BIOLOGICAL ACTIVITIES OF SPECIAL OZONIZED NATURAL SUBSTANCES. FUNDAMENTALS AND APPLICATIONS

"INFORMATION FOR PROFESSIONALS"

It is presented the rationale for the bioactivity of ozone and some of its derivatives, based on its primary interaction with certain very reactive substances present in all living tissues. Justification for the systemic effect of ozone via ozone metabolites is referred. Following previous statements, also rationale is presented for the feasibility to obtain such ozone metabolitesin vitro by means of adequate model substances and coadjuvants in precise reactions conditions. The achievement of such goal can be reached by the special and careful ozonisation of certain natural substances. Such products have been submitted to the fundamental tests established for determination of bioactivity, stability and toxicity, to assess their main beneficial properties, so as stability and innocuity. Some of their properties, documented through different levels testing are also presented.

Different applications of ozonised substances for different treatments, so as the main therapeutic properties of ozone can be reviewed in the "Clinical Tests" section, where a set of clinical controlled studies on several applications of ozonised substances is presented. They include data about sample selection and distribution, initial diagnostics, tabulated results and conclusions.

Ozone is known as the Life Gas, due to the basic role it plays making possible the existence of living organisms on earth surface, thanks to the protection against lethal UV Sun radiation. Nevertheless, and it is still less widely known, also properly administered ozone offer to superior aerobic living organisms protection against deleterious free radical oxidation (ageing). So it is capable to revitalise and stimulate vital and protecting natural enzymatic processes and scavengers of cells.

Ozone therapy is a very valuable medical technology, highly useful in several fields and for many pathologies (Mattassi R. 1981, Quiñones M., 1988, Kramer F., Santiesteban R. 1990, Rilling, S.1983).

Ozone is a gas at normal atmospheric conditions and very unstable, due to its high energy level. That is the reason why the application of this technology involves the necessity of sophisticated equipment for generation, delivery and dosing, so as special handling and administration procedures and instruments (Viebahn R. 1983).

Research in the field of chemistry (Gómez M. 1983, Viebahn R. 1975), biochemistry (Rokitansky O. 1969) and fundamentals of the ozone effects on biological systems opened that the existence of ozone itself in any biological system is extremely brief, due to the presence in all this systems of several essential substances which undergo very fast reactions with this gas. Examples of such substances are the wide variety of unsaturated fatty acids (UFA), present in practically all living systems, free or as part of many different lipidic structures. Their double bonds are capable to react with ozone at reaction rates in the order of 10⁶ mole/L. sec. Such reaction rates cause that, in the presence of this kind of substances, the existence of ozone will be of only about a few hundredths of a second (Gómez, M. 1982).

Taking into account the before mentioned characteristics, it is easy to understand that systemic effects of ozone, so as also most of those at local tissue level must be achieved by means of the products of the main reactions, it means, via ozone metabolites. These ozone metabolites are the products of ozone reactions and/or ozonides decomposition in physiological conditions.

These metabolites, arising from chain ruptures in the unsaturated fatty acids, are similar to the endogenous lipid peroxides and hydroperoxides. They are shorter in chain length, so lower in molecular weight, and higher in hydrophilic character. For that reason, they are better able to penetrate cellular membranes. The arrival of such molecules to the citosolic phase produces the activation of Glutathione peroxidase which reduce them to alcohols, at expenses of the reduced glutathione (GSH), which is then oxidised to glutathione (GSSG). This passage occurs with an energy difference of 280 mV and renders available a protonic flow for the coupled reactions. At the same time, also the Glutathione reductase is activated to regenerate the GSH at expenses of the NADPH. These findings are supported by in vivo determinations during experimental tests in animals and humans, so as in ex vivo tests related with the protective effect produced by ozone metabolites in organs submitted to ischemia-reperfussion processes. The first result of this effect is the improvement of the Glutathione peroxidase System which is a key step in the protection of cells against oxidants.

In addition, to neutralise oxidative stress, the rate GSH - GSSG in the cytoplasm is then kept in a value of about 97,4 : 1 by the activated glutathione reductase, coupled with the system NADPH. « NADP. So, a correspondent improvement of the NADPH production will take place proportionally. The main way of production of NADPH is constituted by the Glucose-6-phosphatedehydrogenase in the glycolisis pentoses shunt, and for that reason the glycolisis is also accelerated, so accelerating ATP production and causing an increase in energy availability for the cells. See following Figure 1:

The consequent acceleration of the GSH turnover and of glycolisis is the final consequence of the effects of those mentioned short chain hydroperoxides. These effects render also higher energy availability for the cells in the form of ATP. Also 2,3-DPG production is increased, so improving oxygen release from oxyhemoglobin, therefore enhancing tissue oxygenation.

It is known that the increase in GSH availability and turnover within the cells is an important factor for the effectivity of the mechanisms protecting these cells against free radicals and peroxides. Moreover, it is also known that the redox system GSH dependent is also basically involved in other important processes as:
 

	Protection of microsomal thiolic enzymes and catepsine.
	Protection of exokinase (in conditions of diminished concentration of GSH, this enzyme reduces its activity and so it is also diminished the ATP synthesis).
	Participation in the processes of cellular nuclei breathing through the system GSH-ascorbic acid.

Also GSH is determinantly involved in basic detoxifying cell processes as:
 

	Glyoxalic reaction (hydration of ketoaldehydic substrates, inactivation of pyruvic aldehyde to lactic acid, porphyrins synthesis from g-dioxivalerate).
	Catabolic sequence of aromatic aminoacids.
	Dehydrogenation of formaldehyde to formic acid.
	Detoxification through biosynthesis of mercapturic acids with low ATP involvement.

And in proteic synthesis:
 

	Trans membrane supply of aminoacids through membrane g-glutamyl-transpeptidase.
	Promotion of the reduction of ribonucleotides-deoxiribonucleotides to DNA precursors.
	Stimulation of the own DNA synthesis.
	Maintenance of double helix structure of DNA, matrix of proteic synthesis.

Ozone metabolites existence have been evidenced in many laboratory investigations, so as the fact that many of them are much more stable than ozone itself. On the other hand, it is also known that some products of the normal chemical reactions of ozone with similar substances are less stable than ozone itself, even to the extreme to spontaneously decompose explosively. So, the key of the selective generation of useful ozone metabolites lies on the conditions, coadjuvants, catalysing substances and the order in which the reactions take place.

On the precedent basis, it is perfectly conceivable that it would be possible to generate ozone metabolites also in vitro and that their structure and stability could the closer resemble those of the in vivo ozone metabolites, the better could be possible also reproduce the living tissue conditions for the ozone reactions.

With the aim to determine the optimal chemical-physical characteristics for the obtention of the most stable and biologically active ozone metabolites, extensive research on this direction has been revised (Rainbauer H. 1982 Streichsbier Von F. 1982, Washuttl J. 1982). Many different biological substances, trace catalysers, coadjutants, etc. were combined in multiple forms of reactions with ozone and their products were consequently analysed and submitted to tests of stability, biological activity, so as toxicity, in order to obtain the higher chemical stability and bioactivity, free from toxic side effects.

So, optimised technologies have been designed to obtain formulations of ozone-natural substances derivatives possessing good stability, which have shown remarkable biological activity (Gómez M. 1989), and satisfactorily passed the established toxicity tests (oral & intraperitoneal LD50, ophthalmic & skin irritation, sensitisation, phototoxicity, mutagenicity, teratogenicity, etc.) (Gel A. 1989, Fernandez I. 1988, Rodriguez M. 1990).

Example of this derivatives is a particular kind of the commonly named ozonised oils, specially prepared according to the precedent postulates. It can be prepared in different forms: oleous liquid, lipofilic and hydrophilic emulsions and creams, unguents, ovules, suppositories, soft capsules, etc. They exhibit local biological activities and therapeutic effects similar to those of ozone administered in vivo (Behar R. 1989).

Among the therapeutic properties of this kind of ozonised oil, it can be pointed out, related to some of its local effects, the following:
 

	High general germicidal activity (antimicotic, antibacterial, antiviral).
	Local microcirculation activation.
	Cells oxygen metabolism improvement.
	Stimulation of oxidative defensive enzymatic systems.
	Stimulation of granulation & epithelisation tissue growth.
	Epithelial tissue revitalisation.

This ways of local application of ozone metabolites are very suitable for many of that kinds of pathologies traditionally treated by ozone therapy, achieving similar results although sometimes needing longer treatment periods, probably due to lower oxidant power and/or minor systemic effect. On the other hand, one of the advantages they have over ozone itself is the possibility to apply ozone therapy "at home", not requiring the physical presence of the patients in the places where the ozone gas treatments have to be applied. It can also be combined with ozone gas applications, during the periods between ozone therapy sessions.

Examples of entities in which good results have been achieved are, among others, the following (Cagigas T. 1989, Grillo R. 1990, Hernandez M. 1990, Arias l. 1990, etc.):
 

	Acne.
	Wrinkles attenuation.
	Dermatitis and skin spots.
	Cellulite & deteriorated skin.
	Scars, fistulae & post surgery wounds.
	Gastric ulcers
	Gastric giardiasis
	Gingivostomatitis.
	Lower limbs ulcers (arterial or venous insufficiency).
	Vulvovaginal infections.
	Chronic external otitis.
	Skin burns.
	Recidivant herpes simplex labialis & genitalis.
	Onicomicosis.
	Epidermophytosis.
	Infected teeth radicular conduits.
	Dental hiperstesia.

Important results achieved in several clinical tests performed on different pathologies are presented to support the preceding asserts.

Cell membranes are constituted, among others, also by lipidic structures. The interactions of ozone derivatives with cell membranes breaks excessive electrostatic attraction forces and bridges, so improving their relaxation, flexibility, permeability and deformability.

On Red Blood Cells, it also improves flexibility, deformability and permeability, so as facilitates dissaggregation of the so called "pile of coins aggregates". In this manner, it is achieved better circulation through the narrowest blood vessels (microcapillaries), and better capacity to absorb oxygen in the lungs and liberate it at tissue level for other surrounding body cells. See Figure 2 below:

The ozone metabolites produced by the interaction with the cell membranes are able to penetrate them and there stimulate several basic biochemical processes. One of their effects is to increase the production of 2,3 DPG, which is known to facilitate the liberation of oxygen from oxyhaemoglobin, at tissue level, see Figure 4 (red highlighted):

The experimental demonstration of  these effects is shown in Figure 5, through the results of a clinical study comprising 20 patients treated with ozone. During the study 2,3 DPG in RBC and uric acid (UA) in plasma were measured. UA is a measure of  the circulating oxidants level. As observed, 2,3 DPG significantly increases, which means the oxyhaemoglobin transfers more oxygen to cells, whereas UA diminishes indicating a drop in circulating oxidants level. All this effects imply noticeable improvement of cells condition.

The effect of   more intensive oxygen transference to tissue during the passage of  RBC through the capillary microvessels can be assessed by the evident drop in venous PO2, as seen in Figure 6:

As seen in the figure, the increase in 2,3 DPG facilitates the cession of the Oxygen trapped in oxyhaemoglobin in RBC. So when they pass through the microcapillary vessels of tissue, from the arterial to the venous section, they are better capable to transfer more Oxygen to the circundant tissue. It can be assessed by the drop in oxygen partial pressure in venous blood, which is then better exhausted from the transported Oxygen, so increasing its efficiency.

The ozone metabolites capacity to stimulate enzymes related to the oxidation-reduction processes is very important to enhance the cells protective capacity against aggressive oxidants and free radicals. Ozone metabolites interact with main concatenated enzymatic processes of the cells defensive system and significantly stimulate them. The last link in the defensive chain again oxidants is The Glutathione Redox System, which is activated by ozone metabolites as shown in precedent Figure 4 (blue highlighted). This effect has been measured in different clinical studies. Typical results are shown in Figure 7:

In the example above, it can be seen how at the beginning of the treatment, blood lipoperoxides LPO (red curve) slightly increase up to the fifth day, from which on, due to the activation of glutathione peroxidase GPx (blue curve), which inactivates them, they stop to increase and begin to diminish again, no matter the treatment with ozone is continued every day. The glutathione reductase GRd (green curve) also is stimulated as needed to replenish the reduced glutathione pool, necessary for the GPx enhanced activity. It is easy to observe how the reduced glutathione (yellow curve) keeps practically constant assesing the equilibrium achieved.

Other basic defensive system enzymes as superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, etc. are also consequently stimulated. They are responsible for the metabolisation (inactivation) of superoxide anions, hydrogen peroxide, lipid peroxides, etc., so as for the replenishing of reduced equivalents as NADPH+ and others. In this manner, the general cells capacity to defend themselves from radicals and oxidants is significantly improved, and also the capacity to fight against ageing processes and some diseases.

Skin cell membranes are also constituted, among others, by lipidic structures. The interactions of special ozone derivatives with skin cell membranes break excessive attraction forces and bonds, so as eliminate the effects of the polluted environment aggressivity, so noticeably improving noticeably their relaxation, flexibility, permeability, smoothness, etc.

Specifically in the skin cells it enhances their capacity to absorb nutrients, microelements and oxygen, so as to expulse waste material and end products of metabolism. It improves the skin vitality, so as also its appearance and degree of hydration, as can be seen in Figure 8:

It is noticeable as the Ozonised Cream achieves in the skin a higher long term hydration, as compared with a positive control as Essex Base Cream.

The special ozone metabolites influence on some basic enzymatic processes also involves the stimulation of the Glycolisis, schematically shown in precedent Figure 4 (highlighted in green), which is known to be the most important source of energy in form of ATP for aerobic cells. In this case, such stimulation is achieved through a special kind of chain reactions, it is: the activation of the oxidation of glucose-6phosphate by Glucose-6-phosphate dehydrogenase for the reduction of NADP+ to NADPH+, the last needed for the GRd activity of replenishing the Reduced Glutathione pool, which turnover has been accelerated by the special ozone metabolites, see precedent Figure 4 (highlighted in blue).

The higher availability of ATP allows cells to restore or improve basic functions already lost or deprived. As example, Figure 9 shows the enhancement of fagocitic capacity of mammalian cells by ozone metabolites, under in vivo tests.

As known, the fagocitic activity of certain specialised cells means their ability to trap and inactivate foreign invader microorganisms and extraneous substances to avoid the damage they could do to normal cells. This is a very important part of the organism defences against diseases and general deterioration.

Several studies performed in vitro and in vivo already demonstrated the capacity of ozone metabolites to improve the functions of the immune systems, both cellular and humoral. Enhancement in lymphocytes and macrophages proliferation and activity, so as interleukins, cytokins and immunoglobulins increases under the effect of ozone metabolites have been assessed. Figure 10 shows the normalisation of humoral immunity in 120 immunodeprived patients treated with 3 cycles of ozone therapy. It is noticeable the perdurability of the effects 6 months after the last treatment. These patients had not been improved with other previous treatments.

It is one of the most typical and outstanding properties of special ozone metabolites. Plenty of in vitro, in vivo and clinical tests have been performed during several years, where the general germicidal effect has been demonstrated (see "Clinical Tests"). A short resume of micro-organisms that behaved as sensitive, according most important results of clinical and in vitro tests, is shown in Table 2:

Tissue Regeneration 

The ability of special OZONE metabolites to stimulate tissue reparative processes has been demonstrated, for example, in controlled studies on experimental surgical wounds.

In addition to their anti-inflammatory and disinfectant effects, they are capable to promote neovessels and fibroblasts formation in dermic lesions. In the case of Figures 1 and 2 , corresponding to the study performed in the Alfenas Univ., it was used a particular association of ozonized oils with a-lipoic acid, named Bioperoxoil®, during 7 days. It can be easily observed the higher rate of formation of such structures, as compared with controls. It  accelerates and guarantee healing of such wounds. More details can consulted in the original paper in More Info.

FIGURE 1

FIGURE 2  

Following images show tipical microscopic samples of tissue in Control and Bioperoxoil group animals at 7th day of treatment. It is very noticeable the differences in neovessels presence in tissue among both groups: