Natl. J. Physiol. Pharm. Pharmacol. (2025), Vol. 15(6): 314–318

Original Research

10.5455/NJPPP.2025.v15.i6.3

Antisickling effects of hydroethanolic extract of Oxytenanthera abyssinica leaves

Sène Mbaye^1*, Diouf Ibrahima², Ba Awa³, Ba Fatoumata⁴, Fall Cheikhouna Khadim¹, Diaw Absatou¹, Kane Modou Oumy¹ and Sarr Mamadou¹

¹Laboratory of Pharmaceutical Physiology, Faculty of Medicine, Pharmacy, and Dentistry, Cheikh Anta Diop University, Dakar, Senegal

²Unit of Training and Research in Health Sciences, Assane Seck University, Ziguinchor, Senegal

³Laboratory of Physiologic of Medicine Department, Unit of Training and Research in Health and Sustainable Development, Alioune Diop University, Bambey, Senegal

⁴Unit of Training and Research in Health, Gaston Berger University, Saint-Louis, Senegal

*Corresponding Author: Sène Mbaye. Laboratory of Pharmaceutical Physiology, Faculty of Medicine, Pharmacy, and Dentistry, Cheikh Anta Diop University, Dakar, Senegal. Email: mbaysen [at] yahoo.fr, mbaye2.sene [at] ucad.edu.sn

Submitted: 18/04/2025 Revised: 15/05/2025 Accepted: 23/05/2025 Published: 30/06/2025

---

Abstract

Background: Sickle cell disease is a hereditary hemoglobinopathy. It is characterized by the presence of abnormal hemoglobin S levels at high concentrations in red blood cells.

Aim: This study aimed to investigate the potential of a hydroethanolic extract from Oxytenanthera abyssinica (AO) leaves to inhibit red blood cell sickling in individuals with sickle cell trait (HbAS) and sickle cell disease (HbSS).

Methods: Ten grams of powdered AO leaves were macerated in 100 ml of a 60% hydroethanolic solution. Following filtration, the macerate was concentrated using a rotary evaporator until a dry crude extract was obtained. The dry extract was reconstituted in water to prepare three test solutions at concentrations of 2.5 and 1.25 mg/ml. Emmel tests were performed by incubating these extract solutions with blood samples obtained from individuals with sickle cell trait (HbAS), sickle cell disease (HbSS), and healthy controls (HbAA). The percentage of sickled red blood cells was determined using optical microscopy.

Results: The hydroethanolic extract of AO leaves significantly reduced the percentage of sickled red blood cells in individuals with sickle cell trait (HbAS) and sickle cell disease (HbSS). The antisickling effect exhibited a dosedependent relationship.

Conclusion: Our findings indicate that the crude hydroethanolic extract of AO leaves has significant antisickling activity. This suggests a potential source of bioactive molecules for the development of low-cost therapeutic interventions against sickle cell disease.

Keywords: Sickle cell disease, Antisickling, Oxytenanthera abyssinica.

---

Introduction

Sickle cell disease is a hereditary hemoglobinopathy. It is characterized by the presence of abnormal hemoglobin S (HbS) levels at high concentrations in red blood cells. Autosomal recessive disease of variable expression. The presence of HbS is a consequence of a point mutation at the sixth codon of the β globin gene located on chromosome 11. Specifically, it involves a transversion of adenine to thymine (GAG → GTG), leading to the substitution of hydrophilic glutamic acid with hydrophobic valine at the sixth position of the β-globin chain (Galacteros, 1997; Girot et al., 2003; Janky et al., 2006).

The allele frequency of sickle cell disease exhibits geographical variation, both between continents and within regions of the same continent. In Central Africa, which has the highest prevalence, 10%–40% of the population are heterozygous carriers of the sickle cell trait, whereas less than 2% are present with the homozygous form of the disease. In Senegal, the prevalence of HbS is 10%, with homozygous sickle cell disease occurring in approximately 0.4% of births (Alboury, 1976; Sane, n.d.; Diagne et al., 2006.).

Thus, sickle cell disease is a real public health problem worldwide. Despite advancements in modern medicine, the affordability of effective treatments remains a challenge, particularly in developing nations, including African countries. This limitation has directed research efforts toward identifying the antisickling activity of plant extracts as a potential source of cost-effective therapeutic agents.

It is in this context that our study was conducted with the objective of evaluating the antisickling activity of a hydroethanolic extract of Oxytenanthera abyssinica leaves (AO).

---

Materials and Methods

Study population

This study received ethical approval from the Research Ethics Committee of Cheikh Anta Diop University (CADU) in Dakar. The participants were men and women adults aged 25 to 45 years, recruited from the hematology department of the National Blood Transfusion Center in Dakar after providing free and informed consent. Participants were divided into three groups, each consisting of 15 individuals: healthy controls (hemoglobin genotype AA), sickle cell trait carriers (hemoglobin genotype AS), and individuals with homozygous sickle cell disease (hemoglobin genotype SS).

Blood samples

Blood was collected from an EDTA tube for each patient in accordance with the study protocol. Samples are taken via venous puncture at the fold of the elbow and then kept cool (at + 4°C) if the use of these samples is not immediate.

Plant material

The plant material comprises leaves of AO (A. Riche Munro, whose harvest and authentication were conducted in the botanical garden of the CADU in June 2020. The powder of the drug is obtained after drying, spraying, and storage at room temperature (25° to 30°C) in a ventilated room.

Extraction

Ten grams of leaf powder from AO, obtained after grinding using an RM100 grinder, are introduced into an Erlenmeyer grinder pre-weighed to 0. Then, 100 ml of a hydroethanolic solution (40/60) was measured and added to an Erlenmeyer flask. The latter was vigorously stirred with a magnetized rod for 24 hours. It should be noted that Erlenmeyer is covered with aluminum foil to protect the photosensitive molecules. After maceration, the macerate is recovered and stored at + 4°C to block possible biochemical reactions. After this step, the macerate is filtered using hydrophilic cotton placed in a funnel connected to a suction pump that accelerates filtration. After a few minutes, an exclusively liquid solution is obtained. The resulting filtration was evaporated dry by means of a rotary evaporator under the following conditions: water bath temperature of 40°C, cooling temperature of 21°C, and rotation number of 4,000 tr/min. Thus, the evaporation process produces a hydroethanolic dry raw extract of AO leaves. The dry raw extract was maintained at a temperature of 4°C before the final analysis step.

Preparation of extract solutions

5 mg hydroethanolic dry extract of AO leaves were dissolved in 1 ml physiological saline (0.9% NaCl solution) and subsequently homogenized. This procedure produced a stock solution with a concentration of 5 mg/ml.

Serial dilutions were then performed using physiological saline. A 1:2 dilution of the stock solution produced a working solution of AO extract at a concentration of 2.5 mg/ml. A further 1:2 dilution of this intermediate solution produced a final working solution with a concentration of 1.25 mg/ml. Each dilution was thoroughly mixed.

Characterization of antisickling activity

Emmel tests were performed on participants from all three groups using a 2% sodium metabisulphite (S₂O₅Na₂) solution prepared extemporaneously. 100 µl of whole blood (from individuals with HbAA, HbAS, and HbSS genotypes) were incubated with 100 μl of 5, 2.5, and 1.25 mg/ml OA solutions for 24 hours.

Subsequently, an Emmel test was conducted, followed by sickle cell counting using optical microscopy at ×100 magnification. An average of 500 blood cells (sickled and normal cells) were counted from randomly selected fields of view. Sickle cell counts were performed under basal conditions and in the presence of different concentrations of OA extract (5, 2.5, and 1.25 mg/ml).

The average number of sickled cells per field was calculated. The ratio of sickled cells to the total number of cells counted (500) was used to determine the percentage of sickled cells at baseline and at each extract concentration (1.25, 2.5, and 5 mg/ml). Four independent determinations (n=4) were performed for each participant, and the mean percentage of sickled cells was calculated.

Statistical analysis

The results are expressed as means ± SEM of four experiments. Statistical significance was determined through a one-way analysis of variance, followed by Bonferroni’s test or with Student’s t-test for paired data, as required. Statistical analysis was performed using GraphPad. Prism version 8.0.1^® for Windows (GraphPad Software, San Diego, CA). Values of *p < 0.05 were considered statistically significant.

---

Results

In the basal state

Under basal conditions, the mean percentage of sickled erythrocytes in individuals with homozygous sickle cell disease (HbSS) was significantly higher (40.21%) than in those with sickle cell trait (HbAS) (29.59%). As expected, no sickled cells were observed in healthy controls (HbAA) (Fig. 1).

Effect of AO leaf extract on sickling in HbAS individuals

Leaf extract of AO induces a significant dose-dependent reduction in the percentage of sickled erythrocytes in individuals with sickle cell trait (HbAS). The baseline sickling rate of 29.59% decreased to 26.78%, 24.31%, and 3.03% following incubation with the extract at concentrations of 1.25, 2.5, and 5 mg/ml, respectively (Fig. 2).

Effect of AO leaf extract on sickling in HbSS individuals

Similarly, AO leaf extract induced a significant dose-dependent decrease in the percentage of sickled erythrocytes in individuals with homozygous sickle cell disease (HbSS). The baseline sickling rate of 40.21% decreased to 24.78%, 7.53%, and 4.06% after incubation with the extract at concentrations of 1.25, 2.5, and 5 mg/ml, respectively (Fig. 3).

Fig. 1. Variation in the sickle cell rate at the basal state (BS) in AS and SS subjects. Results are expressed as the average of measurements for different samples. **p < 0.01 for inhibitory effect versus control.

Fig. 2. Variation in the sickle cell rate in the BS and at different concentrations of AO leaf extract in AS subjects. Results are expressed as the average of three measurements for three different samples. ** p < 0.01 for inhibitory effect versus control.

---

Discussion

This study aimed to evaluate the antisickling properties of a hydroethanolic extract of AO leaves in individuals with sickle cell trait (HbAS) and sickle cell disease (HbSS). Antisickling activity was assessed by incubating leaf extract with erythrocytes from individuals with HbAS and HbSS genotypes under hypoxic conditions for 24 hours. The percentages of sickled erythrocytes were determined by optical microscopy following the Emmel test.

Fig. 3. Variation in the sickle cell rate in the BS and at different concentrations of AO leaf extract in subjects with SS. Results are expressed as the average of three measurements for three different samples. ** p < 0.01 for inhibitory effect versus control.

The baseline analysis revealed a higher mean percentage of sickled erythrocytes in individuals with homozygous sickle cell disease (HbSS) (41.21%) compared with those with sickle cell trait (HbAS) (30.39%). This observation is consistent with the understanding that individuals with HbSS primarily possess HbS, whereas those with HbAS possess both hemoglobin A and HbS (Bayeme and Chiabi, 2004).

AO leaf extract demonstrated significant antisickling activity at concentrations of 1.25, 2.5, and 5 mg/ml in both individuals with sickle cell trait (HbAS) and homozygous sickle cell disease (HbSS).

Effect of AO leaf extract on sickling in HbAS individuals

A reduction in the percentage of sickled erythrocytes was observed in HbAS individuals following incubation with AO leaf extract, exhibiting a clear dose-dependent response. The decrease was minimal at the lowest tested concentration (1.25 mg/ml), became more pronounced at 2.5 mg/ml, and was highly significant at the highest concentration of 5 mg/ml. Specifically, the percentage of sickled cells decreased from a baseline of 29.59% to 26.78% (a reduction of 9.53%), 24.31% (a reduction of 17.84%), and 3.03% (a reduction of 89.76%) at extract concentrations of 1.25, 2.5, and 5 mg/ml, respectively.

Effect of AO leaf extract on sickling in HbSS individuals

Similarly, a dose-dependent reduction in the percentage of sickled erythrocytes was observed in HbSS individuals following incubation with AO leaf extract. A considerable decrease in the sickle cell count was noted at 1.25 mg/ml, with a further substantial reduction observed at 2.5 mg/ml, where erythrocytes began to regain their normal morphology. At the highest concentration of 5 mg/ml, the presence of sickled cells was almost completely eliminated. Quantitatively, the percentage of sickled cells decreased from a baseline of 40.21% to 24.78% (a reduction of 38.37%), 7.53% (a reduction of 81.28%), and 4.06% (a reduction of 89.90%) at extract concentrations of 1.25, 2.5, and 5 mg/ml, respectively. These findings strongly suggest the significant in vitro antisickling activity of AO leaf extract.

Phytochemical screening of AO leaves revealed the presence of various polyphenols, including flavonoids and other phenolic compounds such as tannins and anthracenes (Diene, 2005). The observed antisickling activity may be attributed to the presence of these compounds. Several studies employing similar in vitro sickle cell models and extracts with comparable compositions, particularly those rich in phenolic compounds, have also demonstrated antisickling effects (Kane et al., 2013; Wumba et al., 2013; Ibrahima et al., 2020; Diouf et al., 2024). The reported activity of both aqueous and alcoholic extracts suggests that the chemical constituents responsible for this effect exhibit solubility in both polar solvents.

Anthocyanins, a class of phenolic compounds, have been specifically implicated in antisickling activity, according to Yuma et al. (2013). Furthermore, studies conducted by Nirmala et al. (2018) highlighted the potential of AO in mitigating the risk of chronic age-related diseases, including cardiovascular disease, Alzheimer’s disease, Parkinson’s disease, cancer, and diabetes (Nirmala et al., 2018). In our laboratory, we demonstrated the vasorelaxant effects of this extract in a pig model of coronary arteries in pigs (Diouf et al., 2021).

The demonstrated in vitro antisickling activity of AO warrants further investigation. Future studies involving chromatographic separation are necessary to identify the specific bioactive molecules, including anthocyanins, tannins, flavonoids, and cardiotonic heterosides, present in the extract. This will allow for a more precise determination of the fraction(s) exhibiting the highest activity and facilitate the exploration of the underlying mechanisms of action.

---

Conclusion

The findings of this study demonstrate that the hydroethanolic extract of AO leaves effectively inhibits red blood cell sickling, resulting in a significant dose-dependent decrease in the percentage of sickled erythrocytes in vitro. Given the observed dose-dependent antisickling activity, further in vivo investigations, including comprehensive toxicity studies, are warranted to determine the therapeutic index, as well as potential lethal or organ-modifying doses. This preliminary study suggests that AOis a promising avenue for the development of accessible and cost-effective therapeutic strategies for sickle cell disease.

---

Références

Alboury, B. 1976. La drépanocytose au Sénégal: conséquences sociales. Thèse de Doctorat en pharmacie, Montpellier, France.

Bayeme, O.M. and Chiabi, A. 2004. Epidemiologie de la drépanocytose. Clin. Mother Child Health. 1(1), 6–8.

TCHAMAGO CJ. 2006. Dépistage néonatal de la drépanocytose au Sénégal: Etude préliminaire au sein de deux maternités de Dakar (Doctoral dissertation, Thèse de Medecine, UCAD, Dakar, Sénégal)

Diene, G. 2005. Recherche de l’activité antidiabétique d’une plante de la pharmacopée traditionnelle sénégalaise: Oxytenanthera abyssinica Munro (Poacees). Thèse de doctorat en pharmacie, UCAD, Dakar, Senegal.

Diouf, M., Sene, M., Ba, A., Ndiaye, M., Sene, M., Ouedraogo, A., Kane, M.O. and Sarr, M. 2024. Antisickling properties of hydro-alcoholic extract of Ficus abutilifolia leaves. J. Afr. Ass. Physiol. Sci. 12(2), 31–37; doi:10.4314/jaaps.v12i2.4

Diouf, I., Sene, M., Sene, M., Ba, A., Diawṣ, A.N., Kane, M.O. and Sarr, M. 2021. Vasoactive effect of a sheet sample of Oxytenanthera abyssinica (Poaceae) on a model of coronary arteries of pigs. Natl. J. Physiol. Pharm. Pharmacol. 11(10), 1191–1195

Galacteros, F. 1997. Drepanocytose orphanet. Available via http://www.orpha.net/data/patho/FR/fr-drepanocy.pdf (Accessed 19 February 2025)

Girot, R., Begue, P. and Galacteros, F. 2003. La drépanocytose. Paris, France: Editions John Libbey eurotext.

Ibrahima, Diouf., Sene, M., Doudou, D., Diaw, A., Maimouna, T., Mor, D., Khadre, A.S., Oumy, B.K.M., Mamadou, S., Abdoulaye, B., Aminata, D. and Abdoulaye, S. 2020. Study of the antidrepanocytary properties of an extract of Ficus umbellata leaves. Natl. J. Physiol. Pharm. Pharmacol. 10(06), 504–507; doi:10.5455/njppp.2020.10.02054202029042020

Janky, E., Etienne-Julan, M., Kadhel, P.H., Leborgne-Samuel, Y. and Melki, E. 2006. Extrait des Mises à jour en Gynécologie et obstétrique.

Kane, M.O., Sene, M., Diaw, M., Seck, M., Ba, A., Ba, F., Sow, A.K., Samb, A. and Diallo AS. 2013. Effets d’un extrait de Hibiscus sabdariffa sur la viscosité sanguine et la falciformation érythrocytaire chez des sujets drépanocytaires. J. Sci. Pharm. Biol. 14(2), 34–41.

Nirmala, C., Bisht, M.S. and Bajwa, H.K. 2018. Bamboo: a rich source of natural antioxidants and its applications in the food and pharmaceutical industry. Trends Food Sci. Technol. 77, 91–99.

Sane, T. Lutte contre la drépanocytose: une association pour réduire le taux de prévalence en milieu scolaire. Available via http://www.lesoleil.sn/article.php3?idarticle=48139

Wumba, D.M.R., Kambale, J.K. and Mpiana, P.T. 2013. Evaluation in vitro de l’activité antifalcémiante et effet antioxydant des extraits d’Uapaca heudelotii Baill. (Euphorbiaceae). Int. J. Biol. Chem. Sci. 7(2), 523–534.

Yuma, P.M., Mpiana, P.T. and Bokota, M.T. 2013. Étude de l’activité antifalcemiante et de la thermo- et photo-dégradation des anthocyanes de Centella asiatica, Thomandersia hensii et Maesopsis eminii. Int. J. Biol. Chem. Sci. 7(5), 1892–1901.