The world's scientific and social network for malaria professionals
Subscribe to free Newsletter | 10241 malaria professionals are enjoying the free benefits of MalariaWorld today

Hydrogen peroxide and its hidden power

December 30, 2015 - 19:46 -- Pierre Lutgen

H₂O₂ is omnipresent in plants and animals.

ANALYTICAL DATA

If the concentration in human plasma is fairly well known (around 3 micromol/l), the content in plant material has not received much attention. This is to some extend related to the fact that it is difficult to measure micromolar concentrations. There are a few papers but most of them measure concentrations in fresh plant material; for several medicinal herbs they find concentrations which are around 3 micromol/g. This is 1000 times higher than in the human body. Finding data for dried material is almost impossible. Higher hydrogen peroxide concentrations have been noticed in urine after coffee consumption. In a paper on coffee grains the authors report astonishingly high values: up to 2mg/100 g (M Rendon, P Gratao et al., Europ Food Res Technol 2013, 236:753-758). Another paper deals with the dried persimmon, or kaki fruit. The concentrations in fresh material are the same as those in human plasma. In the dried material the concentrations of hydrogen peroxide are much lower in aqueous infusions (10 microg/l) or 1000 times less than in fresh plants (H Arakawa et al.,Biol Pharm Bull 2014, 37, 1119-1123).

HYDROGEN PEROXIDE AND WATER STERILIZATION

Page 1122 of the paper of Arakawa (op cit) is of interest. An excerpt hereafter. “Therefore, the effects of persimmon on eight intestinal bacteria were examined. A control experiment was carried out in a similar manner using a standard hydrogen peroxide solution. As a result, it was found that hydrogen peroxide is generated from persimmon tissues, and the bactericidal activity of persimmon extracts correlated with bacterial sensitivity to hydrogen peroxide”.

In 2008 a research team at Luxembourg had noticed on Microtox equipment, measuring the light emitted by Vibrio fischeri bacteria, that Artemisia annua tea significantly reduced the luminescence of these bacteria. In a second series of experiments the ATPase luminescence technique on a Pallman Luminometer was used. The bactericidal effect of Artemisia annua tea on three different samples of water were tested : water from the sewage plant at Stenay-F, river water from Niederanven-L and tap water. In all cases adding fresh Artemisia annua tea had a stronger bactericidal effect than boiling the contaminated water for 5 minutes or irradiating it for 10 minutes under a 365 nm UV lamp (P.Lutgen et al., Proceedings International Conference, Luxembourg June 3-4 2008). This effect was confirmed by several other laboratories, at the University of Bangui, at the University of Gent, at the University of Antioquia, at the University of Dakar. The bactericidal effect was supposedly related to artemisinin. Until the Universities in Medellin and Bangui found that after 24 hours this bactericidal property had vanished. The bacterial concentration was even increasing in the infusion. In light of the preceding paragraphs it is likely that the bactericidal properties were due to hydrogen peroxide in the infusion. This molecule has a decent half-life but shorter than artemisinin, especially in the presence of other organic molecules or enzymes in a tea infusion in an open glass. Artemisinin in infusions is in fact stable for weeks (unpublished data from the University of Liège). A strange effect had been noticed in our early trials. Tea which had been prepared the day before did not sterilize the water. We also noticed that although fresh tea initially inhibited the bacterial growth, after 8 hours it seemed to loose its power.

The most likely hypothesis to explain all this is hydrogen peroxide. It is formed rapidly (R Weinstain et al, JACS, 2014, 136, 874-877), in infusions in the first minutes of the addition of hot water. In freshly prepared coffee or tea it attains micromolar concentrations (SJ Rinkus et al., Food Chem Toxicol 1990, 28, 323-31).. Hydrogen peroxide is known to be a strong desinfectant. Against Vibrio cholerae it is 10 times stronger and faster than hypochlorite or ozone. (Shikongo-Nambabi et al., Water SA 36-3, 2010). Early this years Dr Jérôme Munyangi found that Artemisia annua and Artemisia afra infusions kill paramecia (personal communication). These protozoans were are also killed by wheat grass powder infusions and it was confirmed that hydrogen peroxide was the toxic substance in the infusion (N Mizobuchi, J Eukaryot Microbiol, 2003, 50, 299-303).

Peroxides are unique in that the oxygen exists in a -1 oxidation state, which lies between the usual states Oᵒ and O¯². It can easily switch to one of these states, which explains its instability. In the human body it appears and disappears so rapidly that it is difficult to measure it. Until 1969 it was considered that H₂O₂ even existed free in a biological milieu (JA Watson et al., J Gen Microbiol 1969, 57, 25-34). We can only speculate on the mechanism which destroys hydrogen peroxide in Artemisia annua tea infusion. In mammals it is the enzyme catalase. In the tea infusion organic molecules, iron or other metal ions probably play a role. Another argument in favor of hydrogen peroxide as active molecule in the killing of bacteria is that the University of Bangui observed that Artemisia annua infusions were only efficient in daylight, not in the dark. The UV/H2O2 system is well known as advanced oxidation process in which hydrogen peroxide is added in the presence of ultraviolet light to generate hydroxyl radicals. The oxidation potential of a hydroxyl radical is much greater than other oxidizing agents such as ozone and chlorine. Hydrogen peroxide strongly absorbs long wave present in daylight.

Nevertheless a freshly prepared liter of Artemisia annua infusion might provide a person with good quality drinking water if it is consumed in the first hours after preparation.

HYDROGEN PEROXIDE AND MALARIA

The susceptibility to oxidant mediated killing of Plasmodium falciparum by exposure to hydrogen peroxide has been assessed. The parasites were most susceptible during maturation, although a reduction in parasite invasion was also observed (S Kamchonwongpaisan et al., Parasitology, 1989, 99, 171-4). In another study, Plasmodium yoelii and Plasmodium berghei were killed in vitro by hydrogen peroxide at concentrations as low as 10¯⁵ M, in the nanomolar range as for artemisinin. Injection of hydrogen peroxide in vivo significantly reduced P yoelii parasitemia but has less effect on P berghei (M Hazel et al., Infection and Immunity, 1983, 39, 456-459). This same effect is described in another paper (IA Clark et al., Clin Exp Immunol, 1984, 56, 524-530). Intravenous injection of t-butyl hydroperoxide rapidly killed Plasmodium vinckei in mice. The injections of the hydroperoxide caused parasites to disintegrate inside erythrocytes The same dose seemed harmless to unparasitized mice. Many synthetic peroxides, like trioxolanes, have been developed as antimalarials.

Although the deleterious effects of H2O2-induced oxidative stress on malaria parasite viability are well established, and there is increasing evidence that some antimalarials exert their effect, at least in part, through oxidative mechanisms, the mechanisms by which oxidative stress disrupts parasite function are not fully understood. A recent study (D van Schalkwyk et al., PlosOne, 2013, DOI 10.1371) has shown that the oxidising agent H2O2, at concentrations comparable to those to which the parasite may be exposed in vivo causes a decrease in parasite ATP levels and a profound disruption of intracellular pH regulation: an acidification of the parasite cytosol and an alkalinisation of the digestive vacuole.

Hydrogen peroxide is able to degrade hemozoin (M Chen et al, Molec and Biochem Parasitol 2001, 113, 1-8). All Plasmodium species produce a brown birefringent crystal known as malarial pigment or hemozoin. Hydrogen peroxide as a test reagent can distinguish the hemozoins from various parasites by different concentrations needed to degrade half of the crystals. These differences have been studied for P falciparum. P malariae, P ovale, P knowlesi, P yoelli et others (GS Noland et al.,Mol Biochem Parasitol 2003, 130, 91-99). Reactions between beta-hematin and H₂O₂ show beta-hematin degradation with increasing concentration of the reactive agent (M Carter, Thesis Aug 2009, Vanderbilt University). Similar effects were seen for NO and it is possible that the small molecules NO and H₂O₂ work their way into the crystal lattice, disrupting the structural integrity of hemozoin from within as well from the surface. Unlike cations these molecules are neutral and can diffuse freely through membranes.

The involvement of H₂O₂ in the mechanism of action of chloroquine is supported by the fact that it enhances the activity against Plasmodium. Chloroquine by itself is a promoter of oxidative stress. The potentiation of chloroquine activity by the peroxidase-hydrogen peroxide system was already described 25 years ago (K Malhotra et al., Antimicrob Agents and Chemother 1990, 34, 1981-1985). Peroxide mediated oxidation of the porphyrin ring leads to its opening and subsequent breakdown. Inhibition of detoxification of ferriprotoporphyrin by hydrogen peroxide could account for the effectiveness of chloroquine in malaria (CD Fitch et al., Life Sciences 25 :42, 1988). Hydrogen peroxide also shows synergism with other antimalarials or antibiotics (S Utaida et al., Southeast Asian J Prop Med Public Health, 2014, 45, 1-5).

Hydrogen peroxide forms strong hydrogen bonds. Raman studies show that in aqueous milieu it has no free, non hydrogen-bonded OH groups (P Guigère et al., J Raman Spectroscopy, 1984, 15-3, 199-204). Hydrogen peroxide forms an iron coordination complex with heme, with one oxygen atom ligating the heme iron and the other forming hydrogen bonds to amino acids. This of course is a perturbing factor for heme crystallization.

A number of processes operate in the food vacuole to decompose the hydrogen peroxide that is produced in parasitized erythrocytes as consequence of the conversion of oxyhemoglobin to methemoglobin in schizonts. No increase in hydrogen peroxide over that observed in uninfected erythrocytes could be detected at the ring stage when host cell digestion is absent (H Atamna et al., Mol Biochem Parasitol 1993, 61, 231-41). Plasmodium falciparum also imports the human protein peroxiredoxin into its cytosol as an enzymatic scavenger for peroxide detoxification (S Koncarevic et al., PNAS, 2009, 106.22). Peroxiredoxin reduces hydrogen peroxide extremely rapidly (P Nagy et al., J Biol Chem. 2011, 286, 18048-55).

And then there is the unresolved issue of breast milk which protects neonates against malaria and other diseases during six months. This has been related to the absence of PABA (para amino benzoic acid) and the high concentrations of arginine and nitrates in mother’s milk. But breastmilk is also very rich in hydrogen peroxide. Colostrum contains up to 24 000 microM/L, thousand times more than in human blood! (E.A Al-Kerwi et al., Asia Pac J Chem Nutr 2005, 14, 428-431).

The University of Liège-Gembloux (Guy Mergeai personal communication) had a strange result on beta-hematin inhibition. Artemisia annua harvested late in the year did not inhibit. A possible hypothesis is that this is related to the decrease in arginine and the increase of proline with senescence. But senescence has other effects: several papers studied the senescence of plants and find for example that the hydrogen peroxide concentration in vine buds is 4 times lower in winter than in summer (S Qsaib et al., Int J Scient.Res Pub. 2014, 4 ISSN 2250-3153). Others have shown that freezing temperatures decrease hydrogen peroxide in the plants.

HYDROGEN PEROXIDE AND THE IMMUNE SYSTEM

Hydrogen peroxide is produced by the body's T-cells for destroying bacteria, viruses and fungi. Blood platelets release hydrogen peroxide when they encounter foreign particles in the blood stream. But there is a hormetic effect or in other words an optimal concentration of hydrogen peroxide in culture media around 50 microM. 5 years ago we found that Artemisia annua from Luxembourg activated lymphocytes and Artemisia annua from Cameroon did not. It is known that hydrogen peroxide is a potent activator of T lymphocytes (M Los et al.,Eur J Immunol 1995, 25, 169-165). It plays an important role as messenger, but the molecular mechanisms behind the activation are less clear (M Reth, Nature Immunology, 2002, 3, 1129-34). Over the years we have been looking at most of the molecules present in Artemisia to explain this difference between Artemisia from Cameroon and Luxembourg . We never considered hydrogen peroxide. But if so that does not yet explain the difference between Cameroon and Luxembourg. It is probably related to the 4x higher scopoletin concentration in Artemisia annua from Cameroon. Scopoletin is a strong antioxidant and scavenger of hydrogen peroxide. Artemisinin and hydrogen peroxide could also be antagonistic. The first activates NFkB and the latter desactivates it (personal unpublished data). Tea from Cameroon is very rich in artemisinin, 10x more than tea from Luxembourg.

Hydrogen peroxide is also generated by Cytochrome P450 enzymes and CYP3A4 have been shown to be the major source of H₂O₂ in liver microsomes (V Mishin et al. Toxicol Sci. 2014, 141, 344-352). In the case of alcolhol abuse the CYP2A1 generates excessive hydrogen peroxide in the liver and disrupts mitochondria.

Many people have noticed an enhancement of therapeutic properties in tea infusions in the presence of stems. Several papers mentioned in this document show that there is more hydrogen peroxide in pulps, skins, veins stems. Maybe the plant needs this enhanced protection in its “skin”.

And the question remains open if grinding of leaves and stems into powder will allow hydrogen peroxide and some monoterpenes to escape or not.

HYDROGEN PEROXIDE AND OTHER PARASITIC DISEASES

Hydrogen peroxide and hydroxyl radicals which are generated by macrophages in a respiratory burst at the beginning of an infection have also been shown to kill protozoa such as Toxoplasma and Leishmania (H Dockrell et al., Infection and Immunity 1984, 43, 451-6). During a straphylococcal infection H₂O₂ production of neutrophils was significantly higher on hour 24 as compared to controls and on day 14 (PV Delebov et al., Bulg J Veterin Med. 2006, 3, 183-188). Leishmania donovani promastigotes introduced into the bloodstream by sandfly vectors, are exposed to H₂O₂ during phagocytosis by the host macrophages. This exposure leads to a 10 fold increase in calcium concentration in the cells. Changes in Ca⁺² homeostasis is a common feature of programmed cell death (M Das et al., J of Cell Science, 2001, 114, 2461-69). Hydrogen peroxide inhibits the differentiation of Leishmania amazonenis promastigotes into amastigotes (D Eluobaju, DePaul Discoveries, 2015, 4, 13).

There is also the astonishing therapeutic effect on acne and warts. Several anecdotic reports on this effect have been received from our partners in Paris, Senegal, Kenya and Luxembourg. The Chinese Pharmacopeia says”: The fresh, crushed herb of Artemisia vulgaris can be applied to warts. When used several times a day in one study on 12 patients, warts fell off within 3-10 days.” The recommendation to use fresh herb could be related to the fact that fresh herb contains more hydrogen peroxide. There is indeed US patent 20040101571 A1 which claims that warts which are a viral infection, can be cured with cutaneous application of diluted hydrogen peroxide.

Hydrogen peroxide kills Salmonella typhimurium eggs (Unlütürk et al., J Appl Bacteriol 1987, 62, 25-8).

It is active against Mycobacterium tuberculosis (Ping Lu et al., Scientific Reports 2015, May 2015, 5-10333).

Artemisia infusions efficiently kill the unicellular organism Paramecium . This is not due to artemisinin because Artemisia afra which does not contain artemisinin has the same effect (Jerome Munyangi, personal communication). It could be due to a synergy between hydrogen peroxide and nickel which is well present in all Artemisias. It was found that nickel treatment caused a notable increase in in the hydrogen peroxide concentration in Paramecium caudatum (Y Takenaka et al. Eukaryotic Cell, 2014, 13, 1181-1190). Nickel, in the presence of hydrogen peroxide, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. Glutathione could completely recover the inhibition by nickel or H2O2 alone but only partially recover the inhibition by nickel plus hydrogen peroxide. This irreversible damage to the proteins involved in DNA repair, replication, recombination, and transcription could be important for the toxic effects of nickel.

A disease where the impact of hydrogen peroxide has been abundantly studied is schistosomiasis. Neutrophils and eosinophils attach themselves to schistosomula, provoke an oxidative burst and produce hydrogen peroxide which kills the parasite. Catalase can inhibit this process (JW Kazura et al., J Clin Invest 1981, 67. 93-102).. Schistosoma manzoni lodges eggs in granuloma in the liver. Around these granulomatous cells hydrogen peroxide can be detected by fluorescence. The parasite even produces its own antioxidants to destroy hydrogen peroxide. It was found that there was a sharp concentration treshold in hydrogen peroxide mediated killing. A depression in the levels of hydrogen peroxide available may be a mechanism by which the parasite can partially evade the host immune system (JM Smith et al., Am J Trop Med Hyg 1989, 40, 186-94). Activated leukocytes attach to the surface of blood fluke Schistosoma mansoni and secrete schistosomicidal substances, like the enzyme elastase. Co-treatment with both elastase and hydrogen peroxide indicated that they exert an additive toxic effect (A Freudenstein-Dan et al., J Parasitol 2003, 89, 1129-35).

Artemisia annua is very rich in bicarbonate. A study from Pakistan analyzing 10 medicinal herbs finds that it is top ranking (I Hussain et al., World Appl Sc J. 2011, 12, 1464-68). An ideal combination with polyphenols to generate H₂O₂. The natural supply of hydrogen peroxide in the body may be exhausted during a viral attack. A supply of hydrogen peroxide from external sources might be beneficial in this case to kill the viruses which are anaerobic.

STABILITY OF HYDROGEN PEROXIDE

Remains obviously the question of stability and homeostasis of hydrogen peroxide in living organisms. Hydrogen peroxide is membrane permeable and diffusible, less reactive and longer-lived than hydroxyl radicals or superoxide. The physiological range of intracellular hydrogen peroxide concentrations appears to be remarkably conserved in different forms of life. Among ROS, hydrogen peroxide is the only species that is generated and removed by several specific enzymes, which suggests that the intracellular concentration of hydrogen peroxide is tightly regulated. Superoxide dismutase SOD can catalyze the dismutation of superoxide radical into hydrogen peroxide and molecular oxygen. Hydrogen peroxide can be produced from glucose by glucose oxidase catalysis which explains that it might be higher in diabetics. The amino acid arginine also may generate superoxide and hydrogen peroxide via the NOS enzymes, glutathione peroxidase GPx and catalase CAT can convert it to water. Hydrogen peroxide may react with transition metals such as iron or copper to produce the highly reactive OH radical. In living organisms, besides its well-known cytotoxic effects, hydrogen peroxide also plays an essential role as a signaling molecule in regulating diverse biological processes such as immune cell activation. And a boost of hydrogen peroxide received from a medicinal herb may enhance this immune system activation.

At a first glance this beneficial role of hydrogen peroxide in living organisms appears to be in contradiction with the antioxidant theories. One could even say, the antioxidant hype and business.

If hydrogen peroxide really has a killing effect on Plasmodium and other parasites, it is important to avoid the consumption of substances which inhibit the peroxide production. It was shown that Vitamin C prevents the generation of hydrogen peroxide in Caco-2 cells. (SC Roques et al., Free Radic Res, 2002, 36, 593-9). ACTs are rendered less effective if taken with multivitamins that contain constituents such as Vitamin C. (S Parapini et al, Antimicrob Agents Chemother April 2015), (N Lindegardh et al., Bioanalysis 2011, 3, 1613.1624). Prudence is thus recommended for the consumption of ascorbic acid alongside Artemisia annua tea.