The discovery of alternate or synergistic therapy for the treatment of SCD due to increasing concerns regarding its effect on the health of individuals worldwide is gaining attention. Higher PCV was observed in healthy male blood compared with healthy female blood (Table ). This confirmed the findings of an earlier report [18
]. The mean PCV values in normal healthy individuals range from 42 to 45% in adult males, 36-48% in adult females, and 32 to 65% in children. Values lower than these indicate an abnormality [20
]. Lower PCV values observed in HbSSM and HbSSF compared with NBM and NBF may be attributed to the high rate of hemolysis in HbSS blood [21
] that leads to a shortened life span (15.2 ± 6.3 days) of sickle cell blood compared with normal blood (90-120 days) [22
]. Similar to the reduction in sickle blood cell counts induced by VerMyg (2.42 ± 1.11%), Cari-oil, SolMon and Ipocrata also effectively reduced the numbers of sickle blood cells in the untreated sickle cell patients’ blood (26.85 ± 3.12%) to that of 1.03 ± 0.16, 1.90 ± 0.43 and 4.04 ± 0.71% respectively. All plant extract treatments had greater antisickling efficacy compared with previous reports using extracts from the antisickling plant (VerMyg). However, these extracts did not completely reverse sickling since sickle blood cells still existed in HbSSM and HbSSF blood compared with normal healthy controls (Table ). Treatment with SolMon, Ipocrata, and Cari-oil extracts had similar antisickling properties to VerMyg since higher normal RBC levels were observed in the treated sickle blood compared with untreated sickle blood. This is evident in the images of Ipocrata (Figure ), SolMon (Figure ) and Cari-oil (Figure ) treated HbSS blood, where fewer numbers of sickle blood cells were observed compared with the large amount of normal RBCs. However, Ipocrata treatment was less effective compared with VerMyg treatment as measured by the number of percentage of sickle cells present in the blood sample (Table ). In this study, three plants (SolMon, Ipomea and Cari-oil) were shown to have novel antisickling properties. The antisickling property of Cari-oil was expected since the antisickling properties of C
fruits was previously reported [15
]. This study also demonstrated that the seed oil from C
also possesses antisickling properties. The levels of aggregation were reduced in all the plant extract-treated HbSS blood compared with that of untreated HbSS blood (Figures , , , and ).
The high prevalence of HbSS diseases and G6PDH deficiency (A-
type) in tropical areas is related to the selective advantage these diseases provide against malaria [23
]. The protective effect of HbSS against malaria has been attributed to heme oxygenase-1 (HO-1), an enzyme whose expression is strongly induced by sickle hemoglobin [24
]. G6PDH deficiency screening among SCD patients has provided the opportunity to administer appropriate preventive and therapeutic measures [25
]. G6PDH activity in HbSSM was higher compared with either healthy male or female donor blood (Table ), suggesting that male HbSS patients may be more susceptible to malaria than female HbSS patients may. A higher prevalence of malaria was reported in males than in females [26
]. Symptomatic patients are almost exclusively male, due to the X-linked pattern of inheritance [29
]. Therefore, G6PDH deficiency may be more prevalent in HbSS females than in HbSS males, and suggests why a higher occurrence of SCD-associated hemolytic crises (anemia, pain, jaundice) often occurs in female HbSS patients [30
An increase in Fe2+/
ratios upon application of a drug or plant extract indicates a reversal of sickling, suggesting conversion of deoxyHbS to oxyHbS [31
ratios were reduced by all methanolic plant extract treatments in HbSS males (Table ), suggesting they may decrease the oxygen affinity of RBCs in HbSS male patients [31
]. All plant extract treatments generally led to an increase in hemoglobin concentrations in HbSS female patients (Table ). Interestingly, SolMon and Ipocrata treatments were more effective than VerMyg for increasing hemoglobin levels in HbSSM. However, only SolMon treatment was more effective than VerMyg in HbSSF.
One particular area of focus in the management of SCDs is the inhibition of sickle cell hemoglobin polymerization. It was hypothesized that an ideal antisickling drug or agent should significantly inhibit polymerization of the sickle cell hemoglobin in SCD blood [32
]. Apart from inhibiting polymerization, the agent should also increase the oxygen affinity of hemoglobin. In this study, polymerization was inhibited by VerMyg plant extract treatment in male SCD patients (Table ). This was expected as the plant had previously been reported to have antisickling properties [14
]. Similarly, SolMon exhibited a remarkable ability to inhibit sickle cell polymerization in male SCD patients (Table ). Of note, all plant extracts tested in this study inhibited sickle cell polymerization in HbSSF. Importantly, the potency of SolMon to inhibit sickle cell hemoglobin polymerization exceeded that of VerMyg in HbSSF as previously described (Table ).
The effect of plant extracts on oxidative stress in Sickle cell patient bloods
LDH is an important enzyme for carbohydrate metabolism and is used as an indicative criterion for exposure to chemical stress [33
]. All plant extracts in this study reduced LDH activity in sickle cell blood (Table ). Reduced LDH activity levels in treated HbSS blood samples (Table ) indicate a lower energy metabolism of RBCs [34
]. In addition, all plant extracts facilitated biochemical changes that enhanced oxidative interactions in HbSS blood, indicating anaerobic fermentation in RBCs [33
]. Therefore, the plants studied here possess the ability to alleviate stress associated with HbSS patients. Hence, the plant extract treatment may enhance HbSS blood stability and patient health. This study also revealed that the antisickling properties of these plants might function by modulating the genes responsible for LDH synthesis in both sexes. The wide variation of LDH activities between both sexes is expected as indicated in the control. Genetic differences between both sexes had been linked to the wide variation [35
]. This wide variation in LDH activity between both sexes was similarly manifested only in the samples treated with Cari-oil (Table ). This is obviously due to the fact that the seed oil could only generate a mild effect in modulating the genes that coded for LDH in male. The same seed oil also generated drastic effect in modulating the genes that code for LDH in female subjects. This insignificant variation in LDH activity in males as against a remarkable variation in females had also been reported [36
The catalase enzyme is an endogenous antioxidant present in all aerobic cells and facilitates the removal of toxic hydrogen peroxide by catalyzing its decomposition to molecular oxygen and water, without the production of free radicals [37
]. It is therefore useful as a defense agent against oxidative stress [37
]. High amounts of free radicals are important in the development of SCD complications and other disease states [38
]. High catalase activity also suggests poor health status of the blood [38
]. Catalase activity is induced by the presence of its substrate, hydrogen peroxide. Thus, a strong antioxidant should reduce catalase activity as shown by glutathione treatment (Table ).
The low catalase activity in NBM compared to that of the NBF in this work is similar to earlier reports [39
]. These difference was attributed to be due to aging since aging decreases catalase gene transcription in male while in female, aging leads to an increase in the translational efficiency of catalase mRNA [41
]. Glutathione and the Ipocrata, VerMyg, Cari-oil and SolMon plant extracts reduced the catalase activity in NBF. However, the Cari-oil, VerMyg, and Ipocrata extracts were less effective than glutathione. Although Cari-oil, Ipocrata and glutathione reduced catalase activity in NBM, VerMyg reduced the antioxidant status of male blood by increasing its catalase activity. The antioxidant potential of glutathione was more effective than SolMon in NBM (Table ).
However, Ipocrata, VerMyg, Cari-oil and SolMon plant extracts had no significant effect on the catalase activity of either male or female sickle cell blood (Table ). The fact that the plant extracts were effective in healthy blood indicates that higher hydrogen peroxide substrate concentrations than were used in this study (0.5% w/v), may be required for plant extracts to be effective in reducing catalase activity in sickle cell blood [42
]. Alternatively, catalase inhibitors such as β-mercaptoethanol and dithiothreitol may be abundantly present in HbSS patients. These inhibitors possess the ability to modify cysteine residues in the catalase enzyme. Some catalase inhibitors are thiol compounds that can also modify the heme group of catalase, and may suggest the cause of enzyme inactivation in sickle cell patient blood [43
Peroxidase activity normally increases in response to increased substrate (hydrogen peroxide) concentrations in the blood [44
]. However, only SolMon exhibited effective antioxidant activity by reducing the peroxidase enzyme levels in NBM and NBF (Table ). Cari-oil and Ipocrata treatment of NBM and NBF blood may encourage free radical generation (H2
) since treatment increased peroxidase activity (Table ). Similar free radical generation effects were also observed for the Cari-oil extract-treated HbSSM and HbSSM blood (Table ).