Antioxidants and clinical outcomes of patients with coronavirus disease 2019: A systematic review of observational and interventional studies

Abstract Coronavirus disease 2019 (COVID‐19) is a newly emerging viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Oxidative stress appears to be a prominent contributor to the pathogenicity of SARS‐CoV‐2. Therefore, we carried out a systematic review of human observational and interventional studies to investigate the role of some antioxidants such as vitamins A, E, D, and C, selenium, zinc, and α‐lipoic acid in the main clinical outcomes of subjects with COVID‐19. Google Scholar, Cochrane Library, Web of Science, Scopus, and Medline were searched using Medical Subject Headings (MeSH) and non‐MeSH terms without restrictions. Finally, 36 studies for vitamins C and D, selenium, and zinc were included in this systematic review; however, no eligible studies were found for vitamins A and E as well as α‐lipoic acid. The results showed the promising role of vitamin C in inflammation, Horowitz index, and mortality; vitamin D in disease manifestations and severity, inflammatory markers, lung involvement, ventilation requirement, hospitalization, intensive care unit (ICU) admission, and mortality; selenium in cure rate and mortality; and zinc in ventilation requirement, hospitalization, ICU admission, biomarkers of inflammation and bacterial infection, and disease complications. In conclusion, it seems that antioxidants, especially vitamins C and D, selenium, and zinc, can improve multiple COVID‐19 clinical outcomes. Nevertheless, more studies are necessary to affirm these results.

tioxidants in favor of the former, has been proposed as a prominent contributor to pathogenicity of SARS-CoV-2 (Cecchini & Cecchini, 2020). It seems that this virus binds to angiotensinconverting enzyme (ACE) 2, disturbs the renin-angiotensin system (RAS), and causes oxidative stress in the body (Silvagno et al., 2020). In addition, although SARS-CoV-2 can affect human beings of all ages, individuals with already elevated levels of oxidative stress including those with old age, obesity, cardiovascular disease, or diabetes mellitus have been reported to be at greater risk for severe COVID-19 (Zhou et al., 2020). It seems that oxidative stress per se can weaken the immune system, induce viral activation, stimulate the production of proinflammatory chemokines and cytokines, and lead to inflammation and cell death in people with COVID-19 (Chernyak et al., 2020;Delgado-Roche & Mesta, 2020). Given these evidences, oxidative stress may have a role in the incidence, severity, and mortality of COVID-19, and therefore, antioxidants could be a potential intervention to control  Antioxidants are endogenous or exogenous substances that prevent, delay, or repair oxidative damage to biological macromolecules (Halliwell, 2007). As shown in Figure 1, these beneficial substances are classified into two groups based on the presence or absence of enzymatic activity. In the group of enzymatic antioxidants, glutathione peroxidase, catalase, superoxide dismutase, and peroxiredoxins are present as examples of primary enzymes, and glutathione reductase, glucose-6-phosphate dehydrogenase, and glutathione S-transferases are present as examples of secondary enzymes (Mehta & Gowder, 2015;Nimse & Pal, 2015;Ratnam et al., 2006). In the group of nonenzymatic antioxidants, there are multiple subgroups that are mainly derived from dietary sources.
Importantly, these pivotal proteins are involved in the host cell recognition, transcription, and replication of SARS-CoV-2 (Cannalire et al., 2020).

F I G U R E 1 Classification of antioxidants
All in all, antioxidants may be helpful for individuals infected with SARS-CoV-2. Therefore, we carried out a systematic review of human observational and interventional studies to investigate the role of some important antioxidants in major clinical outcomes of subjects with COVID-19. In the present review, we focused on six well-known antioxidants including vitamin A, vitamin E, vitamin C, zinc, selenium, and α-lipoic acid as well as one vitamin with recently discovered antioxidant property, i.e., vitamin D.

| Research question
This review was performed according to the guidelines available for systematic reviews of observational (Stroup et al., 2000) and interventional studies (Page et al., 2021). The PECO/PICO approach (participants, exposure/intervention, comparator, and outcome) was used to define the review question (Morgan et al., 2018;Santos et al., 2007). The participants of the studies included in this systematic review were individuals with COVID-19, regardless of their age. In observational studies, different dietary/supplement intake or biological sample levels of vitamins A, C, D, or E, selenium, zinc, or α-lipoic acid were compared with each other. In interventional studies, supplementation with vitamins A, C, D, or E, selenium, zinc, or α-lipoic acid was compared to matched placebo, control group, or none (i.e., without comparator). The outcomes of this systematic review were clinical outcomes of  reported in the included studies (e.g., disease severity, disease manifestations and complications, inflammatory biomarkers, hospitalization, and mortality).

| Study selection
The study selection was done by two independent authors. First, the title and abstract of publications were screened to find pertinent ones. Second, the full text of pertinent articles was screened to discern eligible observational and interventional studies. The inclusion criteria for observational studies were: (1)

| Data extraction
General characteristics of observational and interventional researches were extracted and tabulated by two independent investigators using the Cochrane data collection form. These characteristics include author names, year of publication, study design and location, mean age of patients, total and gender-specific sample size, exposure, intervention and control groups, and main results of COVID-19 clinical outcomes. It is worth mentioning that the level of agreement between investigators for data collection was appropriate (Kappa = 0.81).

| Quality assessment
The quality assessment of studies was conducted by two independent reviewers using the Academy of Nutrition and Dietetics Quality

| Study characteristics and quality
The characteristics and quality of observational and interventional studies are summarized in Tables 1 and 2, respectively.
The included studies were published between 2020 and 2022.
According to the time of conducting the studies, the following SARS-CoV-2 variants are covered in this systematic review: Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Iota, and Kappa (World  Rastogi et al., 2022;Zhang et al., 2021), and pre/post trial (n = 1; Hiedra et al., 2020). All studies were rated as neutral quality except three that were rated as positive quality (Castillo et al., 2020;Rastogi et al., 2022;Zhang et al., 2021).

| Vitamin A
For vitamin A, no observational and interventional studies had criteria for inclusion in this systematic review.

| Vitamin C
For vitamin C, one observational (Arvinte et al., 2020) and two interventional (Hiedra et al., 2020;Zhang et al., 2021) studies were included in this systematic review. Interestingly, although no relationship was found between serum vitamin C levels and mortality in patients with COVID-19 (Arvinte et al., 2020), intravenous supplementation with vitamin C significantly decreased mortality in severe cases (Zhang et al., 2021). In addition, vitamin C supplementation significantly reduced levels of inflammatory biomarkers such as interleukin-6, ferritin, and D-dimer in COVID-19 patients (Hiedra et al., 2020;Zhang et al., 2021). Furthermore, supplementation with vitamin C caused a significant increase in Horowitz index, the ratio of arterial oxygen partial pressure to fractional inspired oxygen (Zhang et al., 2021). Nevertheless, vitamin C supplementation did not affect the length of hospitalization, disease complications, and some other clinical outcomes of subjects infected with SARS-CoV-2 (Hiedra et al., 2020;Zhang et al., 2021; Tables 1 and 2).

| Vitamin E
For vitamin E, no observational and interventional studies had criteria for inclusion in the present systematic review.

| Selenium
For selenium, only two observational studies Jinsong Zhang et al., 2020) were included in the systematic review. In an ecological study, there was a significant positive relationship between mean levels of hair selenium and COVID-19 cure rate, calculated as a percentage of the number of cured COVID-19 patients over the number of confirmed cases (Jinsong Zhang et al., 2020). In a cross-sectional research, serum levels of selenium, selenoprotein P, and glutathione peroxidase-3 were negatively related to COVID-19 mortality  Table 1).

| Zinc
For zinc, five observational (Anuk et al., 2021;Bagheri et al., 2020;Heller et al., 2020;Jothimani et al., 2020;Yasui et al., 2020) as well as three interventional (Carlucci et al., 2020;Derwand et al., 2020;Yao et al., 2021) studies were included in this systematic review. Two observational studies did not find any significant link between zinc status and COVID-19 severity (Anuk et al., 2021;Bagheri et al., 2020), but one observational study reported a significant inverse relationship between serum levels of zinc and severity of COVID-19 (Yasui et al., 2020). In addition, three studies did not detect a role for zinc in COVID-19 survival and mortality (Derwand et al., 2020;Jothimani et al., 2020;Yao et al., 2021), but one study found a significant direct relationship between serum zinc levels and survival of subjects with COVID-19 infection . Moreover, although zinc status was not significantly related to hospitalization and ICU admission (Bagheri et al., 2020;Jothimani et al., 2020), oral zinc sulfate supplementation significantly decreased hospitalization and ICU admission (Carlucci et al., 2020;Derwand et al., 2020). Zinc supplementation also caused a significant reduction in ventilation requirement and a significant increase in the number of COVID-19 patients discharged from hospital to home (Carlucci et al., 2020). Furthermore, serum zinc levels were negatively associated with biomarkers of inflammation and bacterial infection including interleukin-6, erythrocyte sedimentation rate, CRP, and procalcitonin (Anuk et al., 2021). Besides, there was a significant positive association between zinc deficiency and COVID-19 complications (Jothimani et al., 2020). However, zinc did not change the length of hospitalization and some other clinical outcomes of people with COVID-19 (Carlucci et al., 2020;Jothimani et al., 2020; Tables 1 and 2).

| Α-lipoic acid
For α-lipoic acid, no observational and interventional studies had criteria for inclusion in this systematic review.

| Summary of key findings
In this systematic review of primary human studies, we investigated the role of vitamins A, C, D, and E, selenium, zinc, and α-lipoic acid in major clinical outcomes of people with COVID-19. Among the aforementioned seven antioxidants, eligible studies were found only for vitamins C and D, selenium, and zinc. The findings suggest that vitamin C may cause beneficial effects on inflammation status, Horowitz index, and mortality rate of COVID-19 patients. Moreover, vitamin D may have a positive role in the reduction of disease manifestations and severity, inflammatory biomarkers, lung involvement, ventilation requirement, hospitalization, ICU admission, and mortality in individuals with COVID-19. Also, selenium may have the potential to increase and decrease the cure rate and mortality of COVID-19 patients, respectively. Furthermore, zinc may be able to lower hospitalization, ventilation requirement, ICU admission, biomarkers of inflammation and bacterial infection, and disease complications in individuals infected with COVID-19.

| Vitamin A
Although none of the included studies examined the role of vitamin A in subjects with COVID-19, bioinformatics findings proposed that this antioxidant may be beneficial for individuals infected with SARS-CoV-2 . Vitamin A has an important role in enhancing the body's immunity and regulating both cellular and humoral immune responses (Jayawardena et al., 2020). The production of antibodies, also known as immunoglobulins (Ig), is integral to the maintenance of humoral immune responses (Huang et al., 2018). An animal study showed that vitamin A can promote humoral immunity by increasing serum levels of IgG, IgM, and IgA (Ghodratizadeh et al., 2014). Vitamin A also plays a pivotal role in the development of epithelium, which is considered a frontline defense against pathogen invasion (McCullough et al., 1999). As vitamin A enhances mucin secretion in the respiratory tract and intestine, it is able to improve the antigen nonspecific immunity function of these tissues (Huang et al., 2018). Moreover, vitamin A may inhibit inflammatory processes induced by COVID-19 through the regulation of multiple key genes including mitogen-activated protein kinase 1 and 14, interleukin-10, epidermal growth factor receptor, protein kinase C beta type, intercellular adhesion molecule 1, and catalase .

| Vitamin C
The results of this systematic review indicated that vitamin C may exert favorable effects on clinical outcomes of COVID-19 patients.
Vitamin C acts as a powerful antioxidant, especially for epithelial cells of the lungs (Farjana et al., 2020). It appears to scavenge reactive oxygen species (ROS) and inhibit pathways involved in neutrophil extracellular trap formation and cytokine storms (Cerullo et al., 2020). Moreover, vitamin C can suppress lactate production.
This can be of great importance because serum and tissue concentrations of lactate are elevated in critically ill patients with COVID-19 (Earar et al., 2020). Lactate weakens the host immune system by decreasing the production of type I interferon and limiting viral clearance (Lottes et al., 2015;Zhang et al., 2019).

| Vitamin D
The findings of this systematic review showed that vitamin D may play a positive role in improvement of COVID-19 clinical outcomes. It seems that antioxidative, antiinflammatory, and immunomodulatory properties of vitamin D can be involved in this regard (Hajhashemy et al., 2022;Musavi et al., 2020). Besides, some researchers discussed the key role of vitamin D in the RAS (Kumar et al., 2020;Malek Mahdavi, 2020;Musavi et al., 2020). As noted in the introduction, SARS-CoV-2 binds to ACE2, which is expressed on the surface of alveolar epithelial cells (Silvagno et al., 2020). Once the virus is attached, the activity of ACE2 is suppressed, which further enhances the activity of ACE1, that accordingly increases the formation of angiotensin II, leading to intensified pulmonary vasoconstriction and severe COVID-19 reactions (Malek Mahdavi, 2020). In an animal study, the expression of ACE2 in the lungs was significantly elevated by calcitriol, the bioactive form of vitamin D (Xu et al., 2017). Therefore, as a result of vitamin D supplementation, ACE2 may be expressed more, which can decrease lung injury (Imai et al., 2005).
Moreover, vitamin D may reduce the production of angiotensin II and result in less pulmonary vasoconstriction through suppressing renin activity (Kumar et al., 2020).

| Vitamin E
Although none of the included studies investigated the role of vitamin E in individuals with COVID-19, bioinformatics findings suggested that this micronutrient may be beneficial for patients infected with SARS-CoV-2 (Kim et al., 2020). Vitamin E is a lipid-soluble antioxidant with the ability to protect cells from damage caused by ROS, especially in respiratory infections (Lewis et al., 2019).
Moreover, vitamin E is involved in various aspects of the immune response, including but not limited to the production of antibodies, phagocytosis, and T cell function (Akhtar et al., 2021). This vitamin modulates T cell function through affecting T cell membrane integrity, cell division, signal transduction, and several inflammatory mediators such as prostaglandin E 2 and proinflammatory cytokines (Lewis et al., 2019). Furthermore, it seems that vitamin E can induce signals of gene expression that counteract signals associated with COVID-19 (Kim et al., 2020).

| Selenium
The results of this systematic review revealed that selenium may have a promising role in amelioration of COVID-19 clinical outcomes.
As mentioned earlier, COVID-19 increases the production of ROS in host cells, which can cause oxidative stress if not counteracted by the antioxidant defense system (Chernyak et al., 2020). Glutathione peroxidase-1 (GPx1), a cytosolic selenoenzyme with antiviral properties, is considered as a crucial antioxidant defense against ROS (Sajjadi et al., 2022). This selenoprotein catalyzes the detoxification of hydrogen peroxide to water molecules and is particularly involved in protection against viral respiratory infections (Guillin et al., 2019).
There is evidence of an interaction between GPx1 and the main protease of SARS-CoV-2, 3-chymotrypsin-like protease, which is essential for viral replication. This interaction depends on host selenium status to combat SARS-CoV-2 virulence (Seale et al., 2020).
Accordingly, selenium may improve clinical outcomes of patients with COVID-19.

| Zinc
The findings of this systematic review manifested that zinc may have desirable effects on clinical outcomes of COVID-19 patients.
Multiple protective mechanisms of zinc against COVID-19 infection have been proposed in the literature. It seems that SARS-CoV-2 can weaken mucociliary clearance and expose the lungs to further viral and bacterial infections (Koparal et al., 2021). In turn, zinc may enhance mucociliary clearance by improving cilia morphology and increasing cilia beat frequency (Darma et al., 2020).
This mineral can also improve the integrity and barrier function of the respiratory epithelium by increasing its antioxidant activity and upregulating its tight junction proteins such as claudin-1 and zonula occludens-1 (Skalny et al., 2020). In addition, zinc may exert antiviral effects through interference with viral replication cycles (Read et al., 2019). Moreover, zinc can be beneficial for bacterial coinfection in viral pneumonia, because it may inhibit the growth of Streptococcus pneumoniae by modulating bacterial manganese homeostasis (Eijkelkamp et al., 2019). Furthermore, zinc can downregulate the production of proinflammatory cytokines through the inhibition of IκB kinase activity and nuclear factor-κB (NF-κB) signaling (Skalny et al., 2020).

| Α-lipoic acid
Although none of the included studies evaluated the role of α-lipoic acid in patients with COVID-19, some researchers hypothesized that this potent antioxidant may be advantageous for subjects infected with SARS-CoV-2 (Sayıner & Serakıncı, 2021). Α-lipoic acid is able to reduce oxidative stress through the regeneration of other antioxidants and chelation of metal ions. In addition, this quasi-vitamin can inhibit the activation of NF-κB, an inflammatory transcription factor (Tibullo et al., 2017). Furthermore, α-lipoic acid may decrease the activity of a disintegrin and metalloprotease 17 (ADAM17), also known as tumor necrosis factorα-converting enzyme (Cure & Cure, 2020). The lower activity of ADAM17 can reduce the shedding of ACE2 and severity of COVID-19 infection (Peron & Nakaya, 2020). Moreover, α-lipoic acid may increase intracellular pH by activating Na + /K + -ATPase (Cure & Cure, 2020). It seems that higher intracellular pH can inhibit SARS-CoV-2 cellular entry (Petersen et al., 2020). Also, α-lipoic acid has a potential to activate pyruvate dehydrogenase and reduce serum lactate levels (Konrad et al., 1999).

| Limitations
There are several limitations that need to be taken into consideration when interpreting the findings of this systematic review. First, no eligible studies were found for vitamins A and E as well as α-lipoic acid.
Second, a limited number of studies investigated the role of vitamin C, selenium, and zinc in clinical outcomes of COVID-19 patients. In fact, most studies focused on the role of vitamin D. Third, most of the included records were observational studies with neutral quality.
Fourth, the included studies were diverse in terms of study characteristics and methodology. Therefore, more high-quality studies, especially randomized controlled trials, are required for future integration and consensus.

| CON CLUS ION
In conclusion, due to the important role of oxidative stress in the pathogenicity of SARS-CoV-2, antioxidants seem to be beneficial for patients with COVID-19. Particularly, the findings obtained from this systematic review suggest that vitamins C and D, selenium, and zinc can improve some COVID-19 clinical outcomes. Nevertheless, further well-designed and well-reported studies are needed to draw definite conclusions.

This research was financially supported by the Food Security
Research Center of the Isfahan University of Medical Sciences (Grant Number 299234). The authors heartily appreciate all healthcare professionals who dedicatedly take care of patients with COVID-19 throughout the world.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are available on request from the corresponding author.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.