Phytotherapy and the Relevance of Some Endogenous Antioxidant Enzymes in Management of Sickle Cell Diseases Phytotherapy and the Relevance of Some Endogenous Antioxidant Enzymes in Management of Sickle Cell Diseases

Introduction: Sickle cell disease (SCD) is one of the most devastating diseases ravaging most populations. Methodology, results, and discussion: The numerous plants earlier reported to be used for treating SCD were compiled along with their geographical locations (using relevant online databases when not provided in cited articles for each plant) and relative antisickling strength. The process of hemolysis in sickle cell diseases, a brief overview of the current treatments, and management of sickle cell diseases is considered in the chapter. The activities of endogenous antioxidants and some biochemical enzyme markers coupled to these plants’ ability to maintain the integrity of red blood cell membrane are discussed in line with their antisickling health benefits and are also used to proffer more reliable molecular therapeutic strategies for managing sickle cell diseases. Furthermore, the operational principles of some enzymes, as well as their contributions to advancement of knowledge for management of the disease, were examined. Conclusion: Geographical spread of these identified antisickling plants contributes to low levels of sickle cell patients where the potentials are known. More efforts should therefore be channeled toward increasing awareness about the plants, as well as harnessing their active principles to obtain a more lasting solution to sickle cell disease at the molecular level.


Introduction
Sickle cell disease (SCD) is an autosomal recessive genetic disorder that is caused by a mutation in the β-globin gene on chromosome 11q [1]. This mutation involves glutamic acid being substituted with valine at the 6th position along the β-globin chain. α 2 β 2 is expressed as normal hemoglobin, α 2 β S (heterozygote) is expressed as sickle trait, while α 2 S 2 (homozygote recessive) is expressed as sickle cell anemia. Most of the time as a result of repeated series of sickling and unsickling, the erythrocytes become permanently damaged and consequently lyse. Some acute and chronic tissue injuries result when these abnormally shaped red cells impede blood flow through the vessels [2].
Sickle cell disease is one of the most devastating diseases ravaging most populations. It is a disease that affects numerous nations and ethnic groups. It is associated with painful symptoms and is a genetic disease in which an individual inherits the allele for sickle cell hemoglobin from both parents. Patients with this disease possess lower level of erythrocytes than the normal healthy human. In addition to an unusually large number of immature cells such as transferrin receptor-positive, reticulocytes, erythroblasts that sometimes manifest in the form of granular bodies in the cytoplasm of red blood cells, the blood contains many long, thin, crescent-shaped erythrocytes that look like the blade of a sickle [3]. The hemoglobin (hemoglobin S) in blood of patients with sickle cell disease becomes insoluble and forms polymers that aggregate into tubular fibers when deoxygenated. The altered properties of hemoglobin S result from a single amino acid substitution, which leads to the presence of a valine (Val) with no electric charge instead of a glutamate (Glu) residue with a negative charge when pH is 7.4 at position 6 in the two chains, resulting in two fewer negative charges than normal hemoglobin A [3]. Glutamine residue replaces the valine residue at position 6 of β-chain of hemoglobin in the normal blood to form a "sticky" hydrophobic interaction outside the surface of the sickle cell blood. It is the resultant sticky points on the surface of sickle cell blood that makes deoxyhemoglobin S molecules to interact abnormally with each other to form the long, fibrous aggregates peculiar to this disorder that eventually cause the deformation of the normal disc biconcave red blood cell 'RBC' [4].
Polymerization of the sickled cells thereby alters the integrity of the red cell membrane, leading to loss of K + , water, and a corresponding gain of Na + . Increased intracellular free Ca 2+ occurs during sickling, resulting in a loss of K + with accompanying movements of Cland water [5]. The clumping of sickled RBCs leads to blockage of small blood vessels, preventing blood supply to various organs. The deoxygenation process in tissue capillaries causes damage to its endothelium, leading to exudation of plasma into the surrounding soft tissue [6]. The integrity of the red blood cell membrane is maintained by hydration and sickling is generated when there is dehydration of the membrane. It is also believed that increase in synthesis of endogenous nitric oxide may be beneficial to SCD patients by preventing the mopping up of the nitric oxide by the hemoglobin released during hemolysis, which may trigger a cascade of events that ultimately inhibit blood flow [7].
There is high incidence of the sickle cell disease in different parts of the world, especially in Africa and Asia. The traditional people in these regions have learnt to manage the problem using plants which are God's gift of nature, especially among the lower socio-economic class who cannot afford the high cost of western medicine, as well as traditionalists who simply believe in their efficacy [6]. There has been increasing insight into gaining understanding about the management approaches of sickle cell disease in several African countries on the efficacy of conventional and traditional medicines. However, no substantial evidence exists to support the efficacy of herbal medications in actually curing the disease. Research into phytotherapy of diseases is a current trend in the management of sickle cell disease, with the hope of finding inexpensive and less toxic alternative medicines that people can easily access [8].
Nutritional evaluation of S. monostachyus leaves revealed the presence of carbohydrate, protein, ash, fiber, and fat as well as potassium and vitamin C in higher concentrations; calcium, magnesium, vitamin A, vitamin B 6 , vitamin E in lower concentrations; and others in trace quantities. Phytochemical screening revealed the presence of tannins, saponins, alkaloids, flavonoids, cyanogenic glycosides, and phytate [9]. Caffeic acid is one of the bioactive phenolic components of Solenostemon monostachyus leaves (unpublished report). It is a potent antioxidant. The study of antioxidants especially in various antisickling agents is of great significance because antisickling agents vary in their degree of efficacy. Antioxidants constitute a major component of these antisickling agents; thus, it is believed that the higher the antioxidant property of an antisickling agent, the higher its possible antisickling and therapeutic effect. Thus, reducing oxidative stress may ameliorate sickle cell crisis [8].
As a reference point, African/Nigerian medicinal plants are applied in the treatment of diseases, such as HIV/AIDS, malaria, tuberculosis, sickle cell diseases, diabetes, mental disorders, and so on. Research on these medicinal plants has shown various results such as antimicrobial (16%), molluscicidal (11%), antimalarial (7%), plant toxicology (7%), antitumor (4%), and many others. The major challenge with these medicinal plants is the lack of scientifically based evidence, quality standards, and regulations [6]. The antisickling activity of S. monostachyus on human sickle blood cells resulting in the alleviation of SCD symptoms has been reported [10]. Sickle cell disease and thalassemia are hemoglobinopathies characterized by chronic hemolysis [11].

Contribution of phytomedicine in the management of sickle cell diseases
The use of medicinal plants in the control of many diseases such as sickle cell diseases may be useful, especially in developing countries. The cost of treatment provided by orthodox medical practitioners largely contributes to the dependence on the use of traditional medicine in lowincome settings. Much of the medicinal use of plants seems to have been developed through observations of wild animals, and by trial and error. It has been estimated by the World Health Organization that 80% of the world's population relies on traditional medicine to meet their daily health needs. Thiocyanate-rich foods, erythropoietin, nutritional supplements, food extracts, phytochemicals, and synthetic compounds have been tested in vitro and in vivo on their possible roles in the management of sickle cell disease [12]. Many medicinal plants with antisickling properties are indicated in Table 1. The leaves from most of the above-identified plants have been successfully proven to play a role in the management of sickle cell diseases possibly by antioxidant phytochemicals, proximate nutrients, amino acids, and minerals. Phytochemical testing revealed the presence of folic acid, vitamin B12, alkaloids, spooning, glycosides, tannins, and anthraquinones. Studies also indicated the plant extracts contained flavonoids and the antioxidants vitamins A and C [13,14].
A major constituent of a herbal formula (Ajawaron HF) consists of the extracts of the roots of Cssus populnea L. CPK had also been effectively used to reverse sickling in the management of sickle cell disease in south west of Nigeria. The most prominent and widely used of them all is Ciklavit developed by Prof G. Ekeke after 18 years of intensive research in collaboration with Neimeth Pharmaceuticals, Lagos, Nigeria. These efforts led to the development of WHOapproved drugs such as Niprisan and Ciklavit from some of these plants traditionally identified for treating sickle cell diseases [8,20,21].

The role of other components in Ciklavit (apart from Cajanus cajan) is essentially nutritional.
A study on children with sickle cell disease suggests that nutritional supplements can help improve growth and weight gain. It can also boost the immune system and thus help in protecting against bacterial infections. Zinc deficiency is a major nutritional problem seen in sickle cell disease [8,22]. Also reported are amino acids, glycine, phenylalanine, and tyrosine, which have been reported to possess antisickling properties. Particularly, extracts from underutilized plants such as S. monostachyus, Carica papaya seed oil, and Ipomoea involucrata were proven to reverse human sickle cell blood almost completely coupled with the ability to also reduce stress in sickle cell disease patients. Hence, each plant individually or in combination can be used in the management of sickle cell disease [10].
Local mixtures of herbivores, pollinators, and micro-organisms generated from the application of plants usually upregulate or downregulate certain biochemical pathways. These actions are often a result of their secondary metabolites as well as pigments, which can be refined to produce drugs [53]. Many drugs originally derived from plants, such as salicylic acid (a precursor of aspirin) originally derived from white willow bark and the meadowsweet plant, have been developed using this approach. Quinine-antimalarial drug, Vincristine-an anticancer drug, and drugs (morphine, codine, and paregoric) for treating diarrhea were developed from Cinchona bark, periwinkle, and the opium poppy, respectively [54].

Plants as sources of antioxidants in the management of sickle cell diseases
In addition to depletion in iron level, the generation of reactive oxygen species (ROS) in the erythrocytes is a major factor contributing to the occurrence of anemia in sickle cell diseases. ROS are defined as substances generated by one electron reduction of molecular oxygen, including oxygen radicals and reactive nitrogen species (RNS) [55]. Common radical species include peroxide, superoxide, and the hydroxyl radical that contain an unpaired electron and as such are extremely reactive, allowing them to react immediately with any biological molecule to produce cellular damage. ROS contributes to the pathogenesis of several hereditary disorders of erythrocytes, including sickle cell disease, thalassemia, and glucose-6phosphate dehydrogenase (G6PD) deficiency. Oxidative stress is defined as the imbalance between pro-oxidants and antioxidants, which is a result of the formation of reactive oxygen species (ROS) in excess of the capacity of antioxidants to remove them [56].

Antioxidants
Antioxidants are the first line of defense against free radical damage and are critically needed for the maintenance and optimization of human health and well-being. Defence mechanisms against free radical-induced oxidative stress involve: (i) preventative mechanisms, (ii) repair mechanisms, (iii) physical defenses, and (iv) antioxidant defences. The body is also equipped with natural enzymatic antioxidant defences that include superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions (Figure 1). They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols [57].
In order to protect the cells and organ systems of the body against reactive oxygen species, a highly sophisticated and complex antioxidant protection system has been evolved by humans. This involves a variety of components such as nutrient-derived antioxidants, antioxidant enzymes, metal-binding proteins, and numerous other antioxidant phytonutrients, which are both endogenous and exogenous in origin, that function interactively and synergistically to neutralize free radicals [59]. The natural antioxidants are naturally occurring antioxidants having high or low molecular weights and can differ in their physical and chemical properties. The mechanisms by which these antioxidants act at molecular and cellular levels include role in gene expression and regulation, apoptosis, and signal transduction. Thus, antioxidants are involved in fundamental metabolic and homeostatic processes [58]. General patterns of behavior of some endogenous antioxidant enzymes and other relevant enzymes associated with sickle cell disease patients are subsequently described to provide more insight into how to solve the numerous challenges of the disease. Furthermore, introduction to a few selected enzymes that uniquely interact with constituents in these medicinal plants and are more relevant to the advancement of sickle cell diseases are provided subsequently.

Glucose-6-Phosphate Dehydrogenase (G6PD)
Glucose-6-phosphate dehydrogenase (G6PD) is the limiting enzyme that catalyzes the first reaction in the pentose phosphate pathway (Figure 2) in which glucose is converted into the pentose sugars required for glycolysis and various biosynthetic reactions. The pentose phosphate pathway (also known as the HMP shunt pathway) has a major biochemical role of providing reducing power to all cells in the form of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate). This is possible in the presence of enzyme G6PD and 6phosphogluconate dehydrogenase. NADPH enables cells to neutralize oxidative stress often induced by several oxidant agents and to preserve the reduced form of glutathione [57]. The hemoglobin in the blood, enzymes, and other proteins are damaged by the oxidants after all the leftover reduced glutathione is consumed. This leads to the generation of cross-bonding, protein deposition, and electrolyte imbalance in the red cell membranes. The hemoglobin from damaged red blood cells is metabolized to bilirubin that causes jaundice after attaining high concentrations [60]. High incidence of G6PD has been associated with areas of high prevalence of sickle cell disease. G6PD deficiency screening among SCD patients has provided the opportunity to administer appropriate preventive and therapeutic measures. The enzyme is becoming an increasingly strong confirmatory indicator of blood associated with sickle cell diseases and other closely associated ailments like malaria [10,61]. The enzyme provides information on the link between malaria and sickle cell diseases so as to understand strategies for the adoption of resistance of SCD patients to malaria to improve human health.

Heme oxygenase
Heme oxygenases (HO) consists of a family of evolutionarily conserved endoplasmic reticulum (ER) enzymes [62]. Heme oxygenase (HO) plays a central role in regulating the levels of intracellular heme by catalyzing the oxidative degradation of heme into equimolar amounts of biliverdin, carbon monoxide, and iron as shown in Figure 3a and b [63]. They are central in determining what happens with regard to the central components of mammalian stress response and defense against oxidative stress [64]. Heme oxygenase activity is a key determinant of the health status of sickle cell anemia patients. Human sickle blood enhances endothelial heme oxygenase (HO) activity and the positive effects of HO-1 induction, biliverdin, and CO in reducing sickle blood adherence and in promoting vasodilation, indicating the need to further explore the therapeutic potentials of the HO pathway in the treatment of SCD [64]. The human HO-1 is comprised of a protein fold that primarily contains α-helices. The heme is held between two of these helices (Figure 3b). HO-1 acts as a cytoprotective stress protein and provides defense against oxidative stress associated with sickle cell disease by accelerating the degradation of pro-oxidant heme and hemo proteins to the radical scavenging bile pigments, biliverdin, and bilirubin [65]. HO-1 helps the body's defense in response to physical stress. The levels of heme are strictly controlled by the balance between heme biosynthesis and catabolism as indicated in Figure 4 [65]. The key factor in the transcriptional activation of HO-1 is transcription factor Nrf2 (Figure 4). It interacts with many other genes that encode phase II drug-metabolizing enzymes so as to respond to oxidative stress [68].  Sickle hemoglobin induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells through a mechanism that involves the ubiquitination-degradation of Kelch-like ECHassociated protein 1 (Keap1), a cytoplasmic repressor of the transcription factor NF-E2-related factor-2 (Nrf2). Upon nuclear translocation, Nrf2 binds to the stress-responsive elements in the Hmox1 promoter, a regulatory mechanism that plays a central role in the control of Hmox1 expression in response to heme [70]. Moreover, the higher rate of free heme released from sickle versus normal human subjects, in the absence of inflammation, induces HO-1 expression without causing cytotoxicity and this explains how sickle human Hb may also cause the expression of HO-1 in human and mouse peripheral blood mononuclear cells and in human endothelial cells as well [54]. Although a link between sickle cell disease and resistance to severe malaria is well established, the biochemical relationship between the two is unknown.

Inducible nitric oxide synthase
Nitric oxide (NO) also influences the outcome of sickle cell disease. This outcome may sometimes be beneficial to SCD patients, provided there is increase in the production of endogenous NO so as to prevent the release of hemoglobin during hemolysis [7]. Inducible nitric oxide synthase (iNOS) is not normally expressed in the cells, but can be induced by the action of bacterial endotoxins (lipopolysaccharide), cytokines, and other agents. Though it is mainly identified in macrophages, iNOS expression may be stimulated in virtually any cell or tissue type, provided the appropriate inducing agents have been identified [71]. Upon its expression, iNOS remains constantly active and independent of intracellular Ca 2+ concentrations. Cell and tissue damage can be linked to the NO radical itself or NO interaction with O2 -• resulting in the formation of peroxynitrite (ONOO -). Most of the inflammatory and autoimmune lesions are characterized by large amounts of activated macrophages and neutrophils. NO can be secreted in large quantities by the cells, causing damage to the surrounding tissues [72]. Finally, the excessive production of NO by iNOS plays a critical role in septic shock. This condition is characterized by massive microvascular lesions, arteriolar vasodilatation, and hypotension. Symptoms are usually initiated by bacterial endotoxins. Nonetheless, decrease in blood pressure can occur as a result of excessive production of NO by iNOS induced in the vascular wall [73]. In mammals, nitrous oxide (NO) is produced by the calcium-calmodulinregulated constitutive isoenzymes eNOS (endothelial NOS) and nNOS (neuronal NOS), while the inducible isoform, iNOS, binds to calmodulin at physiologically significant concentrations producing NO free radicals as an immune defense mechanism (this is the direct cause of septic shock), and it may also play a role in autoimmune diseases. NOS-derived NO represents most of the NO produced in the vasculature and is associated with plasma membranes around cells including the membranes of red blood cells [71]. The structure and catalytic mechanisms of functional NOS are shown in Figure 5.

Conclusion
In conclusion, it is worthwhile to increase the search for potential plants that could supply bioactive compounds useful for the treatment of sickle cell disease. More so, concerted efforts are needed to further generate drugs to complement the already few drugs in existence, while taking into account the synergistic effect on these bioactives. This will help to standardize the administration of the bioactives to avoid any impediment to health due to overdose. Furthermore, it is necessary to exploit understanding of the interaction of these bioactives with the genes of sickle cell disease patient to increase our chances of getting a permanent solution to the disease. Geographical spread of these identified antisickling plants contributes to low levels of sickle cell patients where the potentials are known. More efforts should therefore be channeled toward increasing awareness about the plants.