Phytochemical Characterization of Three Plants and Their Antisickling Activity in the Management of Sickle Cell Disease

Abstract日本漫画大全之无彩翼漫免费

Natural plant products have been used by the population of the south-eastern part of Côte d’Ivoire in the management of sickle cell anemia. This study was aimed at investigating the antisickling activity of the hydro-ethanolic extract of a combination of the leaves of J. secunda, J. gossypiifolia and P. nigrescens. These three plants species were used in the Ivorian traditional herbal medicine. Preliminary phytochemistry was carried out using standard methods. As for the sickling reversal test, the Hb SS blood sickling was induced with 2% sodium metabisulfite. After 120 minutes of incubation, the plant extract was added. Every 30 minutes for a period of 120 minutes, a drop of the prepared solution was observed at (40×) magnification and the percentage of reversion calculated. The phytochemical analysis revealed the presence of Alkaloids, Flavonoids, Polyphenols, Catechic Tannins, Sterols and Polyterpenes. The results of the reversal test showed that the percentage of sickling reversal effect of the combination of the plants (75.00 ± 4.33b,c) was highly superior than the negative control (10.17 ± 0.55d) but was significantly (p < 0.05) the same as the percentage of the individual plant (J. secunda: 83.50 ± 2.33a; J. gossypiifolia: 78.00 ± 3.67b,c and P. nigrescens: 77.83 ± 2.89b,c) and the positive control (80.66 ± 2.22a,b). From the results, the extracts J. secunda, J. gossypiifolia and P. nigrescens have shown to be therapeutically beneficial to the population. Their use is also justified in the management of sickle cell disease in the south-eastern part of Côte d’Ivoire.

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Kplé, T. , Akakpo-Akue, J. , Golly, J. , Fofie, Y. , Ahon, M. , Kra, M. , Sanogo, I. and Yapo-Crezoit, A. (2020) Phytochemical Characterization of Three Plants and Their Antisickling Activity in the Management of Sickle Cell Disease. Journal of Biosciences and Medicines, 8, 100-112. doi: .

Sickle cell disease (SCD), also known as sickle cell anemia, is an inherited hemoglobinopathy caused by a point mutation. This mutation leads to the substitution of glutamic acid by valine in position 6 within the beta chain of hemoglobin. Under low oxygen pressure conditions, the resulted hemoglobin (HbS) has its affinity for oxygen and its solubility altered. The decreased solubility leads to the polymerization and the precipitation of HbS. This situation causes the blood cells’ sickling [1]. In the homozygous individual, the clinical manifestations of the sickle cell anemia are mostly due to hemolytic anemia and vaso-occlusive crises resulting in pain and vital organ damage [2] and serious frequent infections [3]. Usually, the most frequent infections associated with this disease have been due to Staphylococcus aureus, Salmonella sp. and Escherichia coli日本漫画大全之无彩翼漫免费 [4]. The production of the oxidative stress due to a large number of free radicals is also one of main characters of this pathology [5]. All the complications of this disease listed above are main causes of death of sickle cell patients [6]. Sickle cell disease is one of the most prevalent hereditary disorders with prominent morbidity and mortality [7]. Although the global prevalence is more than 5% [8], the proportion rises from 9% to 16% in West Africa [9]. Indeed, sickle cell disease affects nearly 275,000 newborns each year, 85% of which occur in sub-Saharan Africa [10]. In Côte d’Ivoire, the extend of this disease results in a prevalence rate of more than 14%, of which more than 2% are Homozygous HbSS who are suffering the most from the complications of the pathology [11]. Several treatment options have been considered to relieve patients from sickle cell disease. As a curative therapy, chemotherapy, gene therapy and bone marrow transplantation have been proposed. Although bone marrow transplantation and gene therapy provide satisfactory results [12], the problem of finding suitable donor, makes these highly specialized and expensive therapeutic approaches unavailable in most developing countries. As for the symptomatic treatment, the risks of infections related to transfusion and many pronounced side effects of chemical substances such as Hydroxyurea (HU); Erythropoietin, Tucaresol, CiklervitTM etc., constituted a major obstacle to this approach [13] [14]. As proposed therapies remain very expensive for most African populations, they have recourse to medicinal plants in order to sickle cell disease [15] [16] [17] [18]. As some studies have shown that Ivorian flora has a real therapeutic and nutritional potential that can be used to treat or prevent many diseases [19], herbal therapy might provide adequate treatment for sickle cell disease subjects [20]. Several Medicinal plants used in traditional medicine have been shown to contain antisickling principles in vitro [21] [22] [23] [24]. The present study was performed with the aim of studying the in vitro antisickling activity of the aqueous and ethanolic extracts of J. gossypiifolia, J. secunda and P. nigrescens three plants used by the traditional healers in South-eastern of Côte d’Ivoire. Two points have been targeted:

In vitro sickling reversal test of the hydroethanolic extract of each plant and the combination of the three plants.

2.1. Collection and Preparation of Plant Materials

2.2. Phytochemical Screening

Major groups of secondary metabolites such as sterols, polyterpenes, alkaloids, tannins, polyphenols, flavonoids, quinones, and saponins were characterized by classical methods described by [26]. The details concerning the used reagents and the characteristic reactions of the chemical groups are in Table 1.

Table 1日本漫画大全之无彩翼漫免费. Reagents and tests of characterization of the chemical groups.

2.3.2. Sickling Reversal Test

2.3.4. Reversibility Test

Fifty (50) µL of each of the 4 extracts HEE (Jg), HEE (Js), HEE (Pn) and HEE (M) at different concentrations between 0.05 and 10 mg/mL, were added to the previous solution, after an incubation of 120 min. Phenylalanine was used as a positive control. The SQA Vision microscope readings were performed every 30 minutes up to 120 minutes.

$\text{PRS=}\frac{\text{sickle}\text{\hspace{0.17em}}\text{cells}\text{\hspace{0.17em}}\text{average}\text{\hspace{0.17em}}\text{at}\text{\hspace{0.17em}}{T}_{X}}{\text{sickle}\text{\hspace{0.17em}}\text{cells}\text{\hspace{0.17em}}\text{average}\text{\hspace{0.17em}}\text{at}\text{\hspace{0.17em}}{T}_{0}}$

TX = 0, 30, 60, 90 and 120 minutes ; T0日本漫画大全之无彩翼漫免费 = temps initial.

Experimental data were processed using ANOVA statistical tool. The data obtained were entered through Excel software and then statistically analyzed using the software STATISTICA version 7.1 (processing by ANOVA 1 factor). The averages and standard deviations of the analyzed parameters were categorized using the DUNCAN test at 5%.

3.1. Phytochemical Study

Secondary metabolites identification in the studied plants revealed the presence of flavonoids, terpenoids, polyphenols, alkaloids and catechic tannins (Table 1). However, HEE (Pn) tested negative for the presence of flavonoids. HEE (Jg) was the only extract showing the presence of traces of quinones. But this presence was not revealed in HEE (M) (Table 1). Although HEE (Pn) tested negative for the presence of flavonoids and HEE (Jg) showed the presence of traces of Quinones, HEE (M) continued to show only the presence of the compounds listed above. These new results revealed that the compounds characterized in HEE (Jg), HEE (Js), HEE (Pn) were found in HEE (M). The results of this present study are similar to those of [30] [31] which showed that the hydro-alcoholic extract of J. secunda contained the chemical groups listed above. [32] confirmed their presence in the leaves of P. nigrescens. The phytochemical screening of J. gossypiifolia seeds, leaves and roots by [33] revealed the presence of the same compounds. In addition to the molecules revealed by the hydro ethanol extract, the work of [24] revealed the presence of saponins, quinonic substances and gallic tannins in the aqueous extract of the combination of the three plants. The absence of certain secondary metabolites is explained by the fact that ethanol 70% would not be the extracting solvent of all metabolites from the plant combination.

Several studies have shown the biological activities of certain compounds found in HEE (M). All of these compounds are known for their physiological activities and medicinal properties. [34] has demonstrated that alkaloids, tannins and flavonoids groups of chemicals promote tissue regeneration, decrease the permeability of blood capillaries and strengthen their resistance to hemolysis. Also, their antioxidant properties have been highlighted in [35] work, the anti-inflammatory property in [36] paper, the antimicrobial activity in [37] and [38] study. Among these compounds polyphenols and their derivatives have been cited as having antisickling activity [39]. The presence of sterols and polyterpenes, polyphenols, flavonoids, Catechic tannins and alkaloids could explain partially their use in traditional medicine (Table 2).

3.2. Reversal Test

Figure 1日本漫画大全之无彩翼漫免费 shows the result of the red blood cell count in contact with sodium meta bisulfite as a function of time. This figure shows that the Sickling rate is maximal (100%) after two hours (2 h). This Sickling is a function of time.

Table 2日本漫画大全之无彩翼漫免费. Tri-phytochemicals of individual plants and plant combinations.

Figure 1. Percentage of red blood cells sickling in the presence of meta bisulfite of sodium 2% as a function of time.

3.3. Reversibility of Antisickling by the Combination of Plants

Table 3日本漫画大全之无彩翼漫免费 shows that all the tested extracts of all plants have had a significant reversible effect on sickled cells compared to the negative control (10.17 ± 0.55). Compared to HEE (Jg) (78.00% ± 3.67%), HEE (Pn) (77.83% ± 2.89%), and HEE (M) (75.00% ± 4.33%) the reversible effect on sickled cells of HEE (Js) (83.50% ± 2.33%) had the higher reversible effect on sickled cells. This significant difference was not observed between HEE (Js) (83.50% ± 2.33%) and the positive control represented by phenylalanine (80.66% ± 2.22%).

Figures 2-5 show the evolution of reversible effect of HEE (M) on sickled cells according to its concentration. Indeed, HEE (M) at different concentrations of 0.05; 0.5; 5 and 10 mg/mL displayed a reversion rate of 50%, 63%, 65% and 75% respectively. This reversible effect on sickled cells has depended on the concentration of HEE (M). This normalization of sickle cells occurs rapidly with a high rate in the first 30 minutes; then between 30 and 120 min the reversion is slow. This phenomenon is also well observed for phenylalanine.

Table 3. Percentage of sickle cells reversibility.

Figure 2. Percentage of the reversion rate in the presence of hydro-ethanolic extract at the concentration 0.05 mg/mL.

Figure 3. Percentage of the reversion rate in the presence of hydro-ethanolicextractat the concentration 0.5 mg/mL.

Figure 4. Percentage of the reversion rate in the presence of hydro-ethanolicextractat the concentration 5 mg/mL.

Figure 5. Percentage of the reversion rate in the presence of hydro-ethanolic extract at the concentration 10 mg/mL.

4. Conclusion

This study was conducted to evaluate in vitro the reversible effect of hydroethanol extract from the combination of J. secunda, P. nigrescens and J. gossypiifolia. This work shows that hydro-alcolic extract at the concentration of 10 mg/mL has reversibility activity on red sickled cells. This activity might be due to phenolic compounds in this combination of plants as it has been shown to be one of the main components responsible for anti-sickling activity. In addition, this combination of plants could act in synergy because of the antisickling, anticoagulant, antioxidant and anti-hemolytic activity of each of them. Varying proportions of plants and the extraction solvent could improve the activity of the combination. Further studies will be needed to elucidate the nature of the active compounds, the mechanisms of action and perform the short, medium and long term toxicological study of these three plants.

Acknowledgements

The authors would like to thank the officials and staff of the Hematology Unit of the Yopougon University Hospital, the Pasteur Institute of Côte d’Ivoire, not to mention those of the Pharmacognosy Laboratory of the UFR of Pharmaceutical and Biological Sciences of Félix Houphouet Boigny University in Cocody for their availability and assistance in carrying out the work. There is also a thank you to all of these patients who have agreed to participate in this study.

The authors declare no conflicts of interest regarding the publication of this paper.