Polymerization of deoxygenated sickle hemoglobin in the presence of fractionated leaf extracts of Anacardium occidentale, Psidium guajava, and Terminalia catappa

The present study evaluated levels of polymerization of deoxygenated sickle hemoglobin molecules (poly-dHbS-M) in the presence of fractionated leaf extracts of Anacardium occidentale Linn., Psidium guajava Linn., and Terminalia catappa Linn in vitro as well as identified, quantified, and characterized the phytocomponents from fractionated leaf extracts that exhibited comparatively high potency to impede poly-dHbS-M. Non-hemolyzed sickle erythrocytes were premixed with 40, 60, and 80 mg/100 mL of each of the separate fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa in phosphate-buffered saline (PBS; pH = 7.4), osmotically equivalent to 9.0 g/L NaCl. Poly-dHbS-M was induced by adding 2.0 g/100 mL Na2S2O5 to the erythrocyte suspension. The absorbance of the erythrocyte suspension was measured at regular intervals of 30 s for 180 s. Identification, quantification, and characterization of phytocomponents from fractionated leaf extracts were carried out using GC-MS, FT-IR, and UV-visible systems protocols. The level of poly-dHbS-M of the control sample was significantly higher (p < 0.05) than those of the samples containing 40, 60, and 80 mg/100 mL ethylacetate extracts of A. occidentale at t < 60 s. The relative cumulative polymerization index (RCPI%) of dHbS-M in the presence of fractionated leaf extract of A. occidentale varied within a wide range of 3.8–59.4%. A. occidentale (petroleum ether and ethylacetate extracts), P. guajava (n-hexane, chloroform, and ethylacetate extracts), and T. catappa (ethylacetate extract) exhibited comparatively high potency to inhibit poly-dHbS-M. The fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa exhibited differential capacities to impede poly-dHbS-M. The combinations of aliphatic hydrocarbons, methylated esters, methylated fatty acids, aliphatic alcohols, d-erythro-sphinganine, aromatic derivatives, cycloalkanes, phthalates, isothiocyanates, aminated sugars, cyclo-alcohols, and nitro-compounds impeded poly-dHbS-M.


Background
Hemoglobin, a tetrameric conjugate protein molecule, is an attractive model for the study of the structure/function relationship of macromolecules. The sickle hemoglobin (HbS) variant or sickle erythrocyte hemoglobinopathy (α 2 β 2 s ) is caused by a point mutation affecting the coding sequence of β-globin gene, whereby thymine is replaced by adenine with the concomitant replacement of adenine by uracil in the triplet code (GAA or GAG codon → GUA or GUG codon)-a partially acceptable missense mutation. The mutant gene elicits the substitution of hydrophilic glutamic acid at the β 6 globin position for hydrophobic valine; β 6Glu→Val (Rotter et al. 2005;Bianchi et al. 2009). Hydrophobic β 6 valine (Val-beta6) generates a 'sticky patch' on the β-globin chains of deoxygenated sickle hemoglobin molecules (dHbS-M) (Martins 1983;Rotter et al. 2005). The contact position on dHbS-M is such that the hydrophobic R-group of Val-beta6 appears to fit into a hydrophobic pocket constituted by β 88 leucine (Leu-beta88), β 85 phenylalanine (Phe-beta85), and β 73 aspartic acid (Asp-beta73) residues on adjacent dHbS-M (Adachi et al. 1994;Ferrone et al. 2002;Dash et al. 2013). The hydrophobic interaction is stereospecific of Leu-beta88 side chain in the acceptor pocket regions on adjacent dHbS-M (Adachi et al. 1994). The hydrophilic R-group of β 6 glutamic acid (Glu-beta6) would not easily fit into the hydrophobic pocket explaining at least part of the reasons deoxyHbA does not polymerize (Martins 1983). The inter-hydrophobic interactions promote nucleation of dHbS-M followed by their alignment into microfibrils aggregations of low intracellular solubility, which exert pressure on the interior side of erythrocyte membrane causing mechanical distortion of the erythrocytes (sickle shape) (Rotter et al. 2005). Sodium metabisulfite (Na 2 S 2 O 5 ) is often used to induce poly-dHbS-M in vitro, which by virtue of its reducing property triggers low oxygen tension required for aggregation of dHbS-M, engendering morphologically distorted erythrocytes (Oyewole et al. 2008;Uwakwe and Nwaoguikpe, 2008;Nurain et al. 2017).
Epidemiological surveys showed that sickle cell disease (SCD) exerts an enormous burden on public health care system (Stallworth et al. 2010;Adewoyin et al. 2015). Estimates show that SCD affects 20-25 million people globally (Mulumba and Wilson, 2015) and approximately 300,000 children with SCD are born every year in the world, of which 75% of these births are in Sub-Saharan Africa (Diallo and Tchernia 2002;Weatherall et al. 2006; World Health Organization Regional Offıce for Africa, 2010; Makani et al. 2013). The dilapidating health challenges and disability-adjusted lifestyle of SCD sufferers are mostly impacted in developing countries (Weatherall et al. 2006). In Africa, SCD accounts for 50-90% rate of childhood mortality (Grosse et al. 2011). Global epidemiological surveys and the prevalence of SCD is exhaustively reported elsewhere (Mulumba and Wilson 2015). The pathophysiology of SCD is such that management of the disease is often restricted to the use of prophylaxis in concert with drugs that ameliorate the disease symptoms, which offer no therapeutic benefits in form of radical cure.
Presently, hydroxyurea and 2-imidazolines are notable few clinical useful anti-sickling agents that reduce the frequency and severity of sickle cell crises (Chang et al. 1983;Charache et al. 1995;Stallworth et al. 2010;Makani et al. 2013). Hydroxyurea and 5-azacytidine induce the expression of fetal hemoglobin (HbF) via epigenetic regulation of globin gene expression in adult life (Frenette and Atweh 2007). HbF interferes and disrupts aggregation of dHbS-M in sickle cell anemia patients by virtue of its γ 87 glutamine (Gln-gamma87) that impedes critical lateral contact regions on the double strand of HbS polymer (Charache et al. 1995;Setty et al. 2000;Cokic et al. 2003;Frenette and Atweh 2007;Eaton and Bunn 2017;Kassa et al. 2019). Toxicity associated with the use of hydroxyurea and 5-azacytidine has previously been reported (Eliot et al. 2006;Frenette and Atweh 2007;Oyewole et al. 2008;Kapoor et al. 2018).
Therapeutic approaches to radical cure of SCD, namely bone marrow transplantation, stem cell transplantation, and gene replacement therapy, in developing countries such as Nigeria and elsewhere, are expensive and remain inaccessible to the vast majority of SCD sufferers (Makani et al. 2013). Where the technology and expertise are available, there are still barriers to the suitability of donors, possibility of immunologic transplant rejection, prognostic uncertainty coupled with endorgan dysfunction, as well as long-term adverse outcomes, which is especially problematic for older patients (Frenette and Atweh 2007;Makani et al. 2013;Kapoor et al. 2018).
The use of prenatal prognostic evaluations, such as amniocentesis, as preventive measures against SCD, is not readily available in Sub-Saharan Africa for the fact the application of this technology is often scarce and expensive where available. Regrettably, clinical counseling to prospective biological parents of SCD sufferers, based on amniocentesis outcome, may advise termination of pregnancy before term, which is often untenable because of negative ethical and cultural considerations.
Chromatographic/spectrometric systems such as gas chromatography-mass spectrometry (GC-MS), Fourier transform-infrared spectrometry (FT-IR), and ultravioletvisible spectroscopy (UV-visible) are used for chemical screening or metabolite profiling of herbal extracts (Sasidharan et al. 2011;Rašković et al. 2015;Chikezie et al. 2015;Ighodaro et al. 2016;Hemavathy et al. 2019). Molecular probe on establishing the structural identities of unknown organic molecules in complex mixtures and the vast array of phytochemicals in herbal extracts is achieved by matching the spectra being investigated with reference and standard mass spectra from the library database {National Institute of Standards and Technology (NIST08) library and Wiley7n.l libraries} (Semwa and Painuli 2019). Furthermore, FT-IR and UV-visible protocols are applied in elucidating structural conformations and molecular nature of functional groups of phytochemicals (Karayil et al. 2014;Rašković et al. 2015;Chikezie et al. 2015).
Previous studies, based on in vitro studies, revealed that the use of vast varieties of crude herbal extracts provides an approach to impede poly-dHbS-M (Oyewole et al. 2008;Chikezie, 2011;Dash et al. 2013;Nurain et al. 2017). Because of physicochemical diversity of vast combinations of phytochemicals from crude herbal extracts, we hypothesize that fractionated leaf extracts of cashew (Anacardium occidentale Linn.), guava (Psidium guajava Linn.), and Indian almond (Terminalia catappa Linn.) will exhibit differential capacities to alter the process leading to the poly-dHbS-M. The present study evaluated levels of poly-dHbS-M in the presence of fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa using in vitro models. Furthermore, the phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa that exhibited comparatively high potency to impede poly-dHbS-M, or otherwise, were identified, quantified, and characterized using combined GC-MS, FT-IR and UV-visible systems protocols.

Collection and preparation of leaf samples
Fresh leaves of A. occidentale, P. guajava, and T. catappa were harvested during the wet season (3 rd -7 th April 2019) from private botanical gardens within the environment of Imo State University, Owerri (Latitude 5°30.2237′ N; Longitude 7°2.6277′ E), which lies on the rainforest belt of Nigeria. The seeds of the plants were obtained in the wild as non-commercial materials and permissions were not necessary to collect such samples. The collection of plant materials complied with institutional, national, and international guidelines as well as in accordance with local legislation. The harvested leaves of the selected plants used in the present study were identified and authenticated by Professor F.N. Mbagwu of the Department of Plant Science and Biotechnology, Imo State University, Owerri, Nigeria. The voucher numbers were assigned as follows: A. occidentale: IMSUH-009; P. guajava: IMSUH-010; T. catappa: IMSUH-011 and the plant specimens were deposited in the department herbarium.
Thereafter, the leaves were washed and air-dried at ambient laboratory temperature of 25 ± 5°C pending extraction within 24 h of collection of the leaf samples.
The preparation of the leaves for extraction was according to the methods previously described (Ojiako et al. 2015). Eight hundred grams (800 g) part of the chopped fresh leaves were weighed using a triple beam balance (OHAU 750-50; OHAUS Triple Beam Balance, Model TJ611, Burlington, NC, USA) and dried to constant weight in an oven (WTC BINDER; 7200 Tuttlinge, Germany) at 50°C for 10-12 h as previously described (Ezekwe and Chikezie 2017). Thomas-Willey milling machine (ASTM D-3182; India) was used to grind the dried leaf samples into powder. The powdered leaf samples were sieved on a wire mesh screen (1 × 1 mm 2 ) to remove relatively large particles. Finally, the fine ground leaf samples were stored at 4°C in air-tight screwcapped bottles pending extraction and fractionation.

Extraction and fractionation of leaf extracts
Extraction of 300 g of the dried ground samples was carried out in 2000 mL of ethanol/water mixture; 1:1 v/v using repeated cycles of Soxhlet extraction protocol for 18 h to obtain a final volume of 500 mL of each herbal extracts (Ojiako et al. 2015). Preparation of fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa was according to the methods previously described by Okoye et al. (2010) but with modifications. Separate volumes of the crude hydro-ethanolic leaf extracts were transferred into corresponding separating funnels. Fractionation of the crude hydro-ethanolic leaf extracts was carried out by successive partitioning using equal volumes of solvents in the order of increasing polarities, namely petroleum ether, n-hexane, chloroform, and ethylacetate.
Corresponding fractionated leaf extracts, namely petroleum ether-, n-hexane-, chloroform-, ethylacetate-, as well as the residual aqueous extracts, were concentrated under reduced pressure in a rotary evaporator  for 12 h at 50°C and the residues dried in a vacuum desiccator. The yield of the fractionated leaf extracts was calculated as ratio of dried weight of the extract to 100 g of the dried ground leaf sample. Portions of the fractionated leaf extracts were suspended in measured volumes of phosphate-buffered saline (PBS); pH = 7.4, osmotically equivalent to 9.0 g/L sodium chloride (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 HbS polymerization studies.

Exclusion criteria
The guidelines previously reported (Yamamoto et al. 2014) was used as bases for exclusion criteria for participants. The exclusion criteria include participants who were on routine medications, received blood transfusion and infusion for at least 4 weeks prior to blood sampling. Furthermore, blood samples were thoroughly cross-checked for the presence of clot before used for the experiment.

Collection and preparation of blood samples
Venous blood samples were collected by venipuncture, between 7th of May and 28th of July, 2019, from 108 consenting individuals of homozygous sickle hemoglobin (HbSS) genotype under the auspices of Rehoboth Christian Medical Center, Nwaoruebi and Easter Summit Specialist Clinics and Maternity, Amakohia. The clinics are located in Imo State, Owerri, Nigeria. The blood samples were stored in EDTA anticoagulant tubes. The genotype of the blood samples was further subjected to a confirmatory test using cellulose acetate electrophoretic methods previously described (Bain et al. 2012).
The HbS erythrocytes were washed using centrifugation methods as described (Tsakiris et al. 2005) with modifications according to previous reports (Chikezie, 2011;Chikezie and Uwakwe 2011). Within 2 h of collection of the blood sample, a portion of 4.0 mL of the sample was introduced into a centrifuge test tube containing 4.0 mL of PBS; pH = 7.4. The erythrocytes were separated from plasma by centrifugation at 1200×g for 10 min. The protocol was repeated three times. The erythrocytes were finally re-suspended in 5.0 mL of PBS and used for polymerization studies of dHbS-M.

Sickle hemoglobin polymerization studies
Polymerization studies of dHbS-M were carried out according to the modified methods previously described (Chikezie 2011), whereby non-hemolyzed HbSS erythrocytes were used instead of hemolysate samples. A 0.1 mL of HbSS erythrocyte suspension (10% hematocrit) was mixed with 0.5 mL PBS, followed by the introduction of an additional 1.0 mL of PBS in a test tube. The mixture was transferred into a cuvette and 3.4 mL of 2.0 g/100 mL aqueous solution of Na 2 S 2 O 5 was added. The absorbance of the assay mixture was measured at a maximum wavelength (λmax) = 700 nm, at regular intervals of 30 s for 180 s, using a spectrophotometer (Digital Blood Analyzer; SPECTRONIC 20; Labtech, LabX, Bay Street, Midland, ON, Canada) (control assay). The procedure was repeated substituting the 1.0 mL of PBS with corresponding three increasing concentrations (40, 60, and 80 mg/100 mL) of each of the separate fractionated leaf extracts (test assay). Relative poly-dHbS-M (%) was calculated according to the formula previously described (Chikezie et al. 2010;Chikezie 2011).
where: %RP: relative poly-dHbS-M (%) A t/c : absorbance of test/control sample at a given time (second) A c180 th s : absorbance of control sample at the 180 th second Inhibition/activation of poly-dHbS-M Arithmetically, the percentage inhibition/activation of poly-dHbS-M by the leaf extracts at a given experimental time interval was obtained thus: where: %I\A: percentage inhibition or activation of poly-dHbS-M %PC ct : %RP of the control sample at a given experimental time interval %PT ct : %RP of the test sample at a given experimental time interval corresponding to that of the control sample Note: The algebraic sum of percentage activation of poly-dHbS-M is negative, whereas percentage inhibition of poly-dHbS-M is positive in the presence of the leaf extract.

Cumulative polymerization index
The cumulative inhibition/activation of poly-dHbS-M within the experimental time of 180 s is defined by a measure of the Area under the Curve (AUC) of the plot of %RP versus time (s).
Using the Simpson's rule, thus: This is given by: where: t: time intervals of 30 s x: %RP at corresponding time interval Thus: where: RCPI%: relative cumulative polymerization index Note: A positive RCPI% connotes cumulative inhibition of poly-dHbS-M, whereas negative RCPI% connotes cumulative activation of poly-dHbS-M by the 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/spectrometric protocols, viz. GC-MS systems (Agilent 7890A GC system set up with 5975C VL MSD, Agilent Technologies, Inc., Santa Clara, CA, USA) operated as previously described (Rašković et al. 2015). The MS system was accomplished in electron ionization (EI) mode with selected ion monitoring (SIM). FT-IR and UV-visible instruments (PerkinElmer Spectrophotometer, USA) were performed according to the methods previously described (Ighodaro et al. 2016;Hemavathy et al. 2019).

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. The difference of p < 0.05 was considered statistically significant.

Percentage yields of fractionated leaf extracts
The percentage yields of the fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa are presented in Table 1. The aggregate yields of petroleum ether, nhexane, chloroform, ethylacetate, and residual aqueous fractions of the leaf extracts were 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). The residual aqueous fractions of the leaf extracts gave corresponding highest percentage yields ( Table 1).
Levels of poly-dHbS-M in the presence of fractionated leaf extracts of A. occidentale The control sample exhibited a comparatively higher level of poly-dHbS-M than those of the samples containing 40, 60, and 80 mg/100 mL petroleum ether extracts of A. occidentale within the experimental time range of 0 s ≤ t ≤ 120 s (Fig. 1a).
Specifically, at t = 30 s, 40 mg/100 mL petroleum ether extract of A. occidentale caused significantly lower (p < 0.05) level of poly-dHbS-M than the control sample as well as the samples containing 60 and 80 mg/100 mL petroleum ether extracts of A. occidentale (Fig. 1a). Conversely, at t = 30 s, the levels of poly-dHbS-M of the control sample, as well as the samples containing 60 and 80 mg/100 mL petroleum ether extracts of A. occidentale, showed no significant difference (p > 0.05). Figure 1b showed the levels of poly-dHbS-M of the control sample and samples containing n-hexane extract of A. occidentale. An overview of Fig. 1b showed that the pattern of levels of poly-dHbS-M of the control sample and sample containing 80 mg/100 mL n-hexane extract of A. occidentale were biphasic. For instance, at t < 60 s, the control sample and sample containing 80 mg/100 mL n-hexane extract of A. occidentale exhibited exponential increasing levels of poly-dHbS-M, which was followed by a phase of decreasing levels of poly-dHbS-M. The peak levels of poly-dHbS-M of samples containing 40 and 60 mg/100 mL n-hexane extracts of A. occidentale occurred at t = 90 s, which was followed by moderate decreasing levels of poly-dHbS-M as experimental time progressed. The maximum level of poly-dHbS-M of the control sample, at t = 60 s, was significantly higher (p < 0.05) than those of samples containing 40, 60, and 80 mg/100 mL n-hexane extracts of A. occidentale. Conversely, at t = 180 s, the levels of poly-dHbS-M of the sample containing 40, 60, and 80 mg/100 mL n-hexane extracts of A. occidentale were significantly higher (p < 0.05) than that of the control sample. Figure 1c showed that the pattern of levels of poly-dHbS-M of the control sample, as well as samples containing 40, 60, and 80 mg/100 mL chloroform extracts of A. occidentale, were biphasic. A peak level of poly-dHbS-M of the control sample was at t = 90 s, whereas those of the samples containing 60 and 80 mg/100 mL chloroform extracts of A. occidentale were at t = 60 s. Figure 1c showed that the level of poly-dHbS-M of the sample containing 40 mg/100 mL chloroform extract of A. occidentale peaked at t = 30 s. An overview of Fig. 1c showed that 40 and 60 mg/100 mL chloroform extracts of A. occidentale caused significant higher (p < 0.05) levels of poly-dHbS-M compared with that of the control sample. Additionally, within the experimental time of t < 60 s, the sample containing 80 mg/100 mL chloroform extract exhibited a significant higher (p < 0.05) level of poly-dHbS-M. Conversely, at t > 60 s, 80 mg/100 mL chloroform extract caused significantly lower (p < 0.05) level of poly-dHbS-M compared with the control sample. Figure 1d showed the biphasic pattern of levels of poly-dHbS-M of the control sample as well as that of the samples containing 40, 60, and 80 mg/100 mL ethylacetate extracts of A. occidentale, in which the samples exhibited an exponential increase in their levels of poly-dHbS-M within the experimental time of t < 30 s. Figure 1d showed that the control sample gave a peak level of poly-dHbS-M at t = 60 s, whereas that of 80 mg/ 100 mL ethylacetate extract of A. occidentale peaked at t = 90 s. Additionally, the peak levels of poly-dHbS-M of the samples containing 40 and 60 mg/100 mL ethylacetate extracts of A. occidentale occurred at t = 30 s. The level of poly-dHbS-M of the control sample was significantly higher (p < 0.05) than those of the samples containing 40, 60, and 80 mg/100 mL ethylacetate extracts of A. occidentale at t < 60 s (Fig. 1d). Conversely, at t > 90 s, the sample containing 80 mg/100 mL ethylacetate extract of A. occidentale exhibited significantly higher (p < 0.05) levels of poly-dHbS-M than the control sample and samples containing 40 and 60 mg/100 mL ethylacetate extracts of A. occidentale. The sample containing 40 mg/100 mL ethylacetate extract of A. occidentale gave the lowest level of poly-dHbS-M compared with other experimental samples (p < 0.05). Figure 1e showed that the maximum level of poly-dHbS-M of the control sample was at t = 120 s, whereas those of the sample containing 40, 60, and 80 mg/100 mL residual aqueous extracts of A. occidentale peaked at t = 90 s. Furthermore, the levels of poly-dHbS-M of the sample containing 40, 60, and 80 mg/100 mL residual aqueous extracts of A. occidentale were significantly higher (p < 0.05) than that of the control sample. However, at t > 120 s, the sample containing 60 and 80 mg/ 100 mL residual aqueous extracts of A. occidentale exhibited exponential decreasing levels of poly-dHbS-M, which were significantly lower (p < 0.05) than that of the control sample. The levels of poly-dHbS-M of samples containing 60 and 80 mg/100 mL residual aqueous extracts of A. occidentale gave negative numerical values at approximately t > 120 s and t > 135 s respectively ( Fig.  1e). At the end of the experimental time, the levels of poly-dHbS-M of the sample containing 40, 60, and 80 mg/100 mL residual aqueous extracts of A. occidentale were significantly lower (p < 0.05) than that of the control sample. Within the experimental time range of 90 s ≤ t ≤ 150 s, the level of poly-dHbS-M of the sample containing 40 mg/100 mL residual aqueous extract of A. occidentale was significantly higher (p < 0.05) than that of the control sample.

Levels of poly-dHbS-M in the presence of fractionated leaf extracts of P. guajava
The levels of poly-dHbS-M of the control sample and in the presence of fractionated leaf extracts of P. guajava with experimental time are presented in Fig. 2a-e. Figure 2a showed that the levels of poly-dHbS-M of the control sample and the samples containing 40, 60, and 80 mg/100 mL petroleum ether extracts of P. guajava were biphasic. The first phase showed an exponentially increasing level of poly-dHbS-M of the control sample, which peaked at t = 90 s. The levels of poly-dHbS-M of the test samples peaked at t = 60 s. It is worthwhile to note that the levels of poly-dHbS-M of the samples containing 40, 60, and 80 mg/100 mL petroleum ether extracts of P. guajava showed no significant difference (p > 0.05) within the experimental time of t < 90 s. Figure 2a showed that the second phase of poly-dHbS-M of the control sample and the sample containing 40, 60, and 80 mg/100 mL petroleum ether extracts of P. guajava exhibited decreasing levels of poly-dHbS-M as experimental time increased. Additionally, the decreasing levels of poly-dHbS-M of the test samples were depended on the concentrations of the herbal extracts, which were in the order: 80 mg/100 mL > 60 mg/100 mL > 40 mg/100 mL. Overall, the level of poly-dHbS-M of the control sample was significantly lower (p < 0.05) than those of the test samples. Figure2b showed the levels of poly-dHbS-M of the control and test samples. The pattern of level of poly-dHbS-M of the sample containing 80 mg/100 mL n-hexane extract of P. guajava was biphasic. The levels of poly-dHbS-M of the samples containing 40, 60, and 80 mg/100 mL n-hexane extracts of P. guajava were significantly different (p < 0.05) within the experimental time; except at t = 90 s. Figure 2c showed that, within the experimental time range of 90 s ≤ t ≤ 180 s, the level of poly-dHbS-M of the control sample was significantly higher (p < 0.05) than those of the samples containing 40, 60, and 80 mg/ 100 mL chloroform extracts of P. guajava. Conversely, the levels of poly-dHbS-M of the control and test samples exhibited no significant difference (p > 0.05) within the experimental time of t < 90 s. The pattern of levels of poly-dHbS-M of the test samples was biphasic, which exhibited an exponential increase in the level of poly-dHbS-M at t < 60 s; p > 0.05. Figure 2d showed that the levels of poly-dHbS-M of the samples containing 40 and 80 mg/100 mL ethylacetate extracts of P. guajava were significantly lower (p < 0.05) than that of the control sample. Conversely, the level of poly-dHbS-M of the sample containing 60 mg/100 mL ethylacetate extract of P. guajava was significantly higher (p < 0.05) than that of the control sample. An overview of Fig. 2d showed that the pattern of levels of poly-dHbS-M of 40 and 80 mg/100 mL ethylacetate extracts of P. guajava was biphasic. Additionally, the levels of poly-dHbS-M in the presence of 40 and 80 mg/100 mL ethylacetate extracts of P. guajava showed no significant difference (p > 0.05) within the time range of 60 s ≤ t ≤ 90 s, in which the peak value of poly-dHbS-M was at t = 60 s. The levels of poly-dHbS-M of the control sample and the sample containing 40 mg/100 mL ethylacetate extract of P. guajava showed no significant difference at t < 30 s (p > 0.05). Figure 2e showed that within the experimental time range of 90 s ≤ t ≤ 180 s, the increasing levels of poly-dHbS-M of the test samples were in a concentrationdependent manner; i.e., 80 mg/100 mL > 60 mg/100 mL > 40 mg/100 mL. However, the level of poly-dHbS-M of 40 mg/100 mL residual aqueous extract of P. guajava was significantly lower (p < 0.05) than that of the control sample at t < 120 s. Likewise, the level of poly-dHbS-M of the sample containing 60 mg/100 mL of residual aqueous extract was significantly lower (p < 0.05) than that of the control sample at t = 30 s, which was contrary within the time range of 60 s ≤ t ≤ 120 s; p > 0.05 (Fig. 2e).
Within the experimental time, the level of poly-dHbS-M of the sample containing 80 mg/100 mL residue aqueous extract of P. guajava was significantly higher (p < 0.05) than that of the control sample. Furthermore, at t > 120 s, the levels of poly-dHbS-M of the sample containing 40, 60, and 80 mg/100 mL residue aqueous extracts of P. guajava were significantly higher (p < 0.05) than that of the control sample. The level of poly-dHbS-M of the control sample was biphasic with a peak value at t = 120 s (Fig. 2e).

Levels of poly-dHbS-M in the presence of fractionated leaf extracts of T. catappa
Figure 3a-e showed the levels of poly-dHbS-M of the control sample and the samples containing 40, 60, and 80 mg/ 100 mL of fractionated leaf extracts of T. catappa with experimental time. Figure 3a showed that the pattern of levels of poly-dHbS-M of the test samples was biphasic. The levels of poly-dHbS-M of the samples containing 40, 60, and 80 mg/100 mL petroleum ether extracts peaked at t = 90 s, t = 90 s, and t = 30 s respectively. The levels of poly-dHbS-M of the samples containing 60 and 80 mg/100 mL of petroleum ether extracts were significantly lower (p < 0.05) than that of the control sample at t > 150 s, whereas the sample containing 40 mg/100 mL petroleum ether extract showed no significant difference (p > 0.05) from that of the control sample at t = 180 s. Figure 3b showed that within the experimental time of t < 90 s, the levels of poly-dHbS-M of the samples containing 40, 60, and 80 mg/100 mL n-hexane extracts of T. catappa were significantly higher (p < 0.05) than the control sample. Conversely, at t > 120 s, the levels of poly-dHbS-M of the test samples was significantly lower (p < 0.05) than that of the control sample.
Within the experimental time of t < 180 s, the level of poly-dHbS-M of the sample containing 80 mg/100 mL chloroform extract of T. catappa was significantly lower (p < 0.05) than that of the control sample (Fig. 3c). Likewise, the sample containing 60 mg/100 mL chloroform extract of T. catappa gave significantly lower (p < 0.05) level of poly-dHbS-M within the experimental time range of 60 s ≤ t ≤ 120 s. The levels of poly-dHbS-M of the samples containing 40 and 60 mg/100 mL chloroform extracts of T. catappa were significantly higher (p < 0.05) than that of the control sample at t = 30 s. Additionally, at t > 150 s, the level poly-dHbS-M of the sample containing 40 mg/ 100 mL chloroform extract was significantly higher (p < 0.05) than that of the control sample. Figure 3d showed that within the experimental time, t > 120 s, the level of poly-dHbS-M of the sample containing 40 mg/100 mL ethylacetate extract of T. catappa was significantly higher (p < 0.05) than that of the control sample. The levels of poly-dHbS-M of test samples showed no significant difference (p > 0.05) from that of the control sample at t = 30 s. Furthermore, at t = 60 s, the level of poly-dHbS-M in the presence of the sample containing 40 mg/ 100 mL ethylacetate extract of T. catappa was significantly lower (p < 0.05) than that of the control sample. Within the experimental time, the levels of poly-dHbS-M of the test samples were significantly different (p < 0.05) in a concentration-dependent manner; except between samples containing 40 and 60 mg/100 mL ethylacetate extract of T. catappa at t = 60 s; p > 0.05. Figure 3 showed that within the experimental time, the level of poly-dHbS-M of the sample containing 40 mg/100 mL residual aqueous extract of T. catappa was significantly higher (p < 0.05) than those of the control sample as well as the samples containing 60 and 80 mg/ 100 mL residual aqueous extracts of T. catappa; except at t = 180 s; p > 0.05.
The level of poly-dHbS-M of the sample containing 60 mg/100 mL residue aqueous extract of T. catappa was significantly lower (p < 0.05) than that of the control sample at t < 120 s. The level of poly-dHbS-M of the sample containing 80 mg/100 mL residual aqueous extract of T. catappa was significantly lower (p < 0.05) than that of the control sample at t < 120 s; but was not significantly different (p > 0.05) from that of the control sample at t = 30 s. At the end of the experimental time, the level of poly-dHbS-M of the test samples was significantly higher (p < 0.05) than that of the control sample (Fig. 3e).
Percentage inhibition/activation of poly-dHbS-M in the presence of fractionated leaf extracts Table 2 showed the percentage inhibition/activation of poly-dHbS-M in the presence of varying concentrations   Table 2 showed that 40 and 80 mg/100 mL petroleum ether extracts of A. occidentale inhibited poly-dHbS-M at t < 90 s and t < 120 s respectively. Further increase in the experimental time showed that 40 and 60 mg/100 mL petroleum ether extracts of A. occidentale activated poly-dHbS-M. Among the three concentrations of petroleum ether extracts of A. occidentale, that of 60 mg/ 100 mL concentration exhibited the highest capacity to inhibit poly-dHbS-M; specifically at t = 180 s; inhibition of poly-dHbS-M = 18.65 ± 1.10% (Table 2). The capacity of 40 mg/100 mL n-hexane extract to inhibit poly-dHbS-M ranged between 2.96 ± 0.03% at t = 90 s and18.15 ± 1.21% at t = 60 s (Table 2). Similarly, 60 mg/100 mL n-hexane extract of A. occidentale inhibited poly-dHbS-M within the range of 7.04 ± 1.16% at t = 90 s and 25.19 ± 2.56% at t = 60 s. The highest capacity of n-hexane extract of A. occidentale to inhibit poly-dHbS-M was registered in the presence of 80 mg/100 mL of the extract, which corresponded to 27.41 ± 2.98% at t = 90 s. Conversely, 40 and 60 mg/100 mL n-hexane extracts of A. occidentale activated poly-dHbS-M at t > 90 s, whereas 80 mg/100 mL n-hexane extract of A. occidentale activated poly-dHbS-M at t = 180 s; activation of poly-dHbS-M was 9.26 ± 1.67% (Table 2).
Within the experimental time, 40 and 60 mg/100 mL chloroform extracts of A. occidentale activated poly-dHbS-M and were in the range of 31.25 ± 2.2-87.80 ± 4.90% and 17.55 ± 1.1-31.67 ± 3.0% respectively. Additionally, at t < 60 s, 80 mg/100 mL chloroform extract of A. occidentale activated poly-dHbS-M. Further increase in experimental time, t > 60 s, 80 mg/100 mL chloroform extract of A. occidentale inhibited poly-dHbS-M in the range of 27.38 ± 2.56-46.43 ± 3.56% (Table 2). Table 2 showed that 40 and 60 mg/100 mL ethylacetate extracts of A. occidentale inhibited poly-dHbS-M within the experimental time. The 80 mg/100 mL ethylacetate extract inhibited poly-dHbS-M at t < 60 s, whereas further increase in experimental time, t > 60 s, caused activation of poly-dHbS-M in the range of 5.18 ± 1.01-31.85 ± 2.5%. Table 2 showed that 40 mg/100 mL residue aqueous extract of A. occidentale exhibited decreasing capacity to activate poly-dHbS-M. At t = 180 s, 40 mg/100 mL residual aqueous extract of A. occidentale inhibited poly-dHbS-M by 29.93 ± 2.78%. Furthermore, 60 and 80 mg/ 100 mL residual aqueous extracts of A. occidentale  Table 2). The n-hexane extract of P. guajava inhibited poly-dHbS-M in a concentration-depended manner as the experimental time progressed. Table 2 showed that 40 mg/ 100 mL n-hexane extract of P. guajava exhibited a decreasing capacity to inhibit poly-dHbS-M, whereas 80 mg/100 mL n-hexane extract of P. guajava showed increasing capacity to inhibit poly-dHbS-M with increasing experimental time. The capacity of 40 mg/100 mL nhexane extract of P. guajava to inhibit poly-dHbS-M peaked at t = 30 s; inhibition of poly-dHbS-M was 38.68 ± 2.98%. Peak inhibition in the presence of 80 mg/100 mL n-hexane extract of P. guajava was at t = 180 s; inhibition of poly-dHbS-M was 51.73 ± 4.03%, whereas that of 60 mg/100 mL of the extract was at t = 180 s; inhibition of poly-dHbS-M was 34.53 ± 2.33% ( Table 2).
The chloroform extract of P. guajava activated poly-dHbS-M at t = 30 s. However, as the experimental time progressed, t > 30 s, the chloroform extract of P. guajava inhibited poly-dHbS-M in a time-dependent manner. Specifically, Table 2 showed that 80 mg/100 mL chloroform extract caused the highest inhibition (38.84 ± 2.78%) against poly-dHbS-M. Table 2 showed that ethylacetate extract of P. guajava exhibited the highest capacity to inhibit poly-dHbS-M among the three ethylacetate extract concentrations. Conversely, poly-dHbS-M was activated by 60 mg/100 mL ethylacetate extract of P. guajava throughout the experimental time. Poly-dHbS-M was inhibited by 80 mg/ 100 mL of ethylacetate extract of P. guajava within the range of 9.93 ± 1.02-37.00 ± 3.09% (Table 2). Table 2 showed that 40 and 60 mg/100 mL residual aqueous extracts of P. guajava inhibited poly-dHbS-M within the time range of 0 s ≤ t ≤ 120 s. Further increase in the experimental time, t > 120 s, 40, and 60 mg/100 mL residual aqueous extracts of P. guajava activated poly-dHbS-M in a concentration-depended manner (Table 2). Notably, 80 mg/100 mL residual aqueous extract of P. guajava caused activation of dHbS-M throughout the experimental time. Peak activation of poly-dHbS-M (143.88 ± 9.8%) occurred at t = 180 s in the presence of residual aqueous extract of P. guajava (Table 2). Table 2 showed that 40, 60, and 80 mg/100 mL petroleum ether extracts of T. catappa caused activation of poly-dHbS-M within the experimental time range of 0 s ≤ t ≤ 120 s. Peak activation of poly-dHbS-M (57.07 ± 4.78%) was registered in the presence of 60 mg/100 mL petroleum ether extract of T. catappa at t = 60 s. Further increase in experimental time, t > 120 s, showed that 60 and 80 mg/ 100 mL petroleum ether extracts of T. catappa inhibited poly-dHbS-M in a time-depended manner. However, 40 mg/100 mL petroleum ether extract of T. catappa activated poly-dHbS-M throughout the experimental time.
A cursory look at Table 2 showed that peak inhibition of poly-dHbS-M by 40, 60, and 80 mg/100 mL chloroform extracts of T. catappa occurred at t = 60 s, specifically, 0.98 ± 0.02%, 11.13 ± 1.78%, and 16.02 ± 1.98% respectively. Further increase in experimental time, t > 60 s, showed diminishing capacity of the extract to inhibit poly-dHbS-M. Additionally, 40 mg/100 mL chloroform extract of T. catappa activated poly-dHbS-M except at t = 60 s and t = 120 s. Table 2 showed that 60 and 80 mg/100 mL ethylacetate extracts of T. catappa inhibited poly-dHbS-M within the experimental time with peak value at t = 30 s; inhibition of polymerization was 18.76 ± 1.78% and at t = 90 s; inhibition of polymerization was 40.13 ± 3.11% respectively. However, 40 mg/100 mL ethylacetate extract of T. catappa inhibited poly-dHbS-M within the experimental time; at t < 90 s. Further increase in experimental time caused activation of poly-dHbS-M by 40 mg/100 mL ethylacetate extract of T. catappa (Table 2).
At the given experimental time range of 0 s ≤ t ≤ 180 s, 40 mg/100 mL residue aqueous extract of T. catappa activated poly-dHbS-M with a peak value at t = 180 s; activation of poly-dHbS-M was 46.92 ± 3.55% (Table 2). Conversely, 60 and 80 mg/100 mL residual aqueous extracts of T. catappa inhibited poly-dHbS-M within the experimental time of t < 120 s. Further increase in experimental time caused activation of poly-dHbS-M ( Table 2). Table 3 showed the RCPI% of dHbS-M in the presence of fractionated leaf extracts of A. occidentale, P. guajava,  Table 3 showed that RCPI% of dHbS-M in the presence 60 and 80 mg/100 mL residual aqueous extracts of A. occidentale were comparatively greater than that of the control sample. The RCPI% of dHbS-M of the samples containing 40, 60, and 80 mg/100 mL petroleum ether extracts of P. guajava as well as 80 mg/100 mL residual aqueous extract of P. guajava were indicative of cumulative activation of poly-dHbS-M (Table 3). Additionally, the comparative raised level of RCPI% of dHbS-M in the presence of 80 mg/100 mL n-hexane of P. guajava was indicative of higher capacity of the extract to cumulatively inhibit poly-dHbS-M than other concentrations of fractionated leaf extracts of P. guajava. Table 3 showed that dHbS-M of the samples containing 60 and 80 mg/100 mL ethylacetate of T. catappa gave comparative raised levels of RCPI% indicative of cumulative inhibition of poly-dHbS-M. RCPI% of dHbS-M in the presence of 40 and 60 mg/100 mL petroleum ether extracts of T. catappa as well as 40 mg/100 mL residual aqueous extract of T. catappa were indicative of comparatively raised levels of cumulative activation of poly-dHbS-M.

RCPI% of dHbS-M in the presence of fractionated leaf extracts
The dHbS-M of the sample containing 40 mg/100 mL residual aqueous of A. occidentale gave RCPI% = 90.1, which represented the maximum cumulative activation of poly-dHbS-M compared with other concentrations of fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa.

FT-IR spectra of fractionated leaf extracts
Ethylacetate extract of T. catappa exhibited characteristic alcohol (O-H) stretch around 3410.5 cm −1 (Fig. 5f). Table 4(F) showed that ethylacetate extract of T. catappa contained aldehydes, aromatic and nitro-compounds, as well as phenolics and esters. Peaks around 2981.9 and 2907.0 cm −1 were evidence of the presence of aliphatic compounds in ethylacetate extract of T. catappa. Furthermore, medium band at 1043.7 cm −1 , typified by C-O stretch, was indicative of the presence of alkoxy group in ethylacetate extract of T. catappa. Peaks within the range of (939.3-849.8) cm −1 exhibited aliphatic sp2 C-H bend, which was indicative of the presence of aliphatic compounds in ethylacetate extract of T. catappa.

UV-visible spectra of fractionated leaf extracts
Figure 6a-f showed characteristic patterns of UV-visible spectra of selected fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa. The multiple λmax of petroleum ether extract of A. occidentale was within the range of (224.00-272.00) nm, which suggested the presence of nitrite (-ONO) and nitrate (-NO 3 ) chromophores in the extract (Fig. 6a). Figure 6b showed no evidence of presence of chromophores, within the UVvisible spectra, in ethylacetate extract of A. occidentale. The n-hexane extract of P. guajava gave λmax within the range of (248.00-281.00) nm (Fig. 6c), whereas that of chloroform extract of P. guajava was between 200.00 and 281.00 nm (Fig. 6d). Ethylacetate extracts of P. guajava and T. catappa gave corresponding single λmax at 217.00 nm (Fig. 6e) and 363.00 nm (Fig. 6f) respectively.

Discussion
The solid-liquid solvent extraction protocols are commonly used for the empirical evaluation of phytocomponents. The overall quality, phytochemical profile, and relative quantity (percentage yield) of plant extracts depend on a multitude of intrinsic elements such as age, species, and genetic constitution of the plant in addition to the plant parts of interest for empirical evaluation. The extrinsic factors include but not limited to growth conditions, geographical location, soil chemistry and seasonal period of the harvest of the plant materials (Mburu et al. 2012;Chikezie and Ojiako 2013;Mousavi et al. 2018). For the most part, the polarity of the solvent used in solid-liquid solvent extraction protocol has a bearing on the percentage yield of plant extracts (Mohd et al. 2012) composed of combinations of phytocomponents with diverse physical and chemical properties (Saxena et al. 2013). Furthermore, the phytochemical profile of fractionated extracts is intricately connected with the polarities of solvents of the partitioning cocktails as previously reported Mousavi et al. 2018). Accordingly, fair insights into the physicochemical properties of the phytocomponents in a given plant material provide a guide for selecting the appropriate solvent for extraction protocol in order to achieve the maximum yield of their diverse phytocomponents. The present study showed variability in percentage yields of the fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa, which contained combinations of phytocomponents of diverse biologic and chemical properties. The comparatively high yields of residual aqueous fractions of the plant materials suggested the presence of relatively high levels of hydrophilic phytocomponents in A. occidentale, P. guajava, and T. catappa leaf extracts. It is worthwhile to mention that hydrophilic and hydrophobic phytocomponents exhibit diverse biological activities, in which their ultimate pharmacological actions elicit therapeutic benefits or toxic outcomes .
HbS aggregation and polymerization are pivotal primary events leading to the distortion of sickle erythrocyte morphology and presentation of pathophysiologic indicators of SCD (Vekilov 2007;Uzunova et al. 2010;Piccin et al. 2019). In search of remedies for SCD, using in vitro models, previous empirical studies have demonstrated the capability of varieties of herbal extracts to control and impede HbS aggregation and polymerization (Okpuzor et al. 2008;Uwakwe and Nwaoguikpe 2008;Chikezie et al. 2010;Chikezie 2011;Imaga 2013;Dash Pauline et al. 2013;Nurain et al. 2017). In concord with previous reports, the outcome of the present investigations showed that certain fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa attenuated the tendency of HbS to aggregate and polymerize in vitro. Selected fractionated leaf extracts, namely petroleum ether and ethylacetate extracts of A. occidentale, n-hexane, chloroform, and ethylacetate extracts of P. guajava, as well as ethylacetate extract of T. catappa, contained phytocomponents that attenuated intermolecular aggregation of dHbS-M.
In the absence of impeding molecular species, from plant extract, against HbS aggregation and polymerization, several mechanisms have been ascribed to the tendencies of phytocomponents to attenuate HbS aggregation and polymerization (Chikezie 2011;Syed et al. 2019). Based on previous reports and reviews, the options available include one or combinations of the following mechanisms: i. The propensity of molecular species from plant extract to reversibly interact and interphase with complementary contact regions constituted by Val-beta6 residue of the docking dHbS-M and Leu-beta88, Phe-beta85, and Asp-beta73 residues of adjacent dHbS-M, and thereby, alters and shields the hydrophobic microenvironment of the contact regions required for HbS aggregation and polymerization (Chang et al. 1983;Charache et al. 1995;Abdulmalik et al. 2005;Eaton and Bunn 2017;Syed et al. 2019). ii. The molecular species from plant extract stabilize HbS molecule by reversible non-covalent interactions that thermodynamically favour R-state HbS (Manning and Acharya 1984;Kark et al. 1988;Oyewole et al. 2008;Safo and Kato 2014;Oder et al. 2016;Eaton and Bunn 2017). iii. Chemical modification of HbS molecule by molecular species from plant extract results in HbS derivatives that are adverse to aggregation and polymerization (Manning and Acharya 1984;Xu et al. 1999;Oder et al. 2016).
The major components of results of the present study appeared to suggest that reversible non-covalent interaction between phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa and HbS, for the most parts, was responsible for the capacities of the phytocomponents to attenuate HbS aggregation and polymerization. However, the non-covalent interaction may possibly wane as the experimental time progressed due to relatively transient nature of the interaction. Furthermore, two competing thermodynamic favorable interactions, namely dHbS-M…dHbS-M and dHbS-M…phytocomponent interactions were responsible for the dual behaviors, in certain instances (Tables 3 and 4), of phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa to either exacerbate or attenuate HbS aggregation and polymerization. Precisely, increased entropy kept the interacting species apart, whereas favorable free energy of intermolecular non-covalent interaction, measured by entropy gained, permitted the molecules to associate (Vekilov 2007;Syed et al. 2019). The molecular features of the interacting species and entropy of the interacting environment either exacerbated or attenuated HbS aggregation and polymerization. Accordingly, non-covalent interactions involving dHbS-M and phytocomponents may be disrupted and displaced by more thermodynamic favored interactions with the progression of time, whereby dHbS-M…dHbS-M interactions were favored fostering exacerbated HbS aggregation and polymerization or otherwise. Conversely, dHbS-M…phytocomponents interactions may attenuate HbS aggregation and polymerization provided the molecular configuration of the microenvironment of the contact regions required for HbS aggregation and polymerization was such that negated dHbS-M…dHbS-M interactions but promoted dHbS-M…phytocomponent interactions (Syed et al. 2019).
Another approach to deter HbS aggregation and polymerization involves the use of chemical agents that stabilize relax state (R-state) hemoglobin. The R-state HbS conformation is such that the contact regions required for HbS aggregation and polymerization are shielded, and as a result, do not form fibrous HbS polymers that engender erythrocyte sickling and ensuing clinical crisis (Safo and Kato 2014;Oder et al. 2016;Eaton and Bunn, 2017). It is remarkable to note that the presence of isothiocyanates in ethylacetate extract of A. occidentale obviously contributed, in parts, to impeding HbS aggregation and polymerization, which was in agreement with previous reports (Park et al. 2003;Safo and Kato 2014). Earlier reports showed that the thiols and isothiocyanates formed a covalent adduct with hemoglobin molecules, and thereby, modified the protein to an allosteric state of enhanced oxygen affinity that is adverse to polymerization (Park et al. 2003;Safo and Kato 2014). Specifically, studies showed that aliphatic isothiocyanates bound covalently to β 93 cystine (Cys-beta93) disrupted the native T-state salt-bridge interaction between β 94 aspartate (Asp-beta94) and β 146 histidine (His-beta146), and thereby, destabilized the lower oxygen affinity T-state of HbS that promoted hemoglobin aggregation and polymerization (Safo and Kato 2014;Oder et al. 2016). R-state HbS does not polymerize, whereas T-state HbS forms fibrous polymer (Eaton and Hofrichter 1987).
Additionally, the previous report showed that isoquercitrin (quercetin-3-O-β-D-glucopyranoside) was one of the bioactive components from numerous medicinal plants that readily interacted with HbS and, as a result, impeded HbS aggregation and polymerization (Syed et al. 2019). Using circular dichroism (CD) spectroscopy, Syed et al. (2019) revealed that HbS…isoquercitrin complex exhibited helical structural changes leading to destabilization of HbS polymer as previously described (Hamdani et al. 2009;Ding et al. 2012;Pauline et al. 2013) as well as stabilized R-state of HbS. Their findings were in agreement with the proposed mode of action of phytocomponents investigated in the present study. Accordingly, isothiocyanates from ethylacetate extract of A. occidentale were stabilizers of R-state of HbS in vitro as previously established (Park et al. 2003;Safo and Kato 2014).
Paradoxically, another selected fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa were indicated to exacerbate HbS aggregation and polymerization (Tables 2 and 3), which was in concord with earlier reports . The present results suggest that these groups of fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa, by virtue of their peculiar phytochemical profile, promoted HbS aggregation and polymerization. In a connected research outcome, Uzunova et al. (2010) had previously reported that the addition of 100-260 mM of free heme to dialyzed HbS solutions exacerbated HbS aggregation and polymerization by two orders of magnitude than before dialysis. They noted that the removal of free heme from HbS solutions by dialysis lowered HbS polymerization activity and further proposed that the prevention of free heme accumulation in the erythrocyte cytosol was a therapeutic strategy against SCD.
Chemical modification of HbS molecule by phytocomponents from plant extracts was probably the basis for the rapid and sustained exponential reversion of HbS aggregation and polymerization, typified by HbS polymerization in the presence of residual aqueous of A. occidentale (Fig. 1e). It implied, therefore, that the physicochemical properties of phytocomponents from the fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa had direct bearing on their capacities to alter the process leading to HbS aggregation and polymerization. Since intra-erythrocytic HbS aggregation and polymerization are pivotal to the pathogenesis and pathophysiology of SCD (Uzunova et al. 2010;Piccin et al. 2019), the use of chemical agents that covalently modify HbS molecules has been suggested to be an important approach to impede dHbS-M aggregation and polymerization (Park et al. 2003;Eaton and Bunn 2017;Kassa et al. 2019). Covalent modification of HbS molecules by carbamylation using isothiocyanates, acetylation using methyl acetyl phosphate (MAP), and Snitrosylation of Cys-beta93 has been reported (Xu et al. 1999;Park et al. 2003;Chikezie, 2011;Jana et al. 2018). Furthermore, 5-hydroxymethyl-2-furfural (5HMF) forms a high-affinity Schiff-base adduct with HbS molecules, and thereby impede the tendency of dHbS-M to aggregate and polymerize (Abdulmalik et al. 2005;Safo and Kato, 2014;Oder et al. 2016;Eaton and Bunn, 2017).
However, synthetic covalent modifiers that interrupted HbS aggregation and polymerization might cause undesirable chemical modifications of HbS molecules and other body protein molecules due to their non-specific chemical reactions (Safo and Kato 2014;Eaton and Bunn 2017). Although there are envisaged challenges in applying these options in the management of SCD as a result of a lack of stereo-specificity in dHbS-M…phytocomponents interactions at the complementary contact regions or allosteric sites of dHbS-M, this approach still offers rewarding prospects for alleviation of the sickling phenomena and therefore should not be discounted (Eaton and Bunn 2017).
The present study gave molecular insights into the identities of phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa that attenuated HbS aggregation and polymerization within the experimental time of 180 s. The molecular features of the phytocomponents implicated in this regard, revealed by GC-MS and FT-IR systems analyses, were the aliphatic hydrocarbons, methylated esters, methylated long-chain and short-chain fatty acids, volatile alkanes, short-chain aliphatic alcohols, aromatic derivatives, cycloalkanes, phthalates, isothiocyanates, aminated sugars, cyclo-alcohols, and arachidyl alcohols (Tables 4(A-F) and Table 5(A-F)). Furthermore, UVvisible investigations revealed the presence of nitrocompounds in petroleum ether extract of A. occidentale as a phytocomponent that attenuated HbS aggregation and polymerization (Fig. 6v).
The present study showed that n-hexane extract of P. guajava exhibited the highest capacity to attenuate HbS aggregation and polymerization compared to other fractionated extracts of A. occidentale, P. guajava, and T. catappa. A combined result of GC-MS and FT-IR protocols showed that the phytocomponents from P. guajava that attenuated HbS aggregation and polymerization were viz. D-erythro-sphinganine, nitro compounds, tertiary amine, primary amine, di-, tri-substituted aromatic compounds, cyanate, and esters.
The present study further confirmed the usefulness of GC-MS systems in identification, quantification, and characterization of a mixture of phytocomponents with commensurate reproducibility and reliable outcomes. Accordingly, the application of GC-MS systems protocols, which unravel the nature, quantity, and chemical structures as well as molecular fingerprints of the vast array of phytocomponents from biologic systems have been widely reported (Sasidharan et al. 2011;Sampaio et al. 2011;Rašković et al. 2015;Cyril-Olutayo et al. 2019). To mention but a few, the findings of the present   study, using bioassay-guided approach in vitro in conjunction with the GC-MS and FT-IR systems analyses, suggested that methylated esters such as methyl tetradecanoate, hexadecanoic acid, methyl ester, pentadecanoic acid, 14-methyl-methyl ester, 9, 12-octadecadienoic acid, (Z, Z)-methyl ester, 11-octadecenoic acid, methyl ester, and 9-octadecenoic acid, methyl ester (E)-were phytocomponents from petroleum ether extract of A. occidentale that attenuated HbS aggregation and polymerization. The phytocomponents from fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa that attenuated HbS aggregation and polymerization are summarized (Table 4). There are reports on other biological activities and medicinal properties of these methylated esters. For instance, methyl tetradecanoate is a platelet aggregation inhibitor used for the prevention and treatment of cerebral injuries of hemorrhagic or ischemic origin (Nagarjunakonda et al. 2017). In addition to the potentials of hexadecanoic acid, methyl ester to attenuate HbS aggregation and polymerization, as our present findings suggest, previous studies showed that hexadecanoic acid, methyl ester from calyx of Hibiscus sabdariffa (green roselle) elicited membrane autolysis, inhibited biosynthesis of nitric oxide, phagocytic activity of certain cells, as well as lowered tumor necrosis factor-alpha (TNFα), interleukin-10 (IL-10), and prostaglandin E2 (PGE2) activities and induced dilation of the aorta (Cai et al. 2005;Sarkar et al. 2006;Lin et al. 2009). Methyl stearate is an anti-helminthic, anti-fungal, and anti-nociceptive agent as well as a potent γ-amino butyric acid (GABA) aminotransferase inhibitor, lipid metabolism regulator, gastrin inhibitor, and exhibits antioxidant activity (Pinto et al. 2017;Adnan et al. 2019). Other notable biological activities and medicinal properties of few other phytocomponents, as our present findings suggest, that attenuated HbS aggregation and polymerization are summarized elsewhere: viz. pentadecanoic acid, 14-methyl-, methyl ester (antioxidant) (Vijisaral and Arumugam 2014), phthalates (antibacterial) (Khatiwora et al. 2012), isothiocyanates (antimicrobial) (Dias et al. 2014), and arachidyl alcohols (Garaniya and Bapodra 2014).

Conclusion
The fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa that exhibited comparatively high potency to attenuate HbS aggregation and polymerization were as follows: petroleum ether extract of A. occidentale, ethylacetate extract of A. occidentale, as well as n-hexane extract of P. guajava. Other fractionated leaf extracts that attenuated HbS aggregation and polymerization were chloroform extract of P. guajava, ethylacetate extract of P. guajava, and ethylacetate extract of T. catappa. The fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa exhibited differential capacities to impede HbS aggregation and polymerization in the order of n-hexane extract of P. guajava > ethylacetate extract of A. occidentale > ethylacetate extract of P. guajava > ethylacetate extract of T. catappa > petroleum ether extract of A. occidentale.
The identification, quantitation, and characterization methods using GC-MS in conjunction with FT-IR and UV-visible systems protocols revealed combinations of 53 phytocomponents from the fractionated leaf extracts of A. occidentale, P. guajava, and T. catappa as molecular species that impeded HbS aggregation and polymerization in vitro. In general terms, the phytocomponents from the fractionated leaf extracts that attenuated HbS aggregation and polymerization include the aliphatic hydrocarbons, methylated esters, methylated long-chain and short-chain fatty acids, volatile alkanes, short-chain aliphatic alcohols, D-erythro-sphinganine, aromatic derivatives, cycloalkanes, phthalates, isothiocyanates, aminated sugars, cyclo-alcohols, arachidyl alcohols, and nitro-compounds. The effectiveness of GC-MS, FT-IR, and UV-visible systems protocols notwithstanding, identification of unknown molecular species largely relied on comparison with known molecules from a database/library and established chromatogram and fingerprints patterns. Consequently, the use of these methods for the classification of phytocomponents into functional and structural groups comes with few drawbacks and challenges. Therefore, it is recommended that further investigations should be carried out for such exercise. Additionally, in order to confirm the specific identities of the phytocomponents that attenuated HbS aggregation and polymerization, it is recommended that another study on isolation and purification of the phytocomponents, suggested in the present study, should be applied in further HbS polymerization studies in vitro as well as the use transgenic sickle animal model.