Skip to main content

5-Aminolevulinic acid antiviral efficacy against SARS-CoV-2 omicron variant in vitro


The coronavirus disease 2019 (COVID 19) pandemic continues to pose a threat to global health. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529) has spread rapidly worldwide and became dominant in many countries. A natural 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) has demonstrated antiviral activity in Wuhan, Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 infections in vitro. In this study, we report antiviral activity of 5-ALA, 5-ALA with SFC led to IC50 of 329 and 765/191, respectively after infection with Omicron variant of SARS-CoV-2 in vitro. Our finding suggests that 5-ALA could be used as antiviral drug candidate to treat Omicron variant infected patients.

To the Editor,

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged a year and a half from the onset of coronavirus disease 2019 (COVID-19) pandemic. As of February 2022, Omicron variants have been divided into four distinct sub lineages: BA.1, BA.1.1, BA.2, and BA.3 [1,2,3]. The number of Omicron variant cases has increased in many regions of the world, spreading more easily than previously described SARS-CoV-2 isolates [4]. The Omicron variant has substantial spike protein mutations and is able to escape immune protection elicited by both vaccine and previous infection [5, 6]. Several direct-acting antivirals against COVID-19 have been approved or are under clinical development and can be divided in two categories; small molecules interfering with virus replication machinery, and monoclonal antibodies directed against the spike protein [7]. Aside from improving vaccinations against Omicron and future variants, we must develop new antiviral drugs [8, 9]. Antiviral drugs including remdesivir, molnupiravir, and nirmatrelvir inhibited SARS-CoV-2 Omicron variant infection [7, 10]. A natural amino acid, 5-aminolevulinic acid (5 ALA), is produced from most animals and plants which are present in food. In our previous studies, we reported antiviral activity of 5-ALA with or without sodium ferrous citrate (SFC) against the SARS-CoV-2 Wuhan strain and its variants including Alpha, Beta, Gamma and Delta strains [11, 12]. In this study, we evaluated the antiviral effect of 5-ALA with or without SFC against the SARS-CoV-2 Omicron variant in vitro. Vero E6 cells were treated with remdesivir or 5-ALA with or without SFC for 72 h (h) and then infected with SARS-CoV-2 Omicron variant (TY38-873, BA.1, provided by the Japan National Institute of Infectious Diseases) at a multiplicity of infection of 0.02. After 48 h post infection, the infected cell supernatants were harvested for viral RNA extraction [12, 13]. The SARS-CoV-2 antiviral assay is based on previously established specific quantitative real time PCR (qRT-PCR) [12] using cell supernatant RNA. The antiviral drug effect of remdesivir against the Omicron variant showed an IC50 (virus inhibition by 50%) of 0.3 µM (Fig. 1A). 5-ALA, 5-ALA and SFC inhibited SARS-CoV-2 Omicron variant infection in a dose dependent manner with an IC50 of 329 and 765/191, respectively in vitro (Fig. 1B, C, Table 1). A cell viability assay was conducted in parallel with the antiviral assay [12, 13] and no cytotoxic effects were observed with CC50 (cell survival by 50%) of 5-ALA > 2000 µM and of SFC > 500 µM in Vero E6 cells (Table 1). The Omicron variant which has notable mutations in the receptor binding domain of spike glycoprotein appears to be highly transmissible and less responsive to several of the currently used drugs [14]. Exogenously supplied 5-ALA prompted increased generation of protoporphylin IX (PPIX) and heme inside host cells, likely interfering with interaction of G-quadruplex (G4) structures [15] which inhibited SARS-CoV-2 infection. The G4 structure included in coronaviruses plays a key role in the genome replication/transcription [16]. 5-ALA with SFC is a supplement formulation registered in Japan as a food with functional claims. In a recent clinical study, Japanese patients with COVID-19 who were given 5-ALA and SFC capsules orally experienced a shorter time to recovery than that reported for patients who received only standard care for SARS-CoV-2 infection [17]. Recruitment for clinical trials on the effects of 5-ALA with SFC on COVID-19 outcomes in humans has been completed and the data is now being analyzed (Japan Registry of Clinical Trials CRB 7180001 and 3190006, respectively). Mitochondrial dysfunction has been reported as a cause of disorders in COVID-19 [18]. Given that 5-ALA activates the respiratory chain of mitochondria via heme bio-synthesis, maintenance of mitochondrial function is also expected to play a role in the effect of 5-ALA on the prevention and treatment of COVID and long COVID. In conclusion, we report the antiviral effects of 5-ALA with or without SFC on SARS-CoV-2 Omicron variant in vitro as a potential therapeutic and prophylaxis for COVID-19.

Fig. 1
figure 1

Antiviral effect of remdesivir (A) 5-ALA (B) and 5ALA with SFC (C) against SARS-CoV-2 Omicron variant. Vero E6 cells were pretreated with remdesivir, ALA with and without SFC for 72 h and challenged with SARS-CoV-2. Infected cell supernatants at 48 h pi (MOI 0.02) were quantified by quantitative real time RT-PCR assay. The blue and red lines represent the CC50 and IC50, respectively; the blue squares represent cell viability (%) and the red circles represent SARS-CoV-2 infection inhibition (%). Data were collected from at least two independent experiments (in replicate) and shown data correspond to the mean ± SD

Table 1 IC50 and CC50 values of 5-ALA and 5-ALA with SFC against SARS-CoV-2 Omicron variant

Availability of data and materials

Not applicable.



Severe acute respiratory syndrome coronavirus 2


Coronavirus disease 2019


5-Aminolevulinic acid


Sodium ferrous citrate


Protoporphyrin IX


Quantitative real time reverse-transcription polymerase chain reaction

IC50 :

Virus inhibition by 50%

CC50 :

Cell survival by 50%




  1. World Health Organization. Weekly epidemiological update on COVID-19—8 March 2022

  2. Takashita E, Kinoshita N, Yamayoshi S, et al. Efficacy of antiviral agents against the SARS-CoV-2 omicron subvariant BA.2. N Engl J Med. 2022;386:1475–7.

    Article  Google Scholar 

  3. Yu J, Collier ARY, Rowe M, et al. Neutralization of the SARS-CoV-2 omicron BA.1 and BA.2 Variants. N Engl J Med. 2022.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Callaway E. The race for coronavirus vaccines: a graphical guide. Nature. 2020;580:576–7.

    CAS  Article  Google Scholar 

  5. He X, Hong W, Pan X, et al. SARS-CoV-2 omicron variant: characteristics and prevention. MedComm. 2020;2021(2):838–45.

    Google Scholar 

  6. Zhang L, Li Q, Liang Z, et al. The significant immune escape of pseudotyped SARS-CoV-2 variant omicron. Emerg Microbes Infect. 2022;11:1–5.

    CAS  Article  Google Scholar 

  7. Vangeel L, Chiu W, De Jonghe S, et al. Remdesivir, molnupiravir and nirmatrelvir remain active against SARS-CoV-2 omicron and other variants of concern. Antiviral Res. 2022;198:105252.

    CAS  Article  Google Scholar 

  8. Fang FF, Shi PY. Omicron: a drug developer’s perspective. Emerg Microbes Infect. 2022;11:208–11.

    CAS  Article  Google Scholar 

  9. Bharathi M, Sivamaruthi BS, Kesika P, et al. In silico screening of bioactive compounds of representative seaweeds to inhibit SARS-CoV-2 ACE2-bound omicron B.1.1.529 spike protein trimer. Mar Drugs. 2022;20:148.

    CAS  Article  Google Scholar 

  10. Li P, Wang Y, Lavrijsen M, et al. SARS-CoV-2 omicron variant is highly sensitive to molnupiravir, nirmatrelvir, and the combination. Cell Res. 2022;32:322–4.

    CAS  Article  Google Scholar 

  11. Sakurai Y, Ngwe Tun MM, Kurosaki Y, et al. 5-Amino levulinic acid inhibits SARS-CoV-2 infection in vitro. Biochem Biophys Res Commun. 2021;545:203–7.

    CAS  Article  Google Scholar 

  12. Ngwe Tun MM, Sakura T, Sakurai Y, et al. Antiviral activity of 5-aminolevulinic acid against variants of severe acute respiratory syndrome coronavirus 2. Trop Med Health. 2022;50:6.

    Article  Google Scholar 

  13. Ngwe Tun MM, Toume K, Luvai E, et al. The discovery of herbal drugs and natural compounds as inhibitors of SARS-CoV-2 infection in vitro. J Nat Med. 2022;76:402–9.

    CAS  Article  Google Scholar 

  14. Tiecco G, Storti S, Degli Antoni M, et al. Omicron genetic and clinical peculiarities that may overturn SARS-CoV-2 pandemic: a literature review. Int J Mol Sci. 2022;23:1987.

    CAS  Article  Google Scholar 

  15. Shioda N, Yabuki Y, Yamaguchi K, et al. Targeting G-quadruplex DNA as cognitive function therapy for ATR-X syndrome. Nat Med. 2018;24:802–13.

    CAS  Article  Google Scholar 

  16. Kusov Y, Tan J, Alvarez E, et al. A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication-transcription complex. Virology. 2015;484:313–22.

    CAS  Article  Google Scholar 

  17. Nakajima MK. Safety, Tolerability, and efficacy of 5-aminolevulinic acid phosphate, an inducer of heme oxygenase 1, in combination with sodium ferrous citrate for the treatment of COVID-19 patients. Open COVID J. 2021.

  18. Lei Y, Zhang J, Schiavon CR, et al. SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE2. bioRxiv. 2020

Download references


Not applicable.


This work was supported by grants from Japan Agency for Medical Research and Development (Grant Number JP22wm0125006) and Nagasaki University “Doctoral Program for World-leading Innovative and Smart Education” for Global Health, “Global Health Elite Programme for Building a Healthier World”.

Author information




MMNT, JY, KK, KM: conceptualization, study design. MMNT, TS, YS, YK, DI: data analysis and investigation. MMNT: writing-original draft preparation. MMNT, NS, CS, YS, YK, KK: writing-review and editing. JY, KK, KM: supervision and funding acquisition. All authors read and agreed final version of the manuscript.

Corresponding authors

Correspondence to Mya Myat Ngwe Tun, Jiro Yasuda, Kouichi Morita or Kiyoshi Kita.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

Kita K. is a Scientific Advisor of Neopharma Japan. The other authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ngwe Tun, M.M., Sakura, T., Sakurai, Y. et al. 5-Aminolevulinic acid antiviral efficacy against SARS-CoV-2 omicron variant in vitro. Trop Med Health 50, 30 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • SARS-CoV-2
  • Omicron variant
  • 5-ALA
  • SFC
  • Antiviral activity