ReVIEW

Potential therapeutic effect of oxygen-ozone in

controlling of COVID-19 disease
Bahman Yousefi1, Seyedeh Zahra Banihashemian2, Zahra Khatibiyan Feyzabadi2, Sahar Hasanpour3, Parviz Kokhaei4, 5,
Anna Abdolshahi6, Alireza Emadi7, Majid Eslami8, *
1 Department of Immunology, Semnan University of Medical Sciences, Semnan, Iran
2 Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran
3 Department of Microbiology and Mycology, Science and Research Branch, Islamic Azad University, Tehran, Iran
4 Cancer Research Center, Semnan University of Medical Sciences, Semnan, Iran
5 Department of Oncology-Pathology, BioClinicum, Karolinska University Hospital Solna and Karolinska Institute, Stockholm, Sweden
6 Food Safety Research Center (Salt), Semnan University of Medical Sciences, Semnan, Iran
7 Deputy of Research and Technology, Semnan University of Medical Sciences, Semnan, Iran
8 Department of Bacteriology and Virology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran

*Correspondence to: Majid Eslami, MD, [email protected].

orcid: 0000-0001-6440-4424 (Majid Eslami)

Abstract
Atmospheric ozone is produced when nitrogen oxides react with volatile organic compounds. Severe acute respiratory syndrome corona-
virus 2 (SARS-CoV-2) genome contains a unique N-terminal fragment in the Spike protein, which allows it to bind to air pollutants in the
environment. ’Our approach in this review is to study ozone and its effect on the SARS-CoV-2 virus and patients with coronavirus disease
2019 (COVID-19). Article data were collected from PubMed, Scopus, and Google Scholar databases. Ozone therapy has antiviral proper-
ties, improves blood flow, facilitates the transfer of oxygen in hypoxemic tissues, and reduces blood coagulation phenomena in COVID-19
patients. Ozone has immunomodulatory effects by modulating cytokines (reduction of interleukin-1, interleukin-6, tumor necrosis factor-α,
and interleukin-10), induction of interferon-γ, anti-inflammatory properties by modulating NOD-, LRR- and pyrin domain-containing protein
3, inhibition of cytokine storm (blocking nuclear factor-κB and stimulating nuclear factor erythroid 2-related factor 2 pathway), stimulates
cellular/humoral immunity/phagocytic function and blocks angiotensin-converting enzyme 2. In direct oxygen-ozone injection, oxygen reacts
with several biological molecules such as thiol groups in albumin to form ozonoids. Intravenous injection of ozonated saline significantly
increases the length of time a person can remain hypoxic. The rectal ozone protocol is rectal ozone insufflation, resulting in clinical improve-
ment in oxygen saturation and biochemical improvement (fibrinogen, D-dimer, urea, ferritin, LDH, interleukin-6, and C-reactive protein). In
general, many studies have shown the positive effect of ozone therapy as a complementary therapy in the recovery of COVID-19 patients.
All the findings indicate that systemic ozone therapy is nontoxic and has no side effects in these patients.

Key words: COVID-19; coronavirus; immunity; MERS; ozone; SARS; SARS-CoV-2

doi: 10.4103/2045-9912.325989
How to cite this article: Yousefi B, Banihashemian SZ, Feyzabadi ZK, Hasanpour S, Kokhaei P, Abdolshahi A, Emadi A, Eslami M. Potential
therapeutic effect of oxygen-ozone in controlling of COVID-19 disease. Med Gas Res. 2022;12(2):33-40.

INTRODUCTION of these viruses are associated with viral pneumonia. The

Severe acute respiratory syndrome coronavirus 2 (SARS- principle pathogenesis of COVID-19 involves; severe lung
CoV-2) belongs to the group of beta-coronaviruses and has inflammation and immune deficiency, which are mutually
a diameter in the range of 50–200 nm, which is nearly 500 associated with an excessive inflammatory immune response
and enlarged production of cytokines (cytokine storm) against
times larger than the size of an ozone molecule. The SARS-
viral infection that cause damage to the host cells.5,6
CoV-2 genome is surrounded by a coating of spike proteins.
Currently, ventilation oxygenation and fluid dealing stay the
The main genetic material of this virus is a positive-strand
standard of maintenance for hospitalized COVID-19 patients.
RNA virus that is protected by a nucleocapsid (N), the virus
Numerous clinical trials and healing interventions presently
genome encodes four structural proteins, all of which are
purpose to recognize the most effective drug or combination
required to produce the complete virus.1 It is understood against the disease.7,8 Currently investigated approaches
that interaction between angiotensin-converting enzyme 2 include antiviral (antivirals can be confirmed as harmless and
(ACE2) cell receptor and viral spike protein facilitates the active only in the context of randomized controlled clinical
coronavirus entry into respiratory epithelial cells, and ACE2 trial and anti-proinflammatory cytokines, anti-infectious and
is extremely expressed in these cells. Along with viral-receptor monoclonal antibodies, and passive immunotherapy (plasma
interaction, the proteolytic cleavability of spike protein has therapy), especially in critical patients.9
been considered as the cause of disease strictness.2,3 Epidemiological data available in Tibet, China and high-
The pathogenesis of the SARS-CoV-2 is still not well clear. altitude areas of Bolivia and Ecuador indicate that high-
Coronavirus disease 2019 (COVID-19) is characterized as a altitude inhabitants (2500 m above sea level) are less prone
multi-systemic disease. Most patients suffer a self-limited to severe side effects compared to low-lying areas in severe
course symptom; nevertheless, a few will experience serious SARS-CoV-2 infection.10 The two main findings of these
or even fatal diseases.4 In individuals, the special properties studies include the risk of virus half-life due to the high
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 Yousefi et al. / Med Gas Res www.medgasres.com

altitude of the environment and the second that hypoxia APPLICATION OF OZONE THERAPY IN DISEASES
causes ACE2 to be down-regulated as the primary target for OT has proven, lasting, safe effects with minimal and avoid-
SARS-CoV-2 virus binding in the respiratory epithelium.11,12 able side effects. Ozone is used in medicine to disinfect and
Article data were collected from PubMed, Scopus, and Google treat diseases. Appliances of action of OT include reaction
Scholar databases in 2020–2021 with terms of COVID-19, with polyunsaturated fatty acids and water, ozone creates
coronavirus, ozone, SARS-CoV-2, treatment and therapeutics. H2O2 and at the same time, ozone forms a mixture of lipid
Therefore, this review aims to investigate the physiological ozonation products.21
and immunological mechanisms and beneficial possibilities Its mechanism of action is to inactivate bacteria, viruses,
of ozone on COVID-19 infection and review various clinical fungi, yeasts, and protozoa by stimulating oxygen metabolism
studies in patients with COVID-19. and activating the immune system.15 Newly, the potential
effect of ozone on inactivation of the virus in vivo has been
OZONE recognized, as well as the efficiency and safety of ozone
The ozone molecule, consisting of three oxygen atoms, is the administration on acquired immunodeficiency syndrome, B,
strongest oxidant found in nature, which in gaseous form has and C hepatitis, Ebola, and influenza diseases, however, this
a half-life of about 1 hour at room temperature and returns method along with other managements can be acceptable and
quickly to oxygen.13 The Scripps Institute found that our bod- synergic.22
ies also produce ozone.14 In ozone therapy (OT), 1–5% ozone Ozone can attack proteins, spike lipids, and virus coatings,
in 95–90% oxygen is used as a gas mixture.15 Ozone is the especially tryptophan, methionine, cysteine, arachidonic acid,
strong oxidant following fluorine and persulfate but has higher linoleic acid, and oleic acid.1 If the cytomegalovirus thiol
oxidizing power than oxygen. In OT, the oxygen/ozone gas groups are oxidized, it loses its pathogenicity. Regeneration
mixture is produced using an ozone generator, in which due of thiol groups with dithiothreitol restores 65% of the patho-
to the short half-life of ozone, the concentration of ozone is genicity of the virus. human immunodeficiency virus for
reduced by half in 40 minutes at 20°C and is reduced by half pathogenicity and the Ebola virus also need sulfhydryl groups
in 25 minutes at 30°C.16 to enter cells.15 Cysteine is very susceptible to oxidizing disul-
Ozone has been widely studied in medicine and is presently fide (R-S-S-R) or extra residues, which disrupts the chemical
used in various concentrations in various fields of medicine.17 activity of proteins by altering their three-dimensional struc-
Ozone exhibits contradictory activity in contact with organic ture. If the reduced thiols are oxidized, the enzymes may be
molecules, therefore triggering a strong antioxidant reaction.18 inactivated, and ozone immediately oxidizes the thiol groups
Ozone exerts oxidative preconditioning that can reverse upon contact.15 The ability of ozone to inactivate cysteine-
chronic oxidative stress conditions associated with stimula- containing proteins has been reported as an ozonide attack
tion of the production of radical free scavengers and cell wall on cysteine-dependent papain protease enzyme. It is believed
protectors like glutathione peroxidase, catalase, and superoxide that ozone converts the active sulfhydryl group to sulfenate/
dismutase.19 It also has the distinctive aptitude to inactivate sulfonic acid by oxidation and inactivates the enzyme. Also,
biological contaminants, containing viruses, through the oxi- the spike of coronaviruses is rich in tryptophan, which is after
dation of double bonds. Ozone interrupts the wall integrity of cysteine in terms of vulnerability to oxidation (Figure 1).123
bacterial cells, resulting in lysis and death, and thus effectively High concentrations of ozone can be dangerous to the lungs
controls the growth of spores of various dermatophytes.13 In if inhaled directly for long periods. There are several ways to
minor autohemotherapies, inducing the oxidation of viral-free use ozone that are widely used. While controversial, direct
components extracted from blood samples containing 90 g/mL intravenous injection of ozone gas is the simplest and most ef-
ozone may be practical, which could theoretically be consid- fective technique of ozonation and is widely used. Intravenous
ered as an inactive and immunogenic vaccine. Biochemically, injection of ozone gas does not pose a similar risk because
when the blood is exposed to ozone for a few minutes, it im- it is not composed of 80% nitrogen, and moreover ozone is
mediately reacts with various molecules in biological fluids, highly solvable in blood and plasma. The solubility of ozone
including antioxidants, proteins, carbohydrates, and preferably in water is 10–13 times higher than oxygen. Depending on
unsaturated fatty acids, resulting in leads to the formation temperature and pressure, about 50 mL of ozone gas dissolves
of α-hydroxy hydroperoxides, hydrogen peroxide (H2O2), in 100 mL of water, while only 4 mL of oxygen dissolves
ozonides, and 4-hydroxynonenal. They play a significant role in 100 mL of water.24 Some researchers prefer the usage of
in modulating inflammation, proliferation, growth, and cell extracorporeally ozonated autologous blood transfusion with
death.20 When blood is exposed to ozone, oxygen balances a volume of 100–200 mL, which is extensively recognized as
with extracellular fluid and intracellular fluid before it binds a major autohemotherapy.25 Different procedures for major
to hemoglobin. In contrast, ozone, which has 10 times more auto-hemotherapy typically use only 100–200 mL of blood.
solubility than oxygen, provides a soluble and bimolecular The disadvantage of this method is that it needs blood sam-
composition in biological fluids and dissolves easily in water, pling, heparinization, and ozonation of blood, followed by
reacts immediately with several biomolecules like ascorbic autologous blood transfusion. This procedure is problematic
acid, urate, free cysteine, glutathione, and albumin thiol by the association of heparin with important side effects (al-
groups, and then disappears. The compounds produced in the lergic and thrombocytopenia) as well as the need for personal
reactions are “ozone messengers” and are accountable for their protective equipment against COVID-19.26 In many organs,
biological and therapeutic properties.20 such as the pancreas, peritoneum, liver, mesenteric lymph

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Figure 1: Proposed therapeutic effects of O3 therapy for the treatment of COVID-19.

Note: COVID-19 serves as an ACE2 receptor to enter into human respiratory epithelial cells through its Spike proteins. The transcription factor Nrf2 bound to Keep-1
activated by alkenals. The released Nrf2 translocates into the nucleus and, after binding to Maf, docks on ARE and activates several genes leading to the synthesis of
antioxidant proteins. O3 can inhibit apoptosis and degradation of the cartilage matrix by inhibiting the activation of NF-κB resulting in cell survival. O3 has immunomodulatory
effects by anti-inflammatory properties by modulating NLRP3, inhibition of cytokine storm (reduction of IL-1β, IL-6, TNF-α, IFN-γ, IL-18, IL-12, GM-CSF), O3 can stimulate
cellular and humoral immunity through secondary messengers (H2O2) and NFAT/AP-1 signaling pathway. O3 is a multifunctional drug that can stimulate inflammatory
cytokines (IL-1β, IL-6, IFN-γ, TNF-α), anti-inflammatory cytokines (IL-4, IL-10). Biological responses to Nrf2/ARE activation with mild OT oxidative stress increase the
levels of direct antioxidants such as GSH, CO, and bilirubin through glutathione and thioredoxin reductase. O3 can reduce biomarkers such as CRP, ESR, and uric
acid, which have been shown to reduce CRP in patients with COVID-19. O3 increases oxygenation to the blood and tissues prevents the formation of small thrombosis
by secretion of some prostacyclins such as PGI2. O3 increases the expression of HO-1 in endothelial cells, stimulates 2–3 diphosphoglycerates, so more oxygen to
the delivered tissues in COVID-19 patients. O3 or its mediators (ozonides [ROS, LOPs]) are capable of oxidizing cysteine and tryptophan residues on S-spike protein
and preventing their binding to the ACE2. O2-O3 also affects NO and iNOS signaling pathways. NO, especially by inhibiting palmitoylation of S protein, reduces the
viral replication of COVID-19, thus preventing the virus from binding to the ACE2 receptor. ACE2: Angiotensin-converting enzyme 2; AP-1: activating protein-1; ARE:
antioxidant response element; CO: carbon monoxide; COVID-19: coronavirus disease 2019; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; GM-CSF:
granulocyte-macrophage colony-stimulating factor; GSH: glutathione; H2O2: hydrogen peroxide; HO-1: heme oxygenase-1; IFN-γ: interferon-γ; IL: interleukin; iNOS:
inducible nitric oxide synthase; Keep-1: Kelch like-ECH-associated protein 1; LOP: lipid ozonation product; Maf: musculoaponeurotic fibrosarcoma; NFAT: nuclear factor
of activated T-cells; NF-κB: nuclear factor-κB; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; NO: nitric oxide; Nrf2: nuclear factor erythroid 2-related factor
2; O2: oxygen; O3: ozone; OT: ozone therapy; PGI2: prostaglandin I2; ROS: reactive oxygen species; TNF-α: tomur necrosis factor-α.

nodes, and cecum, OT has been shown to have decreasing in stabilizing the protein structure and replicating the virus.30
properties on bacterial translocation. In an investigational The glycoproteins in the spikes, which cause the virus to en-
sample of necrotizing pancreatitis, OT was found to be more ter cells, are decorated with N-linked heterogeneous glycans
operative in reducing oxidative stress, tissue damage, and and are the chief goal of antibodies.1 Intravenous injection
bacterial translocation than hyperbaric oxygen therapy.27 After of ozonated saline significantly increases the length of time
using intraperitoneal ozone, Schulz et al.28 observed a decline a person can remain hypoxic and delays the lowest point of
in polymicrobial peritonitis. the oxygen saturation curve.31 Ozone is recommended due to
its strong oxidizing power, which can inactivate and destroy
OZONE THERAPY OF COVID-19 PATIENTS SARS-CoV-2. This is justified not only by the fact that ozone
Recently, China, Italy, Spain, and South America have re- is a potent oxidant but also by the fact that SARS-CoV-2 is
ported that the use of the OT method is beneficial in patients a coated virus that is vulnerable to oxidant attack. Ozone
with COVID-19-related acute respiratory failure.29 The lipid can attack viruses at different points in their structure and
structure of SARS-CoV-2 comprises fatty acids, including oxidizing viral capsids and genetic materials prevent them
arachidonic acid, linoleic acid, palmitic acid, and oleic acid, from reproducing and multiplying, which by this mechanism
and is rich in glutamic acid. In particular, arachidonic acid and damages the integrity of viruses. Although any viral structure
linoleic acid have been shown to play a significant role in the can be attacked by ozone, structures with more double bonds
infectivity mechanism of the coronavirus.1 Also, the amino or high electron density groups will be more vulnerable to
acids cysteine and tryptophan are abundant in the structure ozone oxidation.1 Another important feature of OT against
of coronavirus proteins, which are important in membrane COVID-19 infection is the ability of ozone to counteract the
fusion. The amino acid methionine also plays a significant role severe hypoxemia that the virus causes.1,18 There are several

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suggested mechanisms by which OT as an adjunct therapy for unsaturated fatty acids (polyunsaturated fatty acids) and al-
patients with COVID-19 may potentially improve outcomes. dehydes to produce hydroperoxides, especially H2O2, which
spread rapidly through the cells of the immune system. It also
DIFFERENT OZONE ACTIVITY IN COVID-19 PATIENTS regulates biological signal transduction, therefore, increasing
Oxygen-ozone has a high solubility in plasma and produces immune responses, modulating interferon, and interleukins
secondary messengers such as H2O2, ozonoids, and alchenals through NF-κB activation, hence increasing cytokine re-
(Figure 1). These are mainly competent, interacting with lease (Figure 1). The human body can produce ozone to
membrane proteins and cell receptors, especially immune protect itself against infectious agents. This occurs with the
cells. They enter the cells and interact with signal transduc- participation of neutrophils and immune system antibodies
tion proteins in the nucleus and mitochondrial surface. Ozone that kill bacteria and viruses by producing ozone, using its
can inhibit the process of infection by oxidizing S proteins, oxidizing power.1,36 It significantly reduces the concentration
ozone, and its reactive oxygen species can also attack the virus of NADH and contributes to the oxidation of cytochrome C,
coat, and if they can penetrate the virus coat, they can attack thereby stimulating oxygen metabolism, as well as showing
its capsid, genome, and RNA, in which case the virus is not anti-inflammatory function and cellular protection resulting
able to reproduce.32 from interaction with NF-κB and Nrf2.37
The ability of ozone to efficiently inhibit cysteine-dependent Çakır et al.38 described that systemic markers of the in-
proteins has recently been described as an ozone attack on flammatory response (tumor necrosis factor-α (TNF-α) and
cysteine-dependent papain. It is supposed that enzymes can interleukin (IL)-1β levels) decreased after ozone treatment.
be deactivated by oxidation of the active sulfhydryl group Biological responses to Nrf2/antioxidant response element
to sulfate or sulfonic acid. These mechanisms can openly activation with mild OT oxidative stress increase the levels
reduce SARS-CoV2 infection and lead to faster recovery of direct antioxidants such as glutathione, carbon monoxide,
from pneumonia.23,33 Also, coronavirus spike proteins are and bilirubin and stimulate glutathione regeneration through
rich in tryptophan, which, like cysteine, is highly vulnerable glutathione and thioredoxin reductase (Figure 1). It also
to oxidation.34 Also, an interesting conclusion about D-dimer increases the levels of enzymes that detoxify oxidants and
expands the discussion of the role of oxygen-ozone therapy electrophiles and increases the levels of phase II enzymes
in thromboembolism or vascular and thrombotic disorders along with the inhibition of cytokine-mediated inflammation
related to COVID-19, because D-dimer typically increases through the induction of leukotriene B4 reductase, decreas-
during COVID-19 and is greatly reduced in the existence of
ing iron overload and the following oxidative stress. Ozone
ozone. Oxygen-ozone also affects nitric oxide (NO) and induc-
can reduce biomarkers such as C-reactive protein (CRP),
ible nitric oxide synthase signaling pathways. NO, especially
erythrocyte sedimentation rate, and uric acid, which have
by inhibiting palmitoylation of S protein, reduces the viral
been shown to reduce CRP in patients with COVID-19
replication of SARS-CoV2, thus preventing the virus from
(Figure 1).39
binding to the ACE2 receptor (Figure 1). Ozone also increases
Ozone shows strong anti-inflammatory properties by modu-
the expression of inducible nitric oxide synthase, the major
lating NOD-, LRR- and pyrin domain-containing protein 3
enzyme-producing NO in type 2 pneumocytes in rats.35
(NLRP3) inflammation, that plays a vital role in the onset
Immunomodulatory functions and persistence of inflammation in various diseases.40
The inflammatory response is characteristic of severe infec- Ozone is known to have the ability to activate the immune
tion, and the modulation of cytokines is important to prevent system, while ozone is a weak inducer, lymphocytes, and
disease exacerbation. Oxidative stress and innate immunity monocytes reinfused during major autohemotherapy can
play vital roles in lung damage pathways that control the migrate through the lymphatic system and activate other
severity of acute lung cytotoxicity in viral infections such as cells over time, stimulating the immune system. Ozone can
severe acute respiratory syndrome.16 During acute inflamma- stimulate cellular and humoral immunity through secondary
tory procedures, to increase the response rate, nuclear factor- messengers (H2O2) and the signaling pathway nuclear factor
κB (NF-κB) increases the activity of mitochondrial NADPH of activated T-cells and the activator protein 1 pathway. These
oxidase, the major source of endogenous superoxide anion ways are important transcription factors because they induce
radical. There are strong relations amongst the coordinated gene expression for the release of inflammatory cytokines
activity of gene activation by both transcription factors, NF- (IL-2, IL-6, IL-8, TNF-α, and IFN-γ). Ozone can disable the
κB and nuclear factor erythroid 2-related factor 2 (Nrf2), to virus by direct oxidation (ozone) or indirect (reactive oxygen
resolve inflammatory procedures at the cellular and tissue species) and lipid oxidation products and can stimulate the
levels. An imbalance among the NF-κB and Nrf2 pathways cellular and humoral immune systems early in the COVID-19
is also related to COVID-19 (Figure 1).17 infection stage.17
An additional vital role that ozone plays in COVID-19 is its Ozone is a multifunctional drug that can stimulate in-
immunomodulatory properties. The main functional mecha- flammatory cytokines (IL-1β, IL-6, IFN-γ, TNF-α), anti-
nism of oxygen-ozone therapy is active in the proteasome inflammatory cytokines (IL-4, IL-10), nitric oxide secretion
and cascade of inflammation, to control the inflammatory or stem cells and can inhibit the NF-κB pathway, that can
process associated with stimulation of nuclear factor Nrf2 play the main role in stimulating hyper inflammation or
and inhibition of nuclear factor NF-κB.23 Ozone reacts with cytokine storms.41

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Physiological functions divided into ozone autohemotherapy and control groups. This
Ozone has several beneficial properties that can be beneficial treatment contained systemic administration of oxygen-ozone
in the treatment of COVID-19. Systemic oxygen-ozone was twice a day for 7 days. All patients were treated with the best
used to evaluate adjuvant therapy in the initial control of temporary treatment accessible. Mortality within 30 days was
disease development in patients with COVID-19 pneumonia. 3.3% for the ozone group and 10% for the control group. But
The data show that OT can improve the clinical condition of he did not report any toxicity.43
COVID-19 patients in a very short period, and increase many One study found that OT increased the antioxidant action of
gas exchange parameters in people experiencing forced non- Nrf2 and inhibited apoptosis through attenuating nucleotide-
invasive ventilation in intensive care units (ICUs).20 binding receptor domain-like oligomerization, which includes
Ozone can be through; activation of the pentose phosphate NLRP3-mediated inflammation. As a result, given that sys-
pathway, increasing the content of 2,3-diphosphoglyceric acid temic oxygen therapy has all of these positive effects: control-
as well as increasing glucose-6-phosphate dehydrogenase and ling inflammation, stimulating the immune system, antiviral
stimulating the metabolism of oxygen in red blood cells to ability, protecting against ischemia-reperfusion injury, acting
provide sufficient energy and oxygen to the tissues. This may on the proteasome and inflammation. OT can be considered as
allow for a drastic change in functional activity that shifts tis- a new method of immunoceutical therapy. They also showed
sues and organs from hypoxic to normoxic Also, it improves that the protective effect of OT with its anti-inflammatory
blood flow and capillary action, which has been reported to properties was achieved by modulating NLRP3 inflammation.
be beneficial for patients with ischemic vascular disease.17,22 Mixing ozone and oxygen at low concentrations can effectively
Therefore, ozone stimulates the glycolysis of red blood cells improve organ ischemia-reperfusion, which is what happens
and increases the oxygen released to the tissues and stimulates in the lungs of patients with COVID-19 infection. Ischemia-
the Krebs cycle, and thus increases ATP production (Figure reperfusion is the leading cause of lung dysfunction in many
1).13 pathological diseases.23,44
OT stabilizes hepatic metabolism and causes affinity, Tanaka et al.45 showed how influenza viruses are deacti-
plasma levels of fibrinogen, and prothrombin to normal in vated by low concentrations of ozone in the environment and
infected patients, indicating a development in hepatic protein on smooth surfaces. Further studies have shown that ozone
synthesis.29 Ozone has an antiplatelet effect and increases can play a key role in fighting bacteria, viruses, and fungal
the secretion of some prostacyclins such as prostaglandin I2, diseases. Murray et al.46 showed a reduction in viral infection
which is useful for patients with microthrombosis (Figure after ozone exposure. This causes lipid peroxidation in the
1). All of these effects can help reduce the phenomenon of virus capsid, therefore disturbing its reproductive cycle and
hypercoagulation in patients with COVID-19.40 OT rapidly preventing essential interaction between the virus and the
returns patients to normal respiratory physiology of the lungs, receptor. Study has shown how ozone can deactivate strains
reducing inflammation and ischemia and thromboembolic ef- with or without virus coverage.47 Some strains, such as herpes
fect, and ultimately leading to a complete recovery of oxygen simplex virus type 1 and vesicular stomatitis virus decrease af-
saturation parameters.32 ter receiving ozone. Significant showed in infectious particles
in 15 minutes. VAC strains (Elstree strain) and H1N1 strain
CLINICAL PRACTICE OF OZONE IN THE COVID-19 (Influenza A virus) showed a decrease in 40 and 30 minutes,
THERAPY respectively. These outcomes indicate significant changes in
OT was discovered in the mid-1800s and has been used in the morphology of diverse virus species.18
medicine for nearly a century. This substance is used spe- In one study,48 the effect of rectal ozone on COVID-19
cifically in the pre-antibiotic period for infectious diseases. patients with severe pneumonia at the University Hospital of
Ozone improves gas exchange, decreases inflammation, and Santa Cristina in Madrid has been investigated. Four patients
modulates the antioxidant system, making it beneficial for the with severe bilateral pneumonia due to COVID-19 confirmed
inflammatory phase or the cytokine phase, and in the hypox- by reverse transcription polymerase chain reaction (+) for
emia or multifocal stage.42 SARS-CoV2 were treated with rectal ozone. The intra-rectal
Supplemental use of OT for patients with COVID-19 ozone treatment procedure consists of five sessions (one ses-
through auto-hemoinfusion can improve oxygenation of sion per day) in a volume of 100 mL and a concentration of
tissues, reduce lung inflammation and regulate the immune 35 μg/mL. This method improved the oxygen saturation of
response, prevent cytokine storm, slow viral growth, regulate patients. Inflammatory biomarkers such as fibrinogen, D-
pulmonary microcurrents and prevent or reduce the rate of dimer, urea, ferritin, lactate dehydrogenase, IL-6, and CRP
vascular hypertrophy and subsequent hyperemia, especially in were also decreased. In conclusion, radiological symptoms of
the early stages, by counteracting endothelial damage, similar bilateral viral pneumonitis improved by 1 to 2 degrees based
to what occurs in peripheral arterial injuries.29 on Taylor’s radiological scale. Therefore, it was concluded
Evaluating new therapeutic resources to combat COVID-19 that rectal ozone is safe, effective, inexpensive and a simple
is a priority in clinical trials due to the limited number of option that can affect the SARS-CoV-2 virus and is offered as
available options. Probiozovid is a progressive, interven- an adjunctive treatment option in the management of severe
tional, randomized, and prospective, double-arm trial in bilateral pneumonia in COVID-19 patients.48
which COVID-19-induced pneumonia patients participate.43 In a study by Fernández-Cuadros et al.,17 ozone was shown to
In this study, 28 patients were included and were randomly be antiviral, to modulate the immune system, to stimulate cel-

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lular and humoral immunity, and to facilitate oxygen transport which is rich in the amino acid cysteine. These amino acids
in hypoxemic tissues, so it could be used in the treatment of are found in the spike, membrane proteins, and coatings of the
SARS-CoV-2. In Italy, people are treated with ozone via auto- SARS-CoV-2 virus. By oxidizing the reduced thiol groups of
hemotherapy to manage patients with COVID-19 pneumonia. the amino acids cysteine and tryptophan, ozone can prevent
Improvement of general clinical condition, normalization of the virus from entering the host body and exert its lethal effect.
temperature, reduction of CRP, and improvement of oxygen OT against the SARA-CoV-2 virus has antiviral properties,
saturation were observed.17 improves blood flow, facilitates the transfer of oxygen in hy-
A report from a clinical case in China presented that a poxemic tissues, and reduces blood coagulation phenomena
49-year-old patient with severe pneumonia kept in the ICU in patients with COVID-19. Ozone has immunomodulatory
showed evidence of clinical improvement from the outset effects by modulating cytokines (reduction of IL-1, IL-6,
after five sessions of ozone by autohemotherapy. The effect TNF-α, and IL-10) that prevent disease exacerbation, induc-
of OT on tissue oxygenation lasted nearly 9 hours and the tion of INF-γ has anti-inflammatory properties by modulating
patient was effectively transferred to the internal medicine NLRP3, inhibition of cytokine storm (ozone blocks the NF-κB
ward. In addition to the reduction of IL-6 and SARS-CoV-2 pathway and stimulates the Nrf2 pathway), stimulates cellular
negative polymerase chain reaction resulting from nasal swabs, and humoral immunity, stimulates phagocytic function, and
significant reductions in D-dimer, fibrinogen, and CRP were blocks the receptor of the virus, ACE2.
observed.48 In Spain, Hernández et al.49 published the report There are various methods for injecting ozone gas, contain-
of a 49-year-old patient with severe pneumonia. The patient ing penetration into the external ear canal, vagina, rectum, and
needed to use a mechanical ventilator and be admitted to the bladder, as well as direct injection of ozone gas into muscles,
ICU, and was prescribed ozone (autohemotherapy) as his last joint spaces, and discs to relieve back pain. In direct oxygen/
hope for treatment. The patient showed rapid recovery, to the ozone injection, oxygen is equilibrated with extracellular
fluid and intra-erythrocytic before binding to hemoglobin,
point that after two sessions he did not need to be admitted
and ozone, which is more soluble than oxygen, reacts with
to the ICU and after five sessions the need for oxygen was
several biological molecules such as thiol groups in albumin
significantly reduced. Hernández et al.49 emphasized that the
to form ozonoids. Intravenous injection of ozonated saline
clinical improvement was due to the modulation of the immune
significantly increases the length of time a person can remain
system, oxygen delivery, and the antioxidant role of ozone
hypoxic. Many physicians have used intravenous ozonated-
through autohemotherapy in this patient.
saline injections to prevent out-of-body ozonation concerns
OT has been used successfully as an adjunct therapy for
and incompatibilities with heparin. Oxygen-ozone therapy
patients with COVID-19 in China, Spain, Italy, and South
seems to be an effective treatment for COVID-19 patients
America.50 One suggested mechanism is to improve blood/ with severe respiratory failure. The rectal ozone protocol is
tissue oxygenation to prevent multi-organ system failure due intrarectal ozone, which can cause clinical improvement of
to lack of oxygen.26 In three patients with COVID-19-induced oxygen saturation, biochemical improvement (fibrinogen, D-
pneumonia who had respiratory failure, a rapid improvement dimer, urea, ferritin, LDH, IL-6, and CRP), and 1–2 degree
in hypoxia was observed with a decrease in inflammatory improvement of radiological symptoms which are assessed
markers and D-dimer, which was observed after 1–4 sessions based on the Taylor Radiology Scale. Rectal ozone is a safe,
of oxygen-ozone treatment. In these three patients, invasive effective, inexpensive, and simple option that can affect the
mechanical ventilation was not essential. After treatment with SARS-CoV-2 virus and is offered as an adjunctive treatment
oxygen-ozone, all patients were discharged home in 3–4 days. choice in the management of COVID-19 severe bilateral
Oxygen-ozone therapy appears to be an effective treatment pneumonia.
for COVID-19 patients with severe respiratory failure. Many Ozone prevents virus replication and kills the virus, increases
controlled clinical trials are needed to evaluate the efficacy oxygenation to the blood and tissues, stimulates immune func-
and safety of oxygen-ozone therapy compared to the typical tion through Nrf2, inhibits inflammatory pathogens (interleu-
supportive case in patients with COVID-19 in terms of the kins, cytokines, and TNFs), prevents the formation of small
necessity for invasive ventilation and the length of stay in thrombosis, increases the expression of heme oxygenase-1 in
the hospital.40 endothelial cells, stimulates 2–3 diphosphoglycerates, so more
oxygen to the delivers tissues.
CONCLUSION It is important to note that medical ozone should not be
Atmospheric ozone is an air pollutant produced during the administered directly into the bloodstream and should not be
reaction between nitrogen oxides and volatile organic com- mixed in solution with other drugs (other than saline) due to
pounds. The virus genome contains a distinctive N-terminal its oxidizing effect. There are also no studies on OT and preg-
fragment in the Spike protein, which allows the coronavirus to nancy. Therefore, it should be avoided in this case. Another
bind to air pollutants in the environment, although the effect of absolute contraindication is its systemic administration to
the virus spike on the transmission of the infectious agent has people with glucose-6-phosphate dehydrogenase deficiency.
not yet been proven. Some studies support the stimulant effect Medical ozone administration in patients with hyperthyroid-
of ozone on increasing virus replication. Atmospheric oxygen ism can also be harmful to the lungs. In general, many studies
may gradually degrade the thiol groups in the virus and also have shown the positive effect of OT as a complementary
increase the rate of degradation at higher temperatures. Many therapy in the recovery of patients with viral diseases in-
viruses require regenerated thiol groups to enter the host cell, cluding SARS-CoV-2. The results indicate that systemic OT
38 Medical Gas Research ¦ June ¦ Volume 12 ¦ Issue 2
 Yousefi et al. / Med Gas Res www.medgasres.com

is not toxic and has no side effects in patients with severe 9. Gavriatopoulou M, Ntanasis-Stathopoulos I, Korompoki E, et al.
COVID-19. Oxygen–ozone therapy seems to be effective Emerging treatment strategies for COVID-19 infection. Clin Exp
in COVID-19 patients with severe respiratory failure. OT is Med. 2021;21:167-179.
more effective for severe patient’s conditions, and oxygen is 10. Arias-Reyes C, Zubieta-DeUrioste N, Poma-Machicao L, et al.
an oxidizer that acts stronger the higher the temperature, so Does the pathogenesis of SARS-CoV-2 virus decrease at high-
altitude? Respir Physiol Neurobiol. 2020;277:103443.
it can be more effective than existing treatments in critically
11. Semple JL, Moore GWK. High levels of ambient ozone (O3) may
ill patients with fever.
impact COVID-19 in high altitude mountain environments. Respir
Physiol Neurobiol. 2020;280:103487.
Acknowledgements 12. Srivastava S, Garg I, Bansal A, Kumar B. SARS-CoV-2 infection:
We thank Department of Bacteriology and Virology, and Department physiological and environmental gift factors at high altitude. Virus-
of Immunology, Semnan University of Medical Sciences, Semnan, disease. 2020;31:1-3.
Iran for providing research platform. 13. Gavazza A, Marchegiani A, Rossi G, et al. Ozone therapy as a
Author contributions
ME investigate and supervised the findings of this work, wrote the possible option in COVID-19 management. Front Public Health.
article, supervised the project, contributed to the interpretation of the re- 2020;8:417.
sults; BY designed the study, helped supervise the project and conceived 14. Ghude SD, Jena C, Chate DM, et al. Reductions in India’s crop
the original idea; SZB and ZKF developed the theoretical framework; yield due to ozone. Geophys Res Lett. 2014;41:5685-5691.
SH and PK helped supervises the project, conceived the original idea;
AA and AE were responsible for manuscript revision. All authors dis- 15. Rowen RJ, Robins H. A plausible “penny” costing effective treat-
cussed the results and commented on the manuscript, provided critical ment for corona virus - ozone therapy. J Infect Dis Epidemiol.
feedback, and helped shape the research, analysis, and manuscript. The 2020;6:113.
authors contributed to the final version of the manuscript. 16. Martínez-Sánchez G, Schwartz A, Donna VD. Potential cytopro-
Conflicts of interest
The authors have no conflict of interest. tective activity of ozone therapy in SARS-CoV-2/COVID-19. An-
Financial support tioxidants (Basel). 2020;9:389.
This research is not supported by a specific project grant. 17. Fernández-Cuadros ME, Albaladejo-Florín MJ, Peña-Lora D,
Copyright license agreement Álava-Rabasa S, Pérez-Moro OS. Ozone (O3) and SARS-CoV-2:
The Copyright License Agreement has been signed by the author
physiological bases and their therapeutic possibilities according
before publication.
Plagiarism check to COVID-19 evolutionary stage. SN Compr Clin Med. 2020;doi:
Checked twice by iThenticate. 10.1007/s42399-020-00328-7.
Peer review 18. Valdenassi L, Franzini M, Ricevuti G, Rinaldi L, Galoforo AC,
Externally peer reviewed. Tirelli U. Potential mechanisms by which the oxygen-ozone (O2-
Open access statement
This is an open access journal, and articles are distributed under O3) therapy could contribute to the treatment against the coronavi-
the terms of the Creative Commons Attribution-NonCommercial- rus COVID-19. Eur Rev Med Pharmacol Sci. 2020;24:4059-4061.
ShareAlike 4.0 License, which allows others to remix, tweak, and 19. LeBaron TW, Kura B, Kalocayova B, Tribulova N, Slezak J. A
build upon the work non-commercially, as long as appropriate credit new approach for the prevention and treatment of cardiovascular
is given and the new creations are licensed under the identical terms.
disorders. molecular hydrogen significantly reduces the effects of
oxidative stress. Molecules. 2019;24:2076.
20. Cattel F, Giordano S, Bertiond C, et al. Ozone therapy in COV-
References ID-19: A narrative review. Virus Res. 2021;291:198207.
1. Tizaoui C. Ozone: A potential oxidant for COVID-19 virus (SARS- 21. Sagai M, Bocci V. Mechanisms of Action Involved in Ozone Ther-
CoV-2). Ozone Sci Eng. 2020;42:378-385. apy: Is healing induced via a mild oxidative stress? Med Gas Res.
2. Davidson AM, Wysocki J, Batlle D. Interaction of SARS-CoV-2 2011;1:29.
and other coronavirus with ACE (angiotensin-converting en- 22. Zheng Z, Dong M, Hu K. A preliminary evaluation on the effi-
zyme)-2 as their main receptor: therapeutic implications. Hyper-
cacy of ozone therapy in the treatment of COVID-19. J Med Virol.
tension. 2020;76:1339-1349.
2020;92:2348-2350.
3. Yousefi B, Valizadeh S, Ghaffari H, Vahedi A, Karbalaei M, Eslami
23. Ricevuti G, Franzini M, Valdenassi L. Oxygen-ozone immuno-
M. A global treatments for coronaviruses including COVID-19. J
ceutical therapy in COVID-19 outbreak: facts and figures. Ozone
Cell Physiol. 2020;235:9133-9142.
Ther. 2020;5:9014.
4. Le Bert N, Tan AT, Kunasegaran K, et al. SARS-CoV-2-specific T
24. Levanov AV, Isaikina OY, Gasanova RB, Lunin VV. Solubility of
cell immunity in cases of COVID-19 and SARS, and uninfected
ozone and kinetics of its decomposition in aqueous chloride solu-
controls. Nature. 2020;584:457-462.
tions. Ind Eng Chem Res. 2018;57:14355-14364.
5. Chauhan AJ, Wiffen LJ, Brown TP. COVID-19: A collision of
complement, coagulation and inflammatory pathways. J Thromb 25. Viebahn-Hänsler R, León Fernández OS, Fahmy Z. Ozone in medi-
Haemost. 2020;18:2110-2117. cine: clinical evaluation and evidence classification of the systemic
6. Yousefi B, Eslami M. Genetic and structure of novel coronavi- ozone applications, major autohemotherapy and rectal insufflation,
rus COVID-19 and molecular mechanisms in the pathogenic- according to the requirements for evidence-based medicine. Ozone
ity of coronaviruses. Rev Med Microbiol. 2021. doi: 10.1097/ Sci Eng. 2016;38:322-345.
MRM.0000000000000265. 26. Thorp JA, Hollonbeck SA, Viglione DD, et al. Novel therapy for
7. Allam M, Cai S, Ganesh S, et al. COVID-19 diagnostics, tools, and COVID-19 does intravenous ozonated-saline affect blood and tis-
prevention. Diagnostics (Basel). 2020;10:409. sue oxygenation? J Gynecol Res Obstet. 2020;6:046-050.
8. Mark A, Crumley JP, Rudolph KL, Doerschug K, Krupp A. Main- 27. Kapicibaşi HO, Kiraz HA, Demir ET, Adali Y, Elmas S. Pulmo-
taining mobility in a patient who is pregnant and has COVID-19 nary effects of ozone therapy at different doses combined with
requiring extracorporeal membrane oxygenation: a case report. antibioticotherapy in experimental sepsis model. Acta Cir Bras.
Phys Ther. 2021;101:pzaa189. 2020;35:e202000604.

Medical Gas Research ¦ June ¦ Volume 12 ¦ Issue 2 39

 Yousefi et al. / Med Gas Res www.medgasres.com

28. Schulz S, Rodriguez ZZ, Mutters R, Menendez S, Bette M. Repeti- 39. Rosanna DP, Salvatore C. Reactive oxygen species, inflammation,
tive pneumoperitoneum with ozonized oxygen as a preventive in and lung diseases. Curr Pharm Des. 2012;18:3889-3900.
lethal polymicrobial sepsis in rats. Eur Surg Res. 2003;35:26-34. 40. Menendez-Cepero S, Marques-Magallanes-Regojo JA, Hernan-
29. Ranaldi GT, Villani ER, Franza L, Motola G. Devils and an- dez-Martinez A, Tallón FJH, Baeza-Noci aJ. Therapeutic effects
gels: ozonetherapy for microcirculation in COVID-19. Frenxiv. of ozone therapy that justifies its use for the treatment of COV-
2020;doi:10.31226/osf.io/c2jvt. ID-19. J Neurol Neurocrit Care. 2020;3:1-6.
30. de Haan CA, Kuo L, Masters PS, Vennema H, Rottier PJ. Coro- 41. Ferrara F, Pambianchi E, Pecorelli A, et al. Redox regulation of cu-
navirus particle assembly: primary structure requirements of the taneous inflammasome by ozone exposure. Free Radic Biol Med.
membrane protein. J Virol. 1998;72:6838-6850. 2020;152:561-570.
31. Bocci V. Biological and clinical effects of ozone. Has ozone thera- 42. López A, Martinson SA. Respiratory system, mediastinum, and
py a future in medicine? Br J Biomed Sci. 1999;56:270-279. pleurae. Pathologic Basis Vet Dis. 2017;471-560.e1.
32. Franzini M, Valdenassi L, Ricevuti G, et al. Oxygen-ozone 43. Araimo F, Imperiale C, Tordiglione P, et al. Ozone as adjuvant sup-
(O(2)-O(3)) immunoceutical therapy for patients with COV- port in the treatment of COVID-19: A preliminary report of probio-
ID-19. Preliminary evidence reported. Int Immunopharmacol. zovid trial. J Med Virol. 2021;93:2210-2220.
2020;88:106879. 44. Juchniewicz H, Lubkowska A. Oxygen-ozone (O(2)-O(3)) Thera-
33. Obeid BMA. Ozone autohemotherapy: possible mechanisms py in peripheral arterial disease (PAD): a review study. Ther Clin
of anti-viral action and anti oxidative. J Infect Dis Epidemiol. Risk Manag. 2020;16:579-594.
2020;6:117. 45. Hiroshi T, Miei S, Kousuke I, Yoshiaki M. Inactivation of influenza
34. Al-Motawa MS, Abbas H, Wijten P, et al. Vulnerabilities of the virus by ozone gas. IHI Eng Rev. 2009;42:108-111.
SARS-CoV-2 virus to proteotoxicity-opportunity for repur- 46. Murray BK, Ohmine S, Tomer DP, et al. Virion disruption by ozone-
posed chemotherapy of COVID-19 infection. Front Pharmacol. mediated reactive oxygen species. J Virol Methods. 2008;153:74-
2020;11:585408. 77.
35. Fakhrzadeh L, Laskin JD, Laskin DL. Deficiency in inducible ni- 47. Hudson JB, Sharma M, Vimalanathan S. Development of a practi-
tric oxide synthase protects mice from ozone-induced lung inflam- cal method for using ozone gas as a virus decontaminating agent.
mation and tissue injury. Am J Respir Cell Mol Biol. 2002;26:413- Ozone Sci Eng. 2009;31:216-223.
419. 48. Fernández-Cuadros ME, Albaladejo-Florín MJ, Álava-Rabasa S,
36. Suh Y, Patel S, Kaitlyn R, et al. Clinical utility of ozone therapy in et al. Effect of rectal ozone (O(3)) in severe COVID-19 pneumo-
dental and oral medicine. Med Gas Res. 2019;9:163-167. nia: preliminary results. SN Compr Clin Med. 2020;2:1328-1336.
37. Di Mauro R, Cantarella G, Bernardini R, et al. The biochemical 49. Hernández A, Viñals M, Isidoro T, Vilás F. Potential role of oxy-
and pharmacological properties of ozone: the smell of protection gen-ozone therapy in treatment of COVID-19 pneumonia. Am J
in acute and chronic diseases. Int J Mol Sci. 2019;20:634. Case Rep. 2020;21:e925849.
38. Çakır T, Aslaner A, Tekeli S, et al. Effect of ozone on colon anas- 50. Wang Z, Wang J, He J. Active and effective measures for the care
tomoses in rat peritonitis model. Acta Cir Bras. 2016;31:111-118. of patients with cancer during the COVID-19 spread in China.
JAMA Oncol. 2020;6:631-632.

Date of submission: May 13, 2021

Date of decision: May 29, 2021
Date of acceptance: June 20, 2021
Date of web publication: October
19, 2021

40 Medical Gas Research ¦ June ¦ Volume 12 ¦ Issue 2