Page One Featured Paper: Coronavirus: Medicine: Treatment:


Prophylactic Treatment for COVID-19 with Methylene Blue

in the Presence of Ascorbic Acid


by Hiroshi Morooka, M.D., Ph.D., Inst.Gov.

Morooka Neurosurgical & Pediatric Clinic

Okayama Japan


Link for Citation Purposes:


Coronavirus diseases 2019 (COVID-19), some without traceable origin, have been identified and are rapidly expanding in Europe, North America, Asia, and the Middle East with the first confirmed case being identified in Africa and Latin American countries. By March16, 2020, the number of cases of COVID-19 outside China had increased drastically and the number of affected countries, states, or territories reporting infections to WHO was 143. On the basis of alarming levels of spread and severity, and by the alarming levels of inaction on March 11, 2020, the Director-General of WHO characterized Covid-19 situation as a pandemic.


COVID-19 is a novel virus. It causes severe acute respiratory syndrome. Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is the an agent responsible for a surface-to-surface communicable disease that had infected approximately 4.7 million persons on May 17, 2020. It could be transmitted from human to human, via respiratory droplets or close contact. @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@


Health care providers need options to limit and control the spread of the virus between themselves and patients [1,2].


Viral Structure

COVID -19 is an enveloped, positive-sense, single-stranded RNA virus approximately 60 to 140 nm in diameter. The shape is a crown-like appearance under an electron microscope (coronam is the Latin term for crown). The lipid bi-layer envelope of these viruses is relatively sensitive to desiccation, heat, and detergents. The virus spike (S) protein is a large, type1, membrane glycoprotein that contain distinct functional domains near the amino (S1) and carboxy (S2) termini. The S1 firmly binds to the angiotensin converting enzyme 2 receptor in human cell, while the integral membrane S2 portion is required to mediate fusion of viral and cellular membranes.                     


A viral envelope is the outer most larger. The transmembrane domain of the E protein has been found to alter the membrane permeability.


Nucleocapsid (N) or N protein plays a vital role during virion assembly. The process of virion assembly requires efficient packaging of the viral RNA into the virion. For this, N protein associates with the viral genomic RNA, and together they form the ribonucleoprotein.


N protein has also been reported to bind to human cellar heterogeneous nuclear ribonucleoprotein A1 (hn RNPA1) with high affinity. It may be postulated that interaction COVID-19, N protein with hn RNPA1 may play a role in transcription and replication of viral genome[3,4,5] (Fig.1).






There is no specific treatment recommended for COVID-19, and no vaccine is currently available. The treatment is symptomatic, and oxygen therapy represents the first step for addressing respiratory impairment.


Among other therapeutic strategies, although systemic corticosteroids for the treatment of viral pneumonia or acute respiratory distress syndrome (ARDS) were not recommended, in sever ARDS these drugs are used (e.g., dexamethasone 6mg/day for 10 days ).


Preclinical studies suggested that an inhibitor of RNA polymerase, remdesivir as well as favipiravir could be effective.


Preventive measures are focused on the isolation of patients and careful infection control, including appropriate measures to adopt during the diagnosis and the provision of clinical care to an infected patient. For instance, droplet, contact, and airborne precautions should be adopted during specimen collection, and sputum induction should be avoided [6].


Oxygen has the third highest electron affinity and should be considerably more reactive than it is observed to be. Its reactivity masked because oxygen contains two unpaired electrons with the same spin quantum number (parallel spin), and only when this spin restriction is overcome can the true reactivity of oxygen be expressed. When oxygen is reduced by the step-wise addition of electrons, free radicals are formed.


O2 + e+ H„΄¨HO2 ( hydroperoxyl radical)

HO2 ¨H„΄+ O2 ▪—( superoxide radical )

O2 ▪—+ e+ 2H„΄ ¨ H2O2 ( hydrogen peroxide )

H2O2 + e ¨ OH ( hydroxyl radical )

OH+ e + H„΄ ¨ H2O


These free radical oxidation of polyunsaturated fatty acids in biological systems is known as lipid peroxidation.


These short-lived radicals (HO2,O2 ▪— ,H2O2 ,OH) attack biomolecules in a diffusion reaction and are able to crack polysaccharides, proteins and nucleic acid located less than a few nanometers from its site of generation.


First chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid results from the attack by any species with sufficient reactivity to abstract a hydrogen atom (H„΄ ) from a methylene ( — CH2 — ) group. Because a hydrogen atom contain only one electron (e)Aabstraction leaves behind  an unpaired electron on the carbon, —  CH —. The presence of a double bond in the fatty acid weakens the C—H bonds on the carbon atom adjacent to the double bond and thus facilitates H„΄ removal. Extensive lipid peroxidation in biological membranes cause loss of fluidity, falls in membrane potential, increased permeability to H„΄ and other ions, and eventual rapture leading to release of cell and organelle contents[7,8,9].


The production of lipid peroxides and their fragmentation to carbonyl compounds in injured membrane of COVID-19 may help us by killing the virus.


Methylene Blue (MB), following its introduction to biology in the 19th century by Ehrlich has found uses in various area of medicine and biology.


MB is first line of treatment in methemoglobinemias, is used ifosfamide-induced encephalopathy, and is routinely employed as a diagnostic tool in surgical procedures. MB has also been a lead compound in drug research against various bacterial and viral infections[10,11].


In its oxidized state, the color of MB is blue due to the fact that the phenothiazinium molecule absorbs visible light strongly in the region of 600-700nm. In its reduced form (leuco-MB), it is colorless and dose not absorb light in the visible region.


We discovered a new treatment for COVID-19, using the free radical formation through the oxidation of the leuco-MB in the presence of L-ascorbic acid.


The absorbance of oxidized MB was measured at 664nm.


Oxidized MB was reduced gradually to leuco-MB after adding L-ascorbic acid ( Fig.2 ).

( Fig.2 )


The solution of leuco-MB in the presence of L-ascorbic acid worked as the electron donor to oxygen and produced oxygen-free radicals.


When hydrogen peroxide (oxygen-free radical) was added to this solution, oxidized MB increased gradually. This reaction is dependent on the concentrations of hydrogen peroxide which abstract electrons from leuco-MB ( Fig.3 ).    


( Fig.3 )


The solution of colorless leuco-MB was made after adding L-ascorbic acid to blue-oxidized MB.


Cotton swab was dipped in the solution of leuco-MB in the presence of L-ascorbic acid. This swab was inserted into nasal picking-area once between May and August, 2020. 1000 clinical out-patients with lifestyle-related illnesses received this treatment without side effect.


No patients have COVID-19 infection, because oxygen-free radicals may kill the virus and inactivate virus.


Oxidized MB and leuco-MB together form a reversible oxidation-reduction system or electron donor-accepter couple ( Fig.4 ).


( Fig.4 )


MB is a well-known singlet oxygen sensitizer. There is a great deal of evidence suggesting that the photosensitized production of singlet oxygen by MB can cause damage to biochemical and biological systems. There is also evidence that photosensitizers can produce free radicals and that in the presence of ascorbic acid [12].


Coronaviruses as with Hepatitis, Influenza, Herpes, Newcastle Disease and Orthopox are enveloped viruses. Enveloped viruses are viruses that possess an envelope or outer coating that is composed of a lipid layer. Envelope viruses are easily inactivated by routine surface cleaning and disinfection. The inactivation mechanisms of several common virucidal agents have been reported. Treatment with ultraviolet radiation and Aqualution hypochlorous acid usually destroy the viral genome, whereas chlorine dioxide and heat interrupt the process of host cell recognition for virus binding [13]. These substances are not used as an inhalation because of the impairment of the respiratory tract.


The majority of disease-causing viruses and bacteria reach the body through mucosal surfaces, including through the nasal mucosa. The nasal cavity is rich in nasal-associated lymphoid tissue and is also an inductive and effective site of the immune system[14 ].


The nasal application with the solution of leuco-MB in the presence of L-ascorbic acid may kill COVIC-19 virus, and its antigen may work as a vaccine. This solution should be kept in a glass bottle with stopper to avoid oxidation and also in dark place to avoid photosensitization.



[1] D.L.Heymann, N.Shindo (2020) COVID-19: what is next for public health? Lancet, 395, 542

[2] J.Bedford, D.Enria, J. Giesecke, et al. (2020) COVID-19: towards controlling of a pandemic. Lancet, 395, 1015

[3] C.Chang, M. Hou, C.Chang, et al. (2014) The SARS coronavirus nucleocapsid protein-forms and functions. Antiviral. Res. 103, 39

[4] N.Satija, S.K.Lal (2007) The molecular biology of SARS coronavirus. Ann NY Acad Sci. 1, 26

[5] T.M.Gallagher, M.J.Buchmeiert (2001) Coronavirus spike proteins in viral entry and pathogenesis. Virology. 279, 371

[6] M.Cascella, M.Rajnic, A.Cuomo, et al. (2020) Features, evaluation and treatment coronavirus ( COVID-19) Stat Pears (Internet), Treasure Island (FL) Google Scholar.

[7] B.Halliwell, S.Chirico (1993) Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr, 57, 715

[8] J.M.C.Gutterige (1995) Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem,41, 1819

[9] H.Morooka, N.Hirotune, T.Wani, et al. (1994) Histochemical demonstration of free radicals (H2O2) in ischemic brain edema and protective effects of human recombinant superoxide dismutase on ischemic neuronal damage. Acta Neurochir 60, 307

[10] T.Arima, H.Morooka, T.Tanigawa, et al. (1976) Methemoglobinemia induced by chlorphenamidine. Acta Medica Okayama, 30, 57

[11] M.Oz, D.E.Lork, M.Hasan, et al. (2011) Cellular and molecular actions of methylene blue in the nervous system. Med Res Rev, 31, 93

[12] G.R.Buettner, T.P.Doherty, T.D.Bannister (1984) Hydrogen peroxide and by methylene blue in the presence of ascorbic acid. Radiat Environ Biophys, 23, 235

[13] M.R.Lewis (2020) Novel coronavirus (2019—nCoV), scope for aqualution hypochlorous acid solution for effective prevention and control. Electron water hygiene. Co.Uk/ Wp—content/ uplodes. Google Scholar

[14] A.Serralheiro, G.Alves, J.Sousa, et al. (2013) Nose as a route for drug delivery. 191.  T.M.Önerci (ed.), Nasal Physiology and Pathophysiology of Nasal Disorders, Springer-Verlag Berlin Heidelberg.





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