Antisickling and Antihemolytic Mechanism of Spirulina platensis ( Oscillatoriaceae ): A Nutraceutical Commonly Used in Cameroon

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Introduction
Sickle cell disease (SCD) is a serious, inherited, autosomal recessive genetic disorder, in which red blood cells take on a sickle shape instead of their normal disc shape.Tis haemoglobinopathy is caused by the replacement of hydrophilic glutamic acid by hydrophobic valine at position six (6) of the β chain of hemoglobin.Tis substitution alters its afnity for oxygen and its solubility under conditions of low oxygen pressure.Te result is a polymerization of hemoglobin S inside the red blood cells leading to its sickling [1].Tese sickled cells acquire a rigid structure which makes their circulation difcult in the microvessels, causing vasoocclusion and various complications of the disease [2], such as painful crises, increased susceptibility to infections, hyperhaemolysis causing anaemia, splenomegaly, and increased production of endogenous free radicals, mainly the °OH radical which is very abundant in sickle cell patients [3].
Worldwide, this haemoglobinopathy afects more than 50 million people in the homozygous SS form and 250 million heterozygous AS carriers [4].Sickle cell disease is at its highest prevalence in Sub-Saharan Africa and is now a real public health problem for most Black African countries.According to the World Sickle Cell Organization, out of 500,000 sickle cell children born each year, 400,000 are in Africa.In Central and West Africa, 20-40% of people are carriers of the sickle cell trait.In Cameroon, the heterozygous AS prevalence is estimated at 25-30% and 2% in its homozygous SS form [5]. Sickle cell disease remains very deadly; 50%-90% of children with sickle cell disease die before their 5th birthday if they are not supported [6].
In Cameroon, hospitals and healthcare centers are insufcient and an access to medical care is not easy for the rural population.Te management of the disease takes into account the preventive aspects such as the folate supplementation, the prevention of infections, oral hydration with alkaline water, rest, putting on oxygen, keeping warm, prescription of analgesics, and blood transfusion when the hemoglobin level is low [7].Te curative component consists of bone marrow transplantation and genetic therapy [7].However, the various drug managements for this disease which have been recommended to patients demonstrated many limitations both in terms of cost and risks related to incompatibility and drug toxicity problems.
As a result, patients from low-and middle-income countries prefer phytomedicine for the management of the disease.Niprisan ® and Ciklavit ® are a formulation which appeared to be safe and efective in reducing severe painful crises.Tey are reported to inhibit the polymerization of the hemoglobin S and possess antioxidant activities [8].Several plant extracts have demonstrated antisickling properties through the inhibitory efect of hemoglobin S polymerization, the reversibility of sickling, or the increase of the Fe 2+ /Fe 3+ ratio.Tese include the hydroethanolic extract of Ficus sycomorus leaves [9], aqueous extract of Hemodya [10], aqueous extracts of Zanthoxyllum heitzii [11], hydroethanolic extract of Teobroma cacao [12], aqueous extracts of Phaseolus vulgarus L. [13] and Rubia cordifolia L. [14], leaves of Rhaphiostylis beninensis [15], and Woodfordia fruticosa (L.) Kurz leaves [16], among others.Other studies demonstrated that nutraceutic and nutrition supplement have benefcial efects on the management of sickle cell diseases [17,18].Te benefcial efects of these nutraceutic and nutrition in the management of sickle cell disease are attributed both to their higher levels of micronutrients (vitamins and minerals) and bioactive molecules (phenolic compounds and amino acids) composition, as well as the antiradical and antioxidant properties of these bioactive compounds [13][14][15][16][17][18].
Based on a systematic review on Spirulina platensis, it is a blue-green alga of the cyanobacteria family, endowed with nutritional and therapeutic properties [19].Te aqueous extract of Spirulina platensis from Cameroon is rich in macronutrients, micronutrients, and bioactive phytochemicals including polyphenols, favonoids, beta carotene, phycocyanin, phenolic acids, and calcium spirulan endowed with antioxidant activities [20].In addition, a recent in vivo study on sickle cell patients reveals that S. platensis supplementation reduces a number of seizures, blood transfusions, and hospitalizations in children with sickle cell disease [21].However, no study has highlighted in vitro on the antisickling and antihemolytic mechanism of the S. platensis aqueous extract on the red blood cells of sickle cell patients.In order to contribute to the improvement of the management of the disease and demonstrate the in vitro antisickling and antihemolytic activities of S. platensis, we decided to explore the antisickling and antihemolytic properties of the aqueous extract of S. platensis.

Types of Study, Blood Sample Collection, and Ethical
Consideration.Te experimental study was performed after an ethical clearance issued by the Regional Committee for Ethics Research for Human Health Center (clearance N °676./CRERSH/2019) and the patient's written consent was obtained; 27 patients' confrmed sickle cell blood samples were collected including 10 women and 17 men between the ages of 10 and 40 who had been attending routine consultations at the Hemato-Oncology department of the Yaoundé Central Hospital.Te patients who were in a state of crisis and those who have been transfused during the last three (03) months preceding the study were excluded.Te samples were taken from the patients' elbow crease by healthcare personnel and about 5 mL of blood was collected in EDTA tubes.

Preparation of the Extract of S. platensis.
Te green-blue algae (S. platensis) were collected in Nomayos in Yaounde, the capital city of Cameroon.It was oven dried at temperatures between 37 and 45 °C for three days.Fifty grams (50 g) of S. platensis powder were soaked in 1000 mL of distilled water, homogenized for 1 h and kept at room temperature (25 °C) for 24 h before fltration with Whatman paper no. 4. Te residues were reextracted in the same condition.Te total fltrate was lyophilized and the extraction yield was calculated according to the following formula: Te powder of S. platensis obtained was then stored in a refrigerator protected from light for further assays.

Preparation of the Erythrocyte or Hematocrit Suspension.
Te blood sample collected in EDTA tubes was centrifuged at 3000 rpm for 5 min, and the pellet recovered was washed 3 times with iso-saline PBS (10 mM potassium phosphate bufer, pH � 7.4; 154 mM of NaCl) solution.Each step of washing consisted of suspension of the red blood cells (RBCs) into iso-saline phosphate bufer (PBS) followed by centrifugation (3000 rpm; 5 min).After the last centrifugation, the pellet was again suspended in a solution of isosaline PBS to make a 10% hematocrit [22].

Determination of the Antifalcemic Activity of the Aqueous
Extract of S. platensis: In Vitro Induction of Sickling.One hundred (100 μL) microlitres of blood hematocrits were mixed with 100 μL of 2% sodium metabisulfte solution (fnal concentration) and then incubated at room temperature (25 °C). 10 μL of this mixture was diluted in 1990 μL of Marciano liquid and the cells were counted under light microscope 40X continuously during 30 min, 1 h, 1 h 30 min, 2 h, 2 h 30 min, and 3 h.Te initial percentage of sickling cells was obtained by counting the total red blood cells and sickle cells in each sample where the sodium metabisulfte was replaced by 0.9% NaCl.Te percentage of sickling (F) cells was calculated from the following formula: where n = number of sickle cells and Nt = total number of red blood cells counted [23].

Determination of the Inhibitory Activity of the Aqueous
Extract of S. platensis on Sickling.In the tubes, containing 100 μL of the extract at diferent concentrations, 50 μL of SS blood and 50 μL of a 2% sodium metabisulfte (2%, W/V) fnal solution's concentration were added.Ten, they were incubated at room temperature (25 °C) for 2 h 30 min.Total red blood cells and sickle cells were counted under a light microscope (40X) using the Malassez cells.Phenylalanine was used as a positive control at the same concentrations as the extracts.Te inhibition rate (INF) was calculated by the following formula: where f 0 is the percentage of maximum sickling in the presence of HbS + 2% MBS blood and f n is the minimum sickling percentage in the presence of HbS + 2% MBS + extract blood [23].

Determination of the Efect of Extracts of S. platensis on
the Reversibility of Sickling Cells.Te efect of the extract on the reversibility of sickling was achieved by incubating 50 μL of blood at room temperature (25 °C) with 50 μL of the extract at diferent concentrations for 2 h, 4 h, and 24 h.Percent sickling was calculated before and after incubations.Te percentage of sickling was then determined as mentioned above.Te sickling reversibility rates (% R) are calculated according to the following formula: where F 0 is the percentage of initial sickling and F n is the percentage of minimum sickling obtained in the presence of the extract or phenylalanine [23].Te concentrations of the extract which showed the best antisickling activities (800 and 1600 μg•mL − 1 ) were used to investigate the in vitro Fe 2+ /Fe 3+ ratio and the antihemolytic activity of the aqueous extract of spirulina.

Evaluation of the Efect of Extracts of S. platensis on the
Fe 2+ /Fe 3+ Ratio of Hemoglobin S. In the presence of distilled water, the red blood cells undergo considerable osmotic pressure.Te water difuses into the interior of the cell through the membrane and causes hemolysis.Oxyhemoglobin and methemoglobin, constituting the hemolysate, absorb at 540 nm and 630 nm, respectively.Te method was described by the authors in [20] and reported by the authors in [10].
Te percentages of oxyhemoglobin (Fe 2+ ) and methemoglobin (Fe 3+ ) in each tube were calculated using the following formulas: � 100 − % Fe 2+ . ( Te ratio (R) of oxyhemoglobin (Fe 2+ ) to methemoglobin (Fe 3+ ) was calculated as follows: R � %Fe 2+ /%Fe 3+ .Te rate of increase (TA) of the Fe 2+ /Fe 3+ ratio was calculated by the following relationship: 2.5.In Vitro Antihemolytic Property of S. platensis.Te antihemolytic efect of the aqueous extract of S. platensis was achieved at 800 and 1600 μg•mL − 1 , which showed better antisickling activities.Four models of hemolytic inducers were used including the hypotonic medium, salicylic acid, Triton X-100, and H 2 O 2 in the absence of the extract to determine the concentration of the inducer which provided the maximum hemolysis.

Hemolytic Activity Induction of Aspirin and the
Antihemolytic Efect of S. platensis.To 4.5 mL of the solution of NaCl (4.5 mg•mL − 1 ), 50 μL of aspirin at concentrations 0 and 0.7 mg•mL − 1 was added.Te control tube received the Evidence-Based Complementary and Alternative Medicine same volume of PBS and then 500 μL of the erythrocyte suspension was added to each tube, homogenized and incubated at 37 °C for 30 min.After centrifugation (3000 rpm; 5 min), the absorbance of the supernatant was measured at 540 nm [24].
To determine the efect of the Spirulina platensis extract on hemolysis induced by aspirin, fve hundred microlitres of the aqueous extract of S. platensis at diferent concentrations (800 and 1600 μg•mL − 1 ) were added to 250 μL of hematocrit (10%) and incubated for 5 min at room temperature and then 25 μL of aspirin (0.7 mg•mL − 1 ) and 1750 μL of NaCl (0.45%) were added.Te mixture was homogenized, incubated for 1 h at 37 °C, and centrifuged (3000 rpm, 5 min).Te optical density of the supernatants was read at 540 nm against the blank and the standard (quercetin) [25].

Hemolytic Activity Induction of Hypotonic NaCl and the Antihemolytic Efect of S. platensis.
In order to determine the concentration of NaCl which leads to a maximum lysis of red blood cells, 100 μL of hematocrit (10%) was added to 5 mL of NaCl at diferent concentrations (3.5-8.5 mg•mL − 1 ), to a negative control (isotonic NaCl 9 mg•mL − 1 ), and to a positive control (distilled water).After 30 min of incubation at room temperature (25 °C), the mixture was centrifuged (3000 rpm, 10 min) and the supernatant was read at an absorbance of 540 nm [26].
Fifty (50) microlitres of the extract at diferent concentrations (800 and 1600 μg•mL − 1 ) were mixed with 50 μL of hematocrit (10%) after 10 min incubation at room temperature and 2.5 mL of NaCl (0.35%) was added.Te mixture was incubated at 37 °C for 30 min and then centrifuged (3000 rpm, 10 min).Te absorbance of the supernatant was read at 540 nm.For the control, the extract was replaced by the same volume of NaCl (0.35%) and a standard (quercetin) [26].
A volume of 500 μL of extracts (800 and 1600 μg•mL − 1 ) was added to 250 μL of hematocrit (10%), the mixture was incubated for 5 min at room temperature, and then 400 μL of the Triton X-100 (1%) was added.After incubating for one hour at 37 °C followed by centrifugation (3000 rpm; 10 min), the absorbance of the supernatant was read at 540 nm.For the control prepared, the extract was replaced by the same volume in PBS and a standard (quercetin) [27].

Hemolytic Activity Induction of Hydrogen Peroxide
H 2 O 2 and the Antihemolytic Efect of S. platensis.500 μL of H 2 O 2 at concentrations of 9%, 6%, 3%, 1%, 0.5%, 0.3%, and 0.2% were mixed with 250 μL of hematocrit (10%).After 3 h of incubation at 37 °C, this solution was adjusted with 4.500 mL of PBS and centrifuged (3000 rpm, 10 min).Te absorbance of the supernatants was read at 540 nm.Controls were prepared by replacing H 2 O 2 with distilled water for the positive control and PBS for the negative control [28].Te percent hemolysis for all tests was calculated by applying the following formula [29]: where Abs � absorbance.
To determine the efect of S. spirulina on the inhibition activity of H 2 O 2 , 250 μL of hematocrit (10%) and 500 μL of the extract at diferent concentrations (800 and 1600 μg•mL − 1 ) were incubated for 30 min at room temperature followed by 500 μL of H 2 O 2 (9%).After incubating for 3 h at 37 °C, the fnal volume was adjusted to 4500 μL by PBS and centrifuged (3000 rpm; 10 min), and the optical density was read at 540 nm.Te standard was carried out in the same manner using the quercetin as a reference molecule [28].

Statistical Analysis.
Results are presented as the mean ± standard deviation.All the tests were carried out by group of quadruplets.Distritbution test was checked before carryingout analysis.All the data were analyzed using oneway ANOVA followed by Dunnett's post hoc test for multiple comparisons with the software SPPS version 21.0.

In Vitro Induction of Sickling with Sodium Metabisulphite (MBS) 2% and Antisickling
Properties of the Extract of S. platensis.Figure 1(a) shows the kinetic induction of falciformation using 2% metabisulphite.It appears that SS blood added to 2% MBS (W/V) induced an increase in the percentage sickling of red blood cells at a time-dependent manner up to a maximum of 91, 38% after 2 h 30 min (Figure 1(a)).Te treatment of homozygote SS red blood cells with both aqueous extract of S. platensis and phenylalanine after induction with metabisulphite (2%) showed a signifcant and linear reduction of sickling cells (Figure 1(b)).Tere was no signifcant diference in the reduction of falciformation between phenylalanine and the extract of S. platensis (Figure 1(b)).However, there were no similarities on the morphology of red blood cells between the negative control (blood cell induced with MBS 2%) and those induced with MBS 2% and treated with S. platensis or phenylamine (Figure 2).2+ /Fe 3+ Ratio of the Aqueous Extract at Diferent Concentrations.We determined the efect of incubation time and extract concentration of S. platensis on the reversibility of sickling.Te results showed that the reversibility of sickling increases with a time-and concentration-dependent manner of the aqueous extract of S. platensis (Figure 3).Tese results showed that the maximum reversibility (39.75%) of sickling

In Vitro Antihemolytic Property of S. platensis.
Many studies have shown that the plasma membrane of human red blood cells of sickle cell patients was confronted with diferent molecules inducing a variable stability of the latter depending on the environment in which they are found [26].Tus, we have determined the antihemolytic properties of the aqueous extract of S. platensis subjected to diferent mechanisms of hemolysis.Te results obtained (Figure 6(a)) show maximum and signifcant (p < 0.05) hemolysis induction with salicylic acid of 83.94 ± 4.05% at the concentration of 0.7 mg•mL − 1 of salicylic acid compared to the negative control.After the treatment of hemolytic induction with the aqueous extract of S. platensis at the concentrations 800 μg•mL − 1 and 1600 μg•mL − 1 , we noted that the inhibition of the hemolytic activity signifcantly increased (p � 0.0001), respectively, from 53 to 96% with the concentration of S. platensis, while the percentage of inhibition of hemolysis decreased from 76 to 53% with quercetin (Figure 6(b)).
Signifcant variation of sodium chloride homeostasis can induce hemolysis of the red blood cell membrane.Te maximum hemolysis noted (78.05%) with the hypotonic NaCl solution was found at 0.35% (Figure 7(a)).After treatment with S. platensis, we noted that at 800 μg•mL − 1 , we found a higher inhibitory activity (80%) compared to 71% of inhibition at 1600 μg•mL − 1 .Te results demonstrated that S. platensis induced higher inhibition activity at lower concentration.Similar tendency was noted with quercetin (Figure 7(b)).
Hydrogen peroxide is a product of oxidative stress produced in the mitochondria by NAPDH oxidase.Te level of hydrogen peroxide increased in the condition of anoxia.Te efect of the hydrogen peroxide concentration on hemolysis of membrane mitochondria was explored.Our results demonstrated that a maximum membrane hemolysis of red blood cells (82.21%) could be induced by hydrogen peroxide at the concentration of 0.875% (Figure 8(a)).After treatment with the extract of S. platensis, we found an increase in the inhibition percentage from both S. platensis and quercetin as the concentration is increasing (Figure 8(b)).None of these inhibitions of hemolysis activities went beyond 50%.Triton X-100 is a commonly used detergent and it is widely used to lyse cells to extract protein or organelles or to permeabilize the membranes of living cells.We studied the efect of S. platensis on the inhibition of hemolysis with Triton X-100.Te results showed that at the of 1%, the Triton X-100 induces a total hemolysis of red blood cells of sickle cell patients (Figure 9(a)).Te treatment of Triton X-100 hemolysis with S. platensis or quercetin at the concentration of 800 μg•mL − 1 and 1600 μg•mL − 1 signifcantly and gradually reduced hemolysis up to 49-65% for quercetin and 38-46% for S. platensis (Figure 9(b)).

Discussion
Research investigation of antisickling properties of medicinal plants and nutraceutics has increased in recent years.Te alternative management of the disease using phytomedicines has proven to not only reduce crisis but also reverse sickling cells, stabilize the membrane of the red blood cells, and avoid its hemolysis.Antisickling properties of several medicinal plants from Cameroon and Africa have been documented [11,13,23,[30][31][32][33][34].
Extraction is an essential step in the process of the evaluation of the biological activity of natural substance.Many parameters can signifcantly afect the yield of extraction of molecules from natural products.Te yield obtained in our study was 14.015%.Tis value is lower than that obtained by the authors in [20], which, upon aqueous extraction of S. platensis, obtained a yield of 16.84%.Tis variation could be explained by the diference in the level of solubility of solvents used, lyophilization process, degrees of purity, time of agitation, or also the type of fltration which afects the nature of the molecules present in the solution [35,36].
Moreover, the aqueous extract of S. platensis used in this study was previously used for the determination of nutrients, trace elements, carotenoids, phycocyanin, and phytochemical composition using standard methods.Te results showed that S. platensis contains protein (375.5 + 0.7 g/kg•dw), lipids (301.2 + 11.9 g/kg•dw), carbohydrates (243.9 + 9.9 g/kg•dw), and fbers (313.2 g/kg•dw).Te HPLC profle revealed the presence of polyphenols (21.2 + 1.18 mg eq.QE/g Ext.), favonoids (56.4 + 6.47 mg eq.QE/g Ext.), and phenolic acid such as cafeic and coumaric acids.Iron was the micronutrient found in majority but copper, manganese, zinc, and Evidence-Based Complementary and Alternative Medicine selenium were also found.Te percentage of phycocyanin was 16.15% while carotenoids were 3.8% [20].Aqueous extracts of several phytomedicines have demonstrated signifcant in vitro antisickling activity.Te antisickling action mechanism of the medicinal plants in vitro included inhibition of sickling, reversibility of falciformation, increase of the Fe 2+ /Fe 3+ ratio, and osmotic fragility of membrane of red blood cells, as well as the  Evidence-Based Complementary and Alternative Medicine antioxidant activity.Among these, medicinal plants included herbal drugs such as Hemodya and niprisan (renamed Nicosan) with Piper guineense, Pterocapus osun, Eugenia caryophyllum, and Sorghum bicolor as components; Ciklavit (Cajanus cajan seed extract as base), aqueous extracts of Zanthoxylum zanthoxyloides roots, Ajawaron HF complex with Cissus populnea as the main component; aqueous and alcoholic extracts of Terminalia catappa leaves; and Carica papaya unripe fruit and dried leaf extracts [8,10].
Our study demonstrated that the aqueous extract of S. platensis inhibited the sickling cell signifcantly, in a dosedependent concentration of up to 66.09% at 800 μg•mL − 1 .It acts positively on the reversibility of sickling cells in a dosedependent manner and increases the Fe 2+ /Fe 3+ ratio signifcantly.Several studies showed the correlation between the antisickling activity of natural products and its chemical composition.Te aqueous extract of S. platensis possesses a higher level of amino acids like phenylalanine which has been reported to demonstrate signifcant antisickling property [37,38].Active constituents of medicinal plants and naturally occurring compounds, known as antisickling agents, are rich in aromatic amino acids, phenolic compounds, and antioxidant nutrients which are thought to be responsible for their observed antisickling action [39].Studies indicated that in vivo, vitamin and mineral supplements such as vitamins C and E, zinc, and magnesium or treatment with a combination of high dose antioxidants can reduce the percentage of irreversibly sickled cells [40,41].Phenylalanine, tyrosine, and arginine exhibited the antisickling properties which are attributed to their activity of inhibiting the polymerization of hemoglobin S and improving the Fe 2+ /Fe 3+ ratio [42][43][44].Te Fe 2+ /Fe 3+ ratio was Evidence-Based Complementary and Alternative Medicine used to assess an increase or decrease in the afnity of hemoglobin for oxygen.Tis increase of the Fe 2+ /Fe 3+ ratio found in our study due to the presence of S. platensis implies the conversion of deoxyhemoglobin S into oxyhemoglobin S by the extract and therefore promotes antisickling activity.Te higher level of the Fe 2+ /Fe 3+ ratio can be explained by the presence of a higher concentration of micronutrients such as selenium, zinc, copper, vitamins E and A, and other bioactive molecules (polyphenols, favonoids, and carotenoids) present in the Spirulina platensis aqueous extract [20,45].Moreover, the involvement of these bioactive amino acids and their aromatic groups in the inhibition and reversibility of sickling has been demonstrated in some studies [13,46].Tus, the antisickling activity of the extract of S. platensis on the mechanism of the antisickling activity of red blood cells can be attributed to the presence of phenolic compounds and aromatic amino acids such as tyrosine, phenylalanine, and arginine found in a large concentration in S. platensis [20,47].In our study, we found a close result between the activity of the crude extract of S. platensis and that of phenylalanine (standard) but with a signifcantly higher activity of the spirulina extract.Tis could be explained by the fact that the molecules found in the extract act in a synergistic manner either in the inhibition of the polymerization hemoglobin S, the improvement of the Fe 2+ / Fe 3+ ratio, the conversion of methemoglobin into oxyhemoglobin, or the stabilization of the existing oxyhemoglobin form, thus increasing their afnity to oxygen and the reversibility of the sickling cells.Tis corroborates the overlap of curves observed in the reversibility activity we obtained.
Several substances can be used to induce in vitro hemolysis of red blood cells using diferent mechanisms including salicylic acid, Triton X, H 2 O 2 , and hypotonic NaCl.Te movement of fuids in the red blood cell which leads to hemolysis implies osmosis and tonicity of the cell membrane.Hypotonic solutions lead to cell swelling and eventual rupture or lysis if the resultant osmotic movement of water is great enough.In the case of red blood cells, this is referred to as hemolysis.
Te most abundant cells in the human body are the erythrocytes, which can have several biological and morphological characteristics depending on the physiological conditions.Tese cells have been widely exploited in drug transport, gas, and other useful molecules in biological fuids.Te polyunsaturated fatty acids (PUFA) and hemoglobin molecules which are redox active oxygen transport molecules and potent promoters of activated oxygen species mainly target the erythrocytes [48] Oxidative mutilation of the erythrocyte membrane lipids and proteins may be responsible for hemolysis accompanied with several factors of hemoglobinopathies, oxidative drugs, excess transition metals, various radiations, and defciencies in erythrocyte antioxidant coordination [48][49][50].Te magnitude of hemolysis appeared to be much more overwhelming, when red blood cells were exposed to any toxicant like hydrogen peroxide [48].Tese high rates of hemolysis, even at low concentrations of hydrogen peroxide, are thought to be due to the fact that hydrogen peroxide causes oxidative damage to the cytoplasmic membrane following the lipid peroxidation of the polyunsaturated fatty acids present [51].Indeed, when H 2 O 2 crosses the plasma membrane, it can cause the degradation of the heme of hemoglobin by the oxidation of Fe 2+ ions to Fe 3+ , with the production of hydroxyl radicals (HO °) which are very unstable and highly reactive by the Fenton reaction.Tese radicals induce a chain of lipid peroxidation leading to lysis of red blood cells [52].Te Spirulina platensis extract has been shown to protect against hemolysis induced by hydrogen peroxide.In addition,  Evidence-Based Complementary and Alternative Medicine several studies have shown that some of the phenolic compounds, in particular the favonoids, have antifree radical properties by neutralizing or scavenging free radicals [53].Moreover, polyphenols are known to chelate transition metals such as Fe 2+ , thus reducing the rate of the Fenton reaction by electron transfer.Tey can also prevent oxidation caused by the hydroxyl radical [54] and prevent the passage of H 2 O 2 through the erythrocyte membrane and the generation of free radicals [55].
Te results of hemolysis induced by Triton X-100 at diferent concentrations showed signifcant hemolysis of Triton X-100 at the concentrations used.At 1% of Triton X-100 concentration, there is a maximum and signifcant hemolysis (100%).Tis result corroborates with that of [56], who obtained 100% hemolysis at the 1 Mm concentration of Triton X-100.Te total hemolysis may be due to the chemical nature of Triton X-100.Indeed, Triton X-100 has the ability to disrupt the membrane of red blood cells or cells in general.Triton X-100 is a nonionic synthetic detergent which consists of a hydrophilic polar part and a hydrophobic tail.Tis molecule interacts with the hydrophobic parts of the lipids on the erythrocyte membrane until saturation and then causes the disruption of the membrane.At very high concentrations of Triton X-100, the red blood cells will be completely solubilized in the form of micelles or liposomes [27].However, in our study, the antihemolytic properties of the aqueous extract of S. platensis show a signifcant hemolysis inhibitory activity.Te mechanism of its inhibitory efects of the S. platensis extract includes the ability to interact with the external polar poles of the lipid bilayer on the membrane red blood cells and prevent its solubilization and therefore prevent hemolysis.Te aqueous extract of S. platensis has shown the protective efects of the erythrocyte membrane in the presence of several agents inducing hemolysis.Our results corroborate with those of [13], who obtained 80% hypotonic hemolysis in the absence of the extract.Tis is justifed by the fact that in the isotonic medium, the extracellular concentrations of electrolytes are almost equal to its intracellular.Te antihemolytic efect of the S platensis aqueous extract on hemolysis induced by hypotonic solution could be attributed to its richness in mineral salts [20], which establishes a balance of concentration and osmotic pressure through its ability to bind to aquaporin and prevent water from entering the red blood cell [26].Tis higher antihemolytic activity is also due to the presence of antioxidants and antifree radical molecules in S. platensis, mainly its richness in polyphenols [57], favonoids, phycocyanin, carotenoid, and antioxidant vitamins and minerals (vitamin E, selenium, and zinc) [20] which would protect the membrane of red blood cells from lipid peroxidation [58,59].

Conclusion
Te aqueous extract of S. platensis from Nomayos in Cameroon showed antisickling properties and allows the reversibility of sickling cells in a dose-dependent manner.It presented an antihemolytic property at the concentrations 800 μg•mL − 1 and 1600 μg•mL − 1 on various hemolysis inducers.In the future, the investigation of the antisickling mechanism of the diferent fractions of the aqueous extract of S. platensis will be made in order to identify the fraction responsible for the antisickling efect observed in this study.

2
Evidence-Based Complementary and Alternative Medicine R � (mass of the crude extract obtained) (mass of initial powder)   × 100.(1)

Figure 2 :
Figure 2: Images of red blood cells (a) treated with metabisulfte and phenylalanine at the concentration 1600 μg•mL − 1 , (b) treated with metabisulfte and the extract of S. platensis at the concentration 1600 μg•mL − 1 , and (c) treated with metabisulfte, the negative control.

Figure 9 :
Figure 9: Hypotonic hemolysis of Triton X-100: (a) induction of hemolysis and (b) inhibition of hemolysis with S. platensis or quercetin.