ES2323107B1 - PROCEDURE TO DETERMINE ANTIOXIDANT POWER IN MINEROMEDICINAL WATER. - Google Patents

Abstract

Procedure to determine antioxidant power in medicinal mineral water.

A procedure has been developed for determine the antioxidant power in waters Mining-medicinal (M-m). These waters are known for their therapeutic virtues on certain chronic and inflammatory diseases. In addition, they have effects revitalizers on cells and tissues, contributing to the skin repair, reactivating metabolism, eliminating toxins by sweating and facilitating diuresis, attenuating joint conditions such as rheumatic and postoperative processes of the musculoskeletal system and controlling stress, in short, improving the quality of life Although they have developed multitude of methods to determine the antioxidant power of food and drinks, unfortunately most of them are based on  the detection of phenolic compounds and other components antioxidants that do not exist in the water, and also many of them they have been designed to be applied in lipid media, which made its determination impossible in the water.

Description

Procedure to determine antioxidant power in medicinal mineral water.

Technical sector

- Sector according to the technical area: Pharmacist, food, drinks

- Sector according to the area of application of the invention: determination of the antioxidant power of water mining-medicinal application in hydrology medical

State of the art

Oxygen is associated with the conditions of aerobic life (Davis, J. Kelvin (1995). Oxidative stress: the paradox of aerobic life. Biochem Soc. Symp. 61: 1-31) and represents the driving force for the maintenance of metabolism and cell viability at the same time that involves a potential danger because it is responsible for the free radical formation (RL) and reactive oxygen species (ROS). Excessive concentrations of RL and ROS have serious effects adverse effects on living beings, peroxidation may occur of the lipid membranes, the hydroxylation of the bases of the nucleic acids and the oxidation of sulfydryl and other groups sensitive points of proteins.

The generation of these prooxidant substances is controlled by cellular defense mechanisms antioxidants, reducers or sequestrants, which appear strategically distributed in different organelles subcellular in order to minimize the damage that would be caused by an excess of RL and ROS. This antioxidant defense mechanism is composed of enzymatic antioxidants, among which find superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and non-enzymatic antioxidants, among which find vitamins A, C and E. However, when antioxidant defense mechanisms become insufficient to counteract the excessive production of RL and ROS originate cellular alterations that will lead to a stress situation oxidative

In short, both endogenous antioxidants as those that are incorporated into our body in the form of Food, especially fruits and vegetables, take care of counteract the cytotoxic potential of the RL by decreasing the their reactivity by depriving them of the missing electron and transform them to more stable and less reactive species (Fridowich I. (1978). The biology of oxygen radicals. Science 201, 875-880; Halliwell B, Gutteridge JMC. (1989) Free Radicals in Biology and Medicine. Oxford University Press. New York .; Cadenas, E. (1989) Biochemistry of oxygen toxicity. Ann. Rev. Biochem. 58, 79-110 .; Sáez GT, Bannister W, Bannister JV. (1990) Free radicals and thiol compounds. The role of glutathione agaisnt free radical toxicity. In: CRC handbook of physiologycal functions of glutathione. (Viña, J. ed.) CRC Press Inc. Florida. pp 237-254.). Numerous studies have shown that the consumption of antioxidants can reduce the incidence of certain pathologies and improve the disturbances caused in the organism over the years (aging), hence the importance of the study of the antioxidant power of food (especially fruits and vegetables) and drinks (juices, wines, beer, tea, coffee, etc.) and water.

Based on this antioxidant capacity they have developed different evaluation methods of what is called global antioxidant activity, usually based on the evaluation of the ability to capture free radicals or in the evaluation of its reducing capacity. Unfortunately many of these methods have been developed to be applied in media lipids and therefore are not suitable for other types of products.

Among the methodology used to determine Total antioxidant activity can be cited:

a) TRAP test (from total English radical-trapping parameter Wayner DDM, Burton GW, Ingold K. (1985). Quantitative measure of the total, peroxyl radical-trapping capacity of human blood plasmas by controlled peroxidation. FEBS Lett. 187, 33-37.). Currently almost not used.

b) Capture of the superoxide anion (Kanner J, German JB. (1987). Initiation of lipid peroxidation in biological systems. Crit. Rev Food Sci Nutr. 25: 317-364.). Do not a good correlation has been found with the preventive capacity of the lipid oxidation

c) Radical method 2,2-diphenyl-1-picrylhydracil (DPPH) (Kaneda H, Kobayashi N, Furusho S, Sahara H, Koshino S. (nineteen ninety five). Reducing activity and flavor stability of beer. Master Brew Assoc. Am., Tech. Q. 32: 90-94.). Limited by inter-assay reproducibility.

d) FRAP test, (from English ferric-reducing antioxidant power), (Benzie IFF, Strain H. (1996). The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Analytical Biochemistry 239: 70-76). Measure the capacity of reduction that does not necessarily reflect the activity antioxidant

e) ORAC method, (from radical oxygen English absorbance capacity) (Cao G, Sofic E, Prior RL. (1997). Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships. Free Radicals Biol. Med. 22: 749-760.) Each antioxidant analyzed shows very different behaviors.

f) Method of 3-ethylbenzothiazoline-6 sulfonate (ABTS) (Re R, Pellegrini N, Proteggente A., Pannala A, Yang M, Rice-Evans C. (1999). Antioxidant activity applying and improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine 26 (9/10): 1231-1237). The ability to capture free radicals in this method is not a reflection of its true antioxidant activity.

g) Method of N, N-dimethyl-p-phenylenediamine (DMPD) (Fogliano V, Verde V, Randazzo G, Ritieni A. (1999). Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. J. Agric. Food Chem Vol 47, 3: 1935-1940.). Method with high inter-assay reproducibility, useful for non-media lipid

The information that relates to antioxidants with good health it is already known and managed by a wide sector of the population, who cares about acquiring food with high antioxidant content The beneficial effect of food Vegetables, fruits and beverages are mainly attributed to substances with antioxidant activity, such as polyphenolic compounds, the ascorbic acid (vitamin C), carotenoids and vitamin E (Acuña UM, Atha DE, Ma J, Nee MHY, Kenelly EJ. (2002) "Antioxidant Capacities of Ten Edible North Americans Plants", Phytother Res. 16: 63-65, Baolu Z. (2001) "Free Radical Reaction of Green Tea Polyphenols ". In Micronutrients and Health, AOCS. Press, Champaign, Illinois, 60-73, Rice-Evans CA, Miller NJ. (1996) "Antioxidant activities of flavomoids as bioactive components of foods " Biochem Soc. Trans. 20: 790-795). It has been suggested that These substances increase the body's antioxidant defense against "oxidative stress" responsible for different types of cell damage.

Mining and medicinal waters (M-m) are known for their therapeutic virtues about certain chronic and inflammatory diseases. In addition, they have revitalizing effects on cells and tissues, contributing to the skin repair, reactivating metabolism, eliminating toxins by sweating and facilitating diuresis, attenuating joint conditions such as rheumatic and postoperative processes of the musculoskeletal system and controlling stress, in short, improving the quality of life

The antioxidant effects of certain sulfur-rich mineral-medicinal waters and the hydrological techniques that are prescribed with them open new therapeutic pathways to treat inflammation, degenerative processes and the natural aging process itself. As examples of clinical studies we can mention the effect of sulfur Mm waters or thalassotherapy on chronic pharyngitis (Barbieri M, Mora F, Melloni F, Cordone MP, Perottino F, Mora R. Effets des eaux sulfureuses sur les pharyngites chroniques Revue of FMF. 5 July 2002), certain ENT and lung conditions Suskovic S, Hull Z, Mrksic V, Raspor T. Plucne Bolesti. Efficacy of the inhalation of warm mineral water from Moravci well MT-6 in patients with chronic obstructive lung disease. 1991; 43 (1-2): 109-112 (Kurabayashi H, Kubota K, Machida I, Tamura K, Take H, Shirakura T. Effective physical therapy for chronic obstructive pulmonary disease. Pilot study of exercise in hot spring water. Am J Phys Med Rehabil. 1997; 76 (3): 204-207, Tanizaki Y, Kitani H, Okazaki M, Mifune T, Mitsunobu F, Honke N. Arerugi. Clinical effects of complex spa therapy on patients with steroid-dependent intractable asthma (SDIA) 1993; 42 (3Pt): 219-227, Abramo A, Pollastrini L, Cristalli G. Treatment of chronic inflammation of the upper respiratory tract by sulfur-sulfate-bicarbonate-carbonate-alkaline earth mineral water: a immunochemical study of nasal mucus Acta Otorhinolaryngol Ital. 1996 Dec; 16 (6 Suppl 55): 95-100, Staffieri A., Miani C, Bergamin AM, Arcangeli P, Canzi P. Effect of sulfur salt-bromine-iodine-thermal waters on albumin and IgA concentrations in nasal secretions Acta Otorhinolaryngol Ital 1998; 18 (4): 233-238, Cristalli G, Abramo A, Pollastrini. Treatment of chronic inflammation of the upper respiratory airways by inhalation thermal therapy with sulfur-sulfate-bicarbonate-carbonate-alkaline earth mineral water: a study of nasal cytology. Acta Otorhinolaryngol Ital. 1996; 16 (6 Suppl 55): 91-4, Guillemin F, Virion JM, Escudier P, De Talance N, Weryha G. Effect on osteoarthritis of spa therapy at Bourbonne-les-Bains. Joint Bone Spine 2001; 68 (6): 499-503) and especially certain rheumatic conditions (Bouvenot G, Ambrosi P. Evaluation of spa therapy in rheumatology. Joint Bone Spine. 2000; 67 (4): 262-3, Verhagen AP, by Vet HC, by Bie RA, Kessels AG, Boers M, Knipschild PG, Bal-neotherapy for rheumatoid arthritis and osteoarthritis, Cochrane Database Syst Rev. 2000; (2): CD000518, Konrad K. The way forward for balneotherapy. Br J Rheumatol. 1994; 33 (3): 301, Elkayam O, Wigler I, Tishler M, Rosenblum I, Caspi D, Segal R, Fishel B, Yaron M. Effect of spa therapy in Tiberias on patients with rheumatoid arthritis and osteoarthritis J Rheumatol. 1991; 18 (12): 1799-1803, Machtey I. Mineral bath therapy in arthritis. Ann Rheum Dis. 1991; 50 (3): 201, Sukenik S, Buskila D, Neumann L, Kleiner-Baumgarten A , Zimlichman S, Horowitz J. Sulfur bath and mud pack treatment for rheumatoid arthritis at the Dead Sea area. Ann Rheum Dis. 1990; 49 (2): 99-102, Armijo Valenzuela M, San Martín Bacai-
coa J, et al . "Spa and Climate Cures. Thalassotherapy and Heliotherapy". Edit Complutense Madrid. 1994; 18, 255).

Some clinical studies in rheumatology have demonstrated the modulation of serum rates of certain inflammatory parameters (αTNF, PGE2 LTB4) in the course of balnetotherapeutic cures (Bellometti S, Galzigna L, Richelmi P, Gregotti C, Berte F. Both serum receptors of tumor necrosis factor are influenced by mud pack treatment in osteoarthrotic patients Int J Tissue React. 2002; 24 (2): 57-64, Bellometti S, Galzigna L. Serum levels of a prostaglandin and a leukotriene after thermal mud pack therapy. J Investig Med. 1998; 46 (4): 140-145, Bellometti S, Giannini S, Sartori L, Crepaldi G. Cytokine levels in osteoarthrosis patients undergoing mud bath therapy. Int J Clin Pharmacol Res. 1997; 17 (4): 149-53, Bellometti S, Cecchettin M, Lalli A, Galzigna L. Mud pack treatment increases serum antioxidant defenses in osteoarthrosic patients, Biomed Pharmacother. 1996; 50 (1): 37) or thalassotherapy. Other studies on isolated cells (mast cells, eosinophils, monocytes) have revealed the modulation of certain cellular activities by Mm water (Beauvais F, Garcia-Mace JL, Joly F. In vitro effects of Uriage spring water on the apoptosis of human eosinophils, Fundam Clin Pharmacol. 1998; 12 (4): 446-450, Joly F, Charveron M, Arié MF, Bidault J, Kahhak L, Beauvais F, Gall Y. Effect of Avene Spring Water on the Activation of Rat Mast Cell by Substance P or Antigen. Skin Pharmacology and Applied Skin Physiology 1998; 11: 111-116.).

Previous studies carried out by our research group, in spas with sulphured and sulfated mineral-medicinal waters, have shown that these waters produce an antioxidant effect, reducing the urinary elimination of lipoperoxidation products (Hernández Torres A, Ramón Giménez JR, Giralde E Basin, Casado Moragón A, López Fernández E, Guillén J, Chamorro JC, Caballero C. Influence of age and crenotherapy on the human oxidative state Rev Esp Geriatr Gerontol 2002; 37: (S1) 20, Hernández Tones A, "Antioxidant effect of the crenoterapia with mineral-medicinal waters " Anti-Aging Medicine 2003; 1: 55-56 , Hernández Torres A, Ramón Giménez JR, Martell Claros N, Giralde E Basin, Márquez Montes J." Result of the crenotherapeutic action with sulphurated and peloid waters and other non - pharmacological measures blood pressure in the spa "Bull Soc Esp Hidrol Med 2000; 15 (1): 35-46, Ramon Gimenez JR, Hernandez Torres a, Basin Giralde E, Married Moragón A, López Fernández E, Guillén J, Chamorro JC, Caballero C. Influence of different sulfur-rich mining-medicinal waters on blood pressure. Rev Esp Geriatr Gerontol 2002; 37 (S 1): 83-84. Hernández Torres A, Ramón JR, Basin Giralde E, Casado A, López-Fernández E. Antioxidant capacity of balnetotherapy with sulphured and sulfated mineral-medicinal waters. Naturist Medicine 2004; 7: 361-370), but we do not know if other mineral-medicinal waters (with other components, such as carbonates or bicarbonates, for example) are also capable of producing the same antioxidant effect in the body. Therefore, our main objective was to determine the antioxidant power of mineral-medicinal waters and then check how the antioxidant power of these waters varied according to their composition and properties.

Although they have developed multitude of methods to determine the antioxidant power of food and drinks unfortunately most of them are based on the detection of phenolic compounds and other antioxidant components that do not exist in the water, and many of them have also been designed to be applied in lipid media, what it did Impossible its determination in the water.

Description of the invention - Brief description of the invention

The proposed methodological modifications they consist essentially of modifying the dilutions of the sample to analyze (in our case any type of water) with the reagent R1, included in the PAO kit. Reagent R1 contains bathocuproin (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) it is a chromogenic reagent that forms a complex colored with the Cu +, which has a maximum absorbance at 490 nm. The Cu + is generated by the action of the antioxidants in the sample on the Cu ++, present in reagent R2 (also included in the kit)

It is clear, therefore, that for detection of the antioxidant power both elements: sample to value and Chromogenic reagent play an important role; if it does not exist enough sample does not occur the passage from Cu ++ to Cu + and if there is no chromogenic reagent, no complex will originate colored to detect at 490 nm. Hence the importance of optimize the required quantities of both elements: sample and chromogenic reagent (R1) to determine the power total antioxidant With dilution of the sample in reagent R1 proposed in the PAO kit it was not possible to determine the power antioxidant in water, while with the dilution that is proposed if  It has been possible to determine and quantify it.

Description of the figures

Figure 1 calibration curve (included in the kit protocol) representing net absorbance (increase in absorbance) at 490 nm on the ordinate axis against the mM uric acid concentration in the abcissa axis performed with different concentrations of uric acid (2.0; 1.0; 0.5; 0.25; 0.125 and 0.063 mM)

Figure 2 calibration curve (obtained in the laboratory) representing the net absorbance (increase in absorbance) at 490 nm on the ordinate axis against the mM uric acid concentration in the abcissa axis performed with different concentrations of uric acid (2.0; 1.0; 0.5; 0.25; 0.125 and 0.063 mM). This calibration curve has been used to get the results of the samples of the three spas that they show up

- Detailed description

Our solution to determine the power Water antioxidant was to modify the kit's methodology BIOXYTECH® AOP-490TM that markets the house DELTACLON as a PAO kit (English AOP) and based on the reduction from Cu ++ to Cu +, due to the combined action of all Antioxidants present in the sample. The Cu + reacts a Chromogenic reagent containing Bathocuproin (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) which forms a complex colored with Cu +, which has a maximum absorbance at 490 nm.

one

Reagents included in the BIOXYTECH® kit AOP-49O ™

- Reagent 1 (contains Bathcocuproin: 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline)

- Reagent 2 (contains (Cu ++)

- "Stop" solution

- Standard uric acid (lyophilized)

Preparation of the standard solution

Dissolve lyophilized uric acid in 1 ml of 10% NaOH, add 2 ml of deionized water. Adjust the pH to 7.4 with concentrated HC1. Add deionized water (H2O) until get the total volume indicated on the bottle label of the standard uric acid kit. The uric acid concentration of the Standard solution prepared is 2 mM.

The standard solution once reconstituted remains stable for at least one year at -70 ° C.

Obtaining the calibration curve

The calibration curve was performed using a 2 mM concentration of standard uric acid. From this one it prepared decreasing solutions with different concentrations uric acid (2.0; 1.0; 0.5; 0.25; 0.125 and 0.063 mM) as indicated in the following table

2

The results of the analysis can be expressed in "uric acid equivalents in mM", or as "equivalents of copper reduced by µM ". The conversion between the two units it is based on the reduction of 2189 µM Cu ++ to Cu + by 1 mM of uric acid.

Sample Preparation

The samples, before being used, must Store at -70 ° C. Samples with a concentration greater than 2 mM Uric acid equivalents should be diluted with reagent 1 (with Bathocuproina) before the trial.

Test

one.-

Label a test tube for each One of the standards and samples to be determined.

2.-

Dilute each standard or sample with the reagent R1 (contains Bathocuproine) in proportion 1/40 (per example, 15 µl of sample plus 585 µl of R1).

3.-

Add 200 µl of each dilution in each of the microplate wells.

4.-

Read the plate at 490 nm.

5.-

Add 50 µl of reagent 2 (with Cu ++) to each well and mix.

6.-

Incubate for three minutes at room temperature for the reaction to occur

7.-

Add 50 µl of solution "stop" and mix well.

8.-

Read the plate at 490 nm.

Calculations

Net absorbance is calculated by subtracting the initial absorbance read in step 4 to final absorbance read in step 8. With this data, the calibration curve is developed representing net absorbance (increase in absorbance) versus at the concentration of uric acid. For example, a line of Simple calibration is described in Figure 1.

With the calibration curve, you can determine the uric acid equivalents for each sample in function of the net absorbance of the sample, clearing the "x" If the results are preferred to express in "µM Copper equivalent equivalents ", multiply the result for 2,189.

Sample Calculation

As an example, the data obtained in a plasma sample (in duplicate, on the same plate). The absorbance readings are as follows:

3

After calculating the net absorbance for each sample, the average absorbance ÄA_ {490} is determined for the Sample analyzed, which is 0.078. Clearing the "x" in the equation of the calibration line and substituting the value of the net absorbance, the result is:

X = (Y - 0.009) / 0.309 = (0.078 - 0.009) / 0.309 = 0.223 nM equivalent of uric acid

To express the result in µM equivalents of reduced copper:

1 mM ac. Uric = 2.189 µM Cu +: 0.223 x 2,189 = 488 µM equivalent reduced copper

The modification that was introduced in the method was to vary the dilution of the sample (in our case water) with the bathocuproin reagent since with the specifications of the method it was not possible to determine the antioxidant power in water.

Dilution of the sample with reagent R1 (containing bathocuproin) proposed in the Kit is 1/40 (per example, 15 µl of sample plus 585 µl of buffer (R1)). Using this dilution no reaction was observed, so the trial was carried out working with different dilutions:

Dilution 1/20 (12.50 µl shows more 237.50 \ mul buffer)

Dilution 1/10 (25 \ mul shows more 225 \ mul buffer)

Dilution 1/8 (31.25 µl shows more 218.75 \ mul buffer)

Dilution 1/20 (12.50 µl shows more 237.50 \ mul buffer)

Dilution 2/3 (100 µl shows more 150 \ mul buffer)

Dilution 1/1 (125 µl shows more 125 \ mul buffer)

Dilution 3/2 (150 µl shows more 100 \ mul buffer)

finally reaching 4/5 dilution (200 µl of sample plus 50 µl of reagent R1), which allowed determine the antioxidant power in water, taking into account this dilution factor to express the results. The problem is that without that dilution the determination cannot be made and we would give negative results (no antioxidant power). It would not be valid either An alternative solution.

Nothing could be added (phenolic compounds, vitamins, antioxidants ....) first of all by own definition of mineral-medicinal water "that which for its special  characteristics has been credited as a therapeutic agent and has been declared of public utility by the relevant agencies. "And secondly because of the characteristics of the waters mineromedicinals (temperature, pH, ion composition, etc.) given that the use of additives could modify not only the beneficial and therapeutic properties of water mineromedicinals but also of the added compounds, being able to give artifacts that had nothing to do with your actual activity

This dilution gave good results that they were easily reproducible and allowed to demonstrate, first instead, that some waters did have antioxidant power, in second instead, that the antioxidant power of water was related to the content and proportion of different ions (hence some waters have greater antioxidant power than others), and finally He also showed that not all waters have power antioxidant, hence the interest in locating and quantifying those that If they present it.

Example of embodiment of the invention

According to the specifications of the method (BIOXYTECH® AOP-490 ™ kit that sells the house
DELTACLON as a PAO kit) the antioxidant power of different mineral-medicinal waters from different parts of the Spanish geography was determined.

As described in section 4 of this Memory, we proceeded first to reconstitute uric acid standard for obtaining the calibration and straight curve of regression. With the reconstituted standard uric acid not used, they made aliquots that were frozen at -70 degrees for later utilization.

The calibration curve and regression line obtained (Figure 2) presented the following values:

Y = 0.2778x + 0.0292

R2 = 0.9991

In the that:

x are mM uric acid

and it is the difference of absorbances between the 2nd and 1st reading

1 mM uric acid = 2189 µmol / l copper.

We proceeded, secondly, to carry out the test, as previously described (section 4 of this Memory)

one.

Label a test tube for each One of the standards and samples to be determined.

2.

Dilute each standard or sample with the reagent R1 (contains Bathocuproin) in 1/40 ratio (per example, 15 µl of sample plus 585 µl of R1).

3.

Add 200 µl of each dilution in each of the microplate wells.

Four.

Read the plate at 490 nm.

5.

Add 50 µl of reagent 2 (with Cu ++) to each well and mix.

6.

Incubate for three minutes at room temperature for the reaction to occur

7.

Add 50 µl of solution "stop" and mix well.

8.

Read the plate at 490 nm.

None of the samples analyzed resulted positive, since when there was no reaction there was no difference from absorbances between the first and second reading and therefore not Nothing was detected.

The dilutions of the samples with the chromogenic reagent (R1) obtaining a dilution 4/5 (200 µl of sample plus 50 µl of R1) at which previous samples if they gave a positive result.

Calculation of the results obtained:

Sample 1 corresponding to a spa with characteristics of the water: hyperthermal. Weak mineralization. Sulfurized, ions predominant bicarbonate, chloride, sodium. Because of its hardness soft

Y = 0.2778x + 0.0292

R2 = 0.9991

y = 0.191 (absorbance 2nd reading) - 0.044 (absorbance 1st reading) = 0.147

x (in mM uric acid) = \ frac {(0.147-0.0292)} {0.2786} x \ frac {1} {32} = 0.01321 mM Uric acid

F = dilution factor = 1/32

As 1 mM uric acid = 2,189 µm / l copper

0.01321 mM Uric acid x 2.189 = 28.92 µm of reduced copper

The antioxidant power of medicinal mineral water is expressed in mM concentration of uric acid or reduced copper, so the problem water has an antioxidant power of 0.01321 mM Uric acid or 28.92 µm of reduced copper .

Sample 2 corresponding to a spa with characteristics of the water: hyperthermal. Strong mineralization. Bicarbonate Chlorinated Sodium

Y = 0.2778x + 0.0292

R2 = 0.9991

y = 0.121 (absorbance 2nd reading) - 0.036 (absorbance 1st reading) = 0.0558

x (in mM uric acid) = \ frac {(0.0558-0.0292)} {0.2786} x \ frac {1} {32} = 0.00625 mM Uric acid

F = dilution factor = 1/32

As 1 mM uric acid = 2,189 µm / l copper

0.00625 mM Uric acid x 2.189 = 13.687 µm reduced copper

The antioxidant power of medicinal mineral water is expressed in mM concentration of uric acid or reduced copper, so the problem water has an antioxidant power of 0.00625 mM Uric acid or reduced 13.687 copper

Sample 3 corresponding to a spa with characteristics of the Water: Hypothermal. Medium mineralization Sulphurated Radioactive Predominant bicarbonate and sodium ions. Because of its hardness soft

Y = 0.2778x + 0.0292

R2 = 0.9991

y = 0.272 (absorbance 2nd reading) - 0.044 (absorbance 1st reading) = 0.228

x (in mM uric acid) = \ frac {(0,228-0,0292)} {0,2786} x \ frac {1} {32} = 0.02298 mM Uric acid

F = dilution factor = 1/32

As 1 mM uric acid = 2,189 µm / 1 copper

0.02298 mM Uric acid x 2.189 = 48.813 µm reduced copper

The antioxidant power of medicinal mineral water is expressed in concentration of mM of uric acid or reduced copper, so the problem water has an antioxidant power of 0.02298 mM Uric acid or 48.813 µm of reduced copper

Therefore the validity of the method has been demonstrated with samples of various mineral medicinal waters.

Claims (4)

1. Procedure to determine antioxidant power in medicinal mineral water characterized by understanding the following steps

-

Take Reagents and water samples from the refrigerator and keep for at least 3 hours at a stable temperature between 20-25 ° C (unheated).

-

Reconstitute with deionized water the standard uric acid solution and hold for at least 3 hours at a stable temperature between 20-25 ° C (unheated).

-

Add to the water sample Reagent 1 containing Bathcocuproin (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline)

-

Read the liquid at 490 nm.

-

Add 1 volume of reagent 2 (with Cu ++) for every 4 volumes of liquid and mix.

-

Incubate at room temperature (three minutes) for the reaction to occur

-

Optionally you could add 1 volume Additional solution "stop" and mix well.

-

Read to 490 nm

-

Calculate the increase of absorbance

-

Determine the antioxidant power expressed in mM concentration of uric acid in relation to the Curve pattern

2. Method for determining antioxidant power in mineromedicinal water according to claim 1 characterized by preparing the standard curve with 2mM uric acid with the following decreasing solutions with different concentrations of uric acid (2.0; 1.0; 0.5; 0 , 25; 0.125 and 0.063 mM). 3. Procedure for determining antioxidant power in mineromedicinal water according to claims 1 and 2, characterized in that the reagent R1 is used in proportion 4/5. 4. Procedure for determining antioxidant power in mineral-medicinal water according to claims 1 to 3, characterized in that the reagent R1 is used in proportion 4/5 with 200 µl of sample plus 50 µl of R1, use 200 µl of the resulting solution add 50 µl of reagent 2, wait 3 minutes and add 50 µl of the stop solution.