• Química del ozono:

Preliminary study of ozonizated (±) citronellal.
Hernández R., Fernández H., Gómez M. and Díaz M. (Cuba)

The essential oils have been broadly studied due to their antifungical, insecticidal and antimicrobial properties. In this work the ozonization of (±) citronellal with n-hexane and without any diluents at - 60 ^oC, applicating different doses of ozone, was studied. The peroxide index, the acidity index, the iodine index and the antimicrobial activity in front of Pseudomas aeroginosa and Staphilococus aureus , using the agar diffusion technique, was determinated. The peroxide index can reach aproximately 3000 mmol-equiv/kg of active oxygen. An discreet increase in the acidity index and a decrease until values near to five units in the iodine index in both experiments, were observed during the course of ozonization reaction. The (±) citronellal ozonization with n- hexane allows a better advance of the reaction, decreasing the reaction time and without the increase of the viscosity of the reaction medium. The results of the agar diffusion test demonstrated that the ozonizated (±) citronellal presented antimicrobial activity in front of the mentioned microorganisms.

Dissolution of chalcopyrite in acid medium with ozone.
Carrillo F.R., Soria M.J., Martínez A., Gutiérrez E. and Hernández F. (Mexico)

From the environmental point of view, the most attractive process for processing of copper minerals is the acid leaching. Nevertheless this method is slow and requires large amounts of reagents. Recents investigations have been focused on the optimization of this process using ozone as oxidizer, instead of the conventional sulfate or ferric chloride. This work presents two studies on the acid leaching of chalcopyrite (1) using a combination of oxidizers and different acids in according to one mathematic model type Taguchi and (2) one kinetic study in which the best conditions obtained from the first study were used (acid concentration, ozone and conventional oxidizers). The obtained results from these studies show that the use of the ozone as auxiliary oxidizer can help to decrease the consumption of the conventional oxidizers. Also, the use of ozone helps to reduce the time of treatment with regard to the conventional leaching.

Use of the ozone in the treatment of iron and steelmaking dusts.
Soria M.J., Carrillo F.R., Mart&oiacute;nez A. and Salinas E. (Mexico)

Several processes have been developed via aqueous solution to treat the dusts generated in the iron and steel industry, specifically, the dusts from the processes of Blast Furnace and [BOF] (Basic Oxygen Furnace); However, these processes are not efficient to recycling the zinc and alkalis due to the dust contains high concentration of them, which is an economic loss. The efforts to treat these dusts have gone from the conventional technologies, as using sulfuric acid (H₂SO₄), until based ones in the alkaline leaching. This work present a comparison between different treatments via aqueous solution. The main objective is to diminish the zinc and alkalis content into iron and steelmaking dust. Different type of acid and alkaline solutions, and the use of diverse oxidizers, among them the ozone, were used. The obtained results show that the elimination of zinc and alkalis ranging from 5 % to 80 %, depending on the conditions of the treatment (type of solution, the oxidizer and temperature). The results indicate that the use of ozone as oxidizer allow to obtain the biggest decrease of zinc and alkalis into the dust, that is to say, the zinc and alkalis can efficiently be removed. So, the use of ozone as oxidizer is an method alternative for the treatment and recycling of the dust generated in the steel industry where the Blast Furnace and [BOF] processes are used.

Decomposition of Aluminium Di-Stearate in Solid Waste by Ozone.
Poznyak T., López N., Cordova I. and RÍos E. (Mexico)

The aluminium di-stearate is the basic component of the solid waste of the polymer extrusion process. The utilization of this product is a really hard task. The ozonation of aluminium di-stearate (ADS) up to its decomposition in the water emulsion and in the solid states is studied. To realize the preliminary control of the ADS decomposition degree in the water, the UV absorbency is suggested to be used. During the ADS ozonation in the water emulsion the color variation and the dissolving of the initial substance are observed. Under the ozonation of ADS in the solid state the carbonylic compounds are shown to be accumulated. To identify the intermediates and final products formed in the ozonation of ADS in the water emulsion HPLC has been used. For the ozonation products identification with GC technique, the initial and ozonated samples from water with hexane are extracted. The dynamics of the ADS decomposition in the solid state with FTIR are also studied. According to the obtained results, we conclude that in the studied case two different processes (in the liquid and solid states) take place. In each state the different mechanisms of the ADS destruction are realized. In the liquid state the organic acids formation with small hydrocarbon chain very probably takes place. In the solid state the mixture of the carbonylic compounds with large chain is produced.

• Aceites vegetales ozonizados:

Studies of the ozonized vegetable oils used in medicine and cosmetology.
Díaz M. (Cuba)

The ozonation of sunflower oil, coconut oil and theobroma oil have been studied, during several years, in the Ozone Research Centre. These oils have been converted in adequate carrier for ozone therapy, which have a great germicide power, being useful in Dermatology, Parasitology and Cosmetology. When vegetable oils, constituted fundamentally by triglycerides, are ozonized, several substances as ozonides, aldehydes, and peroxides are formed, these could be related with their biological effect. The aim of this work is to show a general panorama of the research in Cuba on ozonized vegetable oils and its use in the fields of medicine and cosmetology.

Ozonation of sunflower oil: Spectroscopic monitoring of the degree of unsaturation.
Soriano N.U., Migo V.P. and Matsumura M. (Japan)

Consumption of ozone by unsaturated fatty acid moieties of sunflower oil (SFO) was monitored by ¹H NMR and FT-IR spectroscopy. Degradation of linoleate was found to be 1.5 and 1.8 times higher than oleate when SFO was ozonized in the absence and presence of water, respectively. Products of ozonation in both cases include aldehyde and ozonide with 1,2,4-trioxolane ring. Hydroxyl containing compounds, which could be carboxylic acids and/or hydroperoxides, were also detected in samples ozonized in the presence of water. The extent of ozonation had very little effect on the aldehyde to ozonide ratio obtained from NMR spectra, especially in the ozonation of neat SFO. The aldehyde to ozonide ratios obtained regardless of the extent of ozonation were 10.5:89.5 and 46.6:53.4 for SFO ozonized in the absence and presence of water, respectively.

Comparative studies of ozonated olive oil and ozonated sunflower oil.
Díaz M., Hernández R., Gómez M., Fernández H., Martínez G., Curtielles V., Vélez H. and Garcés R. (Cuba & Spain)

Prior studies have proven ozonated vegetable oils are useful for medical purposes in different diseases, taking into account their broad spectrum germicidal power. In this study the ozonated olive oil and ozonated sunflower oil are compared chemically and microbiologically. Ozone-oxygen gas was bubbling into the two oils until they were solidifies. The peroxide, acidity and iodine indexes along with antimicrobial activity against Staphylococcus aureus ATCC 6538, Echerichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 27853 and Bacillus subtilis ATCC 6633 were determined. The reaction products were identified using Proton Nuclear Magnetic Resonance (¹H NMR). Ozonation effects on the fatty acid composition of these oils were analyzed using Gas-Liquid Chromatographic Technique (GLC). An increase in peroxidation index was observed in both oils but was higher in ozonized olive oil. The acidity index was similar in both oils and the iodine value was zero in ozonized olive oil whereas in ozonized sunflower was 10.65 Units, this is indicative that all unsaturated group in olive oil reacted with ozone, but not the same in sunflower oil. The highest action spectrum of antimicrobial activity was obtained with the higher peroxide index from both ozonated oils. Signals of oxygenated groups through ¹H NMR were assigned. Composition of fatty acids in both ozonized oils showed gradual decrease in (C18:1, C18:2) unsaturated fatty acids with gradual increase in ozone doses. It was concluded that both ozonated oils have similar germicidal power and physical-chemical characteristics.

Antimicrobial activity of ozonated sunflower oil on Streptococcus mutans.
Fernández I., Curtiellas V. and Sánchez E. (Cuba)

Streptococcus mutans is considered the main ethiological agent of buccal caries. The aim of this work was to measure the lethality action of ozonated sunflower oil (OLEOZON^®) on Streptococcus mutans. The influence of different conditions, like pH, agent concentration and contact time on OLEOZON^® lethality was also evaluated. The values of minimum inhibitory concentration (CMI) and minimum bactericide concentration (CMB) were determined. It was demonstrated that OLEOZON^® activity did not variate significantly with the studied variation of pH, although the microorganism number decreases to pH lightly acid. The study of the influence of the different agent concentrations showed a significant effect of OLEOZON^® in the microorganism lethality, being 287.5 mg/mL the most effective studied concentration. An exposition time, between the agent and the microorganism, of ten minutes, produce a 99,9 % of microorganism lethality. However with only three minutes of exposition time was obtained a reduction of 95,5 % of initial microorganism concentration. These results proved that OLEOZON^® is an effective drug to treat Streptococcus mutans infection.

Physical chemical characteristic of ozonized theobrom oil.
Martínez G., Vargas E. and Díaz M. (Cuba)

Physical chemical characteristic of cosmetic cream with ozonized theobroma oil.
Díaz M., Fernández H., Gómez M., Hernández R., Martínez G., Hernández D., Vargas E., Díaz A., Hasbun S. and Ledea O. (Cuba)

Cosmetic creams with active oxygen present moisturizer and conditioner effects. Therefore, skin oxygenation and microcirculation are increased preventing cutaneous aging. In this work an anti-wrinkle cosmetic cream with ozonized theobroma oil is presented. This cream has been approved for Health Cuban Authorities as anti-wrinkle cosmetic cream with moisturizer and conditioner properties. Chemistry and microbiological stabilities, physical-chemistry properties, rheology and skin and ophthalmic irritably test were performed. The cream is stable for six months. Peroxide values was minor that 65 mmol-equiv O 2 active/kg of sample; acidity was minor that 15 Units, pH values between 4 and 8, and visual evaluation as homogenous consistent solid of white colour. The behaviour of fluidity graphic was plastic with tixotropic value of 14 740. The tests of irritably of the cream were negative. It was concluded, the cream with ozonized theobroma oil maintains their quality parameters during six mounts from be using in cosmetology.

• Caracterización químico-físico y microbiológico del OLEOZON^®:

Physical-chemical aspects of ozonized sunflower oil.
Hernández R., Martínez G. and Díaz M. (Cuba)

The knowledge of the physico-chemical properties of ozonized vegetable oils has a great importance for its characterization and identification. In this study a correlation of the relative density and viscosity with the peroxides index of the ozonized sunflower oil is carried out. Furthermore, main formed oxygenated products are identified after the ozonization by means of Proton Nuclear Magnetic Resonance (¹H-NMR) signals. The peroxides index and the relative density showed a linear relationship with a correlation coefficient of 0.9991 and a determination coefficient of 99.82 %, while the peroxide index and the viscosity showed an exponential relationship with a determination coefficient of 98.52 %. An increase in peroxidation index, in viscosity and oxygenated compounds concentration were observed during the course of ozonization reaction. These results suggest that physical-chemical properties can provide valuable information about of ozonization grade of sunflower oil. This study could also be the base of future chemical investigations of ozonized vegetable oils.

Measurement of hydroperoxides in ozonated sunflower oil.
Martínez G., Hernández R. and Díaz M. (Cuba)

Total hydroperoxides (HPT) concentration using the ferrous oxidation in xylenol orange (OFX) assay and peroxide value (IP) using iodometric assay, were determined in sunflower oil samples ozonated at different ozone dosage. Effect of increasing ozonazed sunflower oil amount was evaluated by assembly assay, where a lineal relationship was obtained between oil amounts from 2 to 17 µg in the assay and absorbance units measured at 560 nm. The OFX reagent calibration and the extinction coefficient calculation were carried out for HPT measurement in ozonazed samples. The correlation established between IP and the concentration obtained by OFX assay showed a lineal relationship (r = 99.29; r² = 98.59 %). These results confirm that this assay is appropriate for HPT measurement in ozonazed sunflower oil.

Variation of OLEOZON^®'s Iodometric Peroxide Value with the Measurement Time.
Ledea O., Díaz M., Vargas E., Martínez G., Hernández R., Fernández H. and Hernández D. (Cuba)

The reaction of ozone with unsaturated triglycerides, present in vegetable oils, produces different types of peroxidic compounds as described by Criegee mechanism. The peroxide value is the method generally employed to determine total peroxidic compounds formed. The United States and British Pharmacopoeias established an iodometric method with a reaction time of two minutes to analyze the samples. Usually, the samples of oils and fats present a low content of peroxidic compounds, but OLEOZON^® contains a great amount and diversity of them, and certainly higher reaction times could be necessary to determine its total peroxide content. In this work, the variation of iodometric peroxide value of OLEOZON^® with the reaction time was evaluated. A representative sample of ten batches of the drugs were randomized chosen for the study, and the reaction times assayed were: 2 min, 4 h, 8 h, 16 h, and 24 h. The peroxide value of OLEOZON^® increases with the reaction time and the increment was determined by the history of each batch. The influence of storage time, viscosity, and peroxide value measured at two minutes reaction time, on peroxide value measured at higher reaction time were evaluated. In general, the variation coefficient of the determination, decrease at the same time that the reaction time increase.

Nuclear Magnetic Resonance spectroscopic characterization of ozonated sunflower oil.
Díaz M., Gavín J., Ledea O., Hernández F., Alaiz M. and Garcés R. (Cuba & Spain)

Ozonation reactions are very important in vegetable oil chemistry since their ozonation products are involved in antimicrobial effect in therapeutical uses for several microbiological etiology diseases. Information on the spectroscopic characterization of the products generated by ozonolysis of sunflower oil is limited. In the present study ozonized sunflower oil with 650 mmol-equiv/g of peroxide index is chemically characterized. Ozonation of sunflower oil produced ozonides, aldehydes and hydroperoxides which were identified by ¹H, ¹³C and two-dimensional ¹H Nuclear Magnetic Resonance (NMR). The virgin sunflower oil and ozonized sunflower oil show very similar ¹H NMR spectra except for the resonances at d = 9.74 and d = 9.63 ppm that correspond to both triplet from aldehydic protons, d = 5.6 ppm (olefinic signal from hydroperoxides), and d = 5.15 ppm (multiplet from ozonides methylic protons). Other resonance assignments are based on the connectivities provided by the proton scalar coupling constants. These are the following: d = 3.15 ppm (doublet from methylenic group in a position respect to olefinic proton), d = 2.45 ppm (multiplet from methylenic group allylic to ozonides methynic protons) and d = 1.62 ppm (multiplet methylenic protons in b position respect to ozonides methynic protons). From the ¹³C NMR and ¹H- ¹³C two-dimensional spectrum of the ozonized sunflower oil, the presence of ozonides was confirmed by the signals d = 103.43 and d = 103.49 ppm respectively. The others new signals found in d = 42.5 and d = 42.76 ppm confirm the presence of methylenic carbons from hydroperoxides and ozonides. These results indicate that the NMR Spectroscopy can provide valuable information about the amount of reaction compounds of ozonized vegetable oil. From the chemical structural elucidation of ozonated sunflower oils, relevant biochemical and chemical information can be achieved.

Identification of OLEOZON^® main peroxidic compounds.
Ledea O., Rosado A., Molerio J., Besada V., Betancourt L., Díaz M., González L., Tambara Y. and Hernández C. (Cuba)

The ozonized sunflower oil for topical applications (OLEOZON^®) is a registered drug obtained through the reaction between ozone and sunflower oil according to a process developed at Ozone Research Center (Cuba). OLEOZON^® has shown antimicrobial effects against virus, bacteria, protozoa, and fungi, but its mechanism of action remains still unknown. The germicide activity of the ozonized vegetable oils has been usually associated to peroxidic compounds formed by the reaction of ozone with unsaturated triglycerides. However, the chemical information available in the specialized literature about the structure of these peroxidic compounds is very limited. In the present work, the electrospray mass spectrometry technique was used to analyze the main peroxidic compounds present in the OLEOZON^®. ¹H and ¹³C Nuclear Magnetic Resonance and Infrared Spectroscopy were also employed. Peracids, a -hydroxy-hydroperoxides, a -acyloxy-hydroperoxides and ozonides, were identified as the main peroxidic compounds in OLEOZON^®. This is the first time than the electrospray mass spectrometry is used to analyze the peroxidic and polymeric compounds present in ozonated or oxidized vegetable oils. Also, the presence of peracids in the ozonated vegetable has not been reported previously and a mechanism for the formation of this peroxidic compound was proposed.

Antibacterial activity of topic OLEOZON^®; packed in glass and polyethylene flasks.
Gómez M., Curtiellas V., Fernández I., Sánchez E., Fernández H. and Díaz M. (Cuba)

Ozonized sunflower oil (Topic OLEOZON^®) presents a high germicidal power. This product is a wide spectrum antimicrobial agent of with inhibitory and lethal activity on bacterial, protozoa and fungi. The aim of this work was to study the microbiological activity from topic OLEOZON^® stored to temperatures from 2 - 8 ^oC. Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633 were selected as test organism. Topic OLEOZON^® was packed in three types of containers like amber colour glass, density low polyethylene and density high polyethylene flasks. Biological evaluation of product was done by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) based on National Committee for Clinical Laboratory Standards (NCCLS) guidelines, for each flask type and storage period (0, 2, 5, 7 and 14 months). Topic OLEOZON^® was effective against all strains tested. MIC values showed an increase of 5 mg/mL to 13 mg/mL that corresponding to 0 and 14 months, MBC values also showed an increase from 30 mg/mL to 148 mg/mL from 0 to 14 months. Taking into account that this product is directly applied on the wounded skin at a concentration near to 950 mg/mL, these results show that Topic OLEOZON^® has high biological activities even when it is stored during fourteen months from 2 - 8 ^oC in all flasks type studied.

New quality system in the Ozone Research Center.
Nicolás P.D., Díaz M., Fernández H., Hernández R., Martínez G., Vargas E., Hernández D., Mora C., Curtiellas V. and Rodríguez M. (Cuba)

Ozonized sunflower oil OLEOZON^® is a wide spectrum antimicrobial product with inhibitory and lethal activity on grampositive and gramnegative bacterias. This product, during several years, has been elaborated in the pilot plant of Ozone Research Center. OLEOZON^® has approved the Medical Registry for the Epidermofitosis disease, pathology of fungicide origin, very frequent in many countries. Also, their production process has been validated according to the norms settled down by the competent health sanitary authorities (CECMED). The new Quality Administration System is conformed by Administration Unit of the Quality that includes following areas: Quality Insurance, Quality Control, Production, Maintenance, Administrative and Security. All these areas are command by Quality Insurance area and the whole System is take command by Center Direction. The codes of the Normalized Procedures of Operation (P.N.O) were changed. In general the system has 166 P.N.O among modified and new. The following support programs were created: Maintenance Program, Calibration and Verification, Hygienic Program, and the modified Training Program. The new Quality Administration System was implanted starting from May of 2003 and the License of Production Renovation was obtained.