Phytocomponents from Anacardium occidentale, Psidium guajava, and Terminalia catappa altered membrane osmotic stability of sickle erythrocytes

The present study identified, quantified, and characterized the combinations of phytocomponents from fractionated leaf extracts of Anacardium occidentale, Psidium guajava, and Terminalia catappa that stabilized sickle erythrocyte membrane against osmotic stress, or otherwise, using standard chromatographic-spectrophotometric techniques, namely GC-MS, FT-IR, and UV-visible systems. The percentage hemolysis of the control sample, in 0.9 g/100 mL NaCl, was 35.08 ± 11.64%, whereas those of the samples containing 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL of ethylacetate extracts of T. catappa ranged between 31.82 ± 8.32 and 39.18 ± 6.94%. Ethylacetate extract of T. catappa contained comparative high quantities of hexadecanoic acid methyl ester, 9,11-octadecadienoic acid, methyl ester, (E, E)-, trans-13-octadecenoic acid methyl ester, and methyl stearate. FT-IR and UV-visible spectra showed that ethylacetate extract of T. catappa contained aromatic compounds as well as nitro-compounds, phenolics, and esters. To mention but a few, the combinations of major phytocomponents that stabilized sickle erythrocyte membrane against osmotic stress were hexadecanoic acid, methyl ester, 11-octadecenoic acid, methyl ester, dibutyl phthalate, pentacosane, trans-13-octadecenoic acid, methyl ester, whereas the minor phytocomponents include methyl tetradecanoate, methoxyacetic acid, 3-pentadecyl ester, methyl stearate, hexadecanoic acid, isoxazole, 4,5-dimethyl-.

When erythrocytes are suspended in hypotonic sodium chloride (NaCl) solution of diminishing osmolality, the uptake of water by the erythrocyte is both instantaneous and quantitative. In practical terms, the quantity of hemoglobin released into the hypotonic solutions following hemolysis is a measure of erythrocyte osmotic fragility index [25][26][27]. Accordingly, osmotic fragility index defines the capacity of the erythrocytes to withstand hemolysis when subjected to osmotic stress [27]. Osmotic fragility test, in which the population of hemolyzed erythrocytes is measured as a function of variability in osmolality of erythrocyte suspension, is primarily diagnostic of hereditary disorders of erythrocyte membrane cytoskeletal structures such as hereditary spherocytosis and other vast groups of the hemolytic anemias [27][28][29][30][31][32]. The extrinsic and intrinsic factors that influence erythrocyte osmotic fragility index are summarized elsewhere [27,30].
Sickle cell disease (SCD) is a hereditary autosomal recessive erythrocyte disorder caused by mutant hemoglobin molecules (sickle cell hemoglobinopathy; α 2 β 2 s ) that polymerize under low oxygen tension within the microvasculature. Intra-erythrocytic polymerization of deoxygenated sickle hemoglobin, in turn, promotes distortion of the erythrocytes that take up the sickle morphology [33][34][35]. The functional and structural defective erythrocytes elicit multiple clinical disorders, notably; severe anemia, vasoocclusion, multiple organ damage, and splenomegaly associated secondary infections [36]. The biological activities and identities of chemical constituents from medicinal plants used for the management of SCD have been exhaustively reviewed elsewhere [37,38].
Sickle erythrocytes are relatively fragile and frequently undergo spontaneous osmotic lysis because of low elasticity and rigidity of their membrane structures [35]. The enhanced membrane fragility index is further exacerbated by high oxidative stressed status of sickle erythrocytes [39][40][41]. Stabilization of erythrocyte membrane structures by herbal extracts is attributable to antioxidant activities and membrane chaotropic agent antagonists of phytocomponents from diverse plants [21,26,[42][43][44]. Because of limited scope of the previous investigations [21,26,43], the combinations of phytocomponents from the herbal extracts that stabilized the erythrocyte membrane against osmotic stress were not identified, quantified, and characterized. Spectrometric methods, viz. Fourier transform-infrared spectroscopy (FT-IR) and ultraviolet-visible spectroscopy (UV-visible) complement gas chromatography-mass spectroscopy (GC-MS) protocols in terms of providing comprehensive insights into the molecular characteristics and quantification of phytocomponents from plant materials [21,[45][46][47][48][49].
In view of the established relatively high fragility of sickle erythrocytes and medicinal usefulness of vast array of phytocomponents, the present study investigated the capacities of fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa to stabilize sickle erythrocyte membrane against osmotic stress using in vitro models. Furthermore, the present study identified, quantified, and characterized the combinations of phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa that stabilized sickle erythrocyte membrane against osmotic stress, or otherwise, using standard chromatographic-spectrophotometric techniques, namely GC-MS, FT-IR, and UV-visible systems.

Preparation, extraction, and fractionation of leaf extracts
The preparation and extraction of the plant materials was according to the methods previously described [50]. Three hundred grams of the ground sample was subjected to repeated cycles of Soxhlet extraction protocol in 2000 mL of ethanol/water mixture; 1:1 v/v for 18 h. The hydro-ethanolic leaf extracts were fractionated according to the modified methods previously described [51]. The crude hydro-ethanolic leaf extracts were fractionated by progressive partitioning with equal volumes of solvents in the order of increasing polarities; viz. petroleum ether, n-hexane, chloroform, and ethylacetate.
The fractionated leaf extracts were concentrated under reduced pressure for 24 h at 50°C using a rotary evaporator (Büch Rotavapor R-200). The separate residues were finally dried in anhydrous calcium chloride (desiccant) embedded desiccator. The yield of the extract per 100 g dry leaf sample was calculated thus: The fractionated leaf extracts were suspended in given volumes of phosphate-buffered saline (PBS; pH = 7.4), osmotically equivalent to 9.0 g/L NaCl {9.0 g NaCl, 1.71 g Na 2 HPO 4 .2H 2 O, and 2.43 g NaH 2 PO 4 .2H 2 O per liter} to give standard solutions of the extracts used for osmotic stability studies of sickle erythrocyte membrane.

Exclusion criteria
Exclusion criteria for participants were according to guidelines previously proposed [52]. Participants on medications for at least 4 weeks prior to blood sampling were excluded from this study. Additionally, the collected blood samples were thoroughly checked for the presence of clot and hemolysis before used for the experiment.

Collection and preparation of blood samples
One hundred eight participants of HbSS genotype donated 5.0 mL of venous blood samples. All blood donors were consenting individuals attending clinics between the months of 7th of May and 28th of July, 2019 at Rehoboth Christian Medical Center, Nwaoruebi and Easter Summit Specialist Clinics and Maternity, Amakohia. The clinics are located in Owerri, Imo State, Nigeria. Venous peripheral blood was drawn from the forearm of participants using vacuum blood sampling disodium ethylene diamine tetraacetate (EDTA-Na 2 ) tubes [52]. The confirmatory test of the blood samples was carried out using electrophoretic methods previously described [53]. The preparation of blood samples was according to previous reports [24]. The erythrocytes were finally re-suspended in 5.0 mL of PBS and used for osmotic stability studies of the sickle erythrocyte membrane.

Membrane osmotic stability studies
Erythrocyte osmotic fragility index was measured according to the method described [26]. In a total of four sets (T1-T4) of three test tubes each, 5.0 mL of PBS of varying concentrations equivalent to 0.9-0.3 g/100 mL NaCl were introduced into the corresponding test tubes, such that T1 = 0.9 g/100 mL, T2 = 0.6 g/100 mL, and T3 = 0.3 mg/100 mL. A 5.0 mL of distilled water was added in the fourth set of test tubes (T4). For the test analyses, 0.5 mL of three increasing concentrations (40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL) of each of the separate fractionated leaf extracts were added in different sets of test tubes in a corresponding order of concentrations of the fractionated leaf extracts. To each test tube, 0.1 mL of HbSS erythrocyte suspension (10% hematocrit) was added and mixed thoroughly by inverting the tubes several times. For the control experiment, the same procedure was repeated but devoid of suspensions of the fractionated leaf extracts, which was replaced by PBS concentration equivalent to 0.9 g/100 mL NaCl. The erythrocyte suspensions were allowed to stand for 30 min at room temperature (24-27°C) after which the contents were centrifuged at 1200×g for 10 min. The relative quantity of hemoglobin released into the supernatant was measured at λmax = 540 nm using a spectrophotometer (Digital Blood Analyzer; SPEC-TRONIC 20; Labtech, LabX, Bay Street, Midland, ON, Canada). PBS concentration equivalent to 0.9 g/100 mL NaCl served as blank.
The range of values of percentage hemolysis in PBS of varying concentrations equivalent to 0.9-0.3 g/100 mL NaCl (T1-T3) and in distilled water (T4) were obtained by evaluating the quotient of absorbance of the content of each corresponding test tubes (T1-T3) and the fourth test tube (T4) containing distilled water and multiplied by a factor of 100. Thus: Where; %H T1-T4 : range of values of percentage hemolysis (%). A T1-T3 : absorbance of the supernatant of erythrocyte suspension in PBS of varying concentrations equivalent to 0.9-0.3 g/100 mL NaCl.
A T4 : absorbance of the supernatant of erythrocyte suspension in distilled water.
Note: the erythrocytes suspension in distilled water (T4) served as a 100% hemolysis point.
The corresponding PBS concentration in g/100 mL of NaCl, which caused 50% hemolysis, was the mean corpuscular fragility (MCF) index. The MCF values were interpolated from the cumulative erythrocyte osmotic fragility curves obtained by plotting the percentage hemolysis against PBS concentrations in g/100 mL of NaCl.
The relative capacities of the fractionated leaf extracts to stabilize erythrocyte membrane were evaluated as percentage of the quotient of the difference between the MCF values of the test and control samples to that of the control sample as previously described [26]. Thus: Where; %RMS: relative membrane stability (%). Note: negative value of %RMS is indicative of destabilization by the fractionated leaf extract.

Spectrometry
The identification, quantification, and characterization of phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa were carried out using standard chromatographic-spectrophotometric protocols, viz. GC-MS systems {Agilent 7890A GC system set up with 5975C VL MSD, Agilent Technologies, Inc., Santa Clara, CA, USA; The MS system was accomplished in Electron Ionization (EI) mode with Selected Ion Monitoring (SIM)}as well as FT-IR and UV-visible instruments (PerkinElmer Spectrophotometer, USA) according to the methods previously described [48,54,55]. The capillary column specification of the GC system was DB-5MS (30 m × 0.25 mm, film thickness of 0.25 μm) with a temperature program set as follows: initial temperature 50°C held for 1.00 min, 5°C per min to 100°C, 9°C per min to 200°C held for 7.89 min, and the total run time was 30 min. The injection mode and volume were set at 20:1 and 1.0 μL respectively. The flow rate of helium as a carrier gas was set at 0.811851 mL/min. The ion source temperature and quadruple temperature of the MS system were set at 230°C and 150°C, respectively.

Statistical analyses
The data collected were expressed in means (X) ± SD and analyzed in one-way ANOVA and least significance difference (LSD). The comparison was made between groups and significance was established by ANOVA at 95% confidence level. Difference of p < 0.05 was considered statistically significant.

Percentage yields of fractionated leaf extracts
The aggregate yields of the fractionated leaf extracts, which include petroleum ether, n-hexane, chloroform, ethylacetate, and residual aqueous extracts were as follows: A. occidentale (13.017 g per 100 g dry leaf sample), P. guajava (9.627 g per 100 g dry leaf sample), and T. catappa (10.060 g per 100 g dry leaf sample). However, the residual aqueous extract fractions gave relatively high yields in the following proportions: A. occidentale (10.20 g per 100 g dry leaf sample), P. guajava (7.20 g per 100 g dry leaf sample), and T. catappa (9.00 g per 100 g dry leaf sample).   Figure 1a showed that the control sample exhibited 38.15 ± 6.31% hemolysis in PBS concentration equivalent to 0.9 g/100 mL NaCl, whereas 80 mg/100 mL petroleum ether extract of A. occidentale caused 73.53 ± 10.12% hemolysis. The percentage hemolysis in the presence of 40 mg/100 mL petroleum ether extract of A. occidentale in PBS concentration equivalent to 0.9 g/100 mL NaCl was not significantly different (p > 0.05) from that of the control sample. The lowest level of percentage hemolysis in PBS concentration equivalent to 0.9 g/100 mL NaCl was in the presence of 60 mg/100 mL ethylacetate extract of A. occidentale. Additionally, PBS concentration equivalent to 0.3 g/100 mL NaCl caused 92.98 ± 4.45% hemolysis in the sample containing 80 mg/100 mL ethylacetate extract of A. occidentale. Figure 1b showed that in PBS concentrations equivalent to 0.6 g/100 mL and 0.9 g/100 mL NaCl, the percentage hemolysis of the control sample was not significantly different (p > 0.05) from those of the samples containing 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL n-hexane extracts of A. occidentale. Conversely, 60 mg/100 mL nhexane extract of A. occidentale caused 75.53 ± 14.01% hemolysis, which was significantly different (p < 0.05) from the control sample as well as the samples containing 40 mg/100 mL and 80 mg/100 mL n-hexane extracts of A. occidentale.
The percentage hemolysis of the samples containing 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL chloroform extracts of A. occidentale in PBS concentrations equivalent to 0.3-0.9 g/100 mL NaCl were not significantly different (p > 0.05) from the control sample. Specifically, the percentage hemolysis of the control sample and the test samples in PBS concentration equivalent to 0.3 g/ 100 mL NaCl varied within a relatively narrow range of 12.22 ± 5.82-19.60 ± 3.43% (Fig. 1c).
The percentage hemolysis of the samples containing 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL ethylacetate extracts of A. occidentale in PBS concentration equivalent to 0.9 g/100 mL NaCl showed no significant difference (p > 0.05). Likewise, the percentage hemolysis of the control sample in PBS concentration equivalent to 0.3 g/100 mL NaCl was not significantly different (p > 0.05) from the sample containing 80 mg/100 mL ethylacetate extract of A. occidentale (Fig. 1d).
The percentage hemolysis of the control sample in PBS concentrations equivalent to 0.6 g/100 mL and 0.9 g/100 mL NaCl were significantly higher (p < 0.05) than those of samples containing 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL residual aqueous extracts of A. occidentale. Conversely, the percentage hemolysis of the control sample in PBS concentration equivalent to 0.3 g/ 100 mL NaCl was significantly lower (p < 0.05) than that of the sample containing 40 mg/100 mL residual aqueous extract of A. occidentale (Fig. 1e).

Sickle erythrocytes osmotic fragility in the presence of fractionated leaf extracts of P. guajava
Figure 2a-e showed sickle erythrocytes osmotic fragility in PBS concentrations equivalent to 0.3-0.9 g/100 mL NaCl in the presence of fractionated leaf extracts of P. guajava. The percentage hemolysis of the control sample in PBS concentrations equivalent to 0.6 g/ 100 mL and 0.9 g/100 mL NaCl showed no significant difference (p > 0.05) from the samples containing 40 mg/100 mL and 80 mg/100 mL petroleum ether extracts of P. guajava. Conversely, the percentage hemolysis in the presence of 60 mg/100 mL petroleum ether extract of P. guajava in PBS concentrations equivalent to 0.6 g/100 mL and 0.9 g/ 100 mL NaCl were significantly higher (p < 0.05) than that of the control sample (Fig. 2a). Figure 2b showed that the percentage hemolysis of sickle erythrocytes in the presence of 40 mg/100 mL n-hexane extract of P. guajava in PBS concentrations equivalent to 0.6 g/100 mL and 0.9 g/100 mL NaCl were significantly higher (p < 0.05) than that of the control sample as well as the samples containing 60 mg/100 mL and 80 mg/100 mL n-hexane extracts of P. guajava. The percentage hemolysis of the sample containing 80 mg/100 mL n-hexane extract of P. guajava in PBS concentration equivalent to 0.3 g/ 100 mL NaCl was significantly lower (p < 0.05) than that of the control sample as well as the test samples containing 40 mg/100 mL and 60 mg/100 mL n-hexane extracts of P. guajava (Fig. 2b).
The percentage hemolysis, in PBS concentration equivalent to 0.3 g/100 mL NaCl, in the presence of 40 mg/100 mL and 80 mg/100 mL chloroform extracts of P. guajava as well as the control sample showed no significant difference (p > 0.05) but was significantly higher (p < 0.05) than that of the sample containing 60 mg/100 mL chloroform extract of P. guajava. Additionally, the percentage hemolysis of the control sample was significantly lower (p < 0.05) than the test samples (Fig. 2c). Figure 2d showed that the percentage hemolysis of the control sample in PBS concentrations equivalent to 0.3-0.9 g/100 mL NaCl were significantly lower (p < 0.05) than those of the samples containing 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL ethylacetate extracts of P. guajava. Conversely, percentage hemolysis in PBS concentrations equivalent to 0.3-0.9 g/100 mL NaCl in the presence of 40 mg/100 mL and 80 mg/100 mL ethylacetate extracts of P. guajava showed no significant difference (p > 0.05) (Fig. 2d).
The percentage hemolysis in PBS concentration equivalent to 0.9 g/100 mL NaCl of the control and test samples showed no significant difference (p > 0.05) (Fig. 2e). On the contrary, the percentage hemolysis of the control and test samples showed significant difference (p < 0.05) in PBS concentration equivalent to 0.3 g/100 mL NaCl.

Sickle erythrocytes osmotic fragility in the presence of fractionated leaf extracts of T. catappa
Figure 3a-e showed sickle erythrocytes osmotic fragility in varying NaCl concentrations in the presence of fractionated leaf extracts of T. catappa. The percentage hemolysis of the sample containing 60 mg/100 mL petroleum ether extract of T. catappa in PBS concentration equivalent to 0.9 g/100 mL NaCl was not significantly different (p > 0.05) from that of the control sample (Fig. 3a). Furthermore, the percentage hemolysis of the control sample and the sample containing 60 mg/100 mL petroleum ether extract of T. catappa was significantly lower (p < 0.05) than those of the samples, in PBS concentration equivalent to 0.9 g/100 mL NaCl, containing 40 mg/100 mL and 80 mg/ 100 mL petroleum ether extracts of T. catappa (Fig. 3a). Figure 3b showed that the percentage hemolysis of the control sample and the sample containing 80 mg/100 mL n-hexane extract of T. catappa showed no significant difference (p > 0.05). However, the percentage hemolysis of the samples containing 40 mg/100 mL and 60 mg/100 mL n-hexane extracts of T. catappa were significantly higher (p < 0.05) than those of the control sample and the sample containing 80 mg/100 mL n-hexane extract of T. catappa. Figure 3c showed that increasing order of percentage hemolysis, in PBS concentrations equivalent to 0.3-0.9 g/100 mL NaCl, in the presence of 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL chloroform extracts of T. catappa showed no significant difference (p > 0.05). Notably, the percentage hemolysis of the control sample in PBS concentrations equivalent to 0.3-0.9 g/100 mL NaCl were significantly higher (p < 0.05) than those of the test samples. The percentage hemolysis of the control sample, in PBS concentration equivalent to 0.9 g/100 mL NaCl, was 35.08 ± 11.64%, whereas those of the test samples ranged between 31.82 ± 8.32% and 39.18 ± 6.94% (Fig.  3d). The lowest percentage hemolysis occurred in the sample containing 60 mg/100 mL ethylacetate extract of T. catappa in PBS concentration equivalent to 0.6 g/100 mL NaCl. Figure 3e showed that the sample containing 60 mg/ 100 mL and 80 mg/100 mL residual aqueous extracts of T. catappa in PBS concentration equivalent to 0.9 g/100 mL NaCl exhibited no hemolysis. The percentage hemolysis of the control sample and the sample containing 40 mg/100 mL residual aqueous extract of T. catappa were 0.44 ± 0.18% and 0.49 ± 0.09% respectively. The percentage hemolysis of the control sample, in PBS concentration equivalent to 0.3 g/100 mL NaCl, was significantly lower (p < 0.05%) than those of the test samples (Fig. 3e).

MCF and percentage stability of sickle erythrocytes
The MCF and percentage stability of sickle erythrocytes incubated in fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa is presented in Table 1.
The results showed that 60 mg/100 mL and 80 mg/ 100 mL petroleum ether extracts of A. occidentale stabilized sickle erythrocyte membrane, whereas the sample containing 40 mg/100 mL petroleum ether extract caused the destabilization of sickle erythrocyte membrane. The 80 mg/100 mL petroleum ether extract of A. occidentale caused higher erythrocyte membrane stability than that of the sample containing 60 mg/100 mL petroleum ether extract of A. occidentale. The sample containing 40 mg/100 mL n-hexane extract of A. occidentale stabilized sickle erythrocyte membrane by 3.70% (Table 1). Further increase in the concentration of nhexane extract of A. occidentale caused the destabilization of sickle erythrocyte membrane. The 60 mg/100 mL n-hexane extract of A. occidentale exhibited greater capacity to destabilize sickle erythrocyte membrane (Table 1). Table 1 showed that 40 mg/100 mL and 60 mg/100 mL chloroform extracts of A. occidentale exhibited an equal capacity to destabilize sickle erythrocyte membrane. Further increase in the extract concentration exacerbated the destabilizing effect of chloroform extract of A. occidentale on the sickle erythrocyte membrane.
The samples containing 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL ethylacetate extracts of A. occidentale stabilized sickle erythrocyte membrane in a concentrationdependent manner ( Table 1). The capacity of ethylacetate extract to stabilize the sickle erythrocyte membrane was within the range of (7.41-22.22)% ( Table 1).
The 40 mg/100 mL residual aqueous extract of A. occidentale caused destabilization of the sickle erythrocyte membrane by 11.54%, whereas 60 mg/100 mL residual aqueous extract of A. occidentale stabilized sickle erythrocyte membrane by the same magnitude ( Table 1). The capacity of the sickle erythrocyte membrane to withstand osmotic stress was further enhanced in the presence of 80 mg/100 mL residual aqueous extract of A. occidentale (Table 1). Table 1 showed that 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL petroleum ether extracts of P. guajava caused destabilization of the sickle erythrocyte membrane. A cursory look at Table 1 showed that 40 mg/100 mL and 80 mg/100 mL petroleum ether extracts of P. guajava exhibited equal capacity to destabilize sickle erythrocyte membrane. Table 1 showed that 40 mg/100 mL n-hexane extract of P. guajava exhibited greater capacity to destabilize sickle erythrocyte membrane than 60 mg/100 mL n-hexane extract of P. guajava. Further increase in the concentration of n-hexane extract of P. guajava destabilized sickle erythrocyte membrane by 36.17% (Table 1).
The 60 mg/100 mL n-hexane extract of T. catappa caused the greatest destabilizing effect on sickle erythrocyte membrane compared with the fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa ( Table 1). The destabilizing effect of n-hexane extract of T. catappa on sickle erythrocyte membrane was within the range of (21.43-207.14)% (Table 1). In the same manner, 40 mg/100 mL, 60 mg/100 mL, and 80 mg/100 mL chloroform extracts of T. catappa caused destabilization of sickle erythrocyte membrane (Table 1). Table 1 showed that 40 mg/100 mL and 60 mg/100 mL ethylacetate extracts of T. catappa exhibited equal capacity to stabilize sickle erythrocyte membrane. However, further increase in the concentration of ethylacetate extract of T. catappa reduced the capacity of the extract to stabilize sickle erythrocyte membrane ( Table 1).
The samples containing 40 mg/100 mL and 60 mg/100 mL residual aqueous extracts of T. catappa caused equal level of destabilization of sickle erythrocyte membrane ( Table 1). The destabilizing effect of residual aqueous extract of T. catappa on sickle erythrocyte membrane increased with corresponding increase in the concentration of the extract. The fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa that stabilized sickle erythrocyte membrane against osmotic stress, summarized from Table 1, were as follows: A. occidentale (petroleum ether and ethylacetate extracts), P. guajava (n-hexane extract), and T. catappa (petroleum ether and ethylacetate extracts). Table 2(A-E) summarized the combinations of phytocomponents present in petroleum ether extract of A. occidentale, ethylacetate extract of A. occidentale, residual aqueous extract of A. occidentale, n-hexane extract of P. guajava, petroleum ether extract of T. catappa, and ethylacetate extract of T. catappa. Aliphatic compounds in corresponding fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa are also summarized in Table 2(A-E). The corresponding GC-MS chromatograms of the selected extracts are illustrated in Fig. 4a-e.
The five major phytocomponents present in A. occidentale were hexadecanoic acid methyl ester, 11-octadecenoic acid methyl ester, pentadecanoic acid 14-methyl-methyl ester, 9,12-octadecadienoic acid (Z, Z)-methyl ester and 9octadecenoic acid, methyl ester, (E)-(Table 2(A)).   phthalate, tricosane, undec-10-ynoic acid undecyl ester, and 3-cyclopentylpropanoic acid but-3-yn-2yl ester. Table 2(C) showed the presence of D-erythro-sphinganine in n-hexane extract of P. guajava.  The FT-IR spectra of the selected leaf extracts, namely ethylacetate and petroleum ether extracts of A. occidentale, n-hexane extract of P. guajava, and petroleum ether and ethylacetate extracts of T. catappa are illustrated in Fig. 5a-e and summarized in Table 3(A-E). Petroleum ether extract of A. occidentale exhibited characteristic strong and medium bands within the range of (2955.8-2855.1) cm −1 and (905.7-693.3) cm −1 (Fig. 5a), which were indicative of the presence of aliphatic compounds and aromatic mono-and di-substituted aromatic compounds (Table 3(A)). Weak band at 1748.1 cm −1 was indicative of the presence of ester carbonyl group bearing molecules. Other notable molecular constituents present were aldehyde, phenolics, acyl halides, and alkoxy functional groups containing compounds. Figure 5b showed that ethylacetate extract of A. occidentale contained alcohol functional group (O-H) bearing molecules. Peaks within the range of (2989.1-2907.3) cm −1 were indicative of the presence of saturated aliphatic compounds (Table 3(B)). The presence of aromatic compounds in the extract was typified by peaks around 1446.2 cm −1 , 786.5 cm −1 , and 733.0 cm −1 (Table 3(B)). A medium band at 1376.9 cm −1 and peaks within the range of (1889.8-1654.9) cm −1 suggested the presence of acyl group, phenolics, as well as carboxylic acid, esters, and amide functional groups in ethylacetate extract of A. occidentale (Table 3(B)).
The presence of saturated aliphatic and aromatic compounds in n-hexane extract of P. guajava was typified by multiple weak and strong bands within the range of (2952.8-2858.9) cm −1 and (909.5-83.08) cm −1 (Fig. 5c). The presence of aromatic compounds as exemplified by weak band around 1457.4 cm −1 . Furthermore, weak bands within the range of (1990.4-697.0) cm −1 suggested the presence of conjugated alkenes, primary and tertiary amines, di-substituted aromatic compounds, and cis-alkenes (Table 3(C)).
The weak band around 3734.80 denotes N-H and O-H stretch, which suggested the presence of amide and alcohol functional groups bearing molecules (Fig. 5d). Peaks within the range of (3112.3-2851.4) cm −1 were indicative of the presence of aliphatic compounds in petroleum ether extract of T. catappa (Table 3(D)). Petroleum ether extract of T. catappa contained carbonyl and alkoxy functional groups as indicated by weak bands at 1733.2 cm −1 and 1039.9 cm −1 respectively. Additionally, peaks around (887.1-801.4) cm −1 were indicative of the presence of aromatic compounds (Table 3(D)).
The broad band at 3410.5 cm −1 was characteristic of the presence of alcohol functional group (O-H) in ethylacetate extract of T. catappa (Fig. 5e). Additionally, the peaks within the range of (2981.9-2907.3) cm −1 and (939.3-849.8) cm −1 suggested the presence of aliphatic compounds. Table 3(E) showed the presence of aromatic compounds as well as nitro-compounds, phenolics, and esters.

UV-visible of fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa
The selected fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa gave characteristic UV-visible spectra ( Fig. 6a-e). Petroleum ether extract of A. occidentale exhibited multiple λmax around region of 272.00 nm, which suggested the presence of nitro-containing chromophores in the extract (Fig. 6a). The λmax of ethylacetate extract of A. occidentale was at 205.00 nm (Fig. 6b). Figure 6c showed multiple peak values of UV-visible spectra of n-hexane extract of P. guajava. Petroleum ether extract of T. catappa exhibited multiple distinct peaks within a broad range of 204.00 nm and 671.00 nm (Fig. 6d). Ethylacetate extract of T. catappa gave single λmax at 363.00 nm (Fig. 6e).

Discussion
The capacity of phytocomponents to alter erythrocyte membrane stability is measured in vitro when a given population of erythrocyte is suspended in hypotonic solutions of varying NaCl concentrations [25,26,42]. Previous studies had reported distortions in membrane osmotic stability of human erythrocyte genotypes, in health and disease, and the capacities of plant extracts to reverse those alterations [26,42,44,[57][58][59]. Certain phytocomponents are associated with stabilization of erythrocyte membrane integrity or, on the contrary, cause exacerbated hemolysis in hypotonic solutions [44,[60][61][62]. For instance, certain classes of the tannins, aminoglycosides, and saponins from verse collections of plant species have been implicated in eliciting erythrocyte membrane destabilization by their chaotropic interaction with biomembrane components [1,21,57,[62][63][64]. The destabilizing effects elicited by phytochemicals are analogous to hemolytic actions of intra-erythrocytic denatured hemoglobin precipitate-Heinz bodies. According to Jarolim et al. [65], Heinz bodies form an attachment with erythrocyte membrane components, which elicits striking membrane damage, evidenced by increased membrane lipid peroxidation and cross-linked membrane proteins. The damaged sickle erythrocyte membrane is characterized by calcium-activated increased permeability of potassium, phospholipids bilayer  destabilization, decreased deformability and unusual assemblies, and interactions of membrane proteins as well as unusual oxidation of protein thiols and cytoskeletal dysfunction [66,67]. Furthermore, the use of mathematical models had revealed the relationship between sickle hemoglobin (HbS) polymerization and membrane morphology, osmotic properties, viscoelasticity, rheology, and related physicochemical characteristics of sickle erythrocytes as empirically described [67][68][69]. By implication, phytocomponents that deterred HbS aggregation and polymerization improved sickle erythrocyte membrane stability [35,59,70,71] within limits of their permeability across the biomembrane system as previously described [72]. In line with the present findings, the presence of isothiocyanates, a notable anti-HbS polymerization phytochemicals [70,71], in ethylacetate extract of A. occidentale obviously contributed, in parts, in stabilizing the sickle erythrocyte membrane against osmotic stress. Additionally, aromatic compounds such as L-phenylalanine and its derivative L-phenylalanine benzyl ester are effective agents, even at relatively low concentrations, against HbS polymerization and have been proposed to be effective therapeutic agents for the management of SCD [72][73][74][75][76]. The improved sickle erythrocyte osmotic stability in samples containing petroleum ether extract of T. catappa appeared to suggest that aromatic compounds in petroleum ether extract of T. catappa attenuated HbS polymerization-induced hemolysis in hypotonic solutions as previously described [35,59,70,71]. From another perspective, the antioxidant phytocomponents from plant materials viz. the anthocyanins, anthraquinones, flavonoids, and antioxidant vitamins (vitamins C and E) generally stabilized membrane systems against molecular and biological agents that compromised membrane integrity [21,30,42,44,57,59,72].
In that regard, the present study showed membrane osmotic stability of sickle erythrocytes were altered in the presence of fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa, such that the extracts exhibited differential capacities to either stabilize sickle erythrocyte membrane integrity or promote membrane destabilization. Based on the percentile yields of petroleum ether, n-hexane, chloroform, and ethylacetate as well as residual aqueous extracts, the results of the present study indicated relatively high content of hydrophilic phytocomponents in leaf extracts of A. occidentale, P. guajava, and T. catappa. Nevertheless, the findings of the present investigations did not implicate hydrophilic phytocomponents of A. occidentale, P. guajava, and T. catappa as agents that promoted sickle erythrocyte stability since, to a large extent, fractions of their residual aqueous extracts promoted membrane destabilization. Rather, hydrophobic fractions of the leaf extracts, namely petroleum ether extract of A. occidentale, ethylacetate extract of A. occidentale, n-hexane extract of P. guajava, petroleum ether extract of T. catappa, and ethylacetate extract of T. catappa exhibited comparative high capacity to promote membrane osmotic stability of sickle erythrocytes. The present findings were in agreement with previous reports, in which the authors noted that there was an established positive correlation between hydrophobicity of short-chain aliphatic alcohols and their capacity to alter membrane integrity [77]. For instance, ethylacetate extract of A. occidentale contained short-chain aliphatic alcohols, namely 1-hexacosanol, 2hexyl-1-octanol and 4-nonanol 2,6,8-trimethyl-, which further explain, in part, the stabilizing effects of the fraction on sickle erythrocyte membranes subjected to osmotic stress. In concord with the present findings, previous report noted that flavonoids from ethylacetate extract of Lantana camara stabilized normal erythrocyte membrane against osmotic stress [42].
The present study noted that diisooctyl phthalate and aromatic compounds from ethylacetate extract of A. occidentale, in part, stabilized sickle erythrocyte membrane under osmotic stress. The molecular configurations of diisooctyl phthalate and aromatic compounds appeared to have contributed to their stabilizing effect on sickle erythrocyte membrane system. A related study had previously reported that despite inter-species differences in erythrocyte membrane properties, dicarboxylic acids with cyclic hydrocarbons, namely bis (2-ethylhexyl) phthalate, 1,2-cyclohexanedicarboxylic acid, and phthalic acid with a benzene ring significantly stabilized cattle erythrocytes subjected to osmotic pressure due to their wedge-like effect on membrane phospholipids [80].
Additionally, the polarities of antioxidant vitamins and related low molecular weight antioxidants (LMWAs), which protect biological systems against oxidative damage to cellular components, dictate their distribution patterns in cellular regions. For instance, hydrophobic antioxidants such as the tocopherols are restricted to act within the hydrophobic regions of the cell, e.g., biomembrane systems, whereas the hydrophilic antioxidants effect their free radical quenching activities within the aqueous environments of the cell. Accordingly, the current information of cellular distribution of LMWAs suggest that the tendency of phytocomponents from the fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa to interact and interfere with sickle erythrocyte membrane systems appeared to correlate with hydrophobicity of molecular species of the leaf extracts.
The present study of GC-MS system protocol in conjunction with sickle erythrocyte osmotic stability-guided analysis revealed vast array of hydrophobic phytocomponents, namely short-chain aliphatic esters, methylated aromatic hydrocarbons, cycloalkanes, hexahydro-methanoindene, isoxazole, and short-chain aliphatic carboxylic acids as molecular species from leaf extracts of A. occidentale, P. guajava, and T. catappa that caused relative osmotic stability of sickle erythrocyte membrane. Furthermore, FT-IR and UV-visible protocols further confirmed the alkanes, aldehydes, esters, conjugated alkenes, aromatic compounds, aromatic ethers, isothiocyanates, primary and tertiary amine, cyanate, alkynes, amide, alcohols, and nitro compounds as phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa that promoted membrane osmotic stability of sickle erythrocytes.

Conclusion
The selected groups of fractionated leaf extracts that stabilized sickle erythrocyte membrane against osmotic stress were ethylacetate and petroleum ether extracts of A. occidentale, n-hexane extract of P. guajava, and petroleum ether and ethylacetate extracts T. catappa. GC-MS, FT-IR, and UV-visible spectrometry systems protocols indicated that short-chain aliphatic esters, methylated aromatic hydrocarbons, cycloalkanes, hexahydro-methanoindene, isoxazole, and short-chain aliphatic carboxylic acids and alkanes were phytocomponents from leaf extracts of A. occidentale, P. guajava, and T. catappa that stabilized sickle erythrocyte membrane integrity. Other phytocomponents, namely the aldehydes, esters, conjugated alkenes, aromatic compounds, isothiocyanates, primary and tertiary amine, cyanate, alkynes, amide, alcohols, and nitro compounds, were also considered as stabilizers of sickle erythrocyte membrane subjected to osmotic stress.
The present research recommend further study on isolation and purification of the phytocomponents suggested, in the present study, to have stabilized osmotic stressed sickle erythrocytes and apply the isolated and purified phytocomponents in membrane osmotic stability studies in vitro in order to establish their membrane stabilizing activity and, possibly, mode of action.