ACTION OF AN ANTIOXIDANT COMPLEX ON THE ANTIOXIDANT POWER OF SALIVA

Transcription

1 V O L U M E 5 2 S U P P L. 1 T O N o. 2 J U N E ACTION OF AN ANTIOXIDANT COMPLEX ON THE ANTIOXIDANT POWER OF SALIVA U. CORNELLI, G. BELCARO, M. R. CESARONE, M. DUGALL, M. HOSOI, M. G. GROSSI, E. IPPOLITO, A. LEDDA, I. RUFFINI

2 PANMINERVA MED 2010;52(Suppl. 1 to No 2):1-5 Action of an antioxidant complex on the antioxidant power of saliva U. CORNELLI 1, G. BELCARO 2, M. R. CESARONE 2, M. DUGALL 2, M. HOSOI 2, M. G. GROSSI 2, E. IPPOLITO 3, A. LEDDA 2, I. RUFFINI 2 Aim. Based on the results of the soluble antioxidants test (SAT), we have produced a combination of oral antioxidants aimed at increasing the antioxidant power of saliva. Several antioxidants are included in this product (Vit E, beta-carotene, Vit A, Vit C, polihenoles, cathechins, ellagic acid, antocyanins, coenzime Q10 and piridoxin in association with Se, Zn, L-cisteine). The aim of this registry study was to evaluate the efficacy of these antioxidants in saliva, plasma and urines. Methods. MF Odontovis, an antioxidant complex, was administered to healthy subjects in the evening for one week with a final administration in the morning. Results. Plasma, urine and saliva showed an increase in antioxidant power following both the evening administration and the final morning administration. The antioxidant action appeared to be present even at night when salival secretion is lower. Plasma SAT levels in the morning following evening treatment were increased by 21% in comparison with controls. Morning administration increased levels up to 34% when measured 4 hours after treatment. Comparable increases were observed in saliva (morning values +44 %; +58% two hours after morning administration and +28 % after 4 hours). In urine the evening administration caused an increase in antioxidant power (+6%); the morning administration did not cause an increase in antioxidants. Conclusion. This study indicated that antioxidant levels can be increased with specific nutritional supplement. The clinical value of an increased antioxidant power in biological fluids, particularly in saliva, may be relevant for future trials of prevention and treatment. KEY WORDS: Antioxidants - Saliva - Plasma - Urine. Saliva, which acts as a barrier for the external environment, includes a number of protective compounds and activities. 1 One important element is the Received on April 28, Accepted for publication on June 18, 2010 Corresponding author: U. Cornelli, Loyola University School of Medicine, 2160 S. First Avenue, Maywood, IL umbertocornelli@cornelliconsulting.it 1Loyola University School of Medicine, Chicago, IL, USA 2Irvine3 Circulation Vascular Labs Chieti Pescara University, Pescara 3Vascular Surgery, Angiology University of Milan Milan, Italy antioxidant system (SAO), which includes different molecules and enzymes, 2 i.e., uric acid (UA) and peroxidases (POXs), both hydrosolubles. Other antioxidants in saliva are liposoluble (Vit E); in association with other not well known antioxidants it produces some 10% of total antioxidant capacity. 2, 3 UA may produce some 70% of the antioxidant capacity, and the role of ascorbic acid may be of secondary importance in comparison with UA. 3, 4 There is a correlation between UA in saliva and UA in plasma because plasma UA is the source of salivary UA. 5 Ascorbic acid levels may be 3 times higher than plasmatic values. 6 Salival antioxidant power in relation to lipid oxidation is lower than the antioxidant power of plasma. 4 POXs are the most important salival enzymes; other secondary enzymes include catalasis, glutatione peroxidase (glu POx), glutatione reductase (Glu red) and glutatione transferase. 2, 9 The last enzymes support the GSH pathway, one of the most important antioxidant systems, which operates in all cells and is present in any body fluid. Considering weight, POXs represent only 0.01% of the total of salivary proteins, including the actual salivary protein (POX) and myeloperoxidase. This last protein is similar to lactoperoxidase prodoced by lymphocytes in areas of inflammation of the oral cavity. 7, 8 POXs has two main roles: (a) the control of Vol Suppl. 1 to No. 2 PANMINERVA MEDICA 1

3 CORNELLI ACTION OF AN ANTIOXIDANT COMPLEX ON THE ANTIOXIDANT POWER OF SALIVA the levels of H 2 O 2 produced by bacteria and leucocytes, and (b) a specific anti-bacterial action for some bacteria H 2 O 2 also originates with Cl, HOCL in neutrophils; this compound represents an important antibacterial (bactericidal) mechanism in saliva. 14 Therefore, antioxidant protection is an important element in oral physiopathology, and its alterations or reduction in power may be associated to pathologies such as caries and parodontopathies. However, oxidative power may be both beneficial and damaging, and we need to understand and measure these elements to learn how to modulate their actions. The evaluation of antioxidant power in saliva may be obtained using methods comparable to the evaluation of plasmatic antioxidants. 15, 16 Salival antioxidant power has been evaluated 2 measuring. superoxidodismutases, uric acis and TAS (total antioxidant status) and the thiolic groups. To evaluate the antioxidant power of saliva, saliva (SATs), plasma (SATp) and urine (SATu) should be studied. Based on the results of the soluble antioxidants test (SAT), we have produced a combination of oral antioxidants aimed at increasing the antioxidant power of saliva. Several antioxidants are included in this product (Vit E, beta-carotene, Vit A, Vit C, poliphenoles, cathechins, ellagic acid, antocyanins, coenzime Q10 and piridoxin in association with Se, Zn, L-cisteine). The aim of this registry study was to evaluate the efficacy of these antioxidants in saliva, plasma and urines. Materials and methods We used the SAT to evaluate antioxidant power in saliva, plasma and urines. 17 This method utilizes the capacity of tiocyanate (SCN) to react with iron (Fe 3+ ) causing formation of Fe[(SCN) 6 ] 3-. This compound is characterized by a brown-red color. The possible reducing elements within the test fluid transform Fe 3+ into Fe 2+, avoiding the reaction with tiocyanate. The color is revealed at 505 nm. The base reactant including F is [Fe(NO 3 ) 3 ]. However Fe +3 may react with phosphates present in saliva. Therefore it has been modified adding to the fluids under zyrconium salts, which inactivate the phosphates without causing precipitation. This passage avoids centrifugation. This test allows the evaluation of the antioxidant power of saliva and plasma in relation to hydrosoluble antioxidants capable of reducing Fe 3+. TABLE I. Composition of MF Odontovis (MFO). Compound Selenium Vit C Red Fruits, Reach Berry Zn L-cisteine Coenzime Q10 Vit E Pirydoxin Vit A Beta-carotene Quantity 48 mcg 30 mg 90 mg 5 mg 12 mg 10 mg 15 mg 1 mg 1 mg 0.5 mg Subjects We studied 26 subjects (13 males); the age range was between 28 and 34 years. Inclusion criteria were the absence of clinical problems and no treatment with drugs or food supplements. All subjects were nonsmokers. Administration Before their night rest all subjects were asked to drink MF Odontovis ( MFO ), which contains a combination of antioxidants, or a comparable solution without antioxidants for a period of one week. MFO was administered using comparable packaging for the active and non-active formulations (10 ml vials separately including a powder and the relative fluid that were mixed just before drinking). The last administration was made in the morning just before evaluation in the study center. A total of 8 vials (7 in the evening and the 8th in the morning before evaluation) were administered. The formulation of MF Odontovis is shown in Table I. Both treatments (active and non active) were given in comparable boxes (10 vials for the two phases). Compliance was evaluated controlling unused vials. SAT evaluation The test was made for plasma, saliva, and urine at inclusion and after the last administration. Plasma SAT (SATp) was made at 8:00 am (basal value), at 11:00 and 12:00 am (the subjects were fasting since the previous evening meal). A drop of blood was collected from the finger in heparinated cuvettes. These were centrifuged to obtain plasma. The total blood used was less than 0.15 ml. SAT measurement was made on plasma samples of 10 µl. 2 PANMINERVA MEDICA June 2010

4 ACTION OF AN ANTIOXIDANT COMPLEX ON THE ANTIOXIDANT POWER OF SALIVA CORNELLI TABLE II. SAT in (SATp) in the two groups (active and non-active MFO). Mean and SD. Products Time SATp µmol/l After non active MFO 8.00 (basal) 2134± (basal) 2305± (basal) 2398±201.4 After MFO 8.00 (basal) 2574±242.9 b (basal) 2636± (basal) 3450±371.8 ab TABLE IV. Antioxidant capacity in urines (SATu) with MFO. Products Raccolta SATu µmol/l Volumi ml Non active MFO Night urine 2381± ± h after administration 1937± ±23.4 Active MFO Night Urine 2537±264.2 a 570± h after administration 2388±288.7 a 220±28.7 a t-test: non active vs. active (P<0.05). a basal vs. SATp at (P<0.05); b active vs. non-active MFO (P<0.05). TABLE III. SATs values (Ìmol /L) in the two groups. Time Non active MFO Active MFO ± ±133.2 a ±190.3 b 2340±127.7 ab ±185.4 b 2498±237.6 ab ± ±139.5 ab ± ±157.5 ab a t-test: non active vs. active MFO vs. AO; b t-test: values in comparison with basal result. Salival SAT (SATs) was made every hour from 8:00 a.m. (at least 30 minutes after teeth brushing) and at 12. Saliva was collected with a defined procedure: subjects were asked to chew a standard cube of medical cotton (150±20 mg) for one minute. The cotton ball was collected in a plastic cup of known weight to obtain the weight of the saliva included in the cotton ball. If salival weight was <0.7 ml/min or >1.5 ml/min, the test was repeated. All measurements were obtained with volume/weights of saliva bewteen 1.1 e 1.3 ml/min. Urine SAT (SATu). Urine was collected the evening before testing using the night urine (between 11:00 pm and 8:00 am). Morning urine was also collected in another container between 8:00 and 12:00. Therefore SATu was the expression of night urine and morning urine (after the last administration). Statistical analysis Mean and standard deviation (SD) were evaluted for all measurements. To evaluate the differences in the effects of the administration of MFO we used both parametric test (Student T-test) and non parametric evaluation (AVOVA, Mann-Whitney) considering independent data for variations before and after treatment. Considering previous SAT evaluations (unpublished data on file) for α value of 0.05 and 1-β 0.90 the difference of one SD in homogeneous groups including 20 subjects should be able to discriminate differences due to treatment. Considering possible dropouts (at a dimension of 30% of included subjects) the dimension of the groups was defined with a total of 26 subjects. Results All subjects completed the study. Compliance was very good (>98 %), and no tolerabilty problems were reported. After aministration of MFO (Table II), plasma antioxidant capacity increased at 4 hours after treatment (P<0.05) with minimal effects at 2 hours. Basal SATp following evening administration of MF Odontovis for seven days was significantly higher (P<0.05) in comparison with controls. This indicates that there is a persistent residual effect of MFO up to the following morning. Non-significant effects were observed with the non-active compound. Saliva was correctly collected in all subjects (all volumes were between 1.1 e 1.3 ml/min). Data concerning SATs is reported in Table III. Evening treatment with MFO increases the antioxidant power of saliva. To evaluate the acute effects of MFO, the treatment was administered in the morning after the last evening administration. An increase of the antioxidant power was shown within one hour after the morning administration, which remained higher than the baseline value for at least 4 hours. In Table IV the values of urinary excretion of antioxidants have been divided into night excretion after the evening administration and morning excretion after the morning administration. SATu values after MFO treatment were significantly higher (P<0.05) in comparison with controls both considering night excre- Vol Suppl. 1 to No. 2 PANMINERVA MEDICA 3

5 CORNELLI ACTION OF AN ANTIOXIDANT COMPLEX ON THE ANTIOXIDANT POWER OF SALIVA tion and the daily excretion within 5 hours after administration. There is no significant difference in urine volumes between the two groups. Discussion The value of these observations is somewhat limited due to the limited time of administration. The primary aim of the study was to evaluate saliva for the cinetic value of an antioxidant compound. It is possible to increase the antioxidant effects of saliva using different types of antioxidants. The types of antioxidants include all 4 categories: circulating antioxidants, membrane antioxidants, intracellular antioxidants (pyridoxine and coenzime Q 10 ), and those defined as system antioxidants. All these antioxidants may generate a protective shield. However, it is important to underline the importance of circulating antioxidants that may, more easily, reach saliva. The presence of antocyanins, catechols, polyphenols and ellacic acid in the red-fruit extract has an important ant-bacterial action (i.e. against Streptococcus mutans and other patogenic bacterial elements of the oral cavity) Whether the action is effective against bacterial adhesion (either bacteriostatic or bactericidal) will have to be determined in specific studies involving caries or paradontosis or other oral infective processess. MF Odontovis transports into saliva an antioxidant complex rather than a single product/compound. As shown by the antioxidant action in other clinical and preventive applications, the combination of these elements appears to be synergic. Dosages aimed at producing antioxidant action are kept low on purpose, and compounds including RDA have been kept at 100% RDA to avoid an unwanted pro-oxidant action that might result from using doses much higher than normal physiological intake. The concept of having different synergic antioxidant actions is produced using different antioxidant products. 21 Whether this combined antioxidant and, possibly, anti-inflammatory action may be enough to produce an antibacterial, anti-infective action should be evaluated in longer prospective studies. Morning SATp levels following the evening treatment increased on average 21% (P<0.05) in comparison with controls. Morning administration increased levels up to 34% (4 hours after administration; P<0.05); the increase was clear one hour after administration (showing a fast absorption). Comparable increases were observed in saliva (SATs); morning values were increased up to 44%; after 2 hours they increased 58%; after 4 hours the increase was 28%. Therefore, hydrosoluble antioxidants are quickly transferred into saliva. In urine (SATu) evening administration caused an increase in night secretion of +6%; morning administration did not show any change in urinary antioxidants. This may indicate that the excretion of metabolites without antioxidant power is prevalent in urine. With a daily production of about 1.5L/per day, saliva is basically recycled as antioxidants flow again into the intestinal tract and follow reabsorbtion and metabolization. This causes an antioxidant overload in the evening, which is shown in urine. The clinical value of an increased antioxidant power in biological fluids, particularly in saliva, may be relevant for future trials including both prevention and treatment. The SAT is very important to measure in a simple way the levels of antioxidants. These tests may indicate how antioxidant levels can be increased with the use of specific nutritional supplements within physiological modulation. References 1. Humphfrey SP, Williamson RT. A review of saliva: normal composition, flow, and function. J Prost Den 2001;85: Nagler RM, Klein I, Zarhevsky N, Drigues N, Reznick AZ. Characterization of the differentiated antioxidant profile in human saliva. Free Rad Biol Med 2002;32: Moore S, Calder KAS, Miller NJ, Rice-Evans CA. Antioxidant activity of saliva and periodontal disease. Free Rad Res 1994;21: Terao J, Nagao A, Yuki H, Itoh Y. Reduction of fatty acid hydroperoxides by human parotid saliva. Lipids 1993;28: Kondorova I, Lissi EA, Pizarro M. Total antioxidant potential in human saliva of smokers and non smokers. Biochem Mol Biol Int 1999;47: Meyle J, Kapitza K. Assay of ascorbic acid in human crevicular fluid from clinically healthy gingival sites by high-performance liquid chromatography. Arch Oral Biol 1990;35: Iwamoto Y, Nakamura R, Watanabe T, Tsunemitsu A. Heterogenity of peroxidase related to antibacterial activity in human parotid saliva. J Dent res 1972;51: Kowolik MJ, Grant MA. Myeloperoxidase activity in human gingival crevicular neutrophils. Arch Oral Biol 1983;28: Pereslegina IA. The activity of antioxidant enzymes in the saliva of normal children. Lab Delo 1989;11: Grisham MB, Ryan EM. Cytotoxic properties of salivary gland. Am J Physiol 1990;258:C115-C Thomas EL, Aune TM. Lactoperoxidase, peroxide, thiocynate antimicrobial system: correlation of sulfhydryl oxidation with antimicrobial action. Infect Imm 1978;20: PANMINERVA MEDICA June 2010

6 ACTION OF AN ANTIOXIDANT COMPLEX ON THE ANTIOXIDANT POWER OF SALIVA CORNELLI 12. Thomas EL, Milligan TW, Joyner RE, Jefferson MM. Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci. Infect Immun 1994;62: Kou F, Takahama U. Hydrogen peroxide-induced luminescence and evolution of molecular oxygen saliva. Arch Oral Biol 1995;40: Miyasaki KT. The neutrophil: mechanism of controlling periodontal bacteria. J Periodontol 1991;62: Magalhães LM, Segundo MA, Reis S, Lima JLFC. Methodological aspects about in vitro evaluation of antioxidant properties. Anal Acta 2008;613: Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. Agr Food Chem 2005;53: Cornelli U. A simple method to determine antioxidant activity in saliva. J Dent Res [in press] 18. Ferrazzano GF, Amato I, Igenito A, De Natale A, Pollio A. Anti-cariogenic effects of polyphenols from plant stimulating beverages (cocoa, coffee, tea). Fitoterapia 2009;80: Petti S, Scully C. Polyphenopls, oral health and disease: A review. J Dent 2009;37: Smullen J, Koutsou GA, Foster HA, Zumbé A, Storey DM. The antibacterial activity of plant extract containing polyphenols agaist Streptococcus mutans. Caries Res 2007;41: Cornelli U, Terranova R, Luca S, Cornelli M, Alberti A. Bioavailability and antioxidants activity of some food supplements in men and women using D-Roms test as a marker of oxidative stress. J Nutr 2001;131: Battino M, Ferreiro MS, Gallardo I, Newman HN, Bullon P. The antioxidan capacity of saliva. J Clin Periodontol 2002;29: Vol Suppl. 1 to No. 2 PANMINERVA MEDICA 5