Systemic hyper inflammation, prominent in the lungs and vascular endothelium, is the hallmark of severe coronavirus disease 2019 (COVID-19). There is currently no specific and conclusively proved treatment available for COVID-19. Anti-inflammatory (immunomodulatory) agents including corticosteroids (W.H.O. Rapid Evidence Appraisal for COVID-19 Therapies Working Group, 2020; RECOVERY Collaborative Group, 2021a) and interleukin (IL)-6 receptor inhibitor tocilizumab (RECOVERY Collaborative Group, 2021b) have been shown to reduce mortality, while Janus kinase (JAK) inhibitors (Kalil et al., 2021) aid rapid clinical improvement in severe COVID-19 illness. Recently, molnupiravir, sotrovimab, casirvimab/imdevimab, and nirmatrelvir/ritonavir have emerged as an early-stage COVID-19 antiviral therapies; however, these medications are costly and only available in the United Kingdom and the United States of America. A combination of early-stage therapeutic interventions that can interfere with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and simultaneously modulate the host’s hyperinflammatory immune response, is considered “crucial” in preventing the progression to severe COVID-19 illness. There is currently an urgent need for safe, effective, and affordable therapeutic agents that are available worldwide to help manage the early-stage COVID-19 infection in vulnerable patients. In the present circumstances, integrative medications with anti-inflammatory, immunomodulatory, or antiviral scientific rationale may offer a suitable choice of treatment of early-stage COVID-19 infection. Among such complementary agents, natural polyphenols curcumin, quercetin, and vitamin D3 (cholecalciferol), hereafter combinedly referred to as CQC, have attracted scientific interest as possible early-stage adjuvant therapies for COVID-19 due to their pharmacological effects.

Curcumin, the yellow pigment of the spice turmeric, is the major active polyphenolic compound extracted from the roots of the rhizome plant Curcuma longa L. (Zingiberaceae). Curcumin has a wide range of in vivo pharmacological activities such as anti-inflammatory, antiviral, antioxidant, antimicrobial, antithrombotic, antitumor, cardioprotective, and hepatoprotective (Aggarwal and Sung, 2009; Bandyopadhyay, 2014; Nabavi et al., 2014; Perrone et al., 2015; Padmanaban and Rangarajan, 2016; Kunnumakkara et al., 2017; Boroumand et al., 2018; Giordano and Tommonaro, 2019; Karimi et al., 2019; Patel et al., 2020). For centuries, turmeric has been used in Ayurveda, Siddha, and Traditional Chinese Medicines to treat diverse diseases associated with infection and inflammation. Curcumin is also an effective antiviral agent against many viruses including dengue virus, human immunodeficiency virus (HIV), Kaposi sarcoma-associated herpesvirus, enterovirus, Zika virus, Chikungunya virus, vesicular stomatitis virus, human respiratory syncytial virus, influenza A virus, hemorrhagic septicemia virus, herpes simplex type 2, norovirus, hepatitis C virus, hepatitis B virus, SARS-CoV-1, and SARS-CoV-2 (Wen et al., 2007; Praditya et al., 2019; Jennings and Parks, 2020; Bormann et al., 2021; Marin-Palma et al., 2021; Bahun et al., 2022). Curcuminoids have an excellent safety profile with “Generally Recognized As Safe” (GRAS) status received from the USA Food and Drug Administration (FDA) (Basnet and Skalko-Basnet, 2011; Gupta et al., 2013). Curcumin doses from 4 to 8 g/day for 3 months in patients with preinvasive malignant or high-risk premalignant conditions have failed to produce any adverse effects (Cheng et al., 2001).

Quercetin, one of the most abundant dietary flavonoids, occurs naturally in a wide range of fruits and vegetables, such as red grapes, citrus fruits, tomatoes, broccoli, and green leafy vegetables. Quercetin supplements are widely used for improving immunity and maintaining general well-being. Extensive in vivo studies have revealed the anti-inflammatory, antiviral, immunoprotective, and antioxidant effects of quercetin (Nair et al., 2002; Robaszkiewicz et al., 2007; Uchide and Toyoda, 2011; Colunga Biancatelli et al., 2020; Salehi et al., 2020). Studies have also shown quercetin as cardioprotective, anticancer, antitumor, antiulcer, antiallergy, antidiabetic, gastroprotective, antihypertensive, and anti-infective/antimicrobial agent (Lakhanpal and Rai, 2007; Salehi et al., 2020; Yang et al., 2020). Quercetin is also a broad-spectrum antiviral and has shown inhibitory activity against many viruses including HIV, herpes simplex virus (type 1 and 2), poliovirus (type 1), parainfluenza (type 3), hepatitis C virus, human respiratory syncytial virus, Sindbis virus, vaccinia virus, SARS-CoV-1, and SARS-CoV-2 (Andersen et al., 1991; Kim et al., 2001; Semple et al., 2001; Yi et al., 2004; Gonzalez et al., 2009; Abian et al., 2020; Lopes et al., 2020; Rizzuti et al., 2021; Bahun et al., 2022). Quercetin has a well-established safety profile and doses up to 1 g/day for 3 months have not resulted in any significant adverse effects (Harwood et al., 2007; Andres et al., 2018). The USA FDA has approved quercetin as a safe agent (GRAS status) in dietary products for human use.

Vitamin D3 deficiency (VDD) is considered as a possible risk factor for susceptibility to COVID-19 infection and/or progression to severe illness. A number of published studies have reported significantly low serum vitamin D3 i.e. (1α,25(OH)₂D3 levels in severely-ill COVID-19 patients, implying a possible association between VDD and the development of severe COVID-19 illness (Ilie et al., 2020; Meltzer et al., 2020; Mercola et al., 2020; Merzon et al., 2020). The precise role of vitamin D3 in COVID-19 remains uncertain. Vitamin D3 has well-documented immunomodulatory and anti-inflammatory effects, particularly in viral infections (Prietl et al., 2013; Tamer and Mesçi, 2013; Grant et al., 2020; Charoenngam et al., 2021). Due to the extreme toll of COVID-19 on the immune system, there has been a significant interest in the potential of vitamin D3 supplementation to ameliorate or prevent detrimental immune responses in the early stage of COVID-19 infection (Charoenngam et al., 2021).

The demonstrated anti-inflammatory (immunomodulatory), antiviral, and antioxidant activities of CQC, prompted us to investigate a combination of these agents as an adjuvant therapy for early-stage COVID-19 infection. In the present study, we conducted a pilot open-label, randomized controlled clinical trial to assess the possible treatment benefits of an oral formulation containing CQC supplements as an add-on to routine care in mild to moderately symptomatic COVID-19 outpatients. It is speculated that the co-supplementation of CQC in a single combined formulation could produce a more effective treatment, resulting from the synergistic anti-inflammatory (immunomodulatory), antiviral, and antioxidant effects of CQC. The synergistic mechanisms can effectively target the early stage of SARS-CoV-2 infection. This can be achieved by interfering with SARS-CoV-2 viral replication and simultaneously modulating the host’s hyperinflammatory response.

Table 1 below shows the patient’s baseline characteristics by treatment group. Overall, the median interquartile range (IQR) age was 44.0 (34.8, 52.0) years and included 25 (50.0%) males. The most common acute COVID-19 symptoms at enrollment were fever [40 (80.0%)], pharyngitis [26 (52.0%)], cough [27 (54.0%)], and myalgia [24 (48.0%)]. Less common symptoms were dyspnea and flu, each in seven (14.0%) patients. At entry, 33 (66.0%) patients had three or more COVID-19-associated symptoms, 13 (26.0%) had two symptoms, and four (16.0%) patients had a single symptom. A total of 21 (58.0%) patients had a history of pre-existing health conditions, and of these nine (36.0%) patients had a single, and 12 (24.0%) patients had two or more conditions. The most common comorbidities were diabetes mellitus (DM) and hypertension (HTN), found in 17 (34.0%), and 14 (28.0%) patients, respectively. Amongst the total 50 patients, 29 (52.0%) were COVID-19 vaccinated with at least two doses. The two treatment groups were comparable in terms of the proportion of men and the median (IQR) for age was only slightly lower in the CQC arm, i.e., 43.0 (32.0, 51.0) vs. 45.0 (37.0, 52.0) years of the control arm. The groups were similar in terms of numbers of COVID-19-associated symptoms; the number (%) of patients reporting three or more symptoms were 17 (68.0%) and 16 (64.0%) in the CQC and control arms, respectively. The control arm had slightly more COVID-19 vaccinated patients, i.e., 16 (64.0%) vs. 13 (52.0%) in the CQC arm, while the CQC arm had slightly fewer patients with DM and/or HTN, i.e., eight (32.0%) vs. 12 (48.0%) patients in the control arm. In both treatment groups, a similar number of patients had received antibiotic azithromycin as part of the SOC, i.e., 17 (34.0%) vs. 16 (32.0%) in the CQC and control arms, respectively. The randomized groups were considered reasonably balanced in terms of baseline characteristics.

This pilot randomized controlled clinical trial assessed the possible treatment benefits of CQC in a single oral formulation, as a complementary therapy to the routine care for early-stage mild to moderate symptoms of COVID-19 in outpatients. In this study, patients who received the tripartite co-supplementation of CQC as an adjuvant therapy, most (60%) of them cleared their SARS-CoV-2 viral infection faster (on day seven of the treatment), as compared to the control arm patients (only 20%). Similarly, more patients (60%) in the CQC arm showed their complete COVID-19 acute symptoms resolution by day seven of the treatment, as compared to the control arm (40%). It is speculated that the speedy clearance of the SARS-CoV-2 in the CQC treated patients may be due to the inhibition of 3C-like protease (3CLpro) also called main protease (Mpro) of the SARS-CoV-2 by curcumin and quercetin as reported by the in vitro studies (Chen et al., 2006; Abian et al., 2020; Bormann et al., 2021; Marin-Palma et al., 2021; Rizzuti et al., 2021; Bahun et al., 2022). 3CLpro is an essential enzyme in the SARS-CoV-2 replication cycle, processing the large viral polyproteins and rendering the individual proteins functional. 3CLpro is highly conserved amongst coronaviruses and is currently the main target for antiviral therapies in COVID-19. The expedited viral clearance of CQC patients could also be due to other antiviral mechanisms associated with curcumin and quercetin. Curcumin can exert antiviral effects directly on the viral particle or at different stages of the replicative cycle by interacting with viral proteins or by interfering with cellular processes or pathways crucial for viral replication (Mazumder et al., 1995; Richart et al., 2018; Jena et al., 2021). Quercetin has been shown to block cell entry of influenza virus, rhinovirus, and SARS-CoV-1 (Yi et al., 2004; Wu et al., 2015), and interferes with the replication of herpesviruses and adenoviruses (Chiang et al., 2003). Quercetin also targets viral polymerases and may disrupt replication via the inhibition of reverse transcriptase and integrase enzymes (Ono and Nakane, 1990). Quercetin has also been shown to alter the expression of 98 of 332 (30%) human genes encoding protein targets of SARS-CoV-2, thus potentially interfering with the functions of 23 of 27 (85%) of the SARS-CoV-2 viral proteins in human cells (Glinsky, 2020). The clinical course of SARS-CoV-2 infection is quite unpredictable and therefore suitable early antiviral therapy is believed to be the key in preventing the progression to severe illness. We can, therefore, infer that the adjuvant therapy of CQC has the potential to target and interfere with the early stages of the SARS-CoV-2 infection (replication) and may possibly help in the speedy recovery of patients.

In addition to faster viral clearance, the co-supplementation of CQC adjuvant therapy also significantly reduced the patients’ hyperinflammatory response as shown by the significantly lowered serum CRP levels (by day seven of treatment) in the CQC arm as compared to the control arm (p = 0.006). We speculate that this significant improvement in the inflammation of patients in the CQC arm may be due to the multiple diverse anti-inflammatory mechanisms reported for curcumin, quercetin, and cholecalciferol. Curcumin has been shown to decrease the secretion of various critical pro-inflammatory cytokines including IL-1, IL-2, IL-6, IL-8, IL-10, IL-11, IL-12, IL-17, tumor necrosis factor alpha (TNF-α), and interferon-gamma (INF-γ), and chemokines such as monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, nuclear factor kappa-light-chain enhancer of activated B cells (NFĸB), cyclooxygenase, plasminogen activator inhibitor-1, and caspase-3 (Babaei et al., 2020; Zahedipour et al., 2020). Curcumin also reportedly inhibits SARS-CoV-2-induced cytokine storm via deactivation of the mitogen-activated protein kinases/NFĸB signaling in the lung and liver epithelial cells (Sharma et al., 2022). Similarly, quercetin also exhibits diverse anti-inflammatory mechanisms, including the inhibition of lipid peroxidation, pro-inflammatory mediators such as lipoxygenase and phospholipase A2 (Colunga Biancatelli et al., 2020), and the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome-mediated IL-1β production (Tozser and Benko, 2016), and diverse proinflammatory cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α (Nair et al., 2002; Chen et al., 2016; Leyva-Lopez et al., 2016). Quercetin in combination with dasatinib has been shown to selectively eliminate virus-induced senescent cells, mitigate COVID-19-reminiscent lung disease, and reduce inflammation in SARS-CoV-2-infected hamsters and mice (Lee et al., 2021). In a meta-analysis study of randomized controlled trials by Mohammadi-Sartang et al. (2017), quercetin diet significant reduced CRP levels, supporting its role as an anti-inflammatory agent. Furthermore, the immunomodulatory role of vitamin D3 is well-known (Bouillon et al., 2019). Vitamin D3 stimulates the expression of both innate and adaptive immune function receptors in the respiratory tract (Gombart et al., 2005; Chen et al., 2013; Brockman-Schneider et al., 2014; Martineau et al., 2017; Greiller et al., 2019). It also supports the innate immunity through the production of several antimicrobial peptides such as cathelicidins, defensivins, and IL-37 and acts on the adaptive immunity by modulating IL-6, IFN-γ, TNF-α, thus help in reducing lung tissue damage caused by inflammation. Serum CRP level is a crucial indicator of systemic inflammation and an important prognostic marker in patients with COVID-19. Elevated serum CRP levels are negatively correlated with SpO₂ and associated with aggravation in asymptomatic and mildly symptomatic cases, and mortality in severely-ill COVID-19 patients, demonstrating lung injury linked disease course (Ali, 2020; Sadeghi-Haddad-Zavareh et al., 2021). Thus, the expedited significant decrease in the CRP levels at day seven indicates a faster clinical recovery (healing) of patients in the CQC arm, as compared to patients in the control arm, showing sustained inflammation. In study by Meng et al. (2020), in Wuhan, China, involving asymptomatic hospitalized COVID-19 pneumonia patients, out of 58 patients, 55 patients (94%) had developed ground glass opacity of lungs as shown by computed tomography scan, indicating a significantly high prevalence of lung involvement. Moreover, studies have shown close association between lung lesions and serum CRP levels in the early stage of COVID-19 infection (Wang, 2020). Therefore, the significantly reduced CRP levels of patients in the CQC arm, may act as an indirect marker of the improved lung pathology (if any) possibly due to the therapeutic effects of CQC adjuvant therapy. Any intervention targeted to reduce lesions/scarring of the lung in patients with COVID-19 infection can improve quality of life in the long term. Therefore, we can speculate that the CQC add-on therapy tested in this study may provide long-term benefits to lung health.

The potential treatment benefits of CQC add-on therapy for COVID-19, as observed in this pilot clinical trial are in line with the results reported by another pilot randomized controlled clinical trials and case series studies that investigated curcumin, quercetin, and cholecalciferol acting either alone or in combination with other dietary supplements as an adjuvant treatment for patients with COVID-19. Studies investigated curcumin as an adjuvant for COVID-19 have revealed multiple therapeutic benefits including early negativization of the SARS-CoV-2, early symptomatic recovery, increased lymphocyte count, less clinical deterioration, shorter hospitalization period, reduce risk and duration of supplementary oxygen, mechanical ventilation, and mortality (Ahmadi et al., 2021; Chabot and Huntwork, 2021; Majeed et al., 2021; Pawar et al., 2021; Saber-Moghaddam et al., 2021; Asadirad et al., 2022). Several clinical trials, in particular, have investigated the anti-inflammatory and immunomodulatory pharmacological effects of curcumin in patients with COVID-19 and revealed improvement in serum CRP levels, a significant reduction in the gene expression, and/or serum levels of IFN-γ, IL-1β, IL-6, IL-10, IL-17, IL-21, IL-23, IL-35, retinoic acid-related orphan receptor-C, forkhead box P3, transforming growth factor beta, granulocyte-macrophage colony-stimulating factor, and frequency of T helper 17 cells (Salehi et al., 2020; Ahmadi et al., 2021; Tahmasebi et al., 2021a; Tahmasebi et al., 2021b; Hassaniazad et al., 2021; Asadirad et al., 2022). Similarly, several randomized clinical trials involving patients with COVID-19 have also revealed multiple treatment benefits for quercetin adjuvant therapy, including faster negativization of the SARS-CoV-2, early amelioration of the acute symptoms, reduction in the serum levels of inflammatory markers such as CRP, LDH, ferritin, and alkaline phosphatase (Kamel et al., 2020; Di Pierro et al., 2021a; Di Pierro et al., 2021b; Onal et al., 2021; Shohan et al., 2022). Moreover, with vitamin D3 adjuvant supplementation, early negativization of the SARS-CoV-2 and improvement in the acute symptoms have also been reported in patients with early-stage of COVID-19 (Opko Press Release, 2021; Sabico et al., 2021; Sanchez-Zuno et al., 2021; Rastogi et al., 2022). Vitamin D3, however, has shown no treatment benefits in patients with severe COVID-19 illness.

Taken together, there is a reasonable body of scientific and clinical evidence to suggest the safety and treatment benefits of curcumin, quercetin, and vitamin D3 for patients with COVID-19. Using a combination of these agents as an adjuvant therapy, the results observed in this pilot clinical trial suggest an expedited clearance of the SARS-CoV-2 viral infection and simultaneous modulation of the host hyperinflammatory response in patients with early mild to moderate symptoms of COVID-19. The faster viral clearance alongside a significant reduction in the acute inflammatory response with CQC adjuvant therapy is possibly due to the synergistic antiviral, anti-inflammatory, and antioxidant mechanisms of the three active agents. One potential serious risk of the currently used anti-inflammatory/immunomodulatory COVID-19 interventions such as corticosteroids, tocilizumab, or bariticinib, particularly in vulnerable and immunocompromised patients, is the immunosuppressive side effect of these drugs, which can further increase the risk of developing superimposed secondary bacterial or fungal infection, and thus progression to severe illness in these patients. In contrast, curcumin, quercetin, and vitamin D3 as anti-inflammatory agents have well-established individual safety profiles and do not suffer from the intrinsic drawback of immunosuppressive effects. Thus, their use as an adjuvant in combination with routine care, represents a safe therapeutic option for early-stage COVID-19 infection, particularly in vulnerable patients. Moreover, the antimicrobial activities of CQC could also prevent the development of such superadded or secondary infections in patients with COVID-19.

Our study was not free of limitations. In the present circumstances, particularly in developing countries, there is a desperate need for safe, cheap, accessible, and effective treatments for early-stage COVID-19 infection. Only clinical trials can identify such agents. The major limitations of our study were the small sample size, and not being a double-blinded and placebo-controlled trial. Large sample size studies are warranted to evaluate the results of our study.

In conclusion, we have carried out a pilot open-label, randomized controlled clinical trial to assess the possible treatment benefits of an oral combination of curcumin, quercetin, and vitamin D3 supplements, in a single-formulation (CQC), as an adjuvant therapy for patients with early-stage mild to moderate symptoms of COVID-19. Our results suggest that the co-supplementation of CQC is a safe intervention and possibly has a therapeutic role in the early stage of COVID-19 including faster clearing of the SARS-CoV-2 viral infection, and rapid improvement of the acute symptoms, and modulation of the early-stage hyperinflammatory response. The synergistic effect of the anti-inflammatory, immunomodulatory, antiviral, and antioxidant activities of CQC is believed to be the driving force for faster COVID-19 recovery of patients observed in this pilot clinical trial. Considering the well-established safety profile, tolerability, worldwide over-the-counter availability, and low cost, the combination of curcumin, quercetin, and vitamin D3 supplements could be used as an adjuvant therapy for the early-stage mild to moderate symptoms of COVID-19 infection.