Viruses neutralization through a oxidation- reduction reaction on the virus envelope

Abstract

The name of the invention: "Neutralization of viruses through oxidation-reduction reaction on the virus envelope" suggests its use in medicine, both in the human and animal fields. This is not only a specific use, but also from the point of view of the dosage regimen, the method of dosage, a new group of patients, of component No. 1 - Cu2+ obtained in a solution of copper sulfate pentahydrate in distilled water, where copper must have at least two free ions and component No. 2 - L-ascorbic acid for neutralization of viruses through oxidation-reduction reaction on the virus envelope, which is characterized by the fact that it ensures the destruction of the virus envelope and thus the inhibition of viruses in humans and other warm-blooded animals. Both substances are administered in a non-toxic concentration appropriate for the human and animal organism and with an appropriate time interval, so that the most suitable conditions for the oxidation-reduction reaction on the virus envelope are created and maintained. The use of these two components establishes a scientifically proven premise for the successful prevention and treatment of infection with various types of enveloped viruses and the diseases they cause.

Description

[0001] Viruses Neutralization Through a Oxidation- Reduction Reaction on the Virus Envelope

[0002] Technical field

[0003] Although viruses have been known and studied since the end of the 19th century, our knowledge about them leaves much to be desired. Thanks to their “efforts,” the evolution of living organisms was successfully carried out on Earth. However, researchers are more focused on viruses that cause illness in living organisms, leading to huge economic losses. Methods to combat pathogenic viruses are quite limited. Let us briefly describe them.

[0004] Backround art

[0005] Vaccination takes first place. The method is old, proven, and often used. Sometimes it is the only method of saving a patient (rabies). A good effect is observed when fighting fairly stable viruses, for example, those that cause measles. The question automatically arises: why were deaths from measles in 2019-2021 numbered in the tens, if not hundreds? Perhaps, in addition to nature, research centers helped the virus mutate. The virus contained proteins that the immune system did not recognize and did not want to react to. A similar reaction of the immune system is observed with viruses like AIDS, COVID- 19, influenza, etc. Well-made vaccines, when the next modification of COVID-19 appears, at best, allow the disease to be transferred in a milder form. We should not forget that in virulent viruses, as a result of mutations, the surface proteins of the envelope can change, and these changes can reach 30%. This is the reason for the loss of vaccine effectiveness, not to mention side effects such as thrombosis, autoimmune disorders, etc.

[0006] The second group of fighters against viral infections are interferons — endogenous low molecular weight proteins (from 15,000 to 25,000) that have antiviral, immunomodulatory, and other biological properties, including antitumor activity. Interferon was discovered in 1957 and actively competes with interferons obtained by genetic engineering.

[0007] The third group is substances isolated from plants: flacoside (from the leaves of Amur velvet and Laval velvet), alpizarin (from the herb of alpine kopek and yellow kopek), helepin, and others.

[0008] The fourth group is synthetic antiviral agents with known and unknown mechanisms of action. Examples include protease inhibitors (indinavir, ritonavir, saquinavir), reverse transcriptase inhibitors (didanosine, stavudine, zalcitabine), influenza virus M2 protein inhibitors (rimantadine, adapromine), neuraminidase inhibitors (zanamivir, oseltamivir), and nonnucleoside reverse transcriptase inhibitors (nevirapine, delavirdine, abacavir) [1].

[0009] Disruption of viral replication does not mean complete victory over it. Using the example of HIV infection or even the common flu, it becomes clear that as a result of mutations, a drug that works great today turns into a dummy tomorrow. We observed a similar picture several decades ago. Sulfonamides, synthetic analogues of folic acid, revolutionized the fight against bacterial infection in their time, but over time, their effectiveness came to naught.

[0010] In the current situation, it makes sense to look for other approaches to combat viral infection. Our group has proposed one of them.

[0011] Living organisms contain millions of proteins, which, despite their exceptional diversity and functions, consist of 26 types of amino acid blocks or linear combinations. Tissues are either in a state of construction or destruction, so amino acids are always present in the blood. As a rule, these are a-amino acids, except proline and oxyproline. In the physiological pH range, the amino group is usually protonated, and the carboxy group is ionized.

[0012] - OH - in serine and tyrosine - COO- - in aspartic and glutamic acid

[0013] - SH- in cysteine - S-S - in cysteine

[0014] The second nitrogen-containing group - in arginine and histidin

[0015] Groups interacting with metal ions [2,4]

[0016] The molecular weight of proteins ranges from several thousand for hormones and enzymes (6500 for insulin) to tens of thousands (hemoglobin - 68000). The polypeptide chain of proteins has the following structure [3]

[0017] O R2 H O R4 H

[0018] II I I II I I

[0019] H2N— CH— C— N— CH— C— N— CH— C— N— CH— C- > - N— CH— COOH

[0020] I I II I I II I

[0021] N - terminal amino acid residue C - terminal amino acid residue Interaction of Metal Ions with Proteins

[0022] It is known that many ions, for example, Zn2+, Cu2+, Fe3 +, and others, are capable of forming complexes with proteins. However, unlike others, Cu2+is fully capable of demonstrating its position in the Periodic Table, as both ns- and (n-l)d-electrons participate in bond formation in copper and its analogues (Ag and Au) [5].

[0023] This kind of reasoning led to the emergence of drugs for which priorities are registered [12, 13]. The correctness of the working hypothesis was confirmed by later works [14, 15, 16]. Zinc and copper, being analogues in chemical properties, in many cases act as competitors in living nature, being part of metalloenzymes. The complexation of zinc with the SARS-CoV-19 virus, which caused the CO VID-19 pandemic, can only slow down the development of the infectious process since the existence of the complex is determined by the stability constant, which is not very high. In a dynamic system, there will always be more suitable ligands, and the virus will continue its harmful effects.

[0024] The works [15, 16] are of much greater interest. They show that even a small amount of copper ions obtained from atmospheric moisture condensate is sufficient to create an equilibrium amount of copper ions that react with CO VID-19 viruses, which leads to their death. The work is devoted to the interaction of bacterial walls with copper ions, which is also typical for the shells of viruses such as RNA viruses, SARS-CoV-2, influenza A viruses, and noroviruses.

[0025] There are at least seven works that are consistent with our approach. The invention

[0017] discloses a composition (with a pH from 3 to 5.5) for the respiratory tract that contains from 0.001 to 20% by weight of a metal compound, for example, copper, and from 0.01 to 10% by weight of an organic acid, for example, ascorbic acid, as well as a polymer that adheres to the mucous membrane. The use of this composition for the treatment of cold and flu symptoms caused by enveloped viruses such as rhinovirus, influenza virus, adenovirus, and coronavirus is described. The composition differs significantly from what we offer. High metal content implies the formation of salts with organic acids, which makes interaction with the virus envelope unlikely. The presence of OH and amino groups in biocompatible polymers prevents the formation of complex compounds with the virus envelope. In the presence of ascorbic acid and copper ions in an aqueous medium, Cu (II) is reduced to Cu (I) by an ionic mechanism. The patent

[0018] discloses a composition with a pH less than 4 for the treatment and prevention of diseases caused by respiratory viruses such as H INI, H7N7, and H5N1. It includes a water- soluble ammonium compound, acid, and water, and may also contain vitamin C. Ammonium ompounds form stable ammonia complexes with copper ions, and in the presence of ascorbic acid, copper (II) is reduced to Cu (I) by an ionic mechanism.

[0026] The article

[0019] states that copper ions have antiviral activity and suppress the infectivity of the avian influenza virus (CuSO4*5H2O was used). Studies have confirmed the possibility of complex formation but not the destruction of the virus.

[0027] The publication

[0020] describes that copper is included in copper-containing enzymes and proteins in the body, mainly in the liver. In the presence of vitamin C, the bond between ceruloplasmin and copper is broken. The extraction of copper from plant raw materials does not exceed 40-60%, which makes it insufficient to combat viral infection.

[0028] The article

[0021] showed that the combination of Cu(II) and ascorbate inhibits herpes simplex virus (HSV), which may be useful in the development of antiviral agents. However, the use of the term “ascorbates” is incorrect since they are not reducing agents by definition.

[0029] The article

[0022] describes the effects of Cu(II) and ascorbate on herpes simplex virus type 2 (HSV-2) in vitro and their protective effect in a mouse model. HSV-2 was completely inactivated in the presence of 2 mM ascorbate and 1 mM Cu(II). This confirms the correctness of our experiments.

[0030] The publication

[0023] reports the inactivation of the rabies virus (CVS) in the presence of ascorbic acid and copper sulfate. In vitro results do not confirm the recovery of rabies-infected animals.

[0031] We list the sources, links, and references named and numbered in the text in numerical order as follows:

[0032] 1. M.D. Mashkovsky. Medicines. Ed. sixteenth. M. New Wave 2010, 1216 p.

[0033] 2. D. Williams. Metals of life. Mir M. 1975, 237 p.

[0034] 3. A.N. Nesmeyanov, N.A. Nesmeyanov. The beginnings of organic chemistry. Book two, M. Chemistry 1970, 824 p.

[0035] 4. Inorganic Biochemistry. Ed. G. Eichhorn. Translated from English. Ed. M.E. Volnina, K.B. Yacimirsky. Mir M. 1978, Vol. 1, 712 s., Vol. 2, 736 s.

[0036] 5. N.S. Akhmetov. General and inorganic chemistry. M. Higher School 1981, 679 p.

[0037] 6. K.B. Yatsimirsky. Introduction to bioinorganic chemistry. Kyiv “Naukova Dumka” 1976,

[0038] 212 p. -

[0039] 7. Chemical Encyclopedia. Vol. 3, p. 4, M. “Big Russian Encyclopedia” 1972.

[0040] 8. A.P. Avtsin. Human microelementoses / / Klin, honey. - 1987 - No. 6, p. 36.

[0041] 9. P.J. Aggett. Physiology and metabolism of essential trace elements: An outline / / Clin Endocrinol. Metab. — 1985 — Vol. 14, No. 3, p. 513-543. B.W.C. Lan, L.M. Klevay. Postheparin plasma lipoprotein lipase in Copper-deficient rats / / J. Nutr. — 1982. — Vol. 112, No. 5, p. 928-933. F.Ya. Berenstain. Microelements in the pathology and physiology of animals. "Urajay" Minsk 1966. Tarasenko V.S., Lisaichuk Yu.S., Noga D.A., Tarasov A.A. “Method for the prevention and treatment of diseases caused by enveloped viruses, including coronaviruses.” Utility model patent No. 144746, October 26, 2020, application filing date April 17, 2020. Gunyan Kumar (IN), Anurag Kumar (IN), Tarasenko V.S. (UA), Noga D.A. (UA), Lisaichuk Yu.S. (UA), Tarasov A.A. (UA). “Method for the prevention and treatment of diseases caused by enveloped viruses, including coronaviruses (variants and a set of pharmaceuticals for its implementation).” Utility model patent No. 144749 with priority under applications No. 2020 02463 dated 04 / 17 / 2020 (UA), No. 2021 01730 dated 04 / 05 / 2021 (UA), No. 202041046651 dated 10 / 26 / 2020 (IN). Mohammad Tariqur Rahman, Syed Zahir Idid. «Can Zn Be a Critical Element in COVID- 19 Treatment?» Biological Trace Element Research (2021) 199:550-558. V. Govind S., Bharadwaj M., R. Sai Ganesh, Jithin Vishnu Karthik, V. Shankar, Balakrishnan Shankar, R. Rajesh. «Antiviral properties of copper and its alloys to inactivate covid-19 virus: a review)) Biometals (2021) 34:1217-1235. Intisar Salaha, Ivan P. Parkina, Elaine Allan. «Copper as an antimicrobial agent: recent advances)) (Review Article) RSC Adv. 2021, 11, 18179-18186. WO 2004 / 064867 Al (Procter & Gamble Company [US]) August 5, 2004 (05.08.2004). WO 2007 / 091037 A2 (Remedy Research Limited [GB]) August 16, 2007 (2007-08-16). Sagripanti, Jose-Luis et al. «Mechanism of Copper-Mediated Inactivation of Herpes Simplex Virus.» Antimicrobial Agents and Chemotherapy, Apr. 1997, Vol. 41, No. 4, p. 812-817. doi: 10.1128 / AAC.41.4.812. Betanzos-Cabrera Gabriel et al. «Inactivation of HSV-2 by ascorbate-Cu(II) and its protective evaluation in CF-1 mice against encephalitis.)) Journal of Virological Methods, 15 Sept. 2004, Vol. 120, No. 2, p. 161-165. doi: 10.1016 / j.jviromet.2004.05.003. Horie M. et al. « Inactivation and morphological changes of avian influenza virus by copper ions.» Archives of Virology, 1 Jul. 2008, Vol. 153, No. 8, p. 1467-1472. doi: 10.1007 / s00705-008-0154-2. Nelson V.A. «Nelson-Town antivirus theory.)) Medical Hypotheses, Jul-Aug. 1978, Vol. 4, No. 4, p. 340-352. doi: 10.1016 / 0306-9877(78)90069-5. 23. Madhusudana Shampur Narayan et al. «In vitro inactivation of the rabies virus by ascorbic acid.» International Journal of Infectious Diseases, Jan. 2004, Vol. 8, No. I, p. 21-25. doi: 10.1016 / j.ijid.2003.09.002.

[0042] Essence and principle of the invention

[0043] Our approach to solving the issue of viruses is their “neutralization through an oxidationreduction reaction on the virus envelope using copper sulfate pentahydrate with the condition Cu 11+ dissolved in distilled water and L-ascorbic acid”.

[0044] In summary, the use of an aqueous solution of copper pentahydrate Cu2+ in combination with L-ascorbic acid is an effective approach to combat viruses. Understanding the mechanisms of interaction of these components opens up new ways of their application. Model systems [4] demonstrated the oxidation of L-ascorbic acid catalyzed by metal ions. The catalytic effect of metal ions in the oxidation of L-ascorbic acid has long been known. In the presence of chelating ligands, the mechanism of action of Cu(II) changes. In this case, the reaction rate does not depend on the oxygen concentration; reduction occurs to distilled water. The reaction rate is limited by the stage of reduction of Cu (II) to Cu (I), forming a semiquinone-type radical. Oxidation of the Cu(I) chelate complex with oxygen occurs rapidly, allowing oxidation of the substrate and the formation of dehydroascorbic acid. If there is not enough space in the coordination sphere of the metal ion for the simultaneous coordination of the substrate molecule and oxygen (which occurs in the presence of a chelating ligand), then the reaction proceeds along the less favorable free radical pathway. This path, as seen in the previously mentioned chemical formula, is possible due to the relative stabilization of the intermediate free radical of L-ascorbic acid.

[0045] It can be expected that a radical mechanism of this type will be hampered in cases where the intermediate free radical of the substrate is not sufficiently stabilized.

[0046] The mechanism studied on model compounds was transferred to a system where the surface proteins of the virus envelope act as the ligand. As we previously indicated, the formation of complexes with biometals is a dynamic process and is characterized by a stability constant [6]. Consequently, the formation of complexes of proteins with biometals can be characterized as a violation of virus replication over time, but not its destruction. Despite the external similarity with some publications in which biomaterials and L-ascorbic acid appear, nowhere is it stated that they deliberately seek to carry out an oxidation-reduction reaction on the surface of the virus envelope by a radical mechanism with the formation of a free oxygen-containing radical of L-ascorbic acid, which destroys the virus envelope. The released peptides and proteins are then inactivated by the immune system as foreign.

[0047] How to Carry Out Such a Promising Reaction?

[0048] Of the 92 naturally occurring elements, 81 are found in the human body. Of these, 15 are recognized as essential, i.e., necessary. Among them is copper. Humans extract copper mainly from plants. The main function of copper is to participate in enzymatic reactions as part of copper-containing enzymes. In animals and humans, copper is present in an amount of 80-120 mg and is involved in processes such as oxidative phosphorylation, antioxidant protection, fatty acid metabolism, synthesis of connective tissues (collagen, elastin), and others [7, 8, 9]. The main absorption processes of copper occur in the stomach and small intestine. The mucous membrane contains metallothionein, which forms complex compounds with copper. In addition to the target chemical reaction, many others occur - with amino acids, bile acids, and other substances - which ultimately leads to the absorption of more than 40-50% of consumed microelements.

[0049] A lack of copper in the body can cause increased levels of triglycerides, lipid metabolism disorders, lack of glucose tolerance, hypercholesterolemia, and as a consequence, coronary heart disease, atherosclerosis, and even death from rupture of an abdominal aortic aneurysm. The normal daily intake of copper is 2-5 mg, while in the United States, no more than 25% of the population consumes 2-3 mg of copper, which is the lower limit of normal

[0010] . Having decided on the amount of Or* ions, which is at least 5 mg (daily norm), and even increasing it by 2-2.5 times in extraordinary cases (for example, in severe forms of COVID- 19 with lang damage up to 80*%), with split injections does not cause gag reflexes or intolerance. These doses are 50 times less than the toxic dose — 500 mg

[0001] . The frequency of administration should take into account the biology of the virus. To prevent the formation of Cm* aqua complexes, the acidity of the solution should be maintained within the pH range of 2.5-3.2, which also facilitates passage through cell membranes and entry into the blood plasma [5, 8 j. The carrier of copper in the blood is serum albumin (molecular weight 66.5 thousand), which includes 585 amino acid residues [2].

[0050] Methods of introducing copper into the body through nutrition or injection are not suitable for our purposes since they do not provide significant amounts of copper tons into the blood and are too long in time. Also, the injection method is unsafe - there have been fatal cases in experiments on animals ( 11].

[0051] The method of using an aqueous solution proposed by our group allows us to solve the problems that have arisen. Dilute solutions (0,05-0.1%) with a pH of 2.5-3, 2 provide long-term storage of CuSO4 -5H2O w i th Cu2*, which corresponds to the acidity of frui ts such as lemon. 1 mi of 0.1% solution contains 0.255 mg Cm * . Dilution in 50-70 ml of distilled water ensures unimpeded passage through the mucous membrane and walls of the esophagus into the blood plasma when taken slowly in small sips over 2-4 minutes. This makes it possible to minimize the binding of copper ions by mucosal proteins and the walls of the esophagus, which makes the method effective. More concentrated solutions stowed down the recovery of patients.

[0052] It is known from chemistry that the rate of a chemical reaction is proportional to the concentrations of the interacting substances (5).

[0053] V - 1 - C ■ f 2 where:

[0054] * .1 is the rate constart of the reaction,

[0055] « and C T are the concenwttons of the reacting substances.

[0056] As Cl > 11 the reaction mte 1" ■ 11

[0057] When taken In small sips and limited quantities, the solution passes through the mucous membrane and walls of the esophagus into the bloodstream without reaching the stomach. The low concentration of the solution minimizes the binding of copper ions by the mucosal proteins and the walls of the esophagus. Using more concentrated solutions slowed down the recovery rate of the patients.

[0058] It has been experimentally shown that 40-60 minutes after taking an aqueous solution of CuSO4-5H2O, an acceptable concentration of Cu2+ions is created in the body, ensuring the formation of chelate complexes with viruses. At this moment, it becomes necessary to take a second reagent — L-ascorbic acid in an amount of 0.5-1.0 g for adults (with a mandatory loading dose of 1.0 g once a day) and for children in accordance with body weight but not more than 0.7 g (up to 14 years) [1]. Thus, the passage of a redox reaction on the virus shell is ensured.

[0059] Example of implementation of the invention

[0060] The above experiments were first theoretically and then practically carried out by the creators of the method of "virus neutralization through oxidation-reduction reaction on the virus envelope" in vitro at the University of Veterinary Medicine and Pharmacy, Kosice, Slovakia, in the Laboratory of Biomedical Microbiology and Immunology.

[0061] In 2023, the creators of the method of neutralizing viruses through an oxidation-reduction reaction on the virus envelope began to establish cooperation with the United Research and Science Center Kosice and the European Association for Science and Research Kosice. The result of this cooperation was that, based on the Memorandum of Understanding from 2019, an Agreement was concluded on the implementation of animal toxicity tests and verification of the effectiveness of the two substances on viruses with the University of Veterinary Medicine and Pharmacy Kosice, and on its basis, the method of neutralizing viruses with a solution in distilled water of 5H2O CuSO4 (CuII+) + L-ascorbic acid was tested at the University of Veterinary Medicine and Pharmacy in Kosice in the fully certified Laboratory of Biomedical Microbiology and Immunology, laboratory level BSL 3, first on a certified encephalitis virus and then on a certified Sars-Cov-2 Omicron with successful results as follows:

[0062] Encephalitis virus (flavivirus - tick borne encephalitis) - individual parts of the method inhibitors inhibited the virus by 55% and their joint use - when administered simultaneously affecting the virus only 43.1%. This test demonstrated the unequivocal correctness of the procedure, when the first inhibitor CuSO4 5H2O administered in a non-toxic solution of distilled water, if administered simultaneously with the second inhibitor L-ascorbic acid, does not achieve the desired effect. On the contrary, the inhibition result is worse than that achieved by each inhibitor separately. Both substances in the verified concentration are non-toxic to animals. This negative definition confirmed the correctness of the procedure of administering active substances with an appropriate time interval.

[0063] Subsequently, the experiment on this virus was repeated with a time interval of 60 minutes between administration of the two active substances, with a result of 78.5% virus inhibition. Sars-Cov-2 virus - the methodology of use consisted in the first step, as with the encephalitis virus, of applying a non-toxic solution in distilled water 5H2O CuSO4 (Cu2+) to the virus and in the second step, after 60 minutes, of using L-ascorbic acid to inhibit the virus. The result of the experiment is an inhibition of the virus by 86.30% in vitro.

[0064] In our opinion, the above result establishes the right to protect this method of neutralizing viruses through an oxidation-reduction reaction on the virus envelope through a patent. All three tests were performed in the fully certified Laboratory of Biomedical Microbiology and Immunology of the University of Veterinary Medicine and Pharmacy in Kosice, BSL level 3, on certified viruses, while samples of active substances, biological material and test results are stored in the same laboratory in accordance with applicable EU legislation for the purposes of further research and possible verification of results.

[0065] The presented works show the interaction of viruses with copper ions and their destruction upon interaction with L-ascorbic acid performed on tissue culture. These studies are the first step in pharmacopoeial testing of this method.

[0066] The work of the group of creators, combined with the work of colleagues from various institutions, but especially from the University of Veterinary Medicine and Pharmacy in Kosice, allows us to confirm the adequacy of the proposed and substantiated concept of combating viral infection caused in general by enveloped viruses, i.e. viruses with a peptide / protein envelope, both RNA and DNA viruses, and especially with already certified tested viruses: SARS-COV-2 Omicron as a respiratory virus and encephalitis as a nervous virus.

[0067] In conclusion, we summarize the formula of the invention as: "a method of neutralizing viral infection by treating viruses with copper ions and subsequently, at a precise time interval, by treating L-ascorbic acid in the body of a human or other warm-blooded animal, characterized in that a redox reaction occurs on the viral envelope with the formation of an oxygencontaining radical that destroys the viral envelope, followed by neutralization of foreign proteins and peptides by the body's immune system and the return of the human or other warm-blooded animal to a normal physiological state".

[0068] For this purpose, basic substances permitted by the pharmacopoeia are introduced into the body, in particular CuSO4*5H2O with Cu2+ in a non-toxic concentration of 0.05-0.1%, in the form of a solution with a pH of 2.5-3.2 using distilled water, which allows overcoming membrane barriers of cellular structures in an amount of copper ions close to the physiological daily requirement and forming chelate compounds with viruses that reach them through the bloodstream, which is characteristic of their natural movement. The subsequent application of L-ascorbic acid is carried out with a discrete time delay in the patient of 40-60 minutes.

[0069] The amount of active ingredients depends on the person's body weight and pharmacologically legal restrictions. For example, in children under 14 years of age, a single dose of L-ascorbic acid should not exceed 0.7 g. The frequency of use of active ingredients, their amount and discretion depend on the nature of the virus, body weight and temperature in the normal state - a characteristic of the metabolic rate - and can be used for the prevention and treatment of humans and various warm-blooded animals.

[0070] Industrial application of the invention

[0071] The invention has so far undergone official in vitro testing. After the application is submitted, further in vitro tests on various viruses will continue, depending on the capacity of Slovak laboratories and the availability of certified viruses. We will then proceed to in vivo testing and preclinical tests, followed by clinical trials. Once the drug is registered, it will be possible to manufacture and sell it to patients as a promising drug for the treatment of diseases caused by enveloped viruses - viruses with an envelope.

Claims

AMENDED CLAIMS received by the International Bureau on 01 March 2026 (01.03.2026)1. Copper 2+ obtained in a non-toxic solution of copper sulfate pentahydrate in distilled water and L-ascorbic acid for use in the prevention and treatment of infections caused by viruses in the form of an oxidation-reduction reaction on the virus envelope, wherein L-ascorbic acid is administered to the patient with a time delay of 40 to 60 minutes after taking a solution of copper sulfate pentahydrate with Cu2+ in distilled water with a pH value higher than 2.5 up to 3.2.

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