand whether the tested extract or compound prevents or reverses falciformation, 3 procedures were assessed: a) A mixture of 950 µL diluted blood and 50 µL Na2S2O5 was incubated for 60 min in a closed vial, then added with 10 µL of DEE, NaCl 9 ‰ or DSCG and homogenized; b) A mixture of 950 µL diluted blood and 10 µL DEE, NaCl 9 ‰ or DSCG was incubated for 60 min in a closed vial, then added with 50 µL Na2S2O5 and homogenized; c) A mixture of 950 µL diluted blood, 10 µL of DEE, NaCl 9 ‰ or DSCG and 50 µL Na2S2O5 was incubated for 60 min in a closed vial. For the different procedures, the proportions of sickle cell inhibition were assessed as previously described. Determination of the Minimum Reversibility Concentrations (MRC) For the MRC determination, the proportions of sickle cell inhibition (= reversibility rate) were measured as described here above by varying the concentrations of DEE and DSCG (50 to 250 g/mL). For each concentration, the proportion of sickle cell inhibition (SI) was calculated as above to determine the rate of reversibility R as: 𝑅 = 𝑆𝐼0 − 𝑆𝐼𝑛 𝑆𝐼0 𝑥 100 Where R is the reversibility rate (%) and 𝑆𝐼0 and 𝑆𝐼𝑛 are the proportions of sickling inhibition for the control (NaCl 9 ‰) and the tested concentration, respectively. Dose-effect curves were obtained by fitting data to the equation y = 𝐴1−𝐴2 1+(𝑥 𝑥0⁄ ) 𝑝 + 𝐴2 using the Origin 8.5 software (OriginLab, Northampton, MA, United States). Inhibition of sickle hemoglobin (HbS) polymerization According to the original method of (29), the HbSS polymerization was assessed at 700 nm from the turbidity of a polymerizing mixture. 200 µL of a red cell pellet were hemolyzed by adding 400 µL of distilled water, incubated for 30 min in the presence or absence of the drug (400 µL) and primed for polymerization by deoxygenating with 3000 µL of a 2 % Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 69 sodium metabisulphite solution. The optical densities were measured after centrifuging at 3500 rpm for 5 min. Their difference yields the measure of turbidity. The rate of polymerization inhibition was estimated by the tangent of the graph "absorbance versus time". The relative polymerization and relative inhibition were determined concerning the control (24) as 𝑅𝑝 = 𝑂𝐷𝑡 − 𝑂𝐷𝑖 𝑡 Where 𝑅𝑝 = rate of polymerization, 𝑂𝐷𝑡 = Optical Density at time t, 𝑂𝐷𝑖 = initial Optical Density, t=time Determination of erythrocyte membrane stability The osmotic fragility of erythrocytes allows the measurement of eventual membrane-stabilizing effects by a 60 min incubation in osmotic stress conditions. 10 µL of a red cell pellet were diluted in 1990 µL of a series of buffered hypotonic saline solutions at different concentrations (0.2 - 0.8 % NaCl), added with 10 µL of DEE, DSCG, or NaCl 9 ‰ and homogenized. The effect of the different extracts on hemolysis was observed in light microscopy with a digital camera (Olympus U-CMAD3). Total cells were counted from 5 different fields across each slide at 0 and 60 min. For each NaCl concentration and extract, the percentage of hemolysis was calculated as follows: % 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 = 𝑁0 − 𝑁60 𝑁0 𝑥 100 where 𝑁0 and 𝑁60 are the numbers of red blood cells at 0 and 60 min, respectively. The median corpuscular fragility (MCF), the NaCl concentration that causes 50 % erythrocyte hemolysis, was estimated from the linear regression "% hemolysis versus NaCl concentration" using the ORIGIN 8.5 software. Determination of methemoglobin concentration The red cell pellet was hemolysed with distilled water in a 1: 2 ratio (v/v) and centrifuged (1500 g, 10 min). The hemolysate was incubated at room temperature in the presence or absence of the DEE/DSCG. The evolution of absorbances was measured at 540 and 630 nm for hemoglobin (Fe2+) and methemoglobin (Fe3+), respectively. The proportion of methemoglobin was calculated at each time as follows: 𝐹𝑒3+ = (𝐴630) 2 (𝐴540) 2 + (𝐴630) 2 𝑥100 𝐹𝑒3+, 𝐴540 and 𝐴630 are the proportion of methemoglobin and the absorbances at 540 and 630 nm, respectively. To appreciate the kinetics of the reaction in the presence or absence of extract, 𝐹𝑒3+-time curves were obtained by fitting data to the equation y = 𝐴1−𝐴2 1+(𝑥 𝑥0⁄ ) 𝑝 + 𝐴2 using the Origin 8.5 software. Statistical analysis All the experiments were conducted in triplicate; the data were expressed as mean ± standard deviation (S.D) and analyzed using Origin 8.5 software with a Chi-square test. The level of significance was classically set at 0.05. Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 70 Results and Discussion Sickling induction Table 1: Sickling induction assay (60 min contact with sodium metabisulphite in air- tight conditions; measurement over 5 microscopic fields) Patient Date of assay Sample code Total red blood cells Sickled red blood cells % Sickling Test considered as 1 31/10/19 SS_70 168 139 80.4 Positive 2 31/10/19 HETERO_40 136 4 4.4 Negative 3 31/10/19 HETERO_58 185 6 3.2 Negative 4 05/11/19 SS_91 189 21 11.1 Negative 5 05/11/19 SS_54 240 240 100.0 Positive 6 08/11/19 SS_40 152 14 9.2 Negative 7 12/11/19 SS_59 144 6 4.2 Negative 8 12/11/19 SS_759 138 14 10.1 Negative 9 12/11/19 SS_36 93 78 83.9 Positive 10 12/11/19 SS_68 127 15 11.8 Negative 11 14/11/19 SS_07 100 13 13.0 Negative 12 18/11/19 SS_396 126 117 92.9 Positive 13 18/11/19 SS_597 195 162 83.1 Positive 14 12/12/19 SS_237 234 207 88.4 Positive Fourteen samples were received from Hôpital Civil Marie Curie and tested for their ability to falciform in deoxygenation conditions (Table 1). Figure 2 shows phenotypic micrographs of representative samples. As expected, the samples from heterozygote patients yielded a very low proportion of sickled cells in our experimental conditions. However, 6 samples from homozygote patients were also weakly falciform, indicating a possible treatment by hydroxyurea (induction of non-sickling fetal hemoglobin) and antioxidants (scavenging Na2S2O5). These heterozygotes and non-falciform samples (<50 % sickling) were not considered for the following experiments. Figure 2. Sickling induction assay. Phenotypic micrographs of representative samples (60 min contact with sodium metabisulphite in air-tight conditions; 500 X) Legends: HETERO: heterozygous blood; SS: SS homozygous blood; 40, 58, 396 are the identification numbers of the samples attributed by the laboratory of the supplying hospitals. Normal RBCs Sickle Cells Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 71 Reversibility assay Table 2 details the sickling reversal of SS patient erythrocytes untreated (control) and treated with DSCG and J. secunda, M. oleifera, and DEE under hypoxic conditions. Figure 3 shows representative phenotypic micrographs of untreated and treated erythrocytes. Figure 3. Morphology of drepanocytes of SS blood (Sample SS_54), A: untreated (0.9% NaCl), and upon treatment with sodium Cromoglycate (B: 250 µg/mL); ethanolic extracts of J. secunda (C), M. oleifera (D) and Drepanolpha® (E) (125 µg/mL); 60 min contact with sodium metabisulphite in air-tight conditions; 500 X) Table 2. Anti-sickling effects on SS erythrocytes (60 min contact with sodium metabisulphite in air-tight conditions; measurement over 5 microscopic fields) Blood sample Negative control Cromoglycate Ethanolic extracts Justicia secunda Moringa oleifera Drepanoalpha® TRB(a) SRB(b) SI(c) TRB SRB SI TRB SRB SI TRB SRB SI TRB SRB SI SS_70 168 139 82.7 159 31 19.5 168 10 6.0 162 11 6.8 156 30 19.2 SS_54 240 240 100.0 186 28 15.1 204 18 8.8 225 20 8.9 219 24 11.0 SS_36 93 78 83.9 93 9 9.7 95 7 7.4 95 7 7.4 93 9 9.7 SS_396 126 117 92.9 177 11 6.2 177 8 4.5 120 8 6.7 102 8 7.8 SS_597 195 162 83.1 174 18 10.3 189 11 5.8 189 18 9.5 183 15 8.2 SS_237 234 207 88.5 183 15 8.2 231 15 6.5 234 20 8.6 210 18 8.6 Mean 88.5 11.5 6.5 8.0 10.8 (SD) (6.9) (4.9) (1.5) (1.2) (4.3) P vs. negative control(e) --- <0.001 <0.001 <0.001 <0.001 Description (a) TRB: Total red blood cell count (b) SRD: Sickled red blood cells count (c) SI: percentage of sickling induction (e) Anova one-way with posthoc t-tests (Tukey); there were no statistical differences between the treatments cromoglycate - Justicia secunda - Moringa oleifera - Drepanoalpha® A B C D E Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 72 Figure 4. Reversibility rate of sickled red blood cells according to the concentration of DEE and DSCG. (60 min contact with sodium metabisulphite in air-tight conditions). (Data from 3 biological tests in triplicate, Bars represent the mean ± SD) Description DEE: Drepanoalpha® ethanolic extract DSCG: disodium cromoglycate MRC: minimum reversibility concentration MRR: maximum reversibility rate Figure 4 presents the reversibility rate of sickled red blood cells according to DEE and cromoglycate concentration. The rate of reversibility of sickle red blood cells in hypoxic conditions increased with the concentration of DEE or DSCG until reaching a maximum threshold (MMR, maximum reversibility rate), above which the reversibility remained constant, regardless of the increase in concentration. The minimum reversibility concentration (MRC) was defined as the extract concentration for which 50 % of the sickled cell population was normalized. MMR and MRC were evaluated by non-linear regression using ORIGIN 8.5 software The capacity of extracts to prevent, reverse, and protect against falciformation To verify whether DEE prevents or reverses falciformation, the SS_54 sample was treated in 3 different protocols (DEE treatment followed by deoxygenation, i.e., prevention; deoxygenation followed by DEE treatment, i.e., reversal; concomitant DEE treatment and deoxygenation, i.e., protection) compared with cromoglycate. Figure 5 shows the phenotypic micrographs of treated samples. Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 73 Figure 5. Morphology of drepanocytes of SS blood (Sample SS_54) upon different schemes of treatment with Drepanolpha® ethanolic extract (DEE) or cromoglycate (DSCG) (125 g/mL; 60 min contact with sodium metabisulphite in air-tight conditions; 500 X) These experiments indicated that the DEE had the ability to prevent, reverse and protect against erythrocyte sickling. Although DSCG also reversed and protected against sickle cell formation, the preventive DSCG treatment appeared inefficient in averting sickling. Inhibition of sickle hemoglobin (HbS) polymerization The polymerization rates of HbS and its inhibition are presented in Table 3 Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 74 Table 3. Polymerization rates of HbS and its inhibition by the Drepanoalpha® extract (DEE) and cromoglycate (DSCG) (n = 3 technical replicates) Sample Rate of polymerization Relative % Inhibition vs. negative control Negative control 0.65  0.01 ---- DSCG 0.17  0.0 74.4  0.0 DEE 0.14  0.02 77.8  0.0 Table 3 indicates that the polymerization of sickle hemoglobin (HbS) is partly inhibited in the presence of either DSCG or DEE in a similar proportion. This property is well known to contribute to antisickling activities. Stabilization of erythrocyte membranes Figure 6 indicates an effective hypoosmolarity-induced lysis of sickle erythrocytes when decreasing the NaCl concentration. Although this effect was less marked in the presence of Drepanoalpha® extract or cromoglycate, the protection afforded was not statistically significant. Figure 6. Lysis susceptibility of sickle erythrocytes, according to osmolarity, in the presence of Drepanoalpha® ethanolic extract or cromoglycate (125 g/mL; 60 min incubation at room temperature) Description MCF: median corpuscular fragility 0,0 0,2 0,4 0,6 0,8 0 25 50 75 100 H e m o ly s is r a te ( % ) NaCl Concentration(%) Negative control : MCF= 0.669 Disodium cromoglycate : MCF= 0.714 Drepanoalpha ® Ethanolic Extract : MCF= 0.732 0.0 0.2 0.4 0.6 0.8 Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 75 Modulation of methemoglobin formation Figure 7 and Table 4 show the evolution of methemoglobin as a function of time; DEE and DSCG significantly reduced the formation of methemoglobin in HbSS blood, preventing the oxidation of Fe2+ into Fe3+. Figure 7. Evolution of methemoglobin proportion versus time. Bars represent the mean ± SD for N=3 technical replicates. Table 4. Modulation of methemoglobin formation in the presence of DEE and DSCG (mean ± SD; n = 3 technical replicates) Sample %Hemoglobin (Fe2+) %Methemoglobin (Fe3+) Fe2+/Fe3+ % increase Negative Control 84.90.3 15.1  0.3 5.6  1.0 ----- DSCG 94.10.4 5.9  0.4 15.9  1.0 64.641.3 DEE 94.70.6 5.3  0.6 18.0  1.0 68.851.0 RESULTS AND DISCUSSION The bioactivity was assessed based on the phenotypic sickling of SS red blood cells (RBCs) in the presence of an oxygen- scavenging agent. The individual plant extracts and DEE displayed remarkable sickling inhibitory effects, reverting sickle erythrocytes to a typically normal morphology in the same proportions as cromoglycate, a well-known positive control (18,24,30). It confirms that the DEE extract conserves the previously shown antisickling effects of Moringa oleifera extracts24 and Drepanoalpha® herbal powder.1 The sickling of red blood cells is a process that results from the polymerization of Hb S under conditions of hypoxia and cellular dehydration by loss of ions (K+, Cl-, Mg++) and water (31,32). The antisickling effect indicated that DEE could rehydrate deoxygenated sicked red cells, thus preventing the increase of intracellular Hb S concentration. Anthocyanins, part of DEE secondary metabolites1, have been shown to be 0 60 120 180 240 4 6 8 10 12 14 16 P ro p o rt io n o f m e th e m o g o lb in ( F e 3 + ) Time (min) Negative controle Disodium cromoglycate Drepanoalpha Ethanolic Extract Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 76 responsible for most plants' biological activities against sickle cell disease in traditional Congolese medicine by weakening hydrophobic interactions at the intermolecular contact sites of different deoxyhemoglobin S molecules.1,15,33–37 The antisickling effect of DEE is higher compared to some antioxidants and micronutrients often combined in the management of sickle cell disease, i.e., magnesium (0.1 mM;48.4 % reversal of in vitro falciformation), zinc (0.1 mM; 89.7 %), vitamins A (100 IU; 30.9 %), C (1 mg/mL; 38.1%) and E (1 mg/mL; 30.9 %) (38). The action of DEE on the inhibition of polymerization could be a synergistic effect of its (phyto)chemical constituents such as anthocyanins, flavonoids and micronutrients, including the Mg++, Zn++, and Vit A .1 However, the copper identified among the micronutrients of this phytomedicine (1) is a negative agent for sickle cell disease whose level should be controlled in the final product. It is well established that dense and dehydrated red blood cells can contain HbS polymers under conditions of moderate hypoxia, and even in arterial blood, due to the particularly high intracellular concentration of HbS. These dense and dehydrated red blood cells thus play a central role in sickle cell disease's acute and chronic manifestations based on reduced blood flow and vaso- occlusions in small vessels (31). The erythrocyte membrane stability test performed on sickled red cells in hypotonic NaCl condition indicates a slight but insignificant increase of sickle cell resistance as measured by the MCF. It would be interesting to repeat the test with the preincubation of erythrocytes before hypotonic stressing to evaluate an eventual membrane protective effect. Given the high red cell oxidative status (39,40), hemoglobin can oxidize to methemoglobin and thus lose the property of combining with oxygen (41– 44). In normal blood, only a very small amount of methemoglobin exists. An effective system based on nicotinamide adenine dinucleotide phosphate (NADPH), methemoglobin reductase and cytochrome B5 to reduce the heme Fe3+ to Fe2+, the metabolic shunt pathway of pentose phosphates in erythrocyte is necessary for the synthesis of NADPH that protects hemoglobin and membrane lipids from oxidation.45 However, this reduction system is less efficient in cases of glucose- 6-phosphate dehydrogenase (G-6-PD) deficiency, an erythrocytic enzymopathy often associated with sickle cell disease. A treatment inducing a decrease in methemoglobin level would indicate an effective antioxidant effect on sickle red blood cells,46 likely to protect from sickling and senescence. As shown here, both DSCG and DEE effectively improve the Fe2+/Fe3+ ratio, a mechanism likely to increase the oxygen affinity of drepanocytes and so to reverse sickle improving the Fe2+/Fe3+ ratio erythrocytes to their original biconcave structure. Our results indicated that, in vitro, DEE effectively prevented both erythrocyte sickling and hemoglobin oxidation. Here again, this activity could be the result of the DEE content in polyphenols, including flavonoids, and trace elements such as Mg++, Zn++ and Vit A (1,38,47). This study will likely impact transfusion treatments as our previous clinical studies on the phytomedicine decoction have shown an increase in Hb level and protection against early hemolysis, which would prevent anemia and avoid transfusion (48,49). Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 77 CONCLUSION This research depicted in vitro anti-sickling activity of Drepanoalpha® ethanolic extract. The results revealed that DEE preventing sickling and reversing sickled HbSS red blood cells had a membrane stabilizing effect on sickled red blood cells, possessed abilities to inhibit sickle cell hemoglobin polymerization, and improved the oxidant status of erythrocytes by increasing the Fe2+/Fe3+ ratio in a sickled red blood cell. It highlighted that this traditional improved herbal formulation had medicinal benefits, confirming its use in managing sickle cell disease (SCD). Future studies are suggested to identify and isolate the active principle of this phytomedicine by bio-guided fractionation, which could enhance the standardization of this anti- sickling recipe. ACKNOWLEDGMENTS This research received funding from the Academy of Research and Higher Education (ARES) in a program entitled "Institutional Support (IA) 2018-2020". Grant number: COOP-CONV-18-004, AI ARES UNIKIN. The authors would like to thank RESUD for providing the herbal material and Dr. Nicole Tasiaux, Mr. Charles Chevalier of “C.H.U. Charleroi” and Mr. Didier BOSOLO EFUNDA «of “Centre de Médecine Mixte et d’Anémie SS” for their collaboration. REFERENCES 1. Gbolo BZ, Asamboa LS, Bongo GN, Tshibangu DST, Kasali F, Memvanga P, et al. Bioactivity and Chemical Analysis of Drepanoalpha : An Anti- Sickle Cell Anemia Poly-Herbal Formula from Congo-Kinshasa. Am J Phytomedicine Clin Ther. 2017;5(1):1–7. https://doi.org/10.9734/JOCAMR/201 7/37350 2. Aufradet E. Drépanocytose et activité physique : conséquences sur les mécanismes impliqués dans l’adhérence vasculaire, l’inflammation et le stress-oxydatif. Thèse en vue de l’obtention d’un doctorat en Sciences et techniques des activités physiques et sportives, Université de Lyon, France. [Lyon]: University of Lyon; 2012. 3. Imaga NA. Phytomedicines and nutraceuticals: Alternative therapeutics for sickle cell anemia. Sci World J. 2013;2013:1–12. https://doi.org/10.1155/2013/269659 4. Okoh MP, Alli LA, Tolvanen MEE, Nwegbu MM. Herbal Drug use in Sickle Cell Disease Management; Trends and Perspectives in Sub- Saharan Africa - A Systematic Review. Curr Drug Discov Technol. 2019;16(4):372–85. https://doi.org/10.2174/157016381566 6181002101611 5. Verlhac S, Kandem A, Bernaudin F, Vasile M. L’accident vasculaire cerebral chez l’enfant drepanocytaire. Efficacite du protocole de prevention par doppler transcranien. J Radiol. 2007;88(10):1453. https://doi.org/10.1016/S0221- 0363(07)81397-7 6. Shongo MY a. P, Mukuku O, Lubala TK asol., Mutombo AM ulang., Kanteng GW akam., Umumbu WS ombod., et al. Sickle cell disease in stationary phase in 6-59 months children in Lubumbashi: epidemiology and https://doi.org/10.9734/JOCAMR/2017/37350 https://doi.org/10.9734/JOCAMR/2017/37350 https://doi.org/10.1155/2013/269659 https://doi.org/10.2174/1570163815666181002101611 https://doi.org/10.2174/1570163815666181002101611 https://doi.org/10.1016/S0221-0363(07)81397-7 https://doi.org/10.1016/S0221-0363(07)81397-7 Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 78 clinical features. Pan Afr Med J. 2014;19(71):1–7. 7. Tshilolo L, Aissi LM, Lukusa D, Kinsiama C, Wembonyama S, Gulbis B, et al. Neonatal screening for sickle cell anaemia in the Democratic Republic of the Congo : experience from a pioneer project on 31 204 newborns. J Clin Pathol. 2009;62:35–8. https://doi.org/10.1136/jcp.2008.058958 8. Abere TA, Egharevba CO, Chukwurah IO. Pharmacognostic evaluation and antisickling activity of the leaves of Securinega virosa Roxb. ex Willd. ( Euphorbiaceae). African J Biotechnol. 2014;13(40):4040–5. 9. Joseph Kahumba, Tsiry Rasamiravaka, Philippe Ndjolo Okusa, Amuri Bakari, Léonidas Bizumukama, Jean-Baptiste Kalonji, Martin Kiendrebeogo, Christian Rabemenantsoa, Mondher El Jaziri, Elizabeth M. Williamson PD. Traditional African medicine: from ancestral know-how to bright future. Science (80- ). 2015;350(6262):871– 871. 10. WHO. WHO Traditional Medicine Strategy: 2014-2023. WHO (World Heal Organ ) Libr Cat Data, Geneva. 2013;78. 11. Manya MH, Keymeulen F, Ngezahayo J, Bakari AS, Mutombo EK, Kahumba BJ, et al. Antimalarial herbal remedies of Bukavu and Uvira areas in DR Congo: An ethnobotanical survey. J Ethnopharmacol. 2020;249:1–28. https://doi.org/10.1016/j.jep.2019.112422 12. Mahavy CE, Duez P, Eljaziri M, Rasamiravaka T. African Plant-Based Natural Products with Antivirulence Activities to the Rescue of Antibiotics. Antibiotics. 2020;9:1–30. https://doi.org/10.3390/antibiotics911 0830 13. Chanda S, Parekh J, Vaghasiya Y, Dave R, Baravalia Y, Nair R. Medicinal Plants - From Traditional Use to Toxicity Assessment:A Review: Int J Pharm Sci Res. 2015;6(7):2652–70. 14. Mpiana PT, Ngbolua K-N, Tshibangu STD. Les alicaments et la drépanocytose : une mini-revue. Comptes Rendus Chim. 2016;19:884–9. https://doi.org/10.1016/j.crci.2016.02.019 15. Ngbolua K, Mpiana PT. The Possible Role of a Congolese polyherbal formula ( Drepanoalpha ) as source of Epigenetic Modulators in Sickle Cell Disease : A Hypothesis. J Adv Med Life Sci Res. 2014;2(1):1–3. 16. Kitadi JM, Lengbiye EM, Gbolo BZ, Inkoto CL, Muanyishay CL, Lufuluabo GL, et al. Justicia secunda Vahl species : Phytochemistry, Pharmacology and Future Directions : a mini-review. Discov Phytomedicine. 2019;6(4):157–71. https://doi.org/10.15562/phytomedici ne.2019.93 17. Koffi NG, Henri KK, Djakalia O. Plants used to treat anaemia , in traditional medicine , by Abbey and Krobou populations , in the South of Côte-d ’ Ivoire. J Appl Sci Res. 2010;6(8):1291– 7. 18. Mpiana PT, Bokota MT, Tshibangu DST, Ngbolua KN, Atibu EK, Kwembe JTK, et al. Antisickling activity of three species of justicia from Kisangani ( DR Congo ): Int J Biol Chem Sci [Internet]. 2010;4(6):1953–61. https://doi.org/10.1136/jcp.2008.058958 https://doi.org/10.1016/j.jep.2019.112422 https://doi.org/10.3390/antibiotics9110830 https://doi.org/10.3390/antibiotics9110830 https://doi.org/10.1016/j.crci.2016.02.019 https://doi.org/10.15562/phytomedicine.2019.93 https://doi.org/10.15562/phytomedicine.2019.93 Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 79 19. Ngbolua K-N. An Updated review on the Bioactivities and Phytochemistry of the Nutraceutical Plant Moringa oleifera Lam (Moringaceae) as valuable phytomedicine of multi- purpose. Discov Phytomedicine. 2018;5(4):52–63. https://doi.org/10.15562/phytomedici ne.2018.71 20. Leone A, Spada A, Battezzati A, Schiraldi A, Aristil J, Bertoli S. Cultivation, genetic, ethnopharmacology, phytochemistry and pharmacology of Moringa oleifera leaves: An overview. Int J Mol Sci. 2015;16(6):12791–835. https://doi.org/10.3390/ijms160612791 21. Maldini M, Maksoud SA, Natella F, Montoro P, Petretto GL, Foddai M, et al. Moringa oleifera: Study of phenolics and glucosinolates by mass spectrometry. J Mass Spectrom. 2014;49(9):900–10. https://doi.org/10.1002/jms.3437 22. Tshingani K, Donnen P, Mukumbi H, Duez P, Dramaix-Wilmet M. Impact of Moringa oleifera lam. Leaf powder supplementation versus nutritional counseling on the body mass index and immune response of HIV patients on antiretroviral therapy: A single- blind randomized control trial. BMC Complement Altern Med. 2017;17(1):1–13. https://doi.org/10.1186/s12906-017- 1920-z 23. Shah SK, Jhade DN, Chouksey R. Moringa oleifera Lam. A study of ethnobotany, nutrients and pharmacological profile. Res J Pharm Biol Chem Sci. 2016;7(5):2158–65. 24. Nwaoguikpe R, Ujowundu C, Igwe C, Dike P. The Effects of Moringa oleifera Leaves Extracts on Sickle Cell Hemoglobin. J Sci Res Reports. 2015;4(2):123–32. https://doi.org/10.9734/JSRR/2015/12905 25. MPiana PT, Misakabu FM, Kitadi JM, Ngbolua KN, Tshibangu DST, Lombe BK, et al. Antisickling activity and physico-chemical stability of anthocyanin extracts from Hypoxis angustifolia Lam (Hypoxidaceae) Bulbs. In: Nohuru Motohashi, editor. Occurrences, Structure, Biosynthesis, and Health Benefits Based on Their Evidences of Medicinal Phytochemicals in Vegetables and Fruits. 3rd ed. Yew York: Nova Science Publishers,Inc; 2014. p. 97–114. 26. Bennett RN, Mellon FA, Foidl N, Pratt JH, Dupont MS, Perkins L, et al. Profiling glucosinolates and phenolics in vegetative and reproductive tissues of the multi-purpose trees Moringa oleifera L. (Horseradish tree) and Moringa stenopetala L. J Agric Food Chem. 2003;51(12):3546–53. https://doi.org/10.1021/jf0211480 27. COE (Council of Europe). High- performance thin-layer chromatography of herbal drugs and herbal drug preparations (2.8.25). 10th ed. EDQM, editor. Vol. 1, European Pharmacopoeia. Strasbourg Cedex: EDQM; 2019. 4370 p. 28. Kotue TC, Djote WNB, Marlyne M, Pieme AC, Kansci G, Fokou E. Antisickling and Antioxidant Properties of Omega-3 Fatty Acids EPA/DHA. Nutr Food Sci Int J https://doi.org/10.15562/phytomedicine.2018.71 https://doi.org/10.15562/phytomedicine.2018.71 https://doi.org/10.3390/ijms160612791 https://doi.org/10.1002/jms.3437 https://doi.org/10.1186/s12906-017-1920-z https://doi.org/10.1186/s12906-017-1920-z https://doi.org/10.9734/JSRR/2015/12905 https://doi.org/10.1021/jf0211480 Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 80 [Internet]. 2019;9(1):555752. Available from: https://juniperpublishers.com/nfsij/N FSIJ.MS.ID.555752.php 29. Noguchi CT, Schechter AN. Inhibition of Sickle Hemoglobin Gelation by Amino Acids and Related Compounds. Biochemistry. 1978;17(25):5455–9. https://doi.org/10.1021/bi00618a020 30. Bizumukama L, Ferster A, Gulbis B, Kumps A, Cotton F. In vitro inhibitory effects of disodium cromoglycate on ionic transports involved in sickle cell dehydration. Pharmacology. 2009;83(5):318–22. https://doi.org/10.1159/000215598 31. Brugnara C, De Franceschi L. New therapeutic approaches to sickle cell disease. Hématologie. 2006;12(4):239–45. 32. Ngbolua KN, Mudogo V, Mpiana PT, Malekani MJ, Herintsoa, Rafatro, Ratsimamanga, S. Urverg, Takoy, L, Rakotoarimana, H and Tshibangu DST. Evaluation de l ’ activité anti- drépanocytaire et antipaludique de quelques taxons végétaux de la République démocratique du Congo et de Madagascar. Ethnopharmacologia. 2013;50:7–12. 33. Ravelojaona M. Analyse histologique des répercussions musculaires , structurales , énergétiques et microvasculaires chez des hommes et des femmes drépanocytaires. Thèse de Sciences Présentée et soutenue publiquement pour l’obtention du diplôme de Doctorat en Biologie Médecine Santé, Spécialité : Biologie et Physiologie de l’Exercice. UNIVERSITÉ JEAN MONNET SAINT ETIENNE, France. [Saint-Etienne]: Université Jean Monnet - Saint- Etienne; 2014. 34. Mpiana PT, Mudogo V, Tshibangu DS., Ngbolua KN, Shetonde OM, Mangwala PK, et al. In vitro antisickling activity of anthocyanins extract of a congolese plant: Alchornea cordifolia. M. Arg. J Med Sci. 2007;7(7):1182–6. https://doi.org/10.3923/jms.2007.1182 .1186 35. Mpiana PT, Tshibangu DST, Shetonde OM, Ngbolua KN. In vitro antidrepanocytary actvity (anti-sickle cell anemia) of some congolese plants. Phytomedicine. 2007;14(2– 3):192–5. https://doi.org/10.1016/j.phymed.200 6.05.008 36. Mpiana PT, Mudogo V, Nyamangombe L, Kakule MK, Ngbolua KN, Atibu EK, et al. Antisickling activity and photodegradation effect of anthocyanins extracts from Alchornea cordifolia ( Schumach & Thonn ) and Crotalaria retusa. Ann Africaines Med. 2009;2(6):239–45. 37. Mpiana PT, Tshibangu DS, Ngbolua K, Tshilanda DD, Atibu EK. Antisickling Activity of Anthocyanins of Jatropha curcas L. In: Plants RP in M, editor. Chemistry and Medicinal Value. RPMP; 2007. p. 101–5. 38. Nwaoguikpe R, Braide W. The antisickling effects of some micronutrients and antioxidant vitamins in sickle cell disease management. J Med Med Sci. https://doi.org/10.1021/bi00618a020 https://doi.org/10.1159/000215598 https://doi.org/10.3923/jms.2007.1182.1186 https://doi.org/10.3923/jms.2007.1182.1186 https://doi.org/10.1016/j.phymed.2006.05.008 https://doi.org/10.1016/j.phymed.2006.05.008 Benjamin Z. Gbolo, Amandine Nachtergael, Damien S. T. Tshibangu,Nicole M. Misengabu,Nsabatien Victoire, Patrick B. Memvanga,et al | In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease 81 2012;3(5):334–40. https://doi.org/10.4314/ijbcs.v3i5.51079 39. Ngbolua KN. Evaluation de l’activité anti-drépanocytaire et anti-paludique de quelques plantes de la République Démocratique du Congo et écotypes de Madagascar. Thèse présentée et soutenue pour obtenir le grade de Docteur en Sciences. Université de Kinshasa, RDC. Kinshasa; 2012. 40. 4Mpiana PT, Ngbolua KTNN, Bokota MT, Kasonga TK, Atibu EK, Tshibangu DST, et al. In vitro effects of anthocyanin extracts from Justicia secunda Vahl on the solubility of haemoglobin S and membrane stability of sickle erythrocytes. Blood Transfus. 2010;8(4):248–54. 41. Abdullahi B. in Vitro Anti-Sickling Effect of Crude and Partially Purified Fractions of Methanolic Extract of Steculia Setigera Leaf on Human Sickled Red Blood Cells. Sci World J. 2018;13(4):81–6. 42. Richard AM, Diaz JH, Kaye AD. Reexamining the risks of drinking- water nitrates on public health. Ochsner J [Internet]. 2014;14(3):392– 8. Available from: http://www.ncbi.nlm.nih.gov/pubme d/25249806%0Ahttp://www.pubmed central.nih.gov/articlerender.fcgi?arti d=PMC4171798 43. Nanfack P, Biapa N, Pieme C, Ama- Moor V, Moukette B, Yonkeu JN. The in vitro antisickling and antioxidant effects of aqueous extracts Zanthoxyllum heitzii on sickle cell disorder. BMC Complement Altern Med. 2013;13(162):1–7. https://doi.org/10.1186/1472-6882-13-162 44. Kiefer I, Prock P, Lawrence C, Bayer P, Rathmanner T, Kunze M, et al. Supplementation with Mixed Fruit and Vegetable Juice Concentrates Increased Serum Antioxidants and Folate in Healthy Adults. J Am Coll Nutr. 2004;23(3):205–11. https://doi.org/10.1080/07315724.200 4.10719362 45. Tomc J, Debeljak N. Molecular pathways involved in the development of congenital erythrocytosis. Genes (Basel). 2021;12(1150):1–20. https://doi.org/10.3390/genes12081150 46. Kambale J, Ngolua K, Mpiana P, Mudogo V, Tshibangu D, Wumba D, et al. Evaluation in vitro de l’activité antifalcémiante et effet antioxydant des extraits d’Uapaca heudelotii Baill. (Euphorbiaceae). Int J Biol Chem Sci. 2013;7(2):523–34. https://doi.org/10.4314/ijbcs.v7i2.9 47. Mpiana PT, Misakabu FM, Tshibangu DST, Ngbolua KN, D.T. M. Antisickling Activity and Membrane Stabilizing Effect of Anthocyanins Extracts Antisickling Activity and Membrane Stabilizing Effect of Anthocyanins Extracts from Adansonia digitata L . Barks on Sickle. Int Blood Res Rev. 2014;2(5):198–212. https://doi.org/10.9734/IBRR/2014/10539 48. Gbolo ZB, Tshibangu STD, Memvanga BP, Bongo NG, Kasali MF, Ngbolua KN, et al. Assessment of the Efficacy and Tolerance of Drepanoalpha® in the Management of Sickle Cell Disease in Kinshasa (DR Congo): About Ten Cases. Int J Med Pharm Case Reports. 2017;9(2):1–10. https://doi.org/10.4314/ijbcs.v3i5.51079 https://doi.org/10.1186/1472-6882-13-162 https://doi.org/10.1080/07315724.2004.10719362 https://doi.org/10.1080/07315724.2004.10719362 https://doi.org/10.3390/genes12081150 https://doi.org/10.4314/ijbcs.v7i2.9 https://doi.org/10.9734/IBRR/2014/10539 Journal of Fundamental and Applied Pharmaceutical Science, 3(2), February 2023 82 https://doi.org/10.9734/IJMPCR/2017/ 33658 49. Gbolo ZB, Tshibangu D, Asamboa L, Bongo G, Kasali F, Feza V, et al. Sickle Cell Anemia Therapeutic Approach Based on Drepanoalpha ® : About 34 Sickle Cell Anemia Therapeutic Approach Based on Drepanoalpha ® : About 34 Cases. J Complement Altern Med Res. 2017;4(2):1–8. https://doi.org/10.9734/JOCAMR/201 7/37350 https://doi.org/10.9734/IJMPCR/2017/33658 https://doi.org/10.9734/IJMPCR/2017/33658 https://doi.org/10.9734/JOCAMR/2017/37350 https://doi.org/10.9734/JOCAMR/2017/37350