The West African Sorghum Bicolor Leaf Sheath Extract Jobelyn ® and Its Diverse 3 Therapeutic Potentials

28 Background: 29 The West-African variety of Sorghum bicolor leaf sheath (SBLS) Jobelyn® is a natural 30 remedy, which has gained international recognition for its anti-anemic effect and energy 31 boosting qualities in debilitating diseases. The widespread use of traditional medicine in 32 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 December 2017 doi:10.20944/preprints201712.0036.v1 © 2017 by the author(s). Distributed under a Creative Commons CC BY license.


Introduction
Sorghum bicolor is an ancient plant that has been cultivated in North-eastern Africa for over 5000 years [1]. This is a cane like grass, up to 6 meters tall with large branched clusters of grains. The individual grains are around 3-4 mm in diameter and vary in colour from white, red, brown, yellow, purple, to black. The leaves resemble those of maize and grow rapidly. Sorghum grains of different cultivars are a rich source of phenolic compounds. These are natural bioactive compounds found in plants, which offer potential health benefits. Examples include secondary metabolites and antioxidants. As plants absorb the sunlight they produce high levels of oxygen and secondary metabolites by photosynthesis, which results in medicinal components being produced and stored in plant leaves. Flavonoids and phenolic acids are the most important group of secondary metabolites and bioactive compounds in plant [2]. In planta, antioxidant polyphenols have a range of roles including protection against herbivores and microbial infection, as allelopathic agents and UV protectants [3]. In humans, there is now increasing evidence on the role of phenolic compounds as protective dietary agents [4]. They are also considered to be natural and antioxidant substances capable of scavenging free superoxide radicals, supporting anti-aging mechanisms and reducing the risk of cancer. The Sorghum grain is a rich and diverse source of phenolic compounds, particularly phenolic acids and flavonoids. Similar to other cereals like maize and wheat, most of the phenols in the Sorghum grain are located in the bran, but differ in that Sorghum grain contains higher levels of phenolic compounds compared to most cereals and even fruits and vegetables depending on their variety [5]. Anthocyanidins are the primary flavonoids found in Sorghum grain and include apigeninidin, apigeninidin 5-glucoside, luteolinidin, luteolinidin 5-glucoside and 3-deoxyanthocyanidins, which is the most common [6]. The biosynthetic pathway that is responsible for the accumulation of 3-deoxyanthocyanins flavonoid compounds is controlled by the yellow seed1 (ys1) gene present in most varieties of Sorghum grains [7]. The uncommonly high levels of flavonoid accumulation in the Sorghum grain differentiates Sorghum from other grains and certainly makes it an interesting grain for healthy dietary applications or a source of bioactive compounds. More intriguing is the fact that the West African variety of Sorghum synthesizes exceptionally high amounts of 3-deoxyanthocyanins pigments in their non-grain tissue [8]. Compared to the Sorghum grain, the leaf sheath and glumes contribute to a greater biomass and may provide an easier and more cost-effective approach to obtain large quantities of stable 3-deoxyanthocyanins pigments.
The intensively coloured leaf sheaths of this wild variety of Sorghum found within the Nigeria flora has been formulated into a commercial pharmaceutical product under the name Jobelyn ® . The immediate interest in this herbal preparation originated from its unique phytochemical profile compared to other variants of Sorghum bicolor as it has been reported to contain significantly high amounts of anthocyanins. Anthocyanins are flavonoids believed to contribute to plant's high antioxidant capacity and provide overall disease protection in vivo through anti-oxidative mechanisms. With regards to the beneficial phytochemicals in medicinal plants and the shift towards natural products in pharmaceuticals, research on medicinal plants particularly is as important as the research on conventional drugs. In this review, we discuss the features of the West African Sorghum bicolor leaf sheath (SBLS) extract on the human health and its diverse therapeutic potentials:

Phytochemical Characterisation of Bioavailable Compounds in the Ethanolic Extract (JE)
To evaluate the qualitative and quantitative presence of various subclasses of flavonoids from the sbls ethanolic extract (JE) was dissolved in methanol (70%) at 10mg/mL, filtered in a vacuum and HPLC analyses was performed as previously described in [9]. Flavonoids were identified by comparison of HPLC retention times, UV Spectra and co-elution with authentic samples analysed in the same condition. The concentration of the measured peaks were determined from the calibration lines by linear regression analysis and the sample accuracy was estimated as the percentage of each measured concentration from the nominal (added) concentrations. Various subclasses of flavonoids, namely apigeninidin (3-deoxyanthocyanidin), luteolinidin (anthocyyanidin), Apigenin and luteolin (flavones), and naringenin (flavanone) were successfully identified and analysed from the ethanolic extract (JE) of the SBLS. Apigeninidin was the predominant compound in the ethanolic extract (JE), which accounted for 83.5% of the total amount of identified phenolic compounds. The proportions of luteolinidin, apigenin, luteolin and naringenin as a percentage of the total quantities of the 5 compounds were 1.0%, 13.7%, 1.5%, and 0.4%, respectively.

Isolation and characterisation of bioavailable compounds in the ethanolic extract (JE)
As previously described in [9] JE of the SBLS was subfractionated adopting a Medium Pressure Liquid Chromatography (MPLC) technique [10]. Fractions of 15 ml each were collected in test tubes and monitored by thin layer chromatography (TLC).
The fractions, which had the same TLC characteristics were bulked as appropriate and concentrated in vacuo to give 4 major fractions labelled JE-5 to JE-8 ( Figure 1). Fractions labelled J, JA, JB, JE and JE-5 to JE-8 were subjected to COX-1 and COX-2 inhibition bioassays and the fraction, JE-5, which had the least ratio of COX-2: COX-1 activity hence suggesting greater anti-inflammatory activity, was subjected to repeated column chromatography to produce two further fractions (monitored on a thin layer plate to be relatively pure components) which were concentrated in vacuo and labelled P8 and P9, respectively. The P8 and P9 were further analysed using an MDS Sciex API QStar Pulsar mass spectrometer with electrospray ionization (AB SCIEX, Foster City, California, USA). Identification of the compounds was based on matching UV-Vis spectra analysis, and MS data with authentic standards. The absorbance profiles of P8 and P9 revealed the presence of 2 peaks in P8 and a single peak in P9. One of the MS data of the compounds in P8 showed m/z 271 and was later identified as apigenin when matched against apigenin standard. The second compound in P8 and the single compound in P9 showed m/z 523 and 509 respectively and were later identified as dimeric flavonoid molecules differing from each other in one methyl group. Using both UV-Vis data and LC elution profiles as described in [9]. the two compounds were inferred to be 3-deoxyanthocyanidin dimers. The second compound in P8 was identified as 7-methoxyflavone-apigeninidin adduct while that in P9 was identified as flavone-apigeninidin adduct [11]. The amount of total flavonoids in the extracts P8 and P9 were measured spectrophotometrically as previously reported in. The quantity of apigenin (29.87±9.85 mg per g of dried leaf sheath) in P8 was approximately ten times that of 7-methoxyflavone-apigeninidin adduct which was 2.8 mg/g while that of flavone-apigeninidin adduct was 7.7 mg/g [12].

Sbls Extract Role in Anemia
The anti-anemic potential of the Sorghum bicolor leaf has been extensively reported [13,14]. In studies involving rats and rabbits made anemic by inoculation with trypanosoma brucei, findings demonstrated significant increases in the red cell count, hemoglobin and packed cell volume within 5 weeks of administration of the SBLS [14,15]. The anti-anemic potential use of the extract was also investigated in a randomized, open label clinical trial in women with preoperative anemia being prepared for myomectomy. A group of the subjects were given SBLS plus hematinics for 4 weeks and the other group was placed on hematinics alone. There was an increase in the red blood cell count, hemoglobin and packed cell volume in the subjects that took SBLS such that there was a 15% increase in subjects that met the criteria for surgery (packed cell volume of 36%). In this trial, there was no evidence of hepatotoxicity or nephrotoxicity. White blood cells and platelet counts were reduced though not significantly and were within the normal ranges [16]. In separate studies SBLS was used on HIV patients to assess its potentials in preventing anemia originating from HIV infection. This was a pilot study conducted on 10 HIV subjects and surprisingly results showed that SBLS not only increased hemoglobin levels but also the CD4 count in antiretroviral naive patients (Table 1). These findings suggest that SBLS could assist as a dietary supplement in the management of anaemic HIV-positive patients to improve anemia, primarily related to iron depletion due to chronic disease status. The early haematological changes observed in this pilot study could serve as basis for a clinical study that would describe the prevalence and characteristics of anemia in a larger cohort of HIV-infected patients.

Sbls Extract Role in Inflammation
In response to fungal infection Sorghum bicolor has been shown to produce a complex mixture of flavonoid secondary metabolites, which are structurally related compounds to 3-deoxyanthocyanidins, apigeninidin, luteolinidin, luteolinidin 5-methylether and apigeninidin 7-methylether. This family of compounds may function as plant pigments [17] or serve as phytoalexins [18]. Phytoalexins are low-molecular weight antimicrobial compounds produced by plants in response to infection or stress [19]. In the Sorghum leaf, these phytoalexins first appear in the cells that are being invaded, where they accumulate inclusions in the cytoplasm [20,21]. The inclusions migrate to the site of attempted penetration, becoming pigmented and losing their spherical shape. Ultimately they release their contents into the cytoplasm, killing the cell and restricting further development of the pathogen. The accumulation of 3-deoxyanthocyanidin phytoalexins is a site-specific response localized around the site of attempted fungal penetration [22] and prevents fungal proliferation through the tissue [23]. Similarly, inflammation in humans is a consequence of release of agents like leukotrienes; prostaglandins; thromboxane and other products of phospholipid metabolism; reactive oxygen species; cytokines like tumour necrotic factors, interleukins-1, -2, -4, -6 and -8; and chemokines like CCL5, CXCL4 and CCL3 by immunocompetent cells. The essence is to increase the blood flow and mobilize immunocompetent cells to sites of tissue damage as a result of invasive organisms, chemical, metabolic, radiation toxicity or physical damage. It is therefore a nonspecific protective event. However, uncontrolled stimulation of inflammation perturbs body hemodynamics, homeostasis and thermoregulation causing tissue damage and pains. The various combinations of these events will define most diseases.

The Effect of Crude and Purified Extracts of SBLS on Prostaglandins
Protaglandins E2 (PGE-2) is a principal mediator of inflammation. Selective cyclooxygenase-2 (COX-2) inhibitors reduce PGE-2 production to diminish inflammation. Sorghum bicolour leaf crude and purified extracts were tested for anti-inflammatory effects based on PGE-2 production from peripheral blood mononuclear cells (PBMC) in the presence of lipopolisacchraride (LPS). Ibuprofen, a non-selective COX-2 inhibitor and CAY10404, an inhibitor with greater COX-2 inhibitory activity compared to COX-1, were used as controls for the study. The crude extract JE5 derived from the ethanol extraction (JE) of SBLS had the greatest inhibitory activity with the least COX2IC50: COX1IC50 ratio and reduced production of PGE2. Of the purified compounds, P8 (purified from JE5) had greater inhibitory activity with the least COX2IC50: COX1IC50 ratio and reduced PGE-2 production from LPS-stimulated PBMC, which was dose dependent. COX-2, the enzyme that catalyses the synthesis of PGE-2 also shows enhanced expression in a number of cell types including circulating coenocytes, tissue macrophages, lymphocytes and neuronal cells during chronic inflammation [24,25]. COX-2-derived PGE-2 production is also dependent on oxidative stress [26] as reports suggest that ROS is an up-regulator of COX-2 expression and activates COX-2 to release PGE-2 [27]. Therefore, the role of SBLS in a) reducing PGE-2 production levels through inhibition of COX-2 expression and b) possibly reducing COX-2 expression through antioxidant capacity of the extract could be used as targets for therapeutic development in the management of chronic inflammation.

Anti-inflammatory effects of the SBLS extracts on cytokines and chemokines
A study on the effects of the West African Sorghum bicolor leaf sheath extracts on LPS-induced cytokine and PGE-2 release in human monocytes was performed and results revealed inhibition of LPS-induced release of cytokines (IL-1β; IL-6; TNFα; IL-8) and PGE-2 [28]. The ability of the SBLS to inhibit these cytokines involved in inflammatory recruitment as well as inhibiting PGE-2 production possibly through inhibition of COX-2 enzyme activity could be explained by the extract's antioxidant properties. We speculate that the antioxidant capacity of the SBLS has the ability to reduce oxidative stress environment thus reducing COX-2 expression in macrophages and other cells, which will in turn reduce the production of PGE-2 as well as limit the release of pro-inflammatory cytokines that assist with immunocompetent cell recruitment during the onset of infection. Further studies are indeed required to validate such theory, however, there seems to be a direct correlation between the SBLS extract's antioxidant capacity: oxidative stress: COX-2 expression: PGE-2 and pro-inflammatory cytokines production during LPSinduced inflammation. In the same study, interferon -alpha (IFN-), an antiviral cytokine showed increased expression by 12-fold following treatment with SBLS extract [29]. There seems to be a correlation between high IFN-production capacities and low HIV viral loads as well as high CD4 cell counts and lack of opportunistic infections [30,31]. This effect can be related to the direct anti-viral role of IFN-on infected HIV cells. LPS-treated mononuclear cells after exposure with the SBLS extract showed a 12-fold increase in the production of IFN-suggesting that the extract may contribute not only in suppressing chronic inflammation originating from HIV infection but possibly in reducing the viral load and increasing CD4 count through IFN-stimulation.

Antioxidant Activities of the SBLS by ORAC
Studies carried out at the Brunswick Laboratories (South borough, MA, United States) have shown that SBLS ranks among the highest Oxygen Radical Absorbance Capacity (ORAC) of all food plants with total ORAC FN of 37,622 µmole TE/g of the dry powder (Figure 1). This variety of Sorghum was found to contain significant levels of antioxidant activities against peroxyl radicals (3,549 µmole TE/g), peroxylnitrite (269 µmole TE/g), hydroxyl radicals (18,387 µmole TE/g), superoxide ions 11,417 µmole TE/g) and singlet oxygen (4,000 µmole TE/g). In fact, the antioxidant capabilities of SBLS extract is greater than that of the anthocyanin-rich acai berry indicating its exceptionally high antioxidant potential (Figure 2). According to these findings it seems that specific varieties of Sorghum like the one described here could positively assist in reducing the severity of several health conditions, as the antioxidant activity is able to contribute to cellular adjustments to oxidative stress.

SBLS Cellular Antioxidant Protection
Apart from ORAC measurements, SBLS was also submitted to relative antioxidant protection capacity analysis, which was carried out by Natural Immune Systems Laboratories (NIS), Oregon, USA. The extract was digested after exposure to gastric acid with pepsin, bile and pancreatic enzymes. Some of the products were further fermented by a blend of common probiotic bacteria for 24 hours. The products were centrifuged and filtrated in a spin column to remove the digestive enzymes. The cellular antioxidant protection (CAP-e) bioassay was performed and results showed that in vitro digestion though reduced the total antioxidant capacity of SBLS, it increased the cellular antioxidant uptake and protection from free radical damage in erythrocytes. In separate animal experiments carried out by [32], SBLS was shown to tolerate oxidative stress as oral administration of the extract (50-200 mg/kg) in rats decreased the levels of malondihydehide (MDA) in the serum suggesting antioxidant property. This variety of Sorghum also elevated the concentrations of reduced glutathione (GSH) in inflammation exudates indicating free radical scavenging activity. Furthermore, it significantly inhibited red blood cells lysis caused by hypotonic medium, suggesting membrane-stabilising property [33].

The Impact of SBLS extract on Aging Protection
Analysis at the Brunswick Laboratories showed that the SBLS causes significant inhibition of collagenase, elastase and protein glycation. Such properties in Sorghum should promote healthy skin and retard aging. Reported collagenase inhibition had 15fold potency to that of vitamin C and 30-fold potency of ferrulic acid while elastic inhibition showed a 22-fold potency to that of vitamin C and 8-fold potency of ferrulic acid ( Table 2). The ongoing attacks of free radicals during HIV infection damages the elastic and collagen fibres [34]. The skin protects itself against these impairments with the aid of radical scavengers. Effective elastase and collagenase inhibition reported with SBLS could be related to its exceptional antioxidant ability although further experiments are required in order to confirm these events. Over the course of a lifetime, significant chronic metabolism disruption may occur when consumption of micronutrient is below the current recommended dietary allowance but above the level that causes acute metabolic symptoms. When, a component of the metabolic network is inadequate, there may be a variety of setbacks in metabolism [35]. As a result, dietary supplements, most commonly multivitamins and minerals are given to fill the nutritional gaps. In HIV infected persons, low serum concentrations of vitamins and nutrients has been associated with an increased risk of HIV disease progression and mortality [36]. In 1996, highly active antiretroviral therapy (HAART) became the new standard for HIV treatment. HAART restores the immunologic function [37], but does not eliminate weight loss, micronutrient deficiency and wasting syndrome [38], which are strong independent predictors of mortality [39].

Mineral Contents in SBLS
SBLS's nutritional contents were assessed by GMP Laboratories of America, Inc., CA, USA. SBLS was proven to be a good source of minerals like calcium (35.2% RDA), magnesium (59.03% RDA), sodium (95.83% RDA), selenium (28.0% RDA), Zinc (13.62% RDA) and copper (127.77% RDA) ( Table 3). More interestingly, it was observed that 100g of this extract provides around 285% of the daily recommended levels of iron (Table 3). HIV infection increases the release of pro-oxidants, cytokines and ROS leading to increased utilization and excretion of proteins and micro minerals such as zinc, iron, selenium manganese and copper. This can result in an imbalance between pro-oxidants and antioxidants which may lead to increased oxidative stress and cause further damage to human cells, proteins and enzymes, thus accelerating HIV replication and mortality of the patient [40,41]. Therefore, vitamins and minerals are crucial in reducing HIV disease progression especially as the cost of effective strategy for reducing HIV disease progression, improving nutritional status and possibly reducing vertical transmission of HIV in low-income countries is high. Anemia, in particular is a common clinical finding in HIV-infected patients and iron deficiency or maldistribution may contribute to the development of low haemoglobin levels. Due to the effects of inflammation in HIV patients, iron is diverted from circulation into the reticulo-endothelial system and other storage sites. Iron maldistribution reduces iron availability in the circulation, increase susceptibility to opportunistic infections and accelerate disease progression [42]. Treating severe anemia in HIV-infected patients is critical because recovery from anemia is associated with increased length of survival, however, excess of iron in the storage sites is associated with an increased viral replication [43]. The SBLS contains large amounts of iron and as such it is used in the treatment of anemia. However, most of the excess iron from SBLS is cheated by the polyphenols in the Sorghum extract and as a result the excess iron is not presented to Observational studies have shown low serum concentration of several micronutrients including selenium and zinc to be associated with low CD4 cell counts, advanced HIV-related disease, faster disease progression or HIV-related mortality [44,45]. Similarly, anemia due to deficiency in iron is more common and more severe with advanced HIV disease progression [46].

Vitamin Contents in SBLS
The West African Sorghum also contains substantial amounts of vital vitamin B 12 (Table 3). Vitamin B 12 , is a water soluble vitamin that acts as a cofactor in the conversion of homocysteine to methionine and methyl tetrahydrofolate to tetrahydrofolate. Polyglutamate is further added tetrahydrofolate to prevent the diffusion of folates out of the cell. Folates are essential for the building blocks of DNA in rapidly regenerating organs like the production of blood while excess homocysteine is known to cause endothelial damage and therefore cardiovascular disease. It is well documented that the body cannot produce B 12 and that it can only be sourced from animal foods or in the form of B 12 vitamin supplement. Deficiency usually results in anemia, impaired brain function, and symptoms of mental disorder [47]. The only good food sources of B 12 are primarily animal foods like meat, fish and eggs producing 2.8 µg/100 1.95 µg/100; 4.15 µg/100 of B12, respectively. Interestingly, SBLS is a good source of B 12 producing 0.83 µg/100 g. This is the first time a plant product has been reported to contain significant amounts (34%) of vitamin B 12 . Low levels of B 12 have been associated with a reduction in CD4 + T cell count in HIV-infected patients [48]. There have also been claims that nutrient supplementation could restore immune function and boost CD4 + T cell counts in people with early stages of HIV infection [49]. Vitamin B 12 could potentially be a useful antioxidant as it directs reaction with reactive oxygen species and through glutathione sparing effect, can modify signaling molecules to decrease oxidative stress and increase total antioxidant capacity [50]. However, such effects are yet to be evaluated in HIV-infected persons.

Fatty Acids Contents in SBLS
SBLS provides an impressive 1:3 ratio of Omega-3 to that of Omega-6 ( Table 3) and as population studies indicate, our diet should contain no more than three parts of Omega-6 to one part of Omega-3, which is ideal for the heart's health [51]. Omega-6 fats have pro-inflammatory effects while omega-3 fats have antiinflammatory effects. The ratio of 3:1 will reduce the chronic inflammation that most people recognize as the root cause of many chronic diseases, including diabetes. Omega-3 fatty acids play an important role in every cell in the body. Omega-3 makes up cell membrane and helps in maintaining membrane fluidity, keeps the nervous system functioning by up regulating brain derived neurotropic factor (BDNF) and modulating neurotransmitters reuptake, degradation, synthesis and anti-apoptotic effects once it gets converted into fatty acids eicosapentaenoic acid (EPA) and later docosahexaenoic acid (DHA). However, Omega-6 inhibits the conversion of Omega-3 into DHA and EPA, therefore, the adequate ratio of 1:2 or 1:3 is usually required to allow conversion of adequate amounts of Omega-3. The Omega balance is critical as Omega-3 and Omega-6 compete for absorption into the cells, and an excess of dietary Omega-6 will result in too few of Omega-6 being incorporated into cell membranes, from where they exert their essential effects [52]. Earlier in the HIV epidemic the observation of increased oxidative stress and elevated cytokines led to a dietary intervention study on whether supplementation with Omega-3 fatty acids would decrease cytokines or markers of inflammation. Omega 3 showed beneficial effects on systemic inflammation mediated my multiple mechanisms including a decrease in the activation of NFkB by inflammatory stimuli. In a pro-oxidative stress environment, ROS has the ability as start a cascade as second messengers for the activation of NFkB, which increase the replication of HIV, because this factor controls the transcription for the HIV viral replication [53]. Thus, the right supplementation of Omega 3 and 6 fatty acids would prove beneficial in a pro-oxidative stress environment as experienced in HIV infection.

The Role of Sbls Extract in Neurocognitive Deficits
SBLS may exert a beneficial role in the treatment of neuropsychiatric symptoms associated with depression, memory deteriorations and psychotic manifestations. Preclinical studies have shown that the extract ameliorated the characteristic feature of immobility in mice subjected to 'forced swimming test' (FST), a well recognized animal model of depression. Mice subjected to FST experienced a period of immobility, which indicates a depressive-like behavior and antidepressant drugs are known to decrease the period of immobility. These findings have provided the impetus for the proposed clinical trials of the Sorghum for treatment of psychotic ailment in Yaba psychiatric hospital Lagos, Nigeria. Results from the clinical study showed that SBLS suppresses amphetamine-induced stereotypy and antagonises hyperlocommotion due to amphetamine injection, which indicate antipsychotic potentials. More importantly, antipsychoticlike activity of the extract was devoid of the adverse effect of cataleptic behavior [54]. In another neurological study conducted by [54]. SBLS was shown to reverse memory impairment induced by scopolamine in mice. Interestingly, the extract was found to attenuate memory deficits in mice subjected to unpredictable chronic mild stress (UCMS), which is an animal model that mimics the pathological changes seen in humans exposed to stress on daily basis [55].
Inhibition of inflammatory cytokines as previously reported by [56] may serve as an important target for development of drugs with potential efficacy against neurological disorders. A recent in vitro study showed that SBLS extract inhibited infiltrations of WBC, release of inflammatory mediators, and formation of free radicals [57], which are the major culprits involved in neurodegenerative diseases. Furthermore, Oyinbo et al. [50] shows that the SBLS extract demonstrated anti-neuroinflammation and reduced the death of astrocytes [58]. Charles et al demonstrates that Jobelyn ® Supplement Lowered Neuronal Degeneration [59]. Omorogbe et al. [52] also shows that Jobelyn attenuates inflammatory responses induced by CFA in mice via inhibition of oxidative stress and release of inflammatory cytokines, [60].
CFA-induced nociception, sensorimotor deficits and depressive-like symptom suggests it might improve the quality of life of patients with arthritic conditions. Specific phytochemicals such as luteolin, naringenin, and apigenin found in this extract have previously demonstrated anti-inflammation activity in cultured cells [61]. Luteolin, in particular, was shown to inhibit nuclear factor-κB (NF-κB) signaling in immune cells, which supports its therapeutic efficacy in conditions associated with chronic inflammation [62].

Hiv and Immunity
The sub-Saharan Africa is home to more than two-thirds (69%) of people living with HIV (World Health Organization (WHO)). Interestingly, it is also a region suitable for the cultivation of a unique variety of Sorghum bicolor. The SBLS extract has gained interest because of its antioxidant capability. Antioxidants are specific compounds that protect human, animal and plants against the damaging effects of free radicals or reactive oxygen species. Imbalance between antioxidants and free radicals results in oxidative stress, which may lead to cellular damage [63]. It has been observed that perturbations in antioxidant defense systems, and consequently redox imbalance, are present in many tissues of HIV-infected patients. Moreover, the level of production of free radical species in HIV infected individuals receiving highly active antiretroviral drugs (HAART) was reported to be higher than those who harbor HIV infection without receiving any treatment or normal and healthy subjects [56]. In HIV infection, the deficiency of total antioxidant status might markedly increase oxidative stress. The increase in reactive oxygen species may enhance viral replication by activating nuclear transcription factors, which ultimately could lead to viral gene expression [57]. Enhanced oxidative stress also occurs after initiating HAART due to persistent tumor necrosis factor-α (TNF-α) activation in HIV-infected patients [58]. Therefore, the antioxidant activity of SBLS may positively act on individual's immunological status and decrease HIV replication. Further, this variety of Sorghum could reduce cellular damage due to oxidative stress originating from HAART treatment. HIV infection causes chronic inflammation, which is beneficial to HIV replication but detrimental for human host leading to senescence of the immune system. The inflammation molecule PGE-2 levels are markedly enhanced during HIV infection as a result of chronic inflammation. COX-2, the enzyme that catalyses the synthesis of PGE-2 also shows enhanced expression in a number of cell types including circulating monocytes, tissue macrophages, lymphocytes and neuronal cells during HIV infection [61]. HIV infection increases the release of pro-oxidants, cytokines and ROS leading to increased utilisation and excretion of proteins and micro minerals such as zinc, iron, selenium manganese and copper. This can result in an imbalance between pro-oxidants and antioxidants which may lead to increased oxidative stress and cause further damage to human cells, proteins and enzymes, thus accelerating HIV replication and mortality of the patient [35,36]. Therefore, vitamins and minerals are crucial in reducing HIV disease progression especially as the cost of effective strategy for reducing HIV disease progression, improving nutritional status and possibly reducing vertical transmission of HIV in low-income countries is high. Anemia, in particular is a common clinical finding in HIV-infected patients and iron deficiency or maldistribution may contribute to the development of low haemoglobin levels. Owing to the effects of inflammation in HIV patients, iron is diverted from circulation into the reticulo-endothelial system and other storage sites. Iron maldistribution reduces iron availability in the circulation, increase susceptibility to opportunistic infections and accelerate disease progression. Such observation suggests that the use of SBLS can help improve hemoglobin concentrations and quality of life in anemic HIV-positive patients as well as reduce several micronutrients deficiencies, which are common in advanced HIV disease.
Multiple studies have shown increased levels of oxidative stress after HIV infection in the Central Nervous System (CNS) [62] and have even correlated increased levels of oxidative stress with the severity of the disease. Neurons are exposed to extensive amounts of oxidative species with the reduced concentration of endogenous antioxidant defenses such as glutathione during HIV neuropathogenesis. Despite the advent of combination of HAART, the CNS complications associated with HIV infection still present a great challenge in the management of neuroAIDS. As survival with chronic HIV-1 infection improves, due to the use of HAART, the number of people harboring the virus in their CNS increases because the brain is largely impervious to HAART. Moreover, it is becoming clear that the brain is an important reservoir for the virus, and that neurodegenerative and neuroinflammatory changes may continue despite HAART and thus, HAND remains a significant independent risk factor for AIDS mortality. Several studies carried out to assess the potentials of SBLS in treating indicators of HIV infection such as chronic inflammation, oxidative stress, HAND, anemia and other micronutrient deficiencies has led to speculate that SBLS extract may contribute to many aspects in the management of HIV infection. As previously described in SBLS role in anemia, a pilot study on 10 HIV subjects was conducted and results showed that SBLS not only increased hemoglobin levels but the CD4 count in antiretroviral naive patients (Table 1). This encouraged a randomised controlled clinical trial in HIV patients on antiretroviral therapy (HAART). Findings from the trial ( Figure  3) show significant increase in CD4 count for HIV patients with initial CD4 count >350/µl and placed on SBLS alone and also significant increase in the CD4 count for patients with initial CD4 count <350/µl placed on SBLS + antiretroviral drugs (AVRs) compared to the CD4 count for patients with initial CD4 count <350/µl placed on antiretroviral drugs alone [63]. Such achievements within 3 months of treatment suggests that the SBLS extract should be evaluated in a long-term clinical disease progression to establish if the supplementary or alternative treatment with SBLS for HIV/Aids patients can serve as an immune system booster and a probable "virus-cidal" factor.

Toxicology
A toxicological evaluation of SBLS on both the acute and shortterm chronic administration in mice was performed. Acute toxicity studies revealed that the LD50 values for oral and intraperitoneal routes were 215.06mg/kg and 193.37mg/k, respectively. SBLS would produce a lethal effect via oral route in about 10 times the maximum recommended dose per day, which are 6 capsules. Behavioral changes including reduced motility and sedation were observed at high doses. Histopathological examination of the liver, lung, spleen and kidney tissues did not indicate any organ damage. At toxic levels, however, there was some degree of congestion in the lungs, liver, spleen and kidney tissues. Short-term chronic studies using sub-lethal administered over 14 days showed no serious behavioral abnormalities or histopathological changes in the lungs, liver, spleen and the kidneys [64].

Conclusion
In summary, it is widely reported that increased levels of oxidative stress and depletion of endogenous antioxidant defense systems are associated with HIV pathology. It is also believed that increased oxidative stress triggers inflammation changes and immunosuppressant that contribute to the severity and symptomatologies of the disease. Therefore, antioxidant supplementation might be a better option in reducing the severity of the devastating effects of HIV on the immune system and body organs including the brain. The standardized dried powder from the West African SBLS described in this review has been shown to possess a unique combination of phytochemicals known to exhibit a wide range of biological activities. Photochemical antioxidants found in SBLS may assist in decreasing oxidative stress-mediated cellular damage and improve immune functions. The nutritional contribution of SBLS will increase hemoglobin concentration and micronutrients deficiencies. While the antiinflammatory effects of the extract will improve neurocognitive activities in psychiatric patients as well as address the uncontrolled inflammation that causes tissue damage and pains in HIV-infected individuals. More importantly, SBLS will promote increase in CD4 count as it targets chronic inflammation, oxidative stress and the destruction of the adaptive immune response (CD4 T cells), which are the most significant factors in the pathogenesis of HIV-AIDS. There are no reported cases of adverse events or allergic reactions during the use of this extract over the years and acute and chronic studies revealed that SBLS was well tolerated by laboratory animals. The high safety profile of this extract coupled with its potent antioxidant properties makes this unique Sorghum-based natural product a potential therapeutic target in the management of HIV-AIDS, chronic inflammatory conditions and anemia.