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Comparative study on the antioxidant capacities of synthetic influenza inhibitors and ellagic acid in model systems
Biomedicine & Pharmacotherapy 83 (2016) 755–762
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Comparative study on the antioxidant capacities of synthetic influenza inhibitors and ellagic acid in model systems Elitsa L. Pavlovaa,* , Nikolay N. Zografova , Lora S. Simeonovab a b
Biophysics & Medical Physics, Sofia University “St. Kliment Ohridski”, 5 James Boucher Blvd., 1164 Sofia, Bulgaria Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Georgi Bonchev Str., 1113 Sofia, Bulgaria
A R T I C L E I N F O
Article history: Received 19 May 2016 Received in revised form 9 July 2016 Accepted 21 July 2016 Nomenclature: Ellagic acid (PubChem CID 5281855) Vitamin E (PubChem CID 20353) Vitamin C (PubChem CID 54670067) Oseltamivir (PubChem CID 65028) Isoprinosine (PubChem CID 37510) Keywords: Oseltamivir Isoprinosine Ellagic acid Vitamin E Vitamin C Luminescence Constant of inhibition
A B S T R A C T
This study compares the antioxidant capacities in vitro of several synthetic and natural compounds applied and researched for influenza treatment – oseltamivir, isoprinosine, ellagic acid, vitamin E and vitamin C. Three chemical systems are utilized for the generation of reactive oxygen species (ROS) at pH 7.4 and pH 8.5: (1) Fenton’s (Fe2+ + H2O2) for OH and OH species (2) H2O2 (3) NADH phenazinemethosulfat, for superoxide radicals (O2 ). The kinetics was evaluated by lucigenin-enhanced chemiluminescence. The calculated constants of inhibition k7 describe the antioxidant capacity at the moment of oxidative burst. Their values do not necessarily correspond to the calculated total antioxidant activity. The obtained results revealed that the synthetic anti-influenza drugs (oseltamivir and isoprinosine) as well as ellagic acid possess pronounced scavenging properties mostly against superoxide radicals, comparable and higher than that of traditional natural antioxidants. Quantitative analysis of the antioxidant effects of the examined synthetic substances was performed. The results compared the corresponding effect of the average physiological concentrations and the applied therapeutic antioxidant dose. With these experiments we registered new aspects of their therapeutic activities, due to antioxidant properties against hydroxyl, superoxide radicals and H2O2 oxidation. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Influenza continues to be a major cause of morbidity and mortality despite the widespread access to vaccines and antivirals [1]. A definitive medical treatment for severe influenza-associated complications has not been established yet. It is suggested that a therapy with antiviral and antioxidant capacity could be a choice for decrease in the disease severity—both by its specific mode of action and by scavenging superoxide anion radicals, generated during influenza pathogenesis [2]. It is well established that the combination of antioxidants with specific antiviral drugs synergistically reduces the lethal effects of influenza virus infections [3–6]. Three substances with proved antioxidant effects (ellagic acid, vitamin E and vitamin C) and two traditional anti-influenza drugs used in clinics (oseltamivir and isoprinosine) were chosen to measure and compare their antioxidant capacities in vitro,
* *Corresponding author. E-mail address: [email protected] (E.L. Pavlova). http://dx.doi.org/10.1016/j.biopha.2016.07.046 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
expressed as constants of inhibition in different systems for the generation of physiologically active reactive oxygen species (ROS). The antioxidant potentials of the synthetic inhibitors had never been studied before. The selection was based on the fact that oseltamivir is currently the most recommended antiviral by the World Health Organization for etiologic treatment of the influenza disease and isoprinosine is a medicine with a broad spectrum of biological effects and is widely prescribed mostly due to its immunomodulation effect. Thus we found it challenging to investigate the potential antioxidant activities of those compounds and to test them for free radicals scavenging capacities in vitro. 1.1. Oseltamivir Oseltamivir is a neuraminidase inhibitor. As a structural analogue of the N-acetylneuraminic acid of the cellular glycoprotein receptors, it binds the viral neuraminidase and blocks the cleavage and release of influenza virus progeny from the surface of the infected cell in the late stages of the replication cycle. The active site of the enzyme is relatively conserved, which makes it active against A, B and C types of the influenza virus [7]. Oseltamivir is a potent influenza inhibitor
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both in vitro and in vivo [8–11]. Due to the increasing rates of resistance to the inhibitor, numerous studies estimated the advantages of the combined therapy of oseltamivir with drugs possessing other active mechanisms [12–14]. 1.2. Isoprinosine Isoprinosine is an inosine-salt complex approved in over 60 countries as an antiviral and an immunomodulator. It is a synthetic compound formed from the p-acetamido benzoate salt of N-N dimethylamino-2-propanol and inosine at a 3:1 molar ratio. It has a thymomimetic immunopharmacology that supports a pro-host action to increase cellular immune response. When applied in vitro, it induces T-cell differentiation and promotes proliferation, cytotoxicity and cytokine production. It also boosts macrophage and granulocyte functions. In vivo these multiple activities translate into enhanced cellular immune responses in infected or cancer-bearing individuals, following burns or in age-related immune-deficiencies [15–18]. Results of many studies indicated beneficial clinical effects of inosine pranobex against several viral diseases, including herpes, influenza, genital warts and type B viral hepatitis [19]. It has also showed beneficial protective effects when studied in combination with specific influenza antivirals (rimantadine) in animal models [20]. 1.3. Ellagic acid Ellagic acid is a polyphenol present in many fruits, such as grapes, strawberries, pomegranates and walnuts, as well as in several medicinal plants [21–23]. It has demonstrated antiviral and antimicrobial capacities, as well as antioxidant, anticancer, antiallergic and anti-inflammatory activities [22–25]. Researches suggest that it has antioxidant properties and inhibits a number of cell-signaling pathways that are important to tumor growth, including inflammatory signaling such as tumor necrosis factor a-induced cyclooxygenase-2 (COX-2) protein expression and nitric oxide synthase inhibition [26,27]. Commercial pomegranate juice shows potent antioxidant and anti-atherosclerotic properties attributed to its high content of polyphenols including ellagic acid in its free and bound forms and other flavonoids (quercetin, kaempferol and luteolin glycosides) [8,22,28–30]. It is proved to be an antioxidant as effective as or better than a-tocopherol or tertiary butylhydroxyanisole and shows inhibitory activity against lipid peroxidation [31,32]. Synergism is found between ellagic acid and current antimalarial drugs (chloroquine, artesunate, mefloquine, and atovaquone). This effect could enable drug-dose reduction during treatment and further limit side effects [31–33]. 1.4. Ascorbic acid Ascorbic acid is a naturally occurring organic compound with antioxidant properties. Its antioxidant effects are only a small part of its vitamin activity. The ascorbate ion is the predominant species at typical biological pH values. It is a mild reducing agent [34,35]. Often the free radicals initiate chain reactions. Ascorbate can terminate these chain radical reactions by electron transfer. The oxidized forms of ascorbate are relatively unreactive and do not cause cellular damage. However, being a good electron donor, the excessive ascorbate, in presence of free metal ions, cannot only promote but also initiate free radical reactions, thus making it a potentially dangerous pro-oxidative compound in certain metabolic contexts. A study of vitamin C in healthy people revealed a sigmoidal relationship between oral dose and plasma/tissue vitamin C concentrations. Hence, optimal dosage is critical to intervention studies using vitamin C [36,37]. Dehydroascorbic acid, the acid with no reducing ability, showed much stronger antiviral
activity than ascorbic acid, indicating that the antiviral activity is due to factors other than the antioxidant mechanism [37–39]. 1.5. Vitamin E Vitamin E is referred to a group of eight fat-soluble compounds that include both tocopherols and tocotrienols. a-tocopherol being the most biologically active form of vitamin E, is the second-most common form of vitamin E in the diet. As a fat-soluble antioxidant it stops the production of ROS formed when fat undergoes oxidation and fights mostly the lipid-oxide-radicals. Vitamin E has also been proved as a commercial antioxidant in ultra-high molecular weight polyethylene. It is known to protect lipids and prevents the oxidation of polyunsaturated fatty acids [40,41]. Vitamin E is not an agent with specific antiviral action but its antioxidant properties are moderating the course of the influenza infection in dose-dependent manner, due to its role as a membrane protector [41,42]. The contemporary approach for the treatment of influenza includes despite the vaccine and antiviral prophylaxis and therapy also the combination and additive effects of antioxidants fighting against the ROS that are accumulated from the infection and the disease itself. For these reasons, the focus of this in vitro research is on the comparison of the antioxidant capacity of oseltamivir, isoprinosine, ellagic acid, vitamin E and vitamin C against ROS, by lucigenin-enhanced chemiluminescence, as calculated constants of inhibition. 2. Materials and methods The kinetics of lucigenin-enhanced chemiluminescence was examined for three chemical systems designed for the generation of ROS. All three systems were tested at 25 C/pH 7.4 – physiological, and 25 C/pH 8.5 – for better differentiation of the chemiluminescent response. The total volume of each sample in the measuring cuvette is 2 ml, and the kinetics was registered for 3 min using chemiluminometer LKB 1251 (Sweden). The first seconds of fast flash luminescence are most informative for the inhibition effects. Each sample is prepared as a mixture, containing the buffer medium, chemiluminescent probe lucigenin, the chemical system for ROS-generation and the tested drug or combinations. The utilized in vitro buffer solutions were prepared from Na2HPO4 [0.05 M] and C8H8O7H2O [0.1 M]. The control systems contained all the presented reagents in the absence of an inhibitor ([InH]0 = 0) – the tested drug or combinations. Chemiluminescent systems for the generation of ROS (given final tested concentrations in a 2 ml sample volume): 1) Fenton’s reagent for the generation of OH and OH species (FeSO4 [5.104 M]-H2O2 [2,5.103 M]) It is well known that the interaction between Fe2+-ions and =2?2 results in highly reactive, short-living ?=-radicals: Fe2+ + H2O2 ! Fe3+ + OH + OH
(1)
Fe3+ + H2O2 ! Fe2+ + OOH + H+
(2)
2) H2O2 oxidation (H2O2 [2,5.103 M]) 3) reduced a-nicotinamide adenine dinucleotide (NADH) [104 M] – phenazinemethosulfat [104 M] – for the generation of superoxide radicals (O2):
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NAD.H + PMS + =+ ! PMS.=2 + NAD+.
(3)
PMS.=2 + PMS ! 2PMS.=
(4)
PMS.= +?2 ! PMS + ?2 + =+
(5) 4
The chemiluminescent probe lucigenin [10 M] is dissolved in DMSO. The tested substances were added as the following final concentrations, as water solutions: - ellagic acid (104 M – equal to the referent dose of vitamin C, accepted as effective antioxidant concentration or - 1,75.105 M, which is equal to 1/6 of the average physiological concentration of vitamin C) - vitamin E, applied as a-tocopherol succinate (2,1.104 M – physiological concentration or 1,75.105 M, which is 1/6 of the average physiological concentrations of vitamin C) - vitamin C (104 M, equal to the referent dose of vitamin C with established antioxidant activity or 1,29.103 M, which is the average physiological concentration) - oseltamivir (104 M, equal to the referent dose of vitamin C, but 750 times lower than the applied daily therapeutic dose) - isoprinosine (104 M, equal to the referent dose of vitamin C, but 1115 times lower than the applied daily therapeutic dose).
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2.2. Calculations The integral evaluation of the content of free radicals and ROS as well as the registration of the kinetic parameters of the oxidation reaction could be easily done by the lucigenin-enhanced chemiluminescent method. The constants of inhibition k7 can be calculated according to the kinetics of chemiluminescence, in correlation to the concentration of ROS [43–45]: ½ROS 2 ðI=I0 Þ ¼ ð6Þ ½ROS0 where I is the measured light intensity [mV], ½ROS0 is the initial concentration of the reactive oxygen species in the absence of inhibitors (t = 0). The constant of inhibition k7 ½l=ðmol:sÞcan be n o calculated from the slope of the linear part II0 ðtÞduring the first period of fast chemiluminescent emission, as follows: dðI=I0 Þ ¼ 2k7 ½InH0 dt
ð7Þ
where I0 is the registered light intensity [mV] at t = 0, and [InH]0 is the initial concentration of the inhibitor (Fig. 1). The accuracy of k7 is determined mainly by the accuracy of the light intensity measurements and less than [InH]0. For convenience, k7 can be expressed in ½l=ðg:sÞ, when taking into account the molecular mass of the inhibitor (Table 1). 2.3. Statistics
2.1. Reagents iron sulphate FeSO4 (Merck, Germany), phenazine methosulfate C13H11N2CH3SO4 (PMS) (N-methyldibenzopyrazine methyl sulfat salt) (Merck, Germany), hydrogen peroxide H2O2 (Boron, Bulgaria), disodium hydrogen phosphate Na2HPO4 (Boron, Bulgaria), citric acid C8H8O7H2O (Boron, Bulgaria), lucigenin C28H22N4O6 (bis-Nmethylacridinium nitrate) (Aldrich, USA), a – nicotinamide adenine dinucleotide, reduced form C21H27N7O14P2Na2 (NAD.=) (Boehringer, Germany), ellagic acid C14H6O8H2O (Alfa Aesar, England), vitamin E (a-tocopherol succinate, C33H54O5) (Aldrich, USA), vitamin C C6H8O6 (Boron, Bulgaria), oseltamivir C16H28N2O8P (F. Hoffmann-La Roche Ltd.), Isoprinosine C52H78N10O17 (Ewopharma AG, Switzerland), dimethyl sulfoxide C2H6OS (DMSO) (Aldrich, USA).
All measurements were made in triplicate, p 0.07, applying Origin 8.5 and Microsoft Office Excel 2010 for the analysis. All described work was carried out in accordance with the Uniform Requirements for manuscripts submitted to biomedical journals. 3. Results and discussion Three stages were defined in the kinetics of the chemiluminescent reaction for generation of ROS: spontaneous (0–20 s), fast flash (20–40 s) and latent period (50–200 s). The interaction between the researched substances and ROS, generated in the system, decreased sharply the chemiluminescent response in system 3, while the observed effects in system 1 and system 2 were mild.
Fig. 1. Fenton’s system for the generation of reactive oxygen species (OH, OH) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 7.4 (p 0.07).
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Table 1 Calculated constants of inhibition (k7) about ellagic acid, oseltamivir, isoprinosine and standard antioxidants (vit. C and vit. E), measured by chemiluminescence in vitro (p 0.07). tested substance
system
type
pH
K7 [l/mol s]
K7 [l/g s]
ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M]
Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS
hydroxyl radicals and hydroxide ions
8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4
3,104 12,809 1,708 3,142 1,810 0,179 1,011 10,278 6,750 32,000 4,536 4,470 3,028 0,176 2,057 25,220 2,620 17,38 0,225 2,110 2,817 0,1809 0,658 2,2143 3,983 20,903 2,839 11,7 13,2 0,316 0,493 23,431 60,800 448,571 85,100 77,750 82,300 3,620 37,762 466,571 27,800 157,428 26,750 29,200 18,800 0,806 11,119 134,857
1,027.102 4,238.102 0,442.102 0,282.102 1,027.102 0,102.102 0,190.102 1,936.102 2,234.102 10,589.102 1,174.102 0,400.102 1,719.102 0,099.102 0,387.102 4,751.102 0,867.102 5,751.102 0,058.102 0,189.102 1,599.102 0,103.102 0,124.102 0,417.102 1,318.102 6,917.102 0,735.102 1,049.102 7,494.102 0,179.102 0,093.102 4,414.102 20,119.102 148,435.102 22,022.102 6,972.102 46,727.102 2,055.102 7,114.102 87,901.102 9,199.102 52,094.102 6,922.102 2,618.102 10,674.102 0,458.102 2,094.102 25,407.102
H2O2 oxidation
superoxide radicals
Usually, higher antioxidant capacity corresponds to lower intensity of chemiluminescence. Best scavenging properties were demonstrated in system 3: all reagents suppressed the reaction (17–82%). Highest constants of inhibition presented ellagic acid, vit.E and vit.C, followed by oseltamivir and isoprinosine (Table 1) at both physiological and pH 8.5. Best scavenging effects towards the generated superoxide radicals exhibited the natural substances with proven antioxidant effect when applied in lower doses (Figs. 5 and 6, Table 1). That phenomenon is explained with the fact that vitamin C and vitamin E are free radical sources when applied at higher dosage, although these radicals are stable and not so aggressive. Some toxicological studies including also our unpublished data show that ellagic acid presents also prooxidant effects when applied at higher concentrations. Oseltamivir and isoprinosine exhibit very good scavenging effect against the superoxide radicals too (Figs. 5 and 6, Table 1). Further studies should clarify if those antioxidant activities are dose-dependent. Ellagic acid, vitamin E and vitamin C are effective antioxidants in the Fenton‘s system for both tested media (Figs. 1 and 2, Table 1).
The antioxidant effect of vitamin C is proportional, dosedependent. The antioxidant effect of vitamin E is inversed, dosedependent (Figs. 1 and 2, Table 1). But the physiological conditions provoke the inhibition features of oseltamivir against the generated hydroxyl radicals. Isoprinosine is an effective antioxidant in both media (Table 1). Against the oxidant H2O2, highest constant of inhibition is calculated about ellagic acid, vitamins C and E for both tested media (Figs. 3 and 4, Table 1). The antioxidant effect of vitamin C is again proportional, dose-dependent. The antioxidant effect of vitamin E is again inversed, dose-dependent (Figs. 3 and 4, Table 1). Very effective against that strong oxidant and ROS – H2O2, at physiological conditions, is also isoprinosine – much more effective than oseltamivir at both pH (Table 1). The calculated constants of inhibition describe the antioxidant capacity at the moment of oxidative burst. Their values do not always correspond to the calculated total antioxidant activity. The application of non-specific immune modifiers and antivirals plays an important role in current immunotherapy. We found that oseltamivir and isoprinosine show pronounced ROS-scavenging
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Fig. 2. Fenton’s system for the generation of reactive oxygen species (OH, OH) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 8.5 (p 0.07).
Fig. 3. H2O2-oxidation system and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 7.4 (p 0.07).
Fig. 4. H2O2-oxidation system and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 8.5 (p 0.07).
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Fig. 5. NAD.H-PMS system for the generation of superoxide radicals (O2 ) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 7.4 (p 0.07).
Fig. 6. NAD.H-PMS system for the generation of superoxide radicals (O2 ) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 8.5 (p 0.07).
properties. These tests register for the first time new aspects of their therapeutic activities, due to antioxidant properties. Designed as a structural analogue of neuraminic acid, oseltamivir competitively binds the active site of the enzyme neuraminidase on the influenza virus surface. Thus the final steps of the viral replication cycle are blocked and the release of viral progeny from the host-cell is inhibited [8,10]. Further research and deeper biochemical analysis via interaction with biological membranes could reveal the precise mechanisms of antioxidant activities of oseltamivir. Inosine pranobex has demonstrated non-specific antiviral effect against several viral models. It acts as a powerful immunostimulant [15]. A potential advantage of isoprinosine is its effectiveness when administered orally and its therapeutic effects when administered after establishing the infection. Previous studies have shown that the combination of pomegranate polyphenol extract and oseltamivir synergistically increase the anti-influenza effect of oseltamivir. The pomegranate polyphenol extract inhibit the replication of human influenza A/ Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potentials especially for influenza epidemics and pandemics strains [46].
The dual antioxidant and pro-oxidant properties of ellagic acid might be interesting for a more profound research. Probably due to chelative and phenol activities, ellagic acid shows strong antioxidant effects (system 3). Its efficacy in the Fenton’s system is better than the antivirals and better or comparable to that of the standard antioxidants—vitamins C and E (Figs. 1 and 2). Ellagic acid can react to free radicals due to its ability to chelate with metal ions [47]. The four rings in its structure give the lipophilic property and the two lactones can act as hydrogen-forming and electron acceptors, respectively, that gives a hydrophilic characteristic [48]. Vitamin E acts as a peroxyl radical scavenger, preventing the propagation of free radicals in tissues by reacting to form a tocopheryl radical, which then is reduced by a hydrogen donor (such as vit. C) and returned to its reduced state. As it is fat-soluble, it is incorporated into cell membranes protecting them from oxidative damage [40]. Here, we have tested the water-soluble form of the vitamin, in order to compare on equal scale its effect in comparison to the other substances. Vit.C typically reacts with ROS, such as the hydroxyl radical. Ascorbic acid also scavenges superoxide anion radicals [34,35].
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Vitamin C exhibits very high total antioxidant capacity but low initial antioxidant inhibition speed, when applied in physiological concentrations. Vit.C presents best antioxidant effect in system 2 and system 3. The lower values obtained for both vitamins can be explained by their specific action mechanisms. Vit.E fights mostly against lipid-oxide-radicals. Vit.C exhibits very high total antioxidant capacity, but low initial antioxidant inhibition when applied in physiological concentrations. The pathology of severe influenza-associated complications results from complex biological phenomena such as apoptosis induction, macrophage activation, oxidative tissue damage and higher contents of pro-inflammatory cytokines [1,6]. The pathogenesis involves not only apoptotic cell death mediated through virus replication in the infected cells, but also the injury of noninfected cells by superoxide anion radicals derived from activated phagocytes infiltrated into the virus-infected organs [5,49]. These superoxide anion-mediated pathways are part of the immune mechanisms of extensive tissue injury observed during severe influenza-induced complications [4,50]. It should be noted, that the treatment with SODs decreased the lethal or toxic effect of influenza virus infection in mouse models but did not inhibit the virus proliferation [51–54]. In particular, oxygen species such as hydrogen peroxide, superoxide anion radicals, singlet oxygen and other radicals, are considered as agents attacking polyunsaturated fatty acid in cell membranes and giving rise to lipid peroxidation [6,55]. Many reports have suggested that lipid peroxidation may result in destabilization and disintegration of cell membranes leading to cell injury and the so-called “free radical” diseases and eventually to aging and susceptibility to cancer. However, healthy cell membranes do not undergo lipid peroxidation so severely in vivo, because of the extremely efficient protective mechanisms against damage caused by active oxygens and free radicals. Such systems include enzymatic inactivation by, for example, superoxide dismutase, glutathione-peroxidase and catalase, as well as nonenzymatic protection of polyunsaturated fatty acid by physiological and biological antioxidants such as vitamin E, vitamin C, ß-carotene, uric acid, etc. [50,55]. In general, antioxidants are compounds that can delay, inhibit or prevent the oxidation of compounds trapping free radicals and reducing the oxidative stress. For these reasons, the combination of antioxidants with anti-influenza drugs could definitely improve the conventional chemotherapy for severe influenza-associated complications [2,5,22,44,56,57] [data in vivo to be published]. The obtained results revealed that the synthetic anti-influenza drugs (oseltamivir and isoprinosine) as well as ellagic acid possess pronounced scavenging properties mostly against superoxide radicals, comparable and higher than that of traditional natural antioxidants (vitamins C and E). With these experiments we registered new aspects of their therapeutic activities, due to antioxidant properties against hydroxyl, superoxide radicals and H2O2 oxidation. Conflict of interest Each author declares no financial or commercial conflicts of interest. Acknowledgements This research is financially supported by the National Science Fund, Ministry of Education and Science, Republic of Bulgaria. Project Contract No: DFNI B01/19 “A Modern Alternative for the Prophylaxis and Treatment of Influenza Virus Infection –
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Multitarget Approaches with Highly Efficient Combinations of Antiviral Chemotherapeutics and Biologically Active Compounds” References [1] T.M. Tumpey, A. García-Sastre, J.K. Taubenberger, P. Palese, D.E. Swayne, M.J. Pantin-Jackwood, S. Schultz-Cherry, A. Solórzano, N. Van Rooijen, J.M. Katz, C.F. Basler, Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice, J. Virol. 79 (2005) 14933–14944. [2] N. Uchide, H. Toyoda, Potential of selected antioxidants for influenza chemotherapy, Anti Infect. Agents Med. Chem. 7 (2008) 73–83. [3] N. Uchide, H. Toyoda, Future target molecules for influenza treatment, Mini Rev. Med. Chem. 8 (2008) 491–495. [4] B. Halliwell, Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 344 (8924) (1994) 721–724. [5] E. Peterhans, Reactive oxygen species and nitric oxide in viral diseases, Biol. Trace Element Res. 56 (1997) 107–115. [6] H. Siess, Oxidative Stress, Academic Press, London, 1985. [7] A. Moscona, Neuraminidase inhibitors for influenza, N. Engl. J. Med. 353 (2005) 1363–1373. [8] S.U. Kim, W. Lew, M.A. Williams, H. Liu, L. Zhang, S. Swaminathan, N. Bischofberger, M.S. Chen, D.B. Mendel, C.Y. Tai, W.G. Laver, R.S. Stevens, Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity, J. Am. Chem. Soc. 119 (1997) 681–690. [9] W. Lew, X. Chen, C.U. Kim, Discovery and development of GS4104 (oseltamivir): an orally active influenza neuraminidase inhibitor, Curr. Med. Chem. 7 (2000) 663–672. [10] D.B. Mendel, R.G., Webster, N.A. Roberts, Inhibition of avian influenza neuraminidases by GS4071 (Ro 64-0802) in vitro, Roche Research Report W-143039 (1999). [11] R.W. Sidwell, J.H. Huffman, D.L. Barnard, K.W. Bailey, M.N. Wong, A. Morrison, T. Syndergaard, C.U. Kim, Inhibition of influenza virus infections in mice by GS4104, an orally effective influenza virus neuraminidase inhibitor, Antivir. Res. 37 (1998) 107–120. [12] N.A. Ilyushina, E. Hoffman, R. Salomon, R.J. Webster, E.A. Govorkova, Amantadine-oseltamivir combination therapy for H5N1 influenza virus in mice, Antivir. Ther. 12 (3) (2007) 363–370. [13] D.F. Smee, M.H. Wong, K.W. Bailey, R.W. Sidwell, Activities of oseltamivir and ribavirin used alone and in combination against infections in mice with recent isolates of influenza A (H1N1) and B viruses, Antivir. Chem. Chemother. 17 (4) (2006) 185–192. [14] A.S. Galabov, L. Simeonova, G. Gegova, Rimantadine and oseltamivir demonstrate synergistic combination effect in an experimental infection with type A (H3N2) influenza virus in mice, Antivir. Chem. Chemother. 17 (4) (2006) 251–258. [15] D.M. Campoli-Richards, E.M. Sorkin, R.C. Heel, Inosine Pranobex. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy, Drugs 32 (1986) 383–424. [16] A. Marton, P. Pacher, K.G. Murthy, Z.H. Nemeth, G. Hasko, C. Szabo, Antiinflammatory effects of inosine in human monocytes, neutrophilis and epithelial cells in vitro, Int. J. Mol. Med. 8 (2001) 617–621. [17] S. Milano, M. Dieli, S. Millott, M.D. Miceli, E. Maltese, E. Cillari, Effect of isoprinosine on IL-2, IFN-gamma and IL-4 production in vivo and in vitro, Int. J. Immunopharmacol. 13 (1991) 1013–1018. [18] R.W. Flo, A. Naess, G. Albrektsen, C.O. Solberg, Isoprinosine stimulates granulocyte chemiluminescence and inhibits monocyte chemiluminescence in vitro, APMIS 102 (1994) 249–254. [19] J. Davidson-Parker, W. Dinsmore, M.H. Khan, D.A. Hicks, C.A. Morris, D.F. Morris, Immunotherapy of genital warts with inosine pranobex and conventional treatment: double blind placebo controlled study, Genitourin. Med. 64 (6) (1988) 383–386. [20] S. Pancheva, G. Gegova, V. Maximova, N. Manolova, The anti-influenza effect of rimantadine and isoprinosine when used in combination in mice, Acta Microbiol. Bulg. 26 (1990) 26–29. [21] I. Barta, P. Smerak, Z. Polivknova, et al., Current trends and perspectives in nutrition and cancer prevention, Neoplasma 53 (1) (2006) 19–25. [22] B. Cerdá, F.A. Tomás-Barberán, J.C. Espín, Metabolism of antioxidant and chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged wine in humans: identification of biomarkers and individual variability, J. Agric. Food Chem. 53 (2) (2005) 227–235. [23] A.P. Rogerio, C. Fontanari, E. Borducchietal, Anti-inflammatory effects of Lafoensia pacariandellagicacidina murine model of asthma, Eur. J. Pharmacol. 580 (1–2) (2008) 262–270. [24] C. Bell, S. Hawthorne, Ellagic acid, pomegranate and prostate cancer—a mini review, J. Pharm. Pharmacol. 60 (2) (2008) 139–144. [25] Y.H. Choi, G.H. Yan, Ellagic acid attenuates immunoglobulin e-mediated allergic response in mast cells, Biol. Pharm. Bull. 32 (6) (2009) 1118–1121. [26] J. Gainok, R. Daniels, D. Golembiowski, P. Kindred, L. Post, R. Strickland, N. Garrett, Investigation of the anti-inflammatory, antinociceptive effect of ellagic acid as measured by digital paw pressure via the Randall-Selitto meter in male SpragueDawley rats, AANA J. 79 (4) (2011) 28–34.
762
E.L. Pavlova et al. / Biomedicine & Pharmacotherapy 83 (2016) 755–762
[27] B. Doyle, L.A. Griffiths, The metabolism of ellagic acid in the rat, Xenobiotica 10 (1980) 247–256. [28] M. Albrecht, W. Jiang, J. Kumi-Diaka, E.P. Lansky, L.M. Gommersall, R.E. Mansel, et al., Pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells, J. Med. Food 7 (2004) 274–283. [29] K.L. Khanduja, R.K. Gandhi, V. Pathania, N. Syanl, Prevention of Nnitrosodiethylamine-induced lung tumorigenesis by ellagic acid and quercetin in mice, Food Chem. Toxicol. 37 (1999) 31–38. [30] M. Toi, H. Bando, C. Ramachandran, S.J. Melnick, A. Imai, R.S. Fife, et al., Preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo, Angiogenesis 6 (2003) 121–128. [31] R.K.Y. Zee-Cheng, C.C.J. Chang, Ellagic acid, Drugs Future 11 (1986) 1029–1033. [32] P. Njomnang Soh, B. Witkowski, D. Olagnier, M. Nicolau, M. Garcia-Alvarez, A. Berry, F. Benoit-Vical, In vitro and In vivo properties of ellagic acid in malaria treatment, Antimicrob. Agents Chemother. 53 (3) (2009) 1100–1106. [33] Y. Tsujimoto, H. Hashizume, M. Yamazaki, Superoxide radical scavenging activity of phenolic compounds, Int. J. Biochem. 25 (1993) 491–494. [34] http://en.wikipedia.org/wiki/Ascorbic_acid. [35] S.J. Padayatty, A. Katz, Y. Wang, P. Eck, O. Kwon, J.H. Lee, S. Chen, C. Corpe, A. Dutta, S.K. Dutta, M. Levine, Vitamin C as an antioxidant: evaluation of its role in disease prevention, J. Am. Coll. Nutr. 22 (1) (2003) 18–35. [36] R.J. Jariwalla, M.W. Roomi, B. Gangapurkar, T. Kalinovsky, A. Niedzwiecki, M. Rath, Suppression of influenza A virus nuclear antigen productionand neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extractand amino acids, Biofactors 31 (2007) 1–15. [37] A. Furuya, M. Uozaki, H. Yamasaki, T. Arakawa, M. Arita, A.H. Koyama, Antiviral effects of ascorbic and dehydroascorbic acids in vitro, Int. J. Mol. Med. 22 (2008) 541–545. [38] D. Banerjee, D. Kaul, Combined inhalational and oral supplementation of ascorbic acid may prevent influenza pandemic emergency: a hypothesis, Nutrition 26 (2010) 128–132. [39] N. Uchide, H. Toyoda, Antioxidant therapy as a potential approach to severe influenza-Associated complications, review, Molecules 16 (2011) 2032–2052. [40] http://en.wikipedia.org/wiki/Vitamin_E. [41] M. Mileva, R. Bakalova, L. Tancheva, A.S. Galabov, S. Ribarov, Effect of vitamin E supplementation on lipid peroxidation in blood and lung of influenza virus infected mice, Comp. Immunol. Microbiol. Infect. Dis. 25 (1) (2002) 1–11. [42] M. Mileva, L. Tancheva, R. Bakalova, A.S. Galabov, V.M. Savov, S. Ribarov, Effect of vitamin E on lipid peroxidation and liver monooxigenase activity in experimental influenza virus infection, Toxicol. Lett. 114 (1–3) (2000) 39–45. [43] I. Rusina, On the absolute values of constants of speed of interoperability of the inhibitors with peroxide radicals, Izvestiya Akademii Nauk SSSR 224 (3) (1975) 646–649 (in Russian).
[44] E. Pavlova, M. Lilova, V. Savov, Study of free radical peroxidation processes In urine by enhanced chemiluminescence, annuaire de LEuniversite de sofia St. kliment ohridski, Faculte de Physique 95 (2002) 57–69. [45] E. Pavlova, V. Savov, Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton’s reaction in urine, Biochemistry (Moscow) 71 (8) (2006) 861–863. [46] M. Haidaria, M. Ali, S.W. Casscells III, M. Madjid, Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir, Phytomedicine 16 (2009) 1127–1136. [47] T. Osawa, M. Namiki, S. Kawakishi, Role of dietary antioxidants in protection against oxidative damage. antimutagenesis and anticarcinogenesis mechanisms II, Basic Life Sci. 52 (1990) 139–153. [48] J.P. Salminen, T. Roslin, M. Karonen, J. Sinkkonen, K. Pihlaja, P. Pulkkinen, Seasonal variation in the content of hydrolyzable tannins, flavonoid glycosides, and proanthocyanidins in oak leaves, J. Chem. Ecol. 30 (9) (2004) 1693–1711. [49] N. Uchide, H. Toyoda, Current status of monocyte differentiation-inducing (MDI) factors derived from human fetal membrane chorion cells undergoing apoptosis after influenza virus infection, Gene Regulation and Systems Biology 1 (2007) 295–302. [50] T. Akaike, M. Suga, H. Maeda, Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO, Proc. Soc. Exp. Biol. Med. 217 (1998) 64–73. [51] T. Oda, T. Akaike, T. Hamamoto, F. Suzuki, T. Hirano, H. Maeda, Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymerconjugated SOD, Science 244 (1989) 974–976. [52] R.W. Sidwell, J.H. Huffman, K.W. Bailey, M.H. Wong, A. Nimrod, A. Panet, Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice, Antimicrob. Agents Chemother. 40 (1996) 2626–2631. [53] J. Serkedjieva, I. Roeva, M. Angelova, P. Dolashka, W.G. Voelter, Combined protective effect of a fungal Cu/Zn-containing superoxide dismutase and rimantadine hydrochloride in experimental murine influenza a virus infection, Acta Virol. 47 (2003) 53–56. [54] T. Akaike, M. Ando, T. Oda, T. Doi, S. Ijiri, S. Araki, H. Maeda, Dependence on O2generation by xanthine oxidase of pathogenesis of influenza virus infection in mice, J. Clin. Inv. 85 (1990) 739–745. [55] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 2nd edn), Clarendon Press, Oxford, UK, 1989. [56] V. Murugan, K. Mukherjee, K. Maiti, P.K. Mukherjee, Enhanced oral bioavailability and antioxidant profile of ellagic acid by phospholipids, J. Agric. Food Chem. 57 (11) (2009) 4559–4565. [57] M. Kaplan, T. Hayek, A. Raz, R. Coleman, L. Dornfield, J. Vayan, et al., Pomegranate juice supplementation to atherosclerotic mice reduces macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis, J. Nutr. 131 (2001) 2082–2089.
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Comparative study on the antioxidant capacities of synthetic influenza inhibitors and ellagic acid in model systems Elitsa L. Pavlovaa,* , Nikolay N. Zografova , Lora S. Simeonovab a b
Biophysics & Medical Physics, Sofia University “St. Kliment Ohridski”, 5 James Boucher Blvd., 1164 Sofia, Bulgaria Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Georgi Bonchev Str., 1113 Sofia, Bulgaria
A R T I C L E I N F O
Article history: Received 19 May 2016 Received in revised form 9 July 2016 Accepted 21 July 2016 Nomenclature: Ellagic acid (PubChem CID 5281855) Vitamin E (PubChem CID 20353) Vitamin C (PubChem CID 54670067) Oseltamivir (PubChem CID 65028) Isoprinosine (PubChem CID 37510) Keywords: Oseltamivir Isoprinosine Ellagic acid Vitamin E Vitamin C Luminescence Constant of inhibition
A B S T R A C T
This study compares the antioxidant capacities in vitro of several synthetic and natural compounds applied and researched for influenza treatment – oseltamivir, isoprinosine, ellagic acid, vitamin E and vitamin C. Three chemical systems are utilized for the generation of reactive oxygen species (ROS) at pH 7.4 and pH 8.5: (1) Fenton’s (Fe2+ + H2O2) for OH and OH species (2) H2O2 (3) NADH phenazinemethosulfat, for superoxide radicals (O2 ). The kinetics was evaluated by lucigenin-enhanced chemiluminescence. The calculated constants of inhibition k7 describe the antioxidant capacity at the moment of oxidative burst. Their values do not necessarily correspond to the calculated total antioxidant activity. The obtained results revealed that the synthetic anti-influenza drugs (oseltamivir and isoprinosine) as well as ellagic acid possess pronounced scavenging properties mostly against superoxide radicals, comparable and higher than that of traditional natural antioxidants. Quantitative analysis of the antioxidant effects of the examined synthetic substances was performed. The results compared the corresponding effect of the average physiological concentrations and the applied therapeutic antioxidant dose. With these experiments we registered new aspects of their therapeutic activities, due to antioxidant properties against hydroxyl, superoxide radicals and H2O2 oxidation. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Influenza continues to be a major cause of morbidity and mortality despite the widespread access to vaccines and antivirals [1]. A definitive medical treatment for severe influenza-associated complications has not been established yet. It is suggested that a therapy with antiviral and antioxidant capacity could be a choice for decrease in the disease severity—both by its specific mode of action and by scavenging superoxide anion radicals, generated during influenza pathogenesis [2]. It is well established that the combination of antioxidants with specific antiviral drugs synergistically reduces the lethal effects of influenza virus infections [3–6]. Three substances with proved antioxidant effects (ellagic acid, vitamin E and vitamin C) and two traditional anti-influenza drugs used in clinics (oseltamivir and isoprinosine) were chosen to measure and compare their antioxidant capacities in vitro,
* *Corresponding author. E-mail address: [email protected] (E.L. Pavlova). http://dx.doi.org/10.1016/j.biopha.2016.07.046 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
expressed as constants of inhibition in different systems for the generation of physiologically active reactive oxygen species (ROS). The antioxidant potentials of the synthetic inhibitors had never been studied before. The selection was based on the fact that oseltamivir is currently the most recommended antiviral by the World Health Organization for etiologic treatment of the influenza disease and isoprinosine is a medicine with a broad spectrum of biological effects and is widely prescribed mostly due to its immunomodulation effect. Thus we found it challenging to investigate the potential antioxidant activities of those compounds and to test them for free radicals scavenging capacities in vitro. 1.1. Oseltamivir Oseltamivir is a neuraminidase inhibitor. As a structural analogue of the N-acetylneuraminic acid of the cellular glycoprotein receptors, it binds the viral neuraminidase and blocks the cleavage and release of influenza virus progeny from the surface of the infected cell in the late stages of the replication cycle. The active site of the enzyme is relatively conserved, which makes it active against A, B and C types of the influenza virus [7]. Oseltamivir is a potent influenza inhibitor
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both in vitro and in vivo [8–11]. Due to the increasing rates of resistance to the inhibitor, numerous studies estimated the advantages of the combined therapy of oseltamivir with drugs possessing other active mechanisms [12–14]. 1.2. Isoprinosine Isoprinosine is an inosine-salt complex approved in over 60 countries as an antiviral and an immunomodulator. It is a synthetic compound formed from the p-acetamido benzoate salt of N-N dimethylamino-2-propanol and inosine at a 3:1 molar ratio. It has a thymomimetic immunopharmacology that supports a pro-host action to increase cellular immune response. When applied in vitro, it induces T-cell differentiation and promotes proliferation, cytotoxicity and cytokine production. It also boosts macrophage and granulocyte functions. In vivo these multiple activities translate into enhanced cellular immune responses in infected or cancer-bearing individuals, following burns or in age-related immune-deficiencies [15–18]. Results of many studies indicated beneficial clinical effects of inosine pranobex against several viral diseases, including herpes, influenza, genital warts and type B viral hepatitis [19]. It has also showed beneficial protective effects when studied in combination with specific influenza antivirals (rimantadine) in animal models [20]. 1.3. Ellagic acid Ellagic acid is a polyphenol present in many fruits, such as grapes, strawberries, pomegranates and walnuts, as well as in several medicinal plants [21–23]. It has demonstrated antiviral and antimicrobial capacities, as well as antioxidant, anticancer, antiallergic and anti-inflammatory activities [22–25]. Researches suggest that it has antioxidant properties and inhibits a number of cell-signaling pathways that are important to tumor growth, including inflammatory signaling such as tumor necrosis factor a-induced cyclooxygenase-2 (COX-2) protein expression and nitric oxide synthase inhibition [26,27]. Commercial pomegranate juice shows potent antioxidant and anti-atherosclerotic properties attributed to its high content of polyphenols including ellagic acid in its free and bound forms and other flavonoids (quercetin, kaempferol and luteolin glycosides) [8,22,28–30]. It is proved to be an antioxidant as effective as or better than a-tocopherol or tertiary butylhydroxyanisole and shows inhibitory activity against lipid peroxidation [31,32]. Synergism is found between ellagic acid and current antimalarial drugs (chloroquine, artesunate, mefloquine, and atovaquone). This effect could enable drug-dose reduction during treatment and further limit side effects [31–33]. 1.4. Ascorbic acid Ascorbic acid is a naturally occurring organic compound with antioxidant properties. Its antioxidant effects are only a small part of its vitamin activity. The ascorbate ion is the predominant species at typical biological pH values. It is a mild reducing agent [34,35]. Often the free radicals initiate chain reactions. Ascorbate can terminate these chain radical reactions by electron transfer. The oxidized forms of ascorbate are relatively unreactive and do not cause cellular damage. However, being a good electron donor, the excessive ascorbate, in presence of free metal ions, cannot only promote but also initiate free radical reactions, thus making it a potentially dangerous pro-oxidative compound in certain metabolic contexts. A study of vitamin C in healthy people revealed a sigmoidal relationship between oral dose and plasma/tissue vitamin C concentrations. Hence, optimal dosage is critical to intervention studies using vitamin C [36,37]. Dehydroascorbic acid, the acid with no reducing ability, showed much stronger antiviral
activity than ascorbic acid, indicating that the antiviral activity is due to factors other than the antioxidant mechanism [37–39]. 1.5. Vitamin E Vitamin E is referred to a group of eight fat-soluble compounds that include both tocopherols and tocotrienols. a-tocopherol being the most biologically active form of vitamin E, is the second-most common form of vitamin E in the diet. As a fat-soluble antioxidant it stops the production of ROS formed when fat undergoes oxidation and fights mostly the lipid-oxide-radicals. Vitamin E has also been proved as a commercial antioxidant in ultra-high molecular weight polyethylene. It is known to protect lipids and prevents the oxidation of polyunsaturated fatty acids [40,41]. Vitamin E is not an agent with specific antiviral action but its antioxidant properties are moderating the course of the influenza infection in dose-dependent manner, due to its role as a membrane protector [41,42]. The contemporary approach for the treatment of influenza includes despite the vaccine and antiviral prophylaxis and therapy also the combination and additive effects of antioxidants fighting against the ROS that are accumulated from the infection and the disease itself. For these reasons, the focus of this in vitro research is on the comparison of the antioxidant capacity of oseltamivir, isoprinosine, ellagic acid, vitamin E and vitamin C against ROS, by lucigenin-enhanced chemiluminescence, as calculated constants of inhibition. 2. Materials and methods The kinetics of lucigenin-enhanced chemiluminescence was examined for three chemical systems designed for the generation of ROS. All three systems were tested at 25 C/pH 7.4 – physiological, and 25 C/pH 8.5 – for better differentiation of the chemiluminescent response. The total volume of each sample in the measuring cuvette is 2 ml, and the kinetics was registered for 3 min using chemiluminometer LKB 1251 (Sweden). The first seconds of fast flash luminescence are most informative for the inhibition effects. Each sample is prepared as a mixture, containing the buffer medium, chemiluminescent probe lucigenin, the chemical system for ROS-generation and the tested drug or combinations. The utilized in vitro buffer solutions were prepared from Na2HPO4 [0.05 M] and C8H8O7H2O [0.1 M]. The control systems contained all the presented reagents in the absence of an inhibitor ([InH]0 = 0) – the tested drug or combinations. Chemiluminescent systems for the generation of ROS (given final tested concentrations in a 2 ml sample volume): 1) Fenton’s reagent for the generation of OH and OH species (FeSO4 [5.104 M]-H2O2 [2,5.103 M]) It is well known that the interaction between Fe2+-ions and =2?2 results in highly reactive, short-living ?=-radicals: Fe2+ + H2O2 ! Fe3+ + OH + OH
(1)
Fe3+ + H2O2 ! Fe2+ + OOH + H+
(2)
2) H2O2 oxidation (H2O2 [2,5.103 M]) 3) reduced a-nicotinamide adenine dinucleotide (NADH) [104 M] – phenazinemethosulfat [104 M] – for the generation of superoxide radicals (O2):
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NAD.H + PMS + =+ ! PMS.=2 + NAD+.
(3)
PMS.=2 + PMS ! 2PMS.=
(4)
PMS.= +?2 ! PMS + ?2 + =+
(5) 4
The chemiluminescent probe lucigenin [10 M] is dissolved in DMSO. The tested substances were added as the following final concentrations, as water solutions: - ellagic acid (104 M – equal to the referent dose of vitamin C, accepted as effective antioxidant concentration or - 1,75.105 M, which is equal to 1/6 of the average physiological concentration of vitamin C) - vitamin E, applied as a-tocopherol succinate (2,1.104 M – physiological concentration or 1,75.105 M, which is 1/6 of the average physiological concentrations of vitamin C) - vitamin C (104 M, equal to the referent dose of vitamin C with established antioxidant activity or 1,29.103 M, which is the average physiological concentration) - oseltamivir (104 M, equal to the referent dose of vitamin C, but 750 times lower than the applied daily therapeutic dose) - isoprinosine (104 M, equal to the referent dose of vitamin C, but 1115 times lower than the applied daily therapeutic dose).
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2.2. Calculations The integral evaluation of the content of free radicals and ROS as well as the registration of the kinetic parameters of the oxidation reaction could be easily done by the lucigenin-enhanced chemiluminescent method. The constants of inhibition k7 can be calculated according to the kinetics of chemiluminescence, in correlation to the concentration of ROS [43–45]: ½ROS 2 ðI=I0 Þ ¼ ð6Þ ½ROS0 where I is the measured light intensity [mV], ½ROS0 is the initial concentration of the reactive oxygen species in the absence of inhibitors (t = 0). The constant of inhibition k7 ½l=ðmol:sÞcan be n o calculated from the slope of the linear part II0 ðtÞduring the first period of fast chemiluminescent emission, as follows: dðI=I0 Þ ¼ 2k7 ½InH0 dt
ð7Þ
where I0 is the registered light intensity [mV] at t = 0, and [InH]0 is the initial concentration of the inhibitor (Fig. 1). The accuracy of k7 is determined mainly by the accuracy of the light intensity measurements and less than [InH]0. For convenience, k7 can be expressed in ½l=ðg:sÞ, when taking into account the molecular mass of the inhibitor (Table 1). 2.3. Statistics
2.1. Reagents iron sulphate FeSO4 (Merck, Germany), phenazine methosulfate C13H11N2CH3SO4 (PMS) (N-methyldibenzopyrazine methyl sulfat salt) (Merck, Germany), hydrogen peroxide H2O2 (Boron, Bulgaria), disodium hydrogen phosphate Na2HPO4 (Boron, Bulgaria), citric acid C8H8O7H2O (Boron, Bulgaria), lucigenin C28H22N4O6 (bis-Nmethylacridinium nitrate) (Aldrich, USA), a – nicotinamide adenine dinucleotide, reduced form C21H27N7O14P2Na2 (NAD.=) (Boehringer, Germany), ellagic acid C14H6O8H2O (Alfa Aesar, England), vitamin E (a-tocopherol succinate, C33H54O5) (Aldrich, USA), vitamin C C6H8O6 (Boron, Bulgaria), oseltamivir C16H28N2O8P (F. Hoffmann-La Roche Ltd.), Isoprinosine C52H78N10O17 (Ewopharma AG, Switzerland), dimethyl sulfoxide C2H6OS (DMSO) (Aldrich, USA).
All measurements were made in triplicate, p 0.07, applying Origin 8.5 and Microsoft Office Excel 2010 for the analysis. All described work was carried out in accordance with the Uniform Requirements for manuscripts submitted to biomedical journals. 3. Results and discussion Three stages were defined in the kinetics of the chemiluminescent reaction for generation of ROS: spontaneous (0–20 s), fast flash (20–40 s) and latent period (50–200 s). The interaction between the researched substances and ROS, generated in the system, decreased sharply the chemiluminescent response in system 3, while the observed effects in system 1 and system 2 were mild.
Fig. 1. Fenton’s system for the generation of reactive oxygen species (OH, OH) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 7.4 (p 0.07).
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Table 1 Calculated constants of inhibition (k7) about ellagic acid, oseltamivir, isoprinosine and standard antioxidants (vit. C and vit. E), measured by chemiluminescence in vitro (p 0.07). tested substance
system
type
pH
K7 [l/mol s]
K7 [l/g s]
ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M] ellagic acid [104 M] ellagic acid [1,75.105 M] oseltamivir [104 M] isoprinosine [104 M] vit.C [104 M] vit.C [1,29.103 M] vit.E [2,1.104 M] vit.E [1,75.105 M]
Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s Fenton‘s H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS
hydroxyl radicals and hydroxide ions
8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4
3,104 12,809 1,708 3,142 1,810 0,179 1,011 10,278 6,750 32,000 4,536 4,470 3,028 0,176 2,057 25,220 2,620 17,38 0,225 2,110 2,817 0,1809 0,658 2,2143 3,983 20,903 2,839 11,7 13,2 0,316 0,493 23,431 60,800 448,571 85,100 77,750 82,300 3,620 37,762 466,571 27,800 157,428 26,750 29,200 18,800 0,806 11,119 134,857
1,027.102 4,238.102 0,442.102 0,282.102 1,027.102 0,102.102 0,190.102 1,936.102 2,234.102 10,589.102 1,174.102 0,400.102 1,719.102 0,099.102 0,387.102 4,751.102 0,867.102 5,751.102 0,058.102 0,189.102 1,599.102 0,103.102 0,124.102 0,417.102 1,318.102 6,917.102 0,735.102 1,049.102 7,494.102 0,179.102 0,093.102 4,414.102 20,119.102 148,435.102 22,022.102 6,972.102 46,727.102 2,055.102 7,114.102 87,901.102 9,199.102 52,094.102 6,922.102 2,618.102 10,674.102 0,458.102 2,094.102 25,407.102
H2O2 oxidation
superoxide radicals
Usually, higher antioxidant capacity corresponds to lower intensity of chemiluminescence. Best scavenging properties were demonstrated in system 3: all reagents suppressed the reaction (17–82%). Highest constants of inhibition presented ellagic acid, vit.E and vit.C, followed by oseltamivir and isoprinosine (Table 1) at both physiological and pH 8.5. Best scavenging effects towards the generated superoxide radicals exhibited the natural substances with proven antioxidant effect when applied in lower doses (Figs. 5 and 6, Table 1). That phenomenon is explained with the fact that vitamin C and vitamin E are free radical sources when applied at higher dosage, although these radicals are stable and not so aggressive. Some toxicological studies including also our unpublished data show that ellagic acid presents also prooxidant effects when applied at higher concentrations. Oseltamivir and isoprinosine exhibit very good scavenging effect against the superoxide radicals too (Figs. 5 and 6, Table 1). Further studies should clarify if those antioxidant activities are dose-dependent. Ellagic acid, vitamin E and vitamin C are effective antioxidants in the Fenton‘s system for both tested media (Figs. 1 and 2, Table 1).
The antioxidant effect of vitamin C is proportional, dosedependent. The antioxidant effect of vitamin E is inversed, dosedependent (Figs. 1 and 2, Table 1). But the physiological conditions provoke the inhibition features of oseltamivir against the generated hydroxyl radicals. Isoprinosine is an effective antioxidant in both media (Table 1). Against the oxidant H2O2, highest constant of inhibition is calculated about ellagic acid, vitamins C and E for both tested media (Figs. 3 and 4, Table 1). The antioxidant effect of vitamin C is again proportional, dose-dependent. The antioxidant effect of vitamin E is again inversed, dose-dependent (Figs. 3 and 4, Table 1). Very effective against that strong oxidant and ROS – H2O2, at physiological conditions, is also isoprinosine – much more effective than oseltamivir at both pH (Table 1). The calculated constants of inhibition describe the antioxidant capacity at the moment of oxidative burst. Their values do not always correspond to the calculated total antioxidant activity. The application of non-specific immune modifiers and antivirals plays an important role in current immunotherapy. We found that oseltamivir and isoprinosine show pronounced ROS-scavenging
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Fig. 2. Fenton’s system for the generation of reactive oxygen species (OH, OH) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 8.5 (p 0.07).
Fig. 3. H2O2-oxidation system and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 7.4 (p 0.07).
Fig. 4. H2O2-oxidation system and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 8.5 (p 0.07).
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Fig. 5. NAD.H-PMS system for the generation of superoxide radicals (O2 ) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 7.4 (p 0.07).
Fig. 6. NAD.H-PMS system for the generation of superoxide radicals (O2 ) and the effects of ellagic acid, vitamin E, vitamin C, oseltamivir and isoprinosine, evaluated by lucigenin-enhanced chemiluminescence, presented at pH 8.5 (p 0.07).
properties. These tests register for the first time new aspects of their therapeutic activities, due to antioxidant properties. Designed as a structural analogue of neuraminic acid, oseltamivir competitively binds the active site of the enzyme neuraminidase on the influenza virus surface. Thus the final steps of the viral replication cycle are blocked and the release of viral progeny from the host-cell is inhibited [8,10]. Further research and deeper biochemical analysis via interaction with biological membranes could reveal the precise mechanisms of antioxidant activities of oseltamivir. Inosine pranobex has demonstrated non-specific antiviral effect against several viral models. It acts as a powerful immunostimulant [15]. A potential advantage of isoprinosine is its effectiveness when administered orally and its therapeutic effects when administered after establishing the infection. Previous studies have shown that the combination of pomegranate polyphenol extract and oseltamivir synergistically increase the anti-influenza effect of oseltamivir. The pomegranate polyphenol extract inhibit the replication of human influenza A/ Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potentials especially for influenza epidemics and pandemics strains [46].
The dual antioxidant and pro-oxidant properties of ellagic acid might be interesting for a more profound research. Probably due to chelative and phenol activities, ellagic acid shows strong antioxidant effects (system 3). Its efficacy in the Fenton’s system is better than the antivirals and better or comparable to that of the standard antioxidants—vitamins C and E (Figs. 1 and 2). Ellagic acid can react to free radicals due to its ability to chelate with metal ions [47]. The four rings in its structure give the lipophilic property and the two lactones can act as hydrogen-forming and electron acceptors, respectively, that gives a hydrophilic characteristic [48]. Vitamin E acts as a peroxyl radical scavenger, preventing the propagation of free radicals in tissues by reacting to form a tocopheryl radical, which then is reduced by a hydrogen donor (such as vit. C) and returned to its reduced state. As it is fat-soluble, it is incorporated into cell membranes protecting them from oxidative damage [40]. Here, we have tested the water-soluble form of the vitamin, in order to compare on equal scale its effect in comparison to the other substances. Vit.C typically reacts with ROS, such as the hydroxyl radical. Ascorbic acid also scavenges superoxide anion radicals [34,35].
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Vitamin C exhibits very high total antioxidant capacity but low initial antioxidant inhibition speed, when applied in physiological concentrations. Vit.C presents best antioxidant effect in system 2 and system 3. The lower values obtained for both vitamins can be explained by their specific action mechanisms. Vit.E fights mostly against lipid-oxide-radicals. Vit.C exhibits very high total antioxidant capacity, but low initial antioxidant inhibition when applied in physiological concentrations. The pathology of severe influenza-associated complications results from complex biological phenomena such as apoptosis induction, macrophage activation, oxidative tissue damage and higher contents of pro-inflammatory cytokines [1,6]. The pathogenesis involves not only apoptotic cell death mediated through virus replication in the infected cells, but also the injury of noninfected cells by superoxide anion radicals derived from activated phagocytes infiltrated into the virus-infected organs [5,49]. These superoxide anion-mediated pathways are part of the immune mechanisms of extensive tissue injury observed during severe influenza-induced complications [4,50]. It should be noted, that the treatment with SODs decreased the lethal or toxic effect of influenza virus infection in mouse models but did not inhibit the virus proliferation [51–54]. In particular, oxygen species such as hydrogen peroxide, superoxide anion radicals, singlet oxygen and other radicals, are considered as agents attacking polyunsaturated fatty acid in cell membranes and giving rise to lipid peroxidation [6,55]. Many reports have suggested that lipid peroxidation may result in destabilization and disintegration of cell membranes leading to cell injury and the so-called “free radical” diseases and eventually to aging and susceptibility to cancer. However, healthy cell membranes do not undergo lipid peroxidation so severely in vivo, because of the extremely efficient protective mechanisms against damage caused by active oxygens and free radicals. Such systems include enzymatic inactivation by, for example, superoxide dismutase, glutathione-peroxidase and catalase, as well as nonenzymatic protection of polyunsaturated fatty acid by physiological and biological antioxidants such as vitamin E, vitamin C, ß-carotene, uric acid, etc. [50,55]. In general, antioxidants are compounds that can delay, inhibit or prevent the oxidation of compounds trapping free radicals and reducing the oxidative stress. For these reasons, the combination of antioxidants with anti-influenza drugs could definitely improve the conventional chemotherapy for severe influenza-associated complications [2,5,22,44,56,57] [data in vivo to be published]. The obtained results revealed that the synthetic anti-influenza drugs (oseltamivir and isoprinosine) as well as ellagic acid possess pronounced scavenging properties mostly against superoxide radicals, comparable and higher than that of traditional natural antioxidants (vitamins C and E). With these experiments we registered new aspects of their therapeutic activities, due to antioxidant properties against hydroxyl, superoxide radicals and H2O2 oxidation. Conflict of interest Each author declares no financial or commercial conflicts of interest. Acknowledgements This research is financially supported by the National Science Fund, Ministry of Education and Science, Republic of Bulgaria. Project Contract No: DFNI B01/19 “A Modern Alternative for the Prophylaxis and Treatment of Influenza Virus Infection –
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Multitarget Approaches with Highly Efficient Combinations of Antiviral Chemotherapeutics and Biologically Active Compounds” References [1] T.M. Tumpey, A. García-Sastre, J.K. Taubenberger, P. Palese, D.E. Swayne, M.J. Pantin-Jackwood, S. Schultz-Cherry, A. Solórzano, N. Van Rooijen, J.M. Katz, C.F. Basler, Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice, J. Virol. 79 (2005) 14933–14944. [2] N. Uchide, H. Toyoda, Potential of selected antioxidants for influenza chemotherapy, Anti Infect. Agents Med. Chem. 7 (2008) 73–83. [3] N. Uchide, H. Toyoda, Future target molecules for influenza treatment, Mini Rev. Med. Chem. 8 (2008) 491–495. [4] B. Halliwell, Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 344 (8924) (1994) 721–724. [5] E. Peterhans, Reactive oxygen species and nitric oxide in viral diseases, Biol. Trace Element Res. 56 (1997) 107–115. [6] H. Siess, Oxidative Stress, Academic Press, London, 1985. [7] A. Moscona, Neuraminidase inhibitors for influenza, N. Engl. J. Med. 353 (2005) 1363–1373. [8] S.U. Kim, W. Lew, M.A. Williams, H. Liu, L. Zhang, S. Swaminathan, N. Bischofberger, M.S. Chen, D.B. Mendel, C.Y. Tai, W.G. Laver, R.S. Stevens, Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity, J. Am. Chem. Soc. 119 (1997) 681–690. [9] W. Lew, X. Chen, C.U. Kim, Discovery and development of GS4104 (oseltamivir): an orally active influenza neuraminidase inhibitor, Curr. Med. Chem. 7 (2000) 663–672. [10] D.B. Mendel, R.G., Webster, N.A. Roberts, Inhibition of avian influenza neuraminidases by GS4071 (Ro 64-0802) in vitro, Roche Research Report W-143039 (1999). [11] R.W. Sidwell, J.H. Huffman, D.L. Barnard, K.W. Bailey, M.N. Wong, A. Morrison, T. Syndergaard, C.U. Kim, Inhibition of influenza virus infections in mice by GS4104, an orally effective influenza virus neuraminidase inhibitor, Antivir. Res. 37 (1998) 107–120. [12] N.A. Ilyushina, E. Hoffman, R. Salomon, R.J. Webster, E.A. Govorkova, Amantadine-oseltamivir combination therapy for H5N1 influenza virus in mice, Antivir. Ther. 12 (3) (2007) 363–370. [13] D.F. Smee, M.H. Wong, K.W. Bailey, R.W. Sidwell, Activities of oseltamivir and ribavirin used alone and in combination against infections in mice with recent isolates of influenza A (H1N1) and B viruses, Antivir. Chem. Chemother. 17 (4) (2006) 185–192. [14] A.S. Galabov, L. Simeonova, G. Gegova, Rimantadine and oseltamivir demonstrate synergistic combination effect in an experimental infection with type A (H3N2) influenza virus in mice, Antivir. Chem. Chemother. 17 (4) (2006) 251–258. [15] D.M. Campoli-Richards, E.M. Sorkin, R.C. Heel, Inosine Pranobex. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy, Drugs 32 (1986) 383–424. [16] A. Marton, P. Pacher, K.G. Murthy, Z.H. Nemeth, G. Hasko, C. Szabo, Antiinflammatory effects of inosine in human monocytes, neutrophilis and epithelial cells in vitro, Int. J. Mol. Med. 8 (2001) 617–621. [17] S. Milano, M. Dieli, S. Millott, M.D. Miceli, E. Maltese, E. Cillari, Effect of isoprinosine on IL-2, IFN-gamma and IL-4 production in vivo and in vitro, Int. J. Immunopharmacol. 13 (1991) 1013–1018. [18] R.W. Flo, A. Naess, G. Albrektsen, C.O. Solberg, Isoprinosine stimulates granulocyte chemiluminescence and inhibits monocyte chemiluminescence in vitro, APMIS 102 (1994) 249–254. [19] J. Davidson-Parker, W. Dinsmore, M.H. Khan, D.A. Hicks, C.A. Morris, D.F. Morris, Immunotherapy of genital warts with inosine pranobex and conventional treatment: double blind placebo controlled study, Genitourin. Med. 64 (6) (1988) 383–386. [20] S. Pancheva, G. Gegova, V. Maximova, N. Manolova, The anti-influenza effect of rimantadine and isoprinosine when used in combination in mice, Acta Microbiol. Bulg. 26 (1990) 26–29. [21] I. Barta, P. Smerak, Z. Polivknova, et al., Current trends and perspectives in nutrition and cancer prevention, Neoplasma 53 (1) (2006) 19–25. [22] B. Cerdá, F.A. Tomás-Barberán, J.C. Espín, Metabolism of antioxidant and chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged wine in humans: identification of biomarkers and individual variability, J. Agric. Food Chem. 53 (2) (2005) 227–235. [23] A.P. Rogerio, C. Fontanari, E. Borducchietal, Anti-inflammatory effects of Lafoensia pacariandellagicacidina murine model of asthma, Eur. J. Pharmacol. 580 (1–2) (2008) 262–270. [24] C. Bell, S. Hawthorne, Ellagic acid, pomegranate and prostate cancer—a mini review, J. Pharm. Pharmacol. 60 (2) (2008) 139–144. [25] Y.H. Choi, G.H. Yan, Ellagic acid attenuates immunoglobulin e-mediated allergic response in mast cells, Biol. Pharm. Bull. 32 (6) (2009) 1118–1121. [26] J. Gainok, R. Daniels, D. Golembiowski, P. Kindred, L. Post, R. Strickland, N. Garrett, Investigation of the anti-inflammatory, antinociceptive effect of ellagic acid as measured by digital paw pressure via the Randall-Selitto meter in male SpragueDawley rats, AANA J. 79 (4) (2011) 28–34.
762
E.L. Pavlova et al. / Biomedicine & Pharmacotherapy 83 (2016) 755–762
[27] B. Doyle, L.A. Griffiths, The metabolism of ellagic acid in the rat, Xenobiotica 10 (1980) 247–256. [28] M. Albrecht, W. Jiang, J. Kumi-Diaka, E.P. Lansky, L.M. Gommersall, R.E. Mansel, et al., Pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells, J. Med. Food 7 (2004) 274–283. [29] K.L. Khanduja, R.K. Gandhi, V. Pathania, N. Syanl, Prevention of Nnitrosodiethylamine-induced lung tumorigenesis by ellagic acid and quercetin in mice, Food Chem. Toxicol. 37 (1999) 31–38. [30] M. Toi, H. Bando, C. Ramachandran, S.J. Melnick, A. Imai, R.S. Fife, et al., Preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo, Angiogenesis 6 (2003) 121–128. [31] R.K.Y. Zee-Cheng, C.C.J. Chang, Ellagic acid, Drugs Future 11 (1986) 1029–1033. [32] P. Njomnang Soh, B. Witkowski, D. Olagnier, M. Nicolau, M. Garcia-Alvarez, A. Berry, F. Benoit-Vical, In vitro and In vivo properties of ellagic acid in malaria treatment, Antimicrob. Agents Chemother. 53 (3) (2009) 1100–1106. [33] Y. Tsujimoto, H. Hashizume, M. Yamazaki, Superoxide radical scavenging activity of phenolic compounds, Int. J. Biochem. 25 (1993) 491–494. [34] http://en.wikipedia.org/wiki/Ascorbic_acid. [35] S.J. Padayatty, A. Katz, Y. Wang, P. Eck, O. Kwon, J.H. Lee, S. Chen, C. Corpe, A. Dutta, S.K. Dutta, M. Levine, Vitamin C as an antioxidant: evaluation of its role in disease prevention, J. Am. Coll. Nutr. 22 (1) (2003) 18–35. [36] R.J. Jariwalla, M.W. Roomi, B. Gangapurkar, T. Kalinovsky, A. Niedzwiecki, M. Rath, Suppression of influenza A virus nuclear antigen productionand neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extractand amino acids, Biofactors 31 (2007) 1–15. [37] A. Furuya, M. Uozaki, H. Yamasaki, T. Arakawa, M. Arita, A.H. Koyama, Antiviral effects of ascorbic and dehydroascorbic acids in vitro, Int. J. Mol. Med. 22 (2008) 541–545. [38] D. Banerjee, D. Kaul, Combined inhalational and oral supplementation of ascorbic acid may prevent influenza pandemic emergency: a hypothesis, Nutrition 26 (2010) 128–132. [39] N. Uchide, H. Toyoda, Antioxidant therapy as a potential approach to severe influenza-Associated complications, review, Molecules 16 (2011) 2032–2052. [40] http://en.wikipedia.org/wiki/Vitamin_E. [41] M. Mileva, R. Bakalova, L. Tancheva, A.S. Galabov, S. Ribarov, Effect of vitamin E supplementation on lipid peroxidation in blood and lung of influenza virus infected mice, Comp. Immunol. Microbiol. Infect. Dis. 25 (1) (2002) 1–11. [42] M. Mileva, L. Tancheva, R. Bakalova, A.S. Galabov, V.M. Savov, S. Ribarov, Effect of vitamin E on lipid peroxidation and liver monooxigenase activity in experimental influenza virus infection, Toxicol. Lett. 114 (1–3) (2000) 39–45. [43] I. Rusina, On the absolute values of constants of speed of interoperability of the inhibitors with peroxide radicals, Izvestiya Akademii Nauk SSSR 224 (3) (1975) 646–649 (in Russian).
[44] E. Pavlova, M. Lilova, V. Savov, Study of free radical peroxidation processes In urine by enhanced chemiluminescence, annuaire de LEuniversite de sofia St. kliment ohridski, Faculte de Physique 95 (2002) 57–69. [45] E. Pavlova, V. Savov, Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton’s reaction in urine, Biochemistry (Moscow) 71 (8) (2006) 861–863. [46] M. Haidaria, M. Ali, S.W. Casscells III, M. Madjid, Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir, Phytomedicine 16 (2009) 1127–1136. [47] T. Osawa, M. Namiki, S. Kawakishi, Role of dietary antioxidants in protection against oxidative damage. antimutagenesis and anticarcinogenesis mechanisms II, Basic Life Sci. 52 (1990) 139–153. [48] J.P. Salminen, T. Roslin, M. Karonen, J. Sinkkonen, K. Pihlaja, P. Pulkkinen, Seasonal variation in the content of hydrolyzable tannins, flavonoid glycosides, and proanthocyanidins in oak leaves, J. Chem. Ecol. 30 (9) (2004) 1693–1711. [49] N. Uchide, H. Toyoda, Current status of monocyte differentiation-inducing (MDI) factors derived from human fetal membrane chorion cells undergoing apoptosis after influenza virus infection, Gene Regulation and Systems Biology 1 (2007) 295–302. [50] T. Akaike, M. Suga, H. Maeda, Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO, Proc. Soc. Exp. Biol. Med. 217 (1998) 64–73. [51] T. Oda, T. Akaike, T. Hamamoto, F. Suzuki, T. Hirano, H. Maeda, Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymerconjugated SOD, Science 244 (1989) 974–976. [52] R.W. Sidwell, J.H. Huffman, K.W. Bailey, M.H. Wong, A. Nimrod, A. Panet, Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice, Antimicrob. Agents Chemother. 40 (1996) 2626–2631. [53] J. Serkedjieva, I. Roeva, M. Angelova, P. Dolashka, W.G. Voelter, Combined protective effect of a fungal Cu/Zn-containing superoxide dismutase and rimantadine hydrochloride in experimental murine influenza a virus infection, Acta Virol. 47 (2003) 53–56. [54] T. Akaike, M. Ando, T. Oda, T. Doi, S. Ijiri, S. Araki, H. Maeda, Dependence on O2generation by xanthine oxidase of pathogenesis of influenza virus infection in mice, J. Clin. Inv. 85 (1990) 739–745. [55] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 2nd edn), Clarendon Press, Oxford, UK, 1989. [56] V. Murugan, K. Mukherjee, K. Maiti, P.K. Mukherjee, Enhanced oral bioavailability and antioxidant profile of ellagic acid by phospholipids, J. Agric. Food Chem. 57 (11) (2009) 4559–4565. [57] M. Kaplan, T. Hayek, A. Raz, R. Coleman, L. Dornfield, J. Vayan, et al., Pomegranate juice supplementation to atherosclerotic mice reduces macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis, J. Nutr. 131 (2001) 2082–2089.