Potential of natural astaxanthin in alleviating the risk of cytokine storm and improve health in COVID-19: A scoping review

Background: Natural astaxanthin as a potent anti-oxidant and broad-spectrum anti-inflammatory bioactive molecule plays important role in modulating the immune response, speculated to be a potential supplement to alleviate cytokine release storm in COVID-19. Objective: Review of published literature to summarize the rationale for possible benefits of natural astaxanthin to support COVID-19 patients. Methods: Retrieved relevant literature from electronic databases including Google scholar, PubMed, Scopus, etc. and reviewed following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Statement. We adapted the article as scoping review. Results: Cytokine release syndrome (CRS) is reported as a common feature in COVID-19, which can lead to potentially fatal, hyper-inflammatory acute respiratory distress condition, diagnosed with elevated serum level of pro-inflammatory cytokines like IL-6, CRP, etc. that positively correlated with disease severity. Anti-inflammatory drug, like tocilizumab, etc. are under clinical trials as anti-CRS therapy. Astaxanthin can potentially alleviate CRS by regulating inflammatory cytokines by inhibiting the activities of NF-kB, TNF-α, JAK/STAT-3, etc. Available pre-clinical and clinical trials data support its excellent safety, and potential therapeutic and health benefits. Conclusions: Natural astaxanthin has tremendous potential as co-adjunctive supplement, desiring necessary clinical supports on its efficacy and beneficial against COVID-19. as a potential co-adjunctive supplement for based on understanding of its putative pathogenesis and clinical in signaling including transcription factor nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) and mitogen activated protein kinases (MAPKs), the production of a variety of inflammatory cytokines (12-13). The excessive of various proinflammatory cytokines mainly including TNF-α, IL-6, IL-1β, IL-12 and IL-8 rapidly initiate a systemic response leading to simulation of adaptive immune response and cytokine storm resulting the acute cellular injury to form sepsis or ALI (6, 12-13). Applications of anti-inflammatory / anti-oxidants candidates to intervene the excessive production of cytokines, including IL6 and TNF-α can be a promising strategy in prevention and treatment of COVID-19 induced ARDS related ALI and sepsis 26, 35). With its proven anti-inflammatory and antioxidant activities nASX may be considered as a promising candidate to be trial against COVID-19. Previous studies reported the potential applications of nASX in the treatment of avian influenza virus infection and stated that nASX can exhibit significant benefit in a combinatorial drug approach along with antiviral compound to be used for the treatment (12-13). Although, detail clinical result is lacking at this moment, pre-clinical trials in animal model was demonstrated with encouraging results. Cai et al . (12) demonstrated that nASX can prevent LPS-induced ALI/ARDS and sepsis by inhibiting the activation of pro- inflammatory signaling pathway of MPAK/NF-κB. Experimental results demonstrated that nASX significantly inhibits the production IL6 and TNF-α. The results showed that treatment with nASX not only significantly reduced the death rate due to sepsis but also exerted protective effects on lung tissues. nASX also wall and lessened the decline in the of pulmonary alveoli in

Study design, search strategy and selection criteria Relevant information on therapeutic and health benefits of astaxanthin, pathogenesis and clinical presentation of COVID-19 were retrieved from Google scholar, PubMed, Scopus, etc. till April 23, 2020. A scoping review of the relevant literature was conducted to summarize the rationale for anticipating nASX as a potential co-adjunctive supplement for COVID-19 based on understanding of its putative pathogenesis and clinical presentations in symptomatic cases following the guidelines stated by Arksey and O'Malley (16). The rationale for potential therapeutic and other health benefits of nASX relevant to COVID-19 were reviewed and reported based on available relevant scientific information following the guidelines of PRISMA (17). No ethical approval was obtained as all relevant information were retrieved from publicly available literature through multiple databases and no primary data was collected or generated during the review process. The studies included with relevant literature describing the clinical characteristics, epidemiology, prevention or control or treatment of COVID-19.
Literature on astaxanthin with relevant information were also retrieved. Here we use specific search phrase like "astaxanthin ARDS", "astaxanthin ALI", "astaxanthin antivirals", "astaxanthin antiinflammatory", etc. We included all relevant scientific publications written in English for this review.
Screening, data extraction and summarizing the findings Eventually after screening and removing duplication, 86 articles were selected for the intended review. Selected articles were categorically reviewed and extracted data were recorded, broadly under the domains: epidemiology, clinical manifestation and diagnosis, and prevention, control and treatment for COVID-19. In case of articles related to nASX were categorized broadly: anti-COVID-19, anti-inflammatory, anti-oxidant, immunomodulator and protection against CRS, ARDS, ALI.
Based on the principal research objective of the study, articles were classified as mentioned above.
Information based on experimental findings and conclusions as stated in the relevant articles supporting as evidence were reported in the review result. All findings and statements regarding potential use of nASX as adjunctive supplement in COVID-19 mentioned herein this review are purely based on published literature as listed in the references. There is no available study related to nASX against coronavirus or SARS-CoV-2 or COVID-19, which we consider as the major limitation of this study. However, presence of multiple pre-clinical and human studies reporting indirect evidences supporting possible therapeutic implications of nASX as anticipated, are the major strength of this review.

Results
The search result revealed that pro-inflammatory cytokine storm is a common feature among severe COVID-19 infection. The reports suggested that CRS mediated hyperinflammatory ARDS, Sepsis, ALI and multiorgan failure are the major cause of death. We included total 86 reports, which were categorized into the selected research domains (S1, Supplementary file). 40.6% of the total articles were related to COVID-19. About 25.5% of the reports were related to the epidemiology, clinical and pathological features including diagnosis of COVID-19. 15.1% of the reports mentioned about possible applications of Chinese Traditional Medicines (CTM) and other natural compound in combination therapy and repurposing antivirals and anti-inflammatory drugs for possible treatment, prevention or control. None of the search result reported any studies directly related to the effectiveness of nASX in treatment of COVID-19, which is understandable as the outbreak is recent our understanding in it is still evolving. However, 59.4% of the total retrieved articles stated potential therapeutic and health benefits of nASX as anti-inflammatory, antioxidant and immunomodulator, which altogether exerts protective activities against cytokine storm and associated diseases.
Available reports suggested that elevated serum concentration of cytokine IL-6 and other 6 inflammatory cytokines are hallmark of CRS, which is common in COVID-19 infection (2)(3)6).
Elevated level of serum IL-6 and CRP that correlate with respiratory failure, ARDS and other clinical adversities, are the biomarkers of severe COVID-19 infection (2-3, 6). Liu et al. (2) reported increased cytokine levels in 69 severe COVID-19 inpatients of median age of 56 years, elevated level of CRP, IL-6, LDH and ferritin were more prominent and significantly corelated to severe immune damage to the lung and multiple other organs. Mild variation in the serum level of IL-2, IL-4, IL-10, IFN-γ, and TNF-α were reported among severe and non-severe patients (2). Intriguingly, based on the comparison of IL-6 level in 30 patients before and after treatment, Liu et al. (2) reported that the IL-6 level was significantly reduced with disease improvement as revealed by CT assessment, while persistent increase of IL-6 was recorded with disease progression. With the remission of disease, the IL-6 level along with D-dimer, CRP and LDH levels were also reported to decrease significantly (2). These observations clearly suggest that lymphocytopenia and CRS are associated with the severity of COVID-19.
In another study, Liu et al. (23) reported that hypoalbuminemia, lymphopenia, decreased percentage of lymphocytes and neutrophils, elevated CRP and LDH, and decreased CD8+ T cell count were common abnormalities among the severe COVID-19 patients developed ARDS.
The CRS triggers a violent attack by the immune system to the body, causing acute lung injury (ALI), RNAaemia, acute cardiac injury, sepsis and multiple organ failure, and finally lead to death in severe cases of SARS-CoV-2 infection (2-3, 5-6, 10, 18-24). The CRS results because of an accentuated immune response triggered by SARS-CoV-2 infection in severe COVID-19 patients, diagnosed with lymphocytopenia, a differential diagnostic criterion for COVID-19 (2-3, 5-6, 22). The CRS caused by SARS-CoV-2 was reported to mediated by leukocytes other than T cells, which may be overcome by blocking IL-1, IL-6 and TNFα (2,6,(22)(23). Hence, destroying the immune evasion of SARS-CoV-2 is imperative in its treatment and specific drug development (6,22). Based on understanding of COVID-19 triggered CRS, it is pivotal to treat the infection (with appropriate anti-viral therapy), and it may be even more critical to treat the host with appropriate therapeutic modalities to dampen the overly exuberant immune responses responsible for CRS-induced multi-organ dysfunction syndrome (MODS) and death (2-3, 5-6, 22-27). Zhou et al. (27) from a retrospective cohort study of 119 hospitalized patients reported that older age with comorbidities and elevated level of d-dimer were at higher risk of COVID-19 fatality. Besides elevated level of IL-6, high-sensitivity cardiac troponin-I, LDH, and lymphopenia in severe COVID-19 illness, occurrences of sepsis was reported to be common complications (27) were also reported under clinical trials (26)(27)29). Zhang et al. (36) proposed melatonin as an adjuvant for COVID-19 based on its known anti-inflammatory and anti-oxidant properties. Of note, the prognosis and recovery from critical hyperinflammation stage of illness is very poor, and prompt recognition and application of preventive therapy may yield better results (6,(26)(27)(34)(35). We anticipated that a range of natural anti-inflammatory and antioxidants as supplements will offer a window of quick recovery of patients by reducing post treatment side-effects.
While the anti-viral approaches and vaccines being considered as immediate countermeasures are unavailable and undergoing intense researches, there may be needed to consider treatment regimens from analogous disease patterns (6,22,26,(29)(30)(31)(34)(35)(36). Matching clinical dispositions may be considered in efforts to develop therapeutic interventions. Additionally, dire outcomes of illness may be overcome with adjunctive measures that do not necessarily cure the underlying disease (6,22,(26)(27)(28)(29)(30)(31). Repurposing an old malaria drug chloroquine or hydroxychloroquine was reported to show potent activity against COVID-19 and reported to use as preventive medicine to treat COVID-19 patients and medical professionals (25,(27)(28)(30)(31). More than a supportive care, an adjunctive measure may assist severe COVID-19 patients health (31). Based on supportive evidences there are multiple natural compound which may have advantages to use as adjunctive countermeasures for rapid recovery of COVID-19 patients (6,26). Herein, we outline the strong evidences of natural astaxanthin as a potent anti-inflammatory and anti-oxidant compound supporting its potential beneficial application as adjunctive in patients with COVID-19.

Rationale for use of astaxanthin as potential adjunctive supplement
Microalgae Haematococcus pluvialis derived natural astaxanthin (nASX) is a unique bioactive molecule with diverse clinical and health benefits (10). Previous studies reported that nASX, which the microalga H. pluvialis accumulated in response to adverse conditions, can play major roles in regulating immunity and disease etiology, suggesting its wide array of potential therapeutic and nutritional supports as antioxidant, immune-booster, anti-inflammatory, neuroprotective, immunomodulatory, anti-proliferative, anti-aging, anti-bacterial, anti-apoptotic, etc., (8-11, 14-15, 37-45). Figure 2, represents an outline of H.pluvialis biomass generation, accumulation of astaxanthin and recovery.
The pathogenesis of SARS-COV-2 is likely involve four major steps: The renin-angiotensin (RAS) 9 signaling pathway, oxidative stress and cell death, cytokine release storm and endothelial dysfunction (46). Based on available published information, Table 1 lists some of the possible roles of nASX for the clinical characteristics of diseases associated with the increased risk of COVID-19 fatalities and how functionally nASX is related to some of them. As there are no direct evidences of applying nASX against COVID-19, herein, we summarize ( Table 2)  increase of Angiotensin II (ANG-II) in COVID-19 severe patients which strongly corelated with multiple organ failure. It is well documented that increased ROS and resulting oxidative stress in ANG-II associated diseases results from NADPH oxidases, mitochondrial dysfunction and inflammation, including reduction of endogenous antioxidants (37,42), which commonly occurs as ANG-II pathological conditions, such as hypertension, atherosclerosis, and diabetes (47). Redox-sensitive transcription factors, such as NF-kB and activator protein-1 were reported to induce the expression of pro-inflammatory cytokines associated with ANG-II related disease, such as cardiovascular disease (48). Furthermore, ANG-II induced ROS stimulate matrix metalloproteinase (MMPs) that regulate the extracellular matrix and vascular remodeling (49). Under normal conditions, the ROS generation is balanced by the rate of elimination through endogenous and dietary antioxidants, which greatly imbalanced by elevated ROS generation with the dysregulation of ANG-II signaling and contributes to cardiovascular dysfunction including heart failure, stroke, hypertension, etc. (42,49). This may also relate the potential COVID-19 high case fatality rate for patients with chronic comorbidities like hypertension (6%) and cardiovascular disease (10.5%) (3,23,50).
Oxidative stress is an important molecular mechanism underlying pulmonary fibrosis. Accumulated evidences suggest that ROS released by activated phagocytes causes lung inflammatory process, leading to lung injury (51)(52). Song et al. (52) demonstrated the anti-oxidative and anti-fibrotic effect of nASX in mouse model in vivo and in vitro and reported that nASX blocks ROS generation and dosedependent apoptosis in alveolar epithelial cells type II, as characterized by inhibition of cytochrome-c (Cyt C) release and the activation caspase-9, caspase-3, Nrf-2 and other cytoprotective genes. With its unique molecular structure nASX stretches through the bilayer membrane, provides resilient protection against oxidative stress (9). Unlike most antioxidants, which work either in the inner (e.g., vitamin E and β-carotene) or the outer side of the membrane (e.g., vitamin C), nASX can scavenge and quench ROS and free radicals (superoxide anion, hydrogen peroxide, singlet oxygen, etc.) in both the inner and outer layers of the cellular membrane (9-10, 38).
It was well documented from studies in animal models that nASX shows significant decrease of important mediators of inflammation including, IL-6, IL-1b, COX-2, CRP, PGE-2, iNOS, and nitric oxide 11 (NO) (12-14, 37-40, 42-46). Research efforts currently have been largely focused on innate immune system and conceptually viewed sepsis and ALI as syndrome of hyperinflammation (2,(5)(6)(18)(19)(20)(21). Evidences from lipopolysaccharide (LPS)-induced acute lung injury and sepsis (12), suggest the activation of proinflammatory signaling pathways, including transcription factor nuclear factor kappa-light chainenhancer of activated B cells (NF-κB) and mitogen activated protein kinases (MAPKs), triggering the production of a variety of inflammatory cytokines (12)(13). The excessive release of various proinflammatory cytokines mainly including TNF-α, IL-6, IL-1β, IL-12 and IL-8 rapidly initiate a systemic inflammatory response leading to simulation of adaptive immune response and cytokine storm resulting the acute cellular injury to form sepsis or ALI (6,(12)(13). Applications of anti-inflammatory / anti-oxidants candidates to intervene the excessive production of cytokines, including IL6 and TNF-α can be a promising strategy in prevention and treatment of COVID-19 induced ARDS related ALI and sepsis (2-6, 26, 35). With its proven anti-inflammatory and antioxidant activities nASX may be the production IL6 and TNF-α. The results showed that treatment with nASX not only significantly reduced the death rate due to sepsis but also exerted protective effects on lung tissues. nASX treatment also decreased alveolar wall swelling and lessened the decline in the number of pulmonary alveoli in lung tissue (12).
With the absence of definitive treatment for acute COVID-19 infection, ARDS/ALI are leading with high case fatality rate (2-3, 5-6, 27, 34). Sepsis syndrome is the most frequent causes of ARDS, leading to increased lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration and respiratory failure causing sudden rise in death toll, as indicated by current pandemic worldwide (21,35,(63)(64)(65). During acute COVID-19 treatment in intensive care unit, high dose of vitamin C was suggested as a "rescue therapy", along with high pressure flow nasal oxygen (63). Administration of anti-inflammatory substance to potentially avert the existing ARDS condition is not known (35). Taken together, we speculate that use of nASX as adjunctive supplement in the treatment of COVID-19 may exert dual purpose of both as anti-oxidant and anti-inflammatory compound with beneficial outcome of reduce fatality and rapid recovery.

Astaxanthin as immunomodulatory and immune booster
Cytokine storms, which rapidly cause life-threatening single or multiple organ failure are, considered to be one the notable causes of death for severe COVID-19. SARS-CoV-2 infection rapidly activate pathogenic T cells and produce granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-6. suggest the importance to block CRS at the right window of time to initiate anti-inflammatory therapy to contain COVID-19 death rate (35).
The immune boosting activities of nASX have been well documented and supported by pre-clinical and clinical trials including human models (9,14,40,45,(68)(69)(70). As a potent antioxidant and antiinflammatory molecule with known immunomodulatory activities nASX may plays a pivotal role in modulating the immune response in COVID-19. Park et al. (14) reported that dietary supplement of nASX stimulate mitogen-induced lymphocyte proliferation, increase natural killer cell cytotoxicity and the delayed-type hypersensitivity response, and increase the number of total T and B cells in the peripheral blood. It was also reported that astaxanthin is absorbed after oral administration subsequently utilized by blood leukocyte subcellular organelles, mostly by the mitochondria (14, 68).
Notably, dramatic decreased of DNA damage biomarker (plasma 8-OHdG), along with significant reduction of plasma C-reactive protein concentrations were reported (14). Lin et al. (70) reported that nASX modulates the production of T helper 1 cytokines, such as IL-2 and IFN-γ, without causing significant cytotoxic effects in primary cultured lymphocytes. Reports also suggest that nASX exerts regulatory actions on the immune system and directly enhances the immune response by improving proliferation and maturation of natural killing cells, T and B lymphocytes, granulocytes and monocytes and nonsteroidal anti-inflammatory drugs (NSAIDs) were reported to be either therapeutically ineffective or exacerbate symptoms in the treatment of sepsis and ALI in acute COVID-19 patients (6,35,74). Application of nASX can be a potential approach to intervene the inflammatory responses for the treatment and prevention of CRS and its associated ailments in COVID-19 patients.

Astaxanthin as neuroprotective molecule
nASX is considered as a broad-spectrum bioactive compound with its myriad of health beneficial activities exerts prudently through multiple pathways. Besides its effectiveness as controlling oxidative stress, hyperinflammation, immune response, etc., as evidenced by multiple studies, nASX is also reported as a potential neuroprotective. In this regard, and mainly due to its capability to cross blood-brain barrier, nASX has gained growing interest as a multi-target pharmacological agent in protection of CNS injury and against neurological disorders including Parkinson's disease, Alzheimer's disease, brain and spinal cord injuries, neuropathic pain, aging, depression, and autism (39-40, 45, 55, 75-76, 80-82).  (77), which suggest early anti-inflammatory intervention may effectively prevent immune damage and reduce of the risk of injury in the nervous system (73,77). Effective therapeutics with the ability of multi-target treatments that simultaneously attenuate neuronal inflammation, oxidative stress and apoptosis are considered effective for treatment of neurological diseases. In this regard, nASX, based on its potent anti-oxidative, anti-inflammatory and anti-apoptotic properties, has gained tremendous interest as a multi-target pharmacological target (75). Clinically, CNS injury is characterized with primary phase involving direct death neuronal cell followed by the secondary phase, consisting of inflammatory, oxidative, apoptotic and other molecular pathways causing further damages to the neuronal cells. Zhang et al. (80) reported that nASX exerts its CNS protective role via decreasing malondialdehyde (MDA) and increasing glutathione (GSH) and SOD in rodent models. Fakhri et al. (81) reported in a rat model that nASX prevented tissue and neuronal damages by downregulating NR2B, TNF-α and p-p38MAPK, and reported to improved sensory-motor function. In another study, Zhang et al. (55) reported that nASX exert anti-inflammatory effect through increasing Sirtuin 1 (SIRT1) and inhibiting the Toll-like receptor 4 (TLR 4) signaling pathway resulting reduction of pro-inflammatory response and secondary brain injury. TLR4 express mainly in microglia and plays a pivotal role in triggering inflammatory response in the CNS (55). Further, in a rat model, it was reported that nASX down-regulated matrix metallopeptidases-9 (MMP-9), which was attributed to the decrement in the level of, infiltering neutrophils, activated microglia, TNF-α and IL-1β (82).
The therapeutic implications of nASX as neuroprotective molecule has been well documented. Its possible role in treatment of CNS injury was demonstrated in animal models. Taken together, it is anticipated that nASX will be useful in developing a potential therapy along with specific antivirals against the potential threat neurological infection by SARS-CoV-2 and its associated disease. Nrf2 and NF-kB pathway. Lung fibrosis is associated with inflammation characterized by the recruitment of macrophages, neutrophils and lymphocytes in the airways. nASX also reported to has therapeutic and prophylactic potential in the airway inflammatory response associated with chronic obstructive pulmonary disease (COPD) (86). Kubo et al (86) demonstrated is mice model that nASX prevent the oxidative damage via activation of Nrf2 pathway. Accumulated evidences based on published reports suggest that nASX may support with preventive measures against COVID-19 induced renal injury, septic cardiomyopathy, and liver injury.
Based on available information it is clearly understandable that astaxanthin can play a very pivotal role in regulating the CRS and may help in preventing the associated disease in multiple way (Fig 3).
We presume, its potent anti-oxidant, anti-inflammatory and immunomodulatory properties support its broad-spectrum benefits as a possible co-adjunctive supplement for COVID-19. However, at this stage we do not know whether nASX has any therapeutic value in the treatment or prevention of novel SARS-CoV-2 infected COVID-19. It has been proposed that nASX may provide protection from further injury by regulating CRS. Lack of any clinical trial related to this is the biggest limitation of the review outcome. 4.7. Food and drug safety of natural astaxanthin for human consumption H. pluvialis sourced natural ASX is reported as safe for human consumption, orally bioavailable, and a natural bio-active compound notified generally recognized as safe (GRAS) and approved by the United States Food and Drug Administration (USFDA) for human consumptions in dosages up to 24 mg per day (41,56). From the therapeutic point of view, nASX has been shown tremendous benefits against multiple disease without any reported side effects. Table 3 lists a few clinical reports involving nASX in human subjects.

Conclusion
COVID-19 caused by the infection of SARS-CoV-2 manifested by fever and pneumonia, leading to hyperinflammatory ARDS at severity is associated with SIRS or CRS, which suggest early prognosis and containment of disease progression. This necessitude a definitive anti-viral treatment along with a timely supportive anti-CRS or anti-inflammatory therapy to reduce the fatality of severe COVID-19 patients. While, several potential drugs for repurposing are undergoing clinical trials worldwide, a definitive COVID-19 treatment is lacking altogether at present. Natural astaxanthin is a potent antiinflammatory and anti-oxidant molecule with proven therapeutic and multiple health benefits anticipated to be useful as a co-adjunctive supplement for COVID-19 patients. Neither astaxanthin has any role as antiviral nor any published evidence is available on its study against COVID-19. However, enough published evidences support its potential application to treat CRS and associated diseases.
There is rationale to be anticipated its possible use as a potent co-adjunctive supplement for COVID-19, which requires further clinical evidences.

Declaration competing interest
All authors declare no competing interests.

Funding
The authors did not receive any funding for the study coagulation, fibrinolysis and platelet aggregation in hyperlipidemic rats.

Supplementary Files
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