# Ethnopharmacological Survey and Antisickling Activity of Plants Used in the Management of Sickle Cell Disease in Kikwit City, DR Congo

Authors: Jules Munganga Kitadi; Prince Pambi Mazasa; Damien Sha-Tshibey Tshibangu; Félicien Mushagalusa Kasali; Dorothée Dinangayi Tshilanda; Koto-Te-Nyiwa Ngbolua; Pius Tshimankinda Mpiana
Journal: Evidence-Based Complementary and Alternative Medicine
 (2020)

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## Abstract

Background. Sickle cell disease or drepanocytosis is the most known hemoglobin abnormality in the world. Recently, many medicinal plants used in the management of sickle cell disease in African traditional medicine have shown in vitro antisickling activity. Objective. This research study aims to document some Congolese plant species used in the management of sickle cell disease in Kikwit city and investigate their pharmacological property in vitro. Methods. A cross-sectional survey was done from June 2015 to March 2016 among 26 traditional healers in Kikwit city, Democratic Republic of Congo. Emmel test was used to assess in vitro antisickling activity. Habitat, morphological, biological types, phytogeographical distribution, local names, and used parts of these plant species were also determined. Results. Obtained results show that 23 plant species belonging to 16 families are used. The leaves represent the most used part (70%). Antisickling investigation showed that aqueous extracts of 18 plants (78%) exhibit a change in the shape of a sickle cell into a normal one with a normalized rate of at least 70%, confirming the in vivo effect observed by traditional healers when used as herbal medicine traditionally in the management of sickle cell disease. The evaluation of perimeter, surface, and radius of untreated and treated sickle red blood cells showed a significant difference (p<0.05) as modification indicators of the red blood cell shape. Alchornea cordifolia, Alternanthera bettzickiana, Annona senegalensis, Dissotis brazzae, Hypoxis angustifolia, and Justicia secunda presented a very high antisickling activity with normalization >70%. Otherwise, Dissotis brazzae was the most active plant with a minimal concentration of normalization (MCN) of 11 μg/mL and a maximal normalization rate (NRmax) of 86%. Conclusion. Almost 80% of the plants studied showed interesting antisickling activity and justified their use in traditional medicine. The isolation of the molecules responsible for the biological activity of the most active plants and the determination of their chemical structures is under investigation.


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## Body

## 1. Introduction

Sickle cell disease (SCD), also called sickle cell anemia or drepanocytosis, is one of the most common hemoglobin abnormalities in the world [1–5]. About 50 million people are affected in the world, mainly sub-Saharan Africans and their immigrants, especially to America. Nevertheless, this hemoglobin disease is also found in some parts of India and Saudi Arabia [6–8]. In certain African regions, carriers of S hemoglobin can reach 20% of the population with a prevalence of 25–30% in Central Africa, including the Democratic Republic of the Congo (RDC). Indeed, in the DRC, about a million and a half people are affected by this genetic disease [9–12].SCD is a painful disease due to a genetic mutation conducting to the substitution of a polar amino acid (glutamic acid) by a nonpolar one (valine) on the sixth position of the beta-globin chain [13]. This change decreases the affinity of hemoglobin for oxygen. Furthermore, this structural modification influences the solubility of hemoglobin conducting abnormal hemoglobin, hemoglobin S (HbS), to polymerize inside the erythrocytes into a gel or further into fibers, causing the change of the red blood cell (RBC) shape from the normal biconcave form into the sickle form in hypoxic conditions. The HbS polymerization and the sickling of the RBC are responsible for sickle cell disease to basic pathological processes, the painful vaso-occlusive crises, and chronic hemolytic anemia. The occlusion of microvessels’ circulation leads to chronic tissue ischemia and infarction affecting the bone, lungs, brain, kidneys, or spleen that conduct to complications in sickle cell disease [13–15].Many therapeutic approaches are proposed to relieve sickle cell disease patients from this painful and chronic disease. These include the medullary transplantation, repeated blood transfusion, and the use of hydroxyurea. These approaches are unfortunately ineffective, expensive for the African population, or present high risk of transmission of other infections [13, 15, 16]. Therefore, there is a need for more affordable and effective treatments for this disease. Medicinal plants and traditional medicine seem to offer an adapted solution to African populations. In fact, according to the World Health Organization (WHO), 80% of the world’s population, mainly in Africa, use medicinal plants for some aspects of primary health care [17, 18]. The DRC is by far the African country with the highest biodiversity with richest flora in the world. Surveys conducted by our research team around big cities revealed more than 120 medicinal plants used in traditional medicine in the management of SCD [4–6, 14, 19–29].The present study aims to document and investigatein vitro the medicinal plants used in Congolese traditional medicine in the management of SCD in Kikwit city.


## 2. Materials and Methods

### 2.1. Study Area

The survey was conducted in Kikwit city, province of Kwilu, located in the southwest part of the Democratic Republic of the Congo at 500 km west of the capital city Kinshasa (Figure1). Kikwit’s population was estimated to be 1,000,000 inhabitants in a total area of 92 km [19].Figure 1
Kikwit city in the Democratic Republic of the Congo.


### 2.2. Ethnopharmacological Survey

The ethnopharmacological survey was carried out from June 2015 to March 2016. Twenty-six traditional healers from different ethnic groups were identified and questioned on a voluntary basis following the principles laid out in the Declaration of Helsinki with minor modification [20]. The data checklist was divided into three sections:Personal information such as name, age, sex, marital status, ethnic group of the informant, and study levelPlant data (scientific and vernacular names, village survey, parts used, and the method of harvesting stage or degree and organ development)Plant therapy data: methods of preparation and administration (transaction and pharmaceutical form, concentration of the organ dose, frequency of taking, and instructions)The identification of collected plants was carried over by comparison with vouchers referenced at the herbarium of Faculty of Sciences, University of Kinshasa. Voucher specimens are on deposit at the same herbarium.


### 2.3. Floristic Characterizations of Collected Plants

The collected medicinal plants are characterized by their biological, morphological, and habitat types and by their phytogeographical distribution [17, 21]. The following biological types were selected: mesophanerophytes (MsPh), microphanerophytes (McPh), nanophanerophytes (NPh), chamaephytes (Ch), therophytes (Th), therophytes scapose (Tsc), erected chamaephytes (Ch er), climbing chamaephytes (Ch cl), and geophytes (G).The morphological types inventoried are tree (T), shrub (Sh), subshrub (Ssh), annual herb (Ah), vivace herb (Vh), perennial herb (Ph), and liana (L).Habitat types are distributed as follows: cultured (Cult), subspontaneous cultured (Cult ssp), forest (Fo), fallow (Fal), and ruderal (Rud).The phytogeographical types of distribution are Afro-tropical (AT), Bas-Guineo-Congolese (BGC), Guineo-Congolese (GC), Guineo-Congolese-Zambezean (GCZ), and pantropical (Pan).


### 2.4. Evaluation of Use Value and Informant Consensus Factor Value

Use valueUV that evaluates the relative importance of each medicinal species based on its relative use among informants was calculated using the following formula:(1)UV=UiNi,where Ui is the number of use reports cited by each informant for a given plant species when Ni is the total number of informants interviewed for a given plant species.The usage value agreement is defined as the relative importance of each plant type known to be used as herbal medicine. This index is useful in identifying plants with the highest use (most frequently mentioned) in the treatment of a disease with a given informant consensus factorICF value. ICF (ranging from 0 to 1) is used to deduce the homogeneity in the data on the use of a specific plant to treat a certain disease. The informant consensus factor value was calculated using the following formula:(2)ICF=Nc−NpsNc−1,where Nc is the number of plant use reports (citation number) per each category and Nps is the number of plant species (taxa used). A high value of ICF (close to 1) shows that a reduced number of plant species are quoted by a large number of informants for a specific type of treatment, indicating the consistence of the use of this medicine [22].


### 2.5. Biological Samples

Blood samples used for antisickling evaluation were collected from known sickle cell disease patients treated in Centre de Médecine Mixte et d’AnémieSS in Kinshasa, DRC. None of the patients had been transfused recently with Hb AA blood. All antisickling experiments were conducted with freshly collected blood. In order to confirm their SS nature, the aforementioned blood samples were first characterized by hemoglobin electrophoresis on cellulose acetate gel, as previously reported [7, 8, 14]. They were found to be SS blood and were then stored at ±4°C in a refrigerator. Informed consent was obtained from all the patients selected in the study. Ethical clearance for the use of sickle blood cells was strictly observed according to international rules.


### 2.6. Antisickling Assay

Antisickling assay was carried out using Emmel test as previously reported [6, 9, 18]. In brief, sickle cell blood was diluted with 150 mM phosphate buffer saline (NaH2PO4 30 mM, Na2HPO4 120 mM, and NaCl 150 mM) and mixed with an equivalent volume of 2% sodium metabisulfite. A drop of the mixture was spotted on a microscope slide in the presence of aqueous plant extracts and covered with a cover slip. Physiologic solution was used as the negative control when parahydroxybenzoic acid was used as the positive control. Paraffin was applied to seal the edges of the cover slip completely to create hypoxia condition.


### 2.7. Data Analysis

Different parameters have been screened in red blood cells, including the area, perimeter, and radius of each RBC using a computer-assisted image analysis system (Motic Images 2000, version 1.3; Motic China Group Co., Ltd), and statistical data analysis was processed using Microcal Origin 8.5 package software.


## 2.1. Study Area

The survey was conducted in Kikwit city, province of Kwilu, located in the southwest part of the Democratic Republic of the Congo at 500 km west of the capital city Kinshasa (Figure1). Kikwit’s population was estimated to be 1,000,000 inhabitants in a total area of 92 km [19].Figure 1
Kikwit city in the Democratic Republic of the Congo.


## 2.2. Ethnopharmacological Survey

The ethnopharmacological survey was carried out from June 2015 to March 2016. Twenty-six traditional healers from different ethnic groups were identified and questioned on a voluntary basis following the principles laid out in the Declaration of Helsinki with minor modification [20]. The data checklist was divided into three sections:Personal information such as name, age, sex, marital status, ethnic group of the informant, and study levelPlant data (scientific and vernacular names, village survey, parts used, and the method of harvesting stage or degree and organ development)Plant therapy data: methods of preparation and administration (transaction and pharmaceutical form, concentration of the organ dose, frequency of taking, and instructions)The identification of collected plants was carried over by comparison with vouchers referenced at the herbarium of Faculty of Sciences, University of Kinshasa. Voucher specimens are on deposit at the same herbarium.


## 2.3. Floristic Characterizations of Collected Plants

The collected medicinal plants are characterized by their biological, morphological, and habitat types and by their phytogeographical distribution [17, 21]. The following biological types were selected: mesophanerophytes (MsPh), microphanerophytes (McPh), nanophanerophytes (NPh), chamaephytes (Ch), therophytes (Th), therophytes scapose (Tsc), erected chamaephytes (Ch er), climbing chamaephytes (Ch cl), and geophytes (G).The morphological types inventoried are tree (T), shrub (Sh), subshrub (Ssh), annual herb (Ah), vivace herb (Vh), perennial herb (Ph), and liana (L).Habitat types are distributed as follows: cultured (Cult), subspontaneous cultured (Cult ssp), forest (Fo), fallow (Fal), and ruderal (Rud).The phytogeographical types of distribution are Afro-tropical (AT), Bas-Guineo-Congolese (BGC), Guineo-Congolese (GC), Guineo-Congolese-Zambezean (GCZ), and pantropical (Pan).


## 2.4. Evaluation of Use Value and Informant Consensus Factor Value

Use valueUV that evaluates the relative importance of each medicinal species based on its relative use among informants was calculated using the following formula:(1)UV=UiNi,where Ui is the number of use reports cited by each informant for a given plant species when Ni is the total number of informants interviewed for a given plant species.The usage value agreement is defined as the relative importance of each plant type known to be used as herbal medicine. This index is useful in identifying plants with the highest use (most frequently mentioned) in the treatment of a disease with a given informant consensus factorICF value. ICF (ranging from 0 to 1) is used to deduce the homogeneity in the data on the use of a specific plant to treat a certain disease. The informant consensus factor value was calculated using the following formula:(2)ICF=Nc−NpsNc−1,where Nc is the number of plant use reports (citation number) per each category and Nps is the number of plant species (taxa used). A high value of ICF (close to 1) shows that a reduced number of plant species are quoted by a large number of informants for a specific type of treatment, indicating the consistence of the use of this medicine [22].


## 2.5. Biological Samples

Blood samples used for antisickling evaluation were collected from known sickle cell disease patients treated in Centre de Médecine Mixte et d’AnémieSS in Kinshasa, DRC. None of the patients had been transfused recently with Hb AA blood. All antisickling experiments were conducted with freshly collected blood. In order to confirm their SS nature, the aforementioned blood samples were first characterized by hemoglobin electrophoresis on cellulose acetate gel, as previously reported [7, 8, 14]. They were found to be SS blood and were then stored at ±4°C in a refrigerator. Informed consent was obtained from all the patients selected in the study. Ethical clearance for the use of sickle blood cells was strictly observed according to international rules.


## 2.6. Antisickling Assay

Antisickling assay was carried out using Emmel test as previously reported [6, 9, 18]. In brief, sickle cell blood was diluted with 150 mM phosphate buffer saline (NaH2PO4 30 mM, Na2HPO4 120 mM, and NaCl 150 mM) and mixed with an equivalent volume of 2% sodium metabisulfite. A drop of the mixture was spotted on a microscope slide in the presence of aqueous plant extracts and covered with a cover slip. Physiologic solution was used as the negative control when parahydroxybenzoic acid was used as the positive control. Paraffin was applied to seal the edges of the cover slip completely to create hypoxia condition.


## 2.7. Data Analysis

Different parameters have been screened in red blood cells, including the area, perimeter, and radius of each RBC using a computer-assisted image analysis system (Motic Images 2000, version 1.3; Motic China Group Co., Ltd), and statistical data analysis was processed using Microcal Origin 8.5 package software.


## 3. Results and Discussion

### 3.1. Ethnobotanical Analysis

Table1 summarizes families, species, vernacular names, used parts, and use values of plants used in the management of sickle cell disease in Congolese traditional medicine in Kikwit city.Table 1
Plants used in the management of sickle cell disease in Kikwit.
FamilyPlant speciesVernacular nameUsed partUse valueFCIAcanthaceaeJusticia secunda Vahl.Matiti ya ba témoins de Jéhovah (Lingala)Leaves3.580.58Rungia grandis T. Ander—Bark3.340.55AmaranthaceaeAlternanthera bettzickiana repens (L.) LinkMatiti ya ba témoins de Jéhovah (Lingala)Leaves5.120.68AnnonaceaeAnnona senegalensis Pers.Kilolo, nlolo (Kikongo)Leaves4.120.58BombaceaeCeiba pentandra (L.) Gaertn.Kapokier, fromager (French), mfuma (Lingala)Leaves4.000.63CaricaceaeCarica papaya L.Payipayi (Lingala)Leaves, fruit5.000.68EuphorbiaceaeAlchornea cordifolia Müll. Arg.Mbunji-mbunji (Lingala), kibunsi (Kikongo)Leaves4.560.7Bridelia ferruginea Benth.Kimuindu ki nseke (Kikongo)Leaves3.670.72FabaceaeVigna unguiculata (L.) Welp.Mbwengi (Lingala, Kikongo)Seeds4.340.68Millettia laurentii De WildKoboto (Kikongo), ntoka (Kikongo), wenge (Lingala)Leaves4.220.62HypoxidaceaeHypoxis angustifolia Lam.Ditungulu ya mfinda (kikongo), litungulu (Lingala)Bulbs4.000.63LamiaceaeLeocus africanus (Bak. ex. Elliot) J. K. MortonZangazanga (Kikongo)Bark4.890.65MelastomataceaeDissotis brazzae Cogn.Mampa ya mfinda (Kikongo)Leaves4.450.67MimosaceaeAlbizia ferruginea (Guill. et Piero) Benth.Sela (Kikongo)Bark4.220.68MoraceaeTreculia africana DecneNgungi (Kikongo)Leaves3.780.58Ficus asperifolia mügKikuya, kuya (Kikongo)Leaves3.890.64Ficus tremula Warb.—Leaves4.340.63NyctaginaceaeBougainvillea sp comm. ex JussBintuntu ya nzanguka (Kikongo)Leaves4.670.63PhyllanthaceaeHymenocardia acida TulDikasu, kigete, ngete (Kikongo)Leaves4.670.59RubiaceaeGardenia leopoldiana De Wild & T. Durand—Bark4.890.65Morelia Senegalensis A. Rich. ex DC—Leaves4.560.65Stipularia africana P. Beauv.—Leaves4.000.63SterculiaceaeSterculia bequaertii De WildMvungela mfinda (Kikongo)Seeds3.440.5423 plant species from 16 botanical families cited in different local names, especially Kikongo (sometimes in Lingala and rarely in French), are used in the management of sickle cell disease in Kikwit city. Figures2 and 3 give the number of species by family used parts, respectively.Figure 2
Distribution of species by family.Figure 3
Percentage of used parts of plants.According to the results (Figure2), Rubiaceae and Moraceae families have been represented by three species. However, three families including Euphorbiaceae, Acanthaceae, and Fabaceae had two species, each one and the remaining possessing only one plant species by family.Figure3 shows that leaves represent the most used part (66.6%) when barks represent 16.6%. The use of leaves could be justified by the abundance of chemical groups they contain. In fact, leaves are known as the main synthesis site of secondary metabolites in plants and are the most commonly used plant parts by traditional medicinal practitioners [17, 21].


### 3.2. Use Value

Eight plants (Justicia secunda, Vigna unguiculata, Alternanthera bettzickiana, Alternanthera bettzickiana, Carica papaya, Annona senegalensis, Alchornea cordifolia, and Hypoxis angustifolia) have presented a use value higher than 0.6. They are the most cited plants, and therefore, they are the most used plants in the management of sickle cell disease in Kikwit. According to previous antisickling studies, all cited plant species, except Alternanthera bettzickiana and Hypoxis angustifolia, are well known [4–6, 14, 23–33].Alternanthera bettzickiana repens (L.) Link displayed both high UV and a large consensus of use among traditional healers indicating the consistence of the use of this plant species as antisickling medicine.


### 3.3. Ecological Status of Plant Species

Ecological characteristics of these plants are given in Table2.Table 2
Ecological status of plants used in the management of sickle cell disease in Kikwit.
Plant speciesHTMTBTPDJusticia secunda VahlCultVhNphPanRungia grandis T. AnderFoVhChPanAlternanthera bettzickiana repens (L.) LinkCultAhNphPanAnnona senegalensis Pers.SavShMcphATCeiba pentandra (L.) Gaertn.Cult sspTMgphPanCarica papaya L.CultTMcphPanAlchornea cordifolia Müll. Arg.FoShMcphATBridelia ferruginea Benth.SavShMcphGCZVigna unguiculata (L.) Welp.CultAhThATMillettia laurentii De WildFoTMgphBGCHypoxis angustifolia Lam.CultPhGPanLeocus africanus (Bak. ex. Elliot) J. K. MortonSav,SshMcphGCDissotis brazzae Cogn.RudSshThPanAlbizia ferruginea (Guill. et Piero) Benth.SavTMsphGCTreculia africana DecneFoTMsphPanFicus asperifolia MügFoSshLphBGCFicus tremula Warb.FoShMcphGCBougainvillea sp comm. Ex JussCultShLphPanHymenocardia acida TulSavShMcphATGardenia leopoldiana De Wild & T. DurandFoShMcphATMorelia senegalensis A. Rich. Ex DCFoShMcphPanStipularia africana P. Beauv.FalSshMcphPanSterculia bequaertii De WildFoTMgphGCHT: habit types, MT: morphological types, BT: biotope types, PD: phytogeographical distribution, Cult: cultured, Cult ssp: subspontaneous cultured, Fo: forest, Fal: fallow, Rud: ruderal, T: tree, Sh: shrub, Ssh: subshrub, Ah: annual herb, Vh: vivace herb, Ph: perennial herb, PsPh: mesophanerophytes, McPh: microphanerophytes, NPh: nanophanerophytes, Ch: chamaephytes, Th: therophytes, G: geophytes, AT: Afro-tropical, BGC: Bas-Guinea-Congolese, GC: Guinea-Congolese, GCZ: Guinea-Congolese-Zambezean, and Pan: pantropical.


### 3.4. Habitat Types

Types of habitat are presented in Figure4. This figure shows that the main habitat type of plants used in the management of sickle cell by traditional healers in Kikwit is forest (39.1%) followed, respectively, by cultured (26.1%) and savanna (21.7%). Fallow, ruderal, and subspontaneous cultured represent each one 4.3%.Figure 4
Habitat types of plant species.


### 3.5. Morphological Types

Figure5 represents morphological types of used plants. Shrub represents the most morphological types (34.8%) followed by tree (26.1%), subshrub (17.4%), and annual herb (8.7%). Vivace herb and perennial herb both represent 13%. Furthermore, 14 plant species, up to 23 representing 61% of species, are trees or shrubs. Only five plant species (21%) are herbs. Thus, a correct use of leaves of these plants can permit a sustainable exploitation of these species in traditional medicine.Figure 5
Morphological types of plants species.


### 3.6. Biological Types

Biological types are listed in Table2 and presented in Figure 6.Figure 6
Biological types of plant species.


### 3.7. Phytogeographical Distribution

Phytogeographical distribution of plants species is given in Figure7.Figure 7
Phytogeographical distribution of plants.Microphanerophyte is the main biological type and represents 43.5% of identified plant species. Pantropical represents the most phytogeographical distribution (47.8%).


### 3.8.In Vitro Antisickling Activity

The results of antisickling evaluationin vitro are represented in Figures 8–11. Figures 8 and 9 represent microscopic images of negative and positive controls when Figures 10 and 11 show morphologies of RBC treated with aqueous plant extracts, Alternanthera bettzickiana, and Dissotis brazzae, respectively.Figure 8
Erythrocytes’ morphology of untreated sickle cell blood (only saline solution, negative control) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure 9
Erythrocytes’ morphology of sickle cell blood in the presence of parahydroxybenzoic acid (0.8μg/mL) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure 10
Erythrocytes’ morphology of sickle cell blood in the presence of the aqueous leaves’ extract ofDissotis brazzae (20 μg/mL) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure 11
Erythrocytes’ morphology of sickle cell blood in the presence of the aqueous leaves’ extract ofAlternanthera bettzickiana leaves (20 μg/mL) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure8 indicates that the majority of red blood cells is sickled. It means and supports that collected blood is from the sickle cell disease patient. Figure 9 indicates that all blood cells have a normal circular form due to the normalization effect of the positive control.Figures10 and 11 show that, in the presence of D. brazzae and A. bettzickiana aqueous extracts, red blood cells (RBCs) have a circular (biconcave) and normal shape indicating the antisickling effect of these extracts. This situation, in vitro activity, confirms the in vivo effect observed by traditional healers when using an aqueous decoction of these medicinal plants in the management of sickle cell disease in Kikwit city.The same antisickling activity was found in the other 21 medicinal plants used in the management of sickle cell disease in Kikwit (Table3).Table 3
In vitro antisickling activity of plant species used in the management of sickle cell disease in Kikwit.
N°Plant speciesAntisickling activity1Albizia ferruginea (Guill. et piero) Benth.+2Alchornea cordifolia Müll. Arg.+++3Alternanthera bettzickiana repens (L.) Link+++4Annona senegalensis Pers.+++5Bougainvillea sp comm. Ex Juss+6Bridelia ferruginea Benth.++7Carica papaya L.++8Ceiba pentandra (L.) Gaertn.+9Dissotis brazzae Cogn.+++10Ficus asperifolia Müg++11Ficus tremula Warb.++12Gardenia leopoldiana De Wild & T. Durand+13Hymenocardia acida Tul++14Hypoxis angustifolia Lam.+++15Justicia secunda Vahl+++16Leocus africanus (Bak. ex. Elliot) J. K. Morton++17Millettia laurentii De Wild+18Morelia senegalensis A. Rich. ex DC++19Rungia grandis T. Ander++20Sterculia bequaertii De Wild++21Stipularia africana P. Beauv.++22Treculia africana Decne++23Vigna unguiculata (L.) Welp++++++: ≥80% of normalization rate; ++: 70% of normalization rate; +: 50% of normalization rate. An extract is considered to possess very high activity (+++) if normalization >70%; high activity (++) if 50< normalization <70%; weak activity (+) if 10< normalization <50%; no activity (−) if normalization <10% (14).Table3 shows that 18 plants (78%) permit a change of shape of a sickle cell into a normal one with a normalized rate of at least 70%. This indicates that the information given by traditional healers is validated by in vitro tests. The same results were found in plants used in the management of sickle disease in other Congolese cities [4–6,14,23–33].Six of these plants (Alchornea cordifolia Müll. Arg., Annona senegalensis; Carica papaya; Hymenocardia acida; Justicia secunda; and Vigna unguiculata) were already cited elsewhere by Congolese traditional healers in the management of sickle cell disease, and they have shown in vitro antisickling activities [4–6, 14, 23–33].Table4 gives use values of medicinal plants used in the management of sickle cell disease in Kikwit city.Table 4
Use values of medicinal plants used in the management of sickle cell disease in Kikwit city.
Plant speciesLocalNLiterature RFCNReferencesAlchornea cordifolia Müll. Arg.0.690.130[14, 23, 31]Annona senegalensis Pers0.7120.087[14, 18]Bougainvillea sp comm. ex Juss0.2210.043[31]Bridelia ferruginea Benth.0.3220.087[14, 31]Carica papaya L.0.7930.130[23, 31, 33]Ceiba pentandra (L.) Gaertn.0.2420.087[14, 31]Dissotis brazzae Cogn0.4810.043[9]Hymenocardia acida Tul0.8120.087[14, 31]Hypoxis angustifolia Lam.0.6810.043[31]Justicia secunda Vahl0.8320.087[23, 31]Treculia africana0.1510.043[31]Vigna unguiculata (L.) Welp0.8130.130[14, 31, 33]Total sources1231Hypoxis angustifolia Lam., Hymenocardia acida Tul, and Vigna unguiculata (L.) Welp are among the most cited species in Kikwit.In order to quantify the observed shape modification, perimeter, surface, and radius of untreated and treated sickle RBCs with plant extracts were evaluated.Table5 shows calculated sickle red blood cell perimeter, surface, and radius modification after treatment of D. brazzae and A. bettzickiana aqueous leaves’ extracts.Table 5
Perimeter, surface, and radius of untreated and treated sickle RBCs with the aqueous extract ofD. brazzae and A. bettzickiana leaves.
SamplesCellular perimeter (μm)Cellular surface (μm2)Cellular radius (μm)Negative control24.9 ± 1.132.5 ± 2.6—Positive control19.0 ± 1.725.1 ± 1.83.5 ± 0.3Extract ofA. bettzickiana18.9 ± 1.724.7 ± 2.43.5 ± 0.4Extract ofD. brazzae19.4 ± 2.125.3 ± 2.03.6 ± 0.5Values (means ± S.D) in Table5 show a significant difference (p<0.05) between the negative control compared to values from the two extracts. This confirms the antisickling activity of these plant aqueous extracts. Furthermore, the values obtained from the two plant extracts are in the same range to those obtained in previous studies [4–6, 23–28, 30–33]. Used software cannot calculate the radius of untreated sickle red blood cells because the shape of cells was elongated, not circular. However, in the presence of plant extracts, cell shape becomes circular, and the radius could be calculated.Indeed, hydrophobic interactions between hemoglobin S (HbS) molecules conduct to intracellular hydrophobic interactions that induce intracellular polymerization. This hemoglobin aggregation modifies the erythrocyte shape conducting to a sickled form. Plant extracts contain molecules that can interact with HbS molecules and compete with their polymerization [12, 32, 33]. This permits sickle red blood cells to return to their normal biconcave form.The normalized rate can be calculated by following the percentage of erythrocytes that regain the normal shape in function of the concentration of plant extracts. Figures12 and 13 give an illustration of the normalization of the aqueous extract of D. brazzae and A. bettzickiana, respectively.Figure 12
Evolution of the normalized rate of the sickle cell shape with the concentration ofD. brazzae aqueous extracts.Figure 13
Evolution of the normalization rate of the sickle cell shape with the concentration of D. brazzae aqueous extracts.These curves show that the percentage of the drepanocytes, which return to the normal shape, increases with the extract concentration indicating that the normalization rate is dose-dependent. The minimal concentration of normalization (MCN) obtained is 0.9 and 11.0μg/mL with a maximal normalization rate (NRmax) of 90 and 86% for D. brazzae and A. bettzickiana, respectively. This indicates that the aqueous extract of the two plants has very high NRmax with low MCN, but D. brazzae is a little more active than A. bettzickiana. The same behavior was found in other Congolese medicinal plants used in the management of sickle cell disease, but the obtained value of MCN and NRmax depends on the plant species [4, 5, 9, 23–33].


## 3.1. Ethnobotanical Analysis

Table1 summarizes families, species, vernacular names, used parts, and use values of plants used in the management of sickle cell disease in Congolese traditional medicine in Kikwit city.Table 1
Plants used in the management of sickle cell disease in Kikwit.
FamilyPlant speciesVernacular nameUsed partUse valueFCIAcanthaceaeJusticia secunda Vahl.Matiti ya ba témoins de Jéhovah (Lingala)Leaves3.580.58Rungia grandis T. Ander—Bark3.340.55AmaranthaceaeAlternanthera bettzickiana repens (L.) LinkMatiti ya ba témoins de Jéhovah (Lingala)Leaves5.120.68AnnonaceaeAnnona senegalensis Pers.Kilolo, nlolo (Kikongo)Leaves4.120.58BombaceaeCeiba pentandra (L.) Gaertn.Kapokier, fromager (French), mfuma (Lingala)Leaves4.000.63CaricaceaeCarica papaya L.Payipayi (Lingala)Leaves, fruit5.000.68EuphorbiaceaeAlchornea cordifolia Müll. Arg.Mbunji-mbunji (Lingala), kibunsi (Kikongo)Leaves4.560.7Bridelia ferruginea Benth.Kimuindu ki nseke (Kikongo)Leaves3.670.72FabaceaeVigna unguiculata (L.) Welp.Mbwengi (Lingala, Kikongo)Seeds4.340.68Millettia laurentii De WildKoboto (Kikongo), ntoka (Kikongo), wenge (Lingala)Leaves4.220.62HypoxidaceaeHypoxis angustifolia Lam.Ditungulu ya mfinda (kikongo), litungulu (Lingala)Bulbs4.000.63LamiaceaeLeocus africanus (Bak. ex. Elliot) J. K. MortonZangazanga (Kikongo)Bark4.890.65MelastomataceaeDissotis brazzae Cogn.Mampa ya mfinda (Kikongo)Leaves4.450.67MimosaceaeAlbizia ferruginea (Guill. et Piero) Benth.Sela (Kikongo)Bark4.220.68MoraceaeTreculia africana DecneNgungi (Kikongo)Leaves3.780.58Ficus asperifolia mügKikuya, kuya (Kikongo)Leaves3.890.64Ficus tremula Warb.—Leaves4.340.63NyctaginaceaeBougainvillea sp comm. ex JussBintuntu ya nzanguka (Kikongo)Leaves4.670.63PhyllanthaceaeHymenocardia acida TulDikasu, kigete, ngete (Kikongo)Leaves4.670.59RubiaceaeGardenia leopoldiana De Wild & T. Durand—Bark4.890.65Morelia Senegalensis A. Rich. ex DC—Leaves4.560.65Stipularia africana P. Beauv.—Leaves4.000.63SterculiaceaeSterculia bequaertii De WildMvungela mfinda (Kikongo)Seeds3.440.5423 plant species from 16 botanical families cited in different local names, especially Kikongo (sometimes in Lingala and rarely in French), are used in the management of sickle cell disease in Kikwit city. Figures2 and 3 give the number of species by family used parts, respectively.Figure 2
Distribution of species by family.Figure 3
Percentage of used parts of plants.According to the results (Figure2), Rubiaceae and Moraceae families have been represented by three species. However, three families including Euphorbiaceae, Acanthaceae, and Fabaceae had two species, each one and the remaining possessing only one plant species by family.Figure3 shows that leaves represent the most used part (66.6%) when barks represent 16.6%. The use of leaves could be justified by the abundance of chemical groups they contain. In fact, leaves are known as the main synthesis site of secondary metabolites in plants and are the most commonly used plant parts by traditional medicinal practitioners [17, 21].


## 3.2. Use Value

Eight plants (Justicia secunda, Vigna unguiculata, Alternanthera bettzickiana, Alternanthera bettzickiana, Carica papaya, Annona senegalensis, Alchornea cordifolia, and Hypoxis angustifolia) have presented a use value higher than 0.6. They are the most cited plants, and therefore, they are the most used plants in the management of sickle cell disease in Kikwit. According to previous antisickling studies, all cited plant species, except Alternanthera bettzickiana and Hypoxis angustifolia, are well known [4–6, 14, 23–33].Alternanthera bettzickiana repens (L.) Link displayed both high UV and a large consensus of use among traditional healers indicating the consistence of the use of this plant species as antisickling medicine.


## 3.3. Ecological Status of Plant Species

Ecological characteristics of these plants are given in Table2.Table 2
Ecological status of plants used in the management of sickle cell disease in Kikwit.
Plant speciesHTMTBTPDJusticia secunda VahlCultVhNphPanRungia grandis T. AnderFoVhChPanAlternanthera bettzickiana repens (L.) LinkCultAhNphPanAnnona senegalensis Pers.SavShMcphATCeiba pentandra (L.) Gaertn.Cult sspTMgphPanCarica papaya L.CultTMcphPanAlchornea cordifolia Müll. Arg.FoShMcphATBridelia ferruginea Benth.SavShMcphGCZVigna unguiculata (L.) Welp.CultAhThATMillettia laurentii De WildFoTMgphBGCHypoxis angustifolia Lam.CultPhGPanLeocus africanus (Bak. ex. Elliot) J. K. MortonSav,SshMcphGCDissotis brazzae Cogn.RudSshThPanAlbizia ferruginea (Guill. et Piero) Benth.SavTMsphGCTreculia africana DecneFoTMsphPanFicus asperifolia MügFoSshLphBGCFicus tremula Warb.FoShMcphGCBougainvillea sp comm. Ex JussCultShLphPanHymenocardia acida TulSavShMcphATGardenia leopoldiana De Wild & T. DurandFoShMcphATMorelia senegalensis A. Rich. Ex DCFoShMcphPanStipularia africana P. Beauv.FalSshMcphPanSterculia bequaertii De WildFoTMgphGCHT: habit types, MT: morphological types, BT: biotope types, PD: phytogeographical distribution, Cult: cultured, Cult ssp: subspontaneous cultured, Fo: forest, Fal: fallow, Rud: ruderal, T: tree, Sh: shrub, Ssh: subshrub, Ah: annual herb, Vh: vivace herb, Ph: perennial herb, PsPh: mesophanerophytes, McPh: microphanerophytes, NPh: nanophanerophytes, Ch: chamaephytes, Th: therophytes, G: geophytes, AT: Afro-tropical, BGC: Bas-Guinea-Congolese, GC: Guinea-Congolese, GCZ: Guinea-Congolese-Zambezean, and Pan: pantropical.


## 3.4. Habitat Types

Types of habitat are presented in Figure4. This figure shows that the main habitat type of plants used in the management of sickle cell by traditional healers in Kikwit is forest (39.1%) followed, respectively, by cultured (26.1%) and savanna (21.7%). Fallow, ruderal, and subspontaneous cultured represent each one 4.3%.Figure 4
Habitat types of plant species.


## 3.5. Morphological Types

Figure5 represents morphological types of used plants. Shrub represents the most morphological types (34.8%) followed by tree (26.1%), subshrub (17.4%), and annual herb (8.7%). Vivace herb and perennial herb both represent 13%. Furthermore, 14 plant species, up to 23 representing 61% of species, are trees or shrubs. Only five plant species (21%) are herbs. Thus, a correct use of leaves of these plants can permit a sustainable exploitation of these species in traditional medicine.Figure 5
Morphological types of plants species.


## 3.6. Biological Types

Biological types are listed in Table2 and presented in Figure 6.Figure 6
Biological types of plant species.


## 3.7. Phytogeographical Distribution

Phytogeographical distribution of plants species is given in Figure7.Figure 7
Phytogeographical distribution of plants.Microphanerophyte is the main biological type and represents 43.5% of identified plant species. Pantropical represents the most phytogeographical distribution (47.8%).


## 3.8.In Vitro Antisickling Activity

The results of antisickling evaluationin vitro are represented in Figures 8–11. Figures 8 and 9 represent microscopic images of negative and positive controls when Figures 10 and 11 show morphologies of RBC treated with aqueous plant extracts, Alternanthera bettzickiana, and Dissotis brazzae, respectively.Figure 8
Erythrocytes’ morphology of untreated sickle cell blood (only saline solution, negative control) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure 9
Erythrocytes’ morphology of sickle cell blood in the presence of parahydroxybenzoic acid (0.8μg/mL) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure 10
Erythrocytes’ morphology of sickle cell blood in the presence of the aqueous leaves’ extract ofDissotis brazzae (20 μg/mL) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure 11
Erythrocytes’ morphology of sickle cell blood in the presence of the aqueous leaves’ extract ofAlternanthera bettzickiana leaves (20 μg/mL) (x500) (NaCl 0.9%; Na2S2O4 2%).Figure8 indicates that the majority of red blood cells is sickled. It means and supports that collected blood is from the sickle cell disease patient. Figure 9 indicates that all blood cells have a normal circular form due to the normalization effect of the positive control.Figures10 and 11 show that, in the presence of D. brazzae and A. bettzickiana aqueous extracts, red blood cells (RBCs) have a circular (biconcave) and normal shape indicating the antisickling effect of these extracts. This situation, in vitro activity, confirms the in vivo effect observed by traditional healers when using an aqueous decoction of these medicinal plants in the management of sickle cell disease in Kikwit city.The same antisickling activity was found in the other 21 medicinal plants used in the management of sickle cell disease in Kikwit (Table3).Table 3
In vitro antisickling activity of plant species used in the management of sickle cell disease in Kikwit.
N°Plant speciesAntisickling activity1Albizia ferruginea (Guill. et piero) Benth.+2Alchornea cordifolia Müll. Arg.+++3Alternanthera bettzickiana repens (L.) Link+++4Annona senegalensis Pers.+++5Bougainvillea sp comm. Ex Juss+6Bridelia ferruginea Benth.++7Carica papaya L.++8Ceiba pentandra (L.) Gaertn.+9Dissotis brazzae Cogn.+++10Ficus asperifolia Müg++11Ficus tremula Warb.++12Gardenia leopoldiana De Wild & T. Durand+13Hymenocardia acida Tul++14Hypoxis angustifolia Lam.+++15Justicia secunda Vahl+++16Leocus africanus (Bak. ex. Elliot) J. K. Morton++17Millettia laurentii De Wild+18Morelia senegalensis A. Rich. ex DC++19Rungia grandis T. Ander++20Sterculia bequaertii De Wild++21Stipularia africana P. Beauv.++22Treculia africana Decne++23Vigna unguiculata (L.) Welp++++++: ≥80% of normalization rate; ++: 70% of normalization rate; +: 50% of normalization rate. An extract is considered to possess very high activity (+++) if normalization >70%; high activity (++) if 50< normalization <70%; weak activity (+) if 10< normalization <50%; no activity (−) if normalization <10% (14).Table3 shows that 18 plants (78%) permit a change of shape of a sickle cell into a normal one with a normalized rate of at least 70%. This indicates that the information given by traditional healers is validated by in vitro tests. The same results were found in plants used in the management of sickle disease in other Congolese cities [4–6,14,23–33].Six of these plants (Alchornea cordifolia Müll. Arg., Annona senegalensis; Carica papaya; Hymenocardia acida; Justicia secunda; and Vigna unguiculata) were already cited elsewhere by Congolese traditional healers in the management of sickle cell disease, and they have shown in vitro antisickling activities [4–6, 14, 23–33].Table4 gives use values of medicinal plants used in the management of sickle cell disease in Kikwit city.Table 4
Use values of medicinal plants used in the management of sickle cell disease in Kikwit city.
Plant speciesLocalNLiterature RFCNReferencesAlchornea cordifolia Müll. Arg.0.690.130[14, 23, 31]Annona senegalensis Pers0.7120.087[14, 18]Bougainvillea sp comm. ex Juss0.2210.043[31]Bridelia ferruginea Benth.0.3220.087[14, 31]Carica papaya L.0.7930.130[23, 31, 33]Ceiba pentandra (L.) Gaertn.0.2420.087[14, 31]Dissotis brazzae Cogn0.4810.043[9]Hymenocardia acida Tul0.8120.087[14, 31]Hypoxis angustifolia Lam.0.6810.043[31]Justicia secunda Vahl0.8320.087[23, 31]Treculia africana0.1510.043[31]Vigna unguiculata (L.) Welp0.8130.130[14, 31, 33]Total sources1231Hypoxis angustifolia Lam., Hymenocardia acida Tul, and Vigna unguiculata (L.) Welp are among the most cited species in Kikwit.In order to quantify the observed shape modification, perimeter, surface, and radius of untreated and treated sickle RBCs with plant extracts were evaluated.Table5 shows calculated sickle red blood cell perimeter, surface, and radius modification after treatment of D. brazzae and A. bettzickiana aqueous leaves’ extracts.Table 5
Perimeter, surface, and radius of untreated and treated sickle RBCs with the aqueous extract ofD. brazzae and A. bettzickiana leaves.
SamplesCellular perimeter (μm)Cellular surface (μm2)Cellular radius (μm)Negative control24.9 ± 1.132.5 ± 2.6—Positive control19.0 ± 1.725.1 ± 1.83.5 ± 0.3Extract ofA. bettzickiana18.9 ± 1.724.7 ± 2.43.5 ± 0.4Extract ofD. brazzae19.4 ± 2.125.3 ± 2.03.6 ± 0.5Values (means ± S.D) in Table5 show a significant difference (p<0.05) between the negative control compared to values from the two extracts. This confirms the antisickling activity of these plant aqueous extracts. Furthermore, the values obtained from the two plant extracts are in the same range to those obtained in previous studies [4–6, 23–28, 30–33]. Used software cannot calculate the radius of untreated sickle red blood cells because the shape of cells was elongated, not circular. However, in the presence of plant extracts, cell shape becomes circular, and the radius could be calculated.Indeed, hydrophobic interactions between hemoglobin S (HbS) molecules conduct to intracellular hydrophobic interactions that induce intracellular polymerization. This hemoglobin aggregation modifies the erythrocyte shape conducting to a sickled form. Plant extracts contain molecules that can interact with HbS molecules and compete with their polymerization [12, 32, 33]. This permits sickle red blood cells to return to their normal biconcave form.The normalized rate can be calculated by following the percentage of erythrocytes that regain the normal shape in function of the concentration of plant extracts. Figures12 and 13 give an illustration of the normalization of the aqueous extract of D. brazzae and A. bettzickiana, respectively.Figure 12
Evolution of the normalized rate of the sickle cell shape with the concentration ofD. brazzae aqueous extracts.Figure 13
Evolution of the normalization rate of the sickle cell shape with the concentration of D. brazzae aqueous extracts.These curves show that the percentage of the drepanocytes, which return to the normal shape, increases with the extract concentration indicating that the normalization rate is dose-dependent. The minimal concentration of normalization (MCN) obtained is 0.9 and 11.0μg/mL with a maximal normalization rate (NRmax) of 90 and 86% for D. brazzae and A. bettzickiana, respectively. This indicates that the aqueous extract of the two plants has very high NRmax with low MCN, but D. brazzae is a little more active than A. bettzickiana. The same behavior was found in other Congolese medicinal plants used in the management of sickle cell disease, but the obtained value of MCN and NRmax depends on the plant species [4, 5, 9, 23–33].


## 4. Conclusion

The traditional healers of Kikwit (DRC) use 23 plant species in the management of sickle cell disease. Eighteen plant species showed a high antisickling activityin vitro with a normalized rate of at least 70%. The present results validate and support the local uses of plants in the treatment of diseases, especially sickle cell disease. Most of these plants have already been cited for their antisickling activity in other parts of the DRC and in the literature. Two species including Alternanthera bettzickiana and Dissotis brazzae deserve in-depth investigations to meet scientific demands for clinical studies. Furthermore, it is crucial to isolate their bioactive compounds in order to understand their pharmacological mechanisms.



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