Journal of Controlled Release 113 (2006) 189 – 207 www.elsevier.com/locate/jconrel

Review

Role of antioxidants in prophylaxis and therapy: A pharmaceutical perspective
D. Venkat Ratnam, D.D. Ankola, V. Bhardwaj, D.K. Sahana, M.N.V. Ravi Kumar ⁎
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Phase-X, S.A.S. Nagar, Mohali, Punjab, India - 160062 Received 31 January 2006; accepted 26 April 2006 Available online 13 May 2006

Abstract Antioxidants are emerging as prophylactic and therapeutic agents. These are the agents, which scavenge free radicals otherwise reactive oxygen species and prevent the damage caused by them. Free radicals have been associated with pathogenesis of various disorders like cancer, diabetes, cardiovascular diseases, autoimmune diseases, neurodegenerative disorders and are implicated in aging. Several antioxidants like SOD, CAT, epigallocatechin-3-O-gallate, lycopene, ellagic acid, coenzyme Q10, indole-3-carbinol, genistein, quercetin, vitamin C and vitamin E have been found to be pharmacologically active as prophylactic and therapeutic agents for above mentioned diseases. Antioxidants are part of diet but their bioavailability through dietary supplementation depends on several factors. This major drawback of dietary agents may be due to one or many of the several factors like poor solubility, inefficient permeability, instability due to storage of food, first pass effect and GI degradation. Conventional dosage forms may not result in efficient formulation owing to their poor biopharmaceutical properties. Principles of novel drug delivery systems need to be applied to significantly improve the performance of antioxidants. Novel drug delivery systems (NDDS) would also help in delivery of these antioxidants by oral route, as this route is of prime importance when antioxidants are intended for prophylactic purpose. Implication of NDDS for the delivery of antioxidants is largely governed by physicochemical characteristics, biopharmaceutical properties and pharmacokinetic parameters of the antioxidant to be formulated. Recently, chemical modifications, coupling agents, liposomes, microparticles, nanoparticles and gel-based systems have been explored for the delivery of these difficult to deliver molecules. Results from several studies conducted across the globe are positive and provided us with new anticipation for the improvement of human healthcare. © 2006 Elsevier B.V. All rights reserved.
Keywords: Antioxidants; Biopharmaceutics; Controlled delivery; Intestinal permeability; Peroral; Pharmacokinetics; Solubility; Targeted delivery

Contents 1. 2. Introduction . . . . . . . . . . . . . . . Antioxidants . . . . . . . . . . . . . . 2.1. Definition and importance . . . . 2.2. Classification of antioxidants . . Role of free radicals and antioxidants in Enzymatic antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . various diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 191 191 192 192 193

3. 4.

Abbreviations: AP, ascorbyl palmitate; CAT, catalase; DCFH-DA, dichlorodihydrofluorescein diacetate; CD, cyclodextrin; CoQ10, coenzyme Q10; CS, chitosan; DNA, deoxyrebose nucleic acid; DMAB, didodecyldimethyl ammonium bromide; DSS, dextran sulfate sodium; EA, ellagic acid; EGCG, epigallocatechin-3-Ogallate; GIT, gastrointestinal tract; GALT, gut associated lymphoid tissue; GPx, glutathione peroxidase; ICAM, intracellular adhesion molecule; IDE, idebenone; I3C, indole-3-carbinol; LAA, lipoamino acid; LAT, liposomal α-tocopherol; NAC, N-acetylcysteine; NDDS, novel drug delivery systems; PECAM, platelet endothelial cell adhesion molecule; PEG, polyethylene glycol; PLGA, poly lactide-co-glycolic acid; PMMA, poly methyl methacrylate; PNC, polymeric nanocarriers; PVA, polyvinyl alcohol; ROS, reactive oxygen species; SEDDS, self-emulsifying drug delivery systems; SLN, solid lipid nanoparticle; SOD, superoxide dismutase; UV, ultraviolet radiation. ⁎ Corresponding author. Tel.: +91 172 2214683 89 2055; fax: +91 172 2214692. E-mail addresses: mnvrkumar@niper.ac.in, mnvrkumar@yahoo.com (M.N.V.R. Kumar). 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.04.015

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Non-enzymatic antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Antioxidants in diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Epidemiology of antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Necessity and superiority of formulated antioxidants over dietary antioxidants 5.4. Physicochemical and biopharmaceutical properties. . . . . . . . . . . . . . . 5.4.1. Dose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2. Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3. Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4. Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.5. Bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Pharmacokinetic parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1. Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2. Distribution, storage and binding . . . . . . . . . . . . . . . . . . . 5.5.3. Metabolism and excretion . . . . . . . . . . . . . . . . . . . . . . . 6. Delivery approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Conventional delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Inclusion complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Chemical modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Novel drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1. Self-emulsifying drug delivery systems (SEDDS). . . . . . . . . . . 6.4.2. Liposomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.3. Microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.4. Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.5. Gel-based systems. . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.6. Other modes of antioxidant delivery . . . . . . . . . . . . . . . . . 6.5. Targeting approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Novel methods to treat oxidative stress . . . . . . . . . . . . . . . . . . . . 7. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction In recent years, antioxidants have gained a lot of importance because of their potential as prophylactic and therapeutic agents in many diseases. The discovery of the role of free radicals in cancer, diabetes, cardiovascular diseases, autoimmune diseases, neurodegenerative disorders, aging and other diseases has led to a medical revolution that is promising a new paradigm of healthcare. Although not many antioxidants are listed in pharmacopoeias, extensive research is being carried out globally on these agents, and most of them have been proven pharmacologically active. Traditionally, herbal medicines with antioxidant properties have been used for various purposes and epidemiological data also points at widespread acceptance and use of these agents. Presently, the active constituents from these herbal sources are extracted, purified and tested for their activities. Results are promising their benefits in prevention and therapy in many of the aforesaid diseases. The global market of antioxidants is increasing rapidly, because of the increased health risk in a constantly polluting environment. These agents also have cosmetic applications, further fuelling research by industry and academia to explore these molecules and their analogues. Free radicals are highly reactive molecules or chemical species containing unpaired electrons that cause oxidative stress, which is defined as “an imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to

damage” [1]. Oxidative stress can damage lipids, proteins, enzymes, carbohydrates and DNA in cells and tissues, resulting in membrane damage, fragmentation or random cross linking of molecules like DNA, enzymes and structural proteins and even lead to cell death induced by DNA fragmentation and lipid peroxidation [2]. These consequences of oxidative stress construct the molecular basis in the development of cancer, neurodegenerative disorders, cardiovascular diseases, diabetes and autoimmune disorders. Human antioxidant defense is equipped with enzymatic scavengers like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase; hydrophilic scavengers like urate, ascorbate, glutathione and flavonoids; lipophilic radical scavengers such as tocopherols, carotenoids and ubiquinol. The defense also comprises enzymes involved in the reduction of oxidized forms of molecular antioxidants like glutathione reductase, dehydroascorbate reductase. Apart from these scavengers, there exists cellular machinery, which maintains a reducing environment, for example regeneration of NADPH by glucose-6-phosphate dehydrogenase. Some of these agents synthesized by cell itself; however, majority including ascorbic acid, lipoic acid, polyphenols and carotenoids are derived from dietary sources. In disease conditions, the defense against ROS is weakened or damaged and the oxidant load increases. In such conditions, external supply of antioxidants is essential to countervail the deleterious consequences of oxidative stress.

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Epigallocatechin-3-O-gallate (EGCG), lycopene, quercetin, genistein, ellagic acid, ubiquinone and indole-3-carbinol are among the major antioxidants apart from the well known antioxidant vitamins ascorbic acid and α-tocopherol. These agents are used as nutritional supplements for prophylaxis or therapy of certain diseases along with the mainstream therapy. However, delivery of these antioxidants using the conventional dosage forms is a challenge due to various reasons like poor solubility, poor permeability, instability and extensive first pass metabolism before reaching systemic circulation. Need of the antioxidants which can penetrate the blood brain barrier has been recognized in some neurodegenerative diseases [3]. Similarly in cancer, the drug targeting to tumors will be beneficial in order to decrease the body burden. More recently, the focus has shifted towards novel drug delivery systems in delivering such difficult to deliver molecules, which would enable development of highly efficient formulations with good patient compliance. Novel drug delivery systems (NDDS) have had an enormous impact on medical technology, significantly improving the performance of drugs in terms of efficacy, safety and patient compliance. NDDS can greatly improve the delivery of drugs which are poorly bioavailable due to their unfavorable physicochemical or pharmacokinetic parameters. NDDS apart from improving the bioavailability of the drug candidates are known for better targeting abilities consequently lowering the

required dose considerably. A molecule with ideal solubility and permeability profile can be administered with a minimum effective dose provided there is no presystemic loss due to metabolism or GI degradation; however, this is not likely with most of these antioxidants. The acceptance of these molecules as prophylactic agents can be increased by reducing the frequency of administration and preferably through oral route. Antioxidants are molecules with multifunctional activities in various diseases, unlike the drugs in current use which serve for specific disease. Therefore, considering the therapeutic potential of the antioxidants, there is every need to implicate novel drug delivery technologies to improve their performance. The present review is an attempt to bring the pharmaceutical issues related to antioxidants into limelight. Focus is on the physicochemical and biopharmaceutical aspects of these compounds, problems associated with their delivery and approaches under evolution to improve their bioavailability and efficacy. 2. Antioxidants 2.1. Definition and importance Antioxidants are substances which counteract free radicals and prevent the damage caused by them. These can greatly reduce the adverse damage due to oxidants by crumbling them

Fig. 1. Classification of antioxidants. Some non-enzymatic antioxidants like uric acid, vitamin E, glutathione and CoQ10 are synthesized in the human body and they can also be derived from dietary sources. Polyphenols are the major class of antioxidants which are derived from diet.

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before they react with biologic targets, preventing chain reactions or preventing the activation of oxygen to highly reactive products [4]. Except for anaerobes, oxygen is vital for all the living systems. However, the paradox of aerobic life is that oxidative damage occurs at the key biological sites, threatening their structure and function. Oxygenic threat is met by an array of antioxidants that evolved in parallel with our oxygenic atmosphere [5]. Our body implements various antioxidants, some of which are dietary-derived antioxidants to help restrain potential free radical damage that could occur in our bodies. If one looks back into the evolution of human diet, it can be observed that in the Paleolithic age human intake of plant-derived antioxidants is considered to have been many times higher than current intake [6]. Organized agriculture has begun approximately 12,000 years ago, and stimulated a pace of dietary and social changes that deprived our biological ability to adapt to the rapid changes in environment. This has led to the hypothesis that various common diseases of civilization are rooted in a chronic divergence between our ancient nutritional programming and our contemporary dietary input [7,8]. A key disparity between dietary supply and physiological need may be in antioxidant nutrients. Adding to this, in modern day environment, people are exposed to a variety of toxins, which can be potent oxidants. If one combines the increasing environmental pressure of oxidant damage with our unbalanced contemporary food supply, the value of antioxidant supplementation becomes apparent. These agents should possess the characteristics like good bioavailability, stability and selectivity to the damaged or transformed cells. Formulating antioxidants is particularly interesting because of their relative lack of toxicity, preventive and therapeutic roles in diverse diseases, and encouraging evidences from epidemiology. 2.2. Classification of antioxidants Antioxidants can be classified into two major groups, i.e., enzymatic and non-enzymatic antioxidants. Some of these antioxidants are endogenously produced which include enzymes, low molecular weight molecules and enzyme cofactors. Among non-enzymatic antioxidants, many are obtained form dietary sources. Dietary antioxidants can be classified into various classes [9], of which polyphenols is the largest class. Polyphenols consist of phenolic acids and flavonoids. The other classes of dietary antioxidants include vitamins, carotenoids, organosulfural compounds and minerals (Fig. 1). 3. Role of free radicals and antioxidants in various diseases Oxidative stress is initiated by ROS such as superoxide anion and hydrogen peroxide. Neither of these ROS is a strong oxidant, but they can be converted into more dangerous oxidants by harmful reactions in tissues [10]. Superoxide can be produced from molecular oxygen by diverse cell types via enzymatic systems including the respiratory chain, xanthine oxidase, cyclo-oxigenase and NADPH-oxidase. It rapidly dismutates into H2O2, either

spontaneously or enzymatically, but if superoxide collides with nitric oxide the formation of peroxynitrite takes place [11]. H2O2 is formed as a product of superoxide dismutation, although some enzymes like monoamine oxidase can produce H2O2 directly from their substrates. Fenton or Haber-Weiss reactions catalysed by transition metals like iron convert H2O2 into extremely strong hydroxyl radicals, while myeloperoxidase produces hypochlorus acid from H2O2. These radicals attack sensitive cellular targets like lipids, proteins and nucleic acids causing their inhibition and accelerated degradation. Thus, oxidative stress inflicts multiple levels of cellular damage, which propagates a vicious cycle. Oxidation of phospholipids and fatty acids produces reactive lipid peroxides, which in turn initiate chain reaction of lipids peroxidation in cellular membranes [12]. These consequences of oxidative stress construct the molecular basis in the development of many diseases. The role of free radicals in various diseases is highlighted in Table 1; on the other hand, the antioxidants that were tested and evaluated for the treatment of oxidative stress are presented in Table 2. Many studies revealed that these antioxidants have moderate efficiency in combating free radical damage without any adverse effects. The reasons could be pharmacokinetic or stability-related loss of potency of the drug. These issues have

Table 1 Role of free radicals in various diseases Diseases Atherosclerosis Myocardial infarction Hypertension Role of free radicals in pathophysiology Superoxide-mediated endothelial dysfunction, activation of macrophages ROS driven ischemic reperfusion injury and myocyte necrosis and/or apoptosis ROS-mediated vascular smooth muscle cell proliferation, oxidant production via NADH/NADPH oxidase and endothelial dysfunction ROS accelerated formation of advanced glycation end products (AGEs) Superoxide-mediated endothelial dysfunction Cell damage and metabolic abnormalities ROS-mediated gene mutations (modification of pyridine and purine bases) and post-translational modifications leading disruption of cellular processes ROS-mediated mitochondrial dysfunction Amyloid peptide and advanced glycation end products ROS-mediated neurotoxicity to hippocampal cells and the synaptosomal membranes ROS-mediated transcriptional dysregulation and mitochondrial impairment ROS-mediated inflammation and tissue destruction Photochemical reactions in the oxygen-rich environment of the outer retina lead to the liberation of cytotoxic (ROS) ROS-mediated inflammation and endothelial dysfunction

Diabetes

Aging Cancer

Parkinson's disease Alzheimer's disease

Huntington's disease Autoimmune disorders Age-related macular degeneration Acute lung injury, acute respiratory distress syndrome, inflammation and hyperoxia

D.V. Ratnam et al. / Journal of Controlled Release 113 (2006) 189–207 Table 2 Some selected antioxidants and their mechanisms of action Antioxidant SOD CAT NAC GSH EGCG Mechanism of action Dismutation of superoxide to H2O2 Decomposes H2O2 to molecular oxygen and water Scavenging of H2O2 and peroxide Deacetylation of precursor for GSH synthesis Intracellular reducing agent Metal chelation Scavenging of superoxide, H2O2, OH and singlet oxygen Tocopherol regeneration Trapping of singlet oxygen Scavenging of H2O2 Stimulation of glutathione-S-transferase Inhibition of lipid peroxidation Reduces mitochondrial oxidative stress Inhibition of DNA-carcinogen adduct formation Suppression of free radical production H2O2 scavenging H2O2 scavenging, one of the potent antioxidant among polyphenols Scavenging of superoxide anion by forming semidehydroascorbate radical which is subsequently reduced by GSH Direct scavenging of superoxide Upregulation of antioxidant enzymes Inhibition of lipid peroxidation

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Lycopene Ellagic acid CoQ10 I3C Genistein Quercetin Vitamin C

Vitamin E

SOD = superoxide dismutase, CAT = catalase, NAC = N-acetyl cysteine, GSH = glutathione, EGCG = epigallocatechin-3-O-gallate, CoQ10 = coenzyme Q10, I3C = indole-3-carbinol.

been described for some selected antioxidants in Sections 4 and 5.4. 4. Enzymatic antioxidants

mitochondria are the major producers and also the main targets of ROS. Accumulated data indicate that an excess production of ROS and free radicals in mitochondria lead to elevated expression of Mn-SOD. The induction of Mn-SOD gene expression under oxidative stress may be one of the selfdefense mechanisms to alleviate oxidative damage to mitochondria. To cope with the oxidative stress elicited by aging or mitochondrial disease, an increase in Mn-SOD must be accompanied by concurrent increase in CAT and/or glutathione peroxidase (GPx) to prevent excessive buildup of H2O2. The imbalance between the production and removal of ROS leads to an elevation of oxidative stress in mitochondria of elderly subjects and patients with mitochondrial diseases. Accumulation of ROS induces mitochondrial permeability transition and disrupts the mitochondrial membrane potential, thereby triggering cells to undergo apoptosis or necrosis. Studies in intact animals and in humans revealed that SOD and CAT afford only modest benefit being potent antioxidants. The potentials of these agents are yet to be developed into effective, reliable and safe antioxidant therapies. The discouraging results of animal and clinical studies can be attributed, at least in part, to unfavorable pharmacokinetic profiles and inadequate delivery of SOD and CAT. For example, the enzyme CAT needs to be delivered to sites where the level of hydrogen peroxide increases in the vicinity of metastasizing tumor cells in order to achieve CAT-based inhibition/prevention of tumor metastasis [15]. Both of these enzymes are poorly absorbed from and rapidly degraded in the GIT. SOD and CAT have extremely short life spans in the blood stream after intravenous administration [16,17]. Hepatic uptake and renal excretion are the major pathways for the elimination of antioxidant enzymes. 5. Non-enzymatic antioxidants

Enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), etc. Antioxidant enzymes, SOD and CAT, are not consumed and have high affinity and rate of reaction with ROS. Therefore, it may be hypothesized that the enzymes afford more effective protection against acute massive oxidative insults, such as hyperoxia or inflammation. Antioxidant enzymes are more potential agents in treating severe acute insults due to oxidative stress [13]. SOD and CAT are among the most potent antioxidants known in nature. There are three types of SODs in humans namely cytosolic CuZn-SOD, mitochondrial MnSOD and extracellular SOD. CAT occurs abundantly in the body, with the highest activity in the liver, followed by erythrocytes, then the lungs. SOD catalyzes dismutation of superoxide into oxygen and hydrogen peroxide and it is widespread in nature in eukaryotic and prokaryotic organisms [14]. CAT protects cells by catalyzing hydrogen peroxide decomposition into molecular oxygen and water with no free radical production. In addition, CAT acts on toxic compounds such as phenols, formic acid, formaldehyde and alcohols by peroxidative reaction. These free radical scavenging enzymes have been found to change qualitatively and quantitatively in various tissues and cells of patients with mitochondrial diseases and elderly subjects. It is now well established that the

5.1. Antioxidants in diet The human diet has evolved over years. Evolution of human diet reveals that the modern day intake of antioxidants is far less from our ancients. The organized agriculture which had instigated some thousands of years ago started depriving us from the antioxidant-rich diet constantly [5]. Human antioxidant defense system is incomplete without dietary antioxidants. At some point in the evolution process, uric acid replaced the ascorbic acid as the major water-soluble antioxidant in human biological fluids. Humans lack the ability to synthesize ascorbic acid endogenously, for which we have an absolute requirement [18]. The ascorbic acid needs can be met only by the dietary sources. Apart from ascorbic acid other antioxidants like vitamin E, CoQ10, carotenoids and polyphenols are obtained from external sources and play an important role in maintaining human health. It has been estimated that more than two-thirds of human cancers, which are contributed by mutations in multiple genes, could be prevented by modification of lifestyle including dietary modification. The supposed mechanisms for prophylaxis may include enhanced enzymatic detoxification of harmful compounds and inhibition of their binding to cellular DNA, their adsorption on

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fiber content and detoxification of radical forms of carcinogens by natural antioxidants when administered as dietary agents. 5.2. Epidemiology of antioxidants Epidemiological studies are the main contributors in enlightening the potentials of these agents in prophylaxis and therapy. Epidemiological studies have indicated a significant difference in the incidence of various diseases among ethnic groups, who have different lifestyles and have been exposed to different environmental factors. The well known French paradox and Spanish Mediterranean diet are better examples to cite for the epidemiological evidences which have proven the efficiency of antioxidants in prevention of diseases. French paradox is about the low cardiovascular mortality in spite of high-fat intake, in the French population, because of antioxidative properties of red wine which is consumed by them [19–22]. Spanish Mediterranean diet is also rich in antioxidant content, which results in the protection from heart diseases. Many of the benefits derived from intake of such diets may be the result of synergism between natural antioxidants and the better known vitamin antioxidants [23,24]. 5.3. Necessity and superiority of formulated antioxidants over dietary antioxidants Efficiency of dietary antioxidants is well described; however, the oral bioavailability issues remain unaddressed. The dietary antioxidant bioavailability is dependent on a number of factors like food processing, food deprivation, stability of the antioxidant, stabilizing effect of food matrix to restrain the release of lipophilic antioxidants, the isomeric form present in it especially in case of carotenoids and the conjugated form in which it is present apart from the physicochemical and biopharmaceutical properties of the active agent. Some polyphenols like flavonols, isoflavones and flavones are usually glycosylated. The linked sugar is usually glucose or rhamnose but occasionally other sugars or groups like malonic acid may be found [25]. The glycosylation influences various physicochemical and biopharmaceutical properties of the polyphenols, for example, solubility and partition coefficient which determine the passive diffusion across the GIT, may change with the type of conjugation present in the polyphenol. For instance quercetin has a partition coefficient (log octanol/water) of 1.2 ± 0.1, where as for quercetin-3-Orhamnoglucoside the value is lower (0.37 ± 0.06), showing greater hydrophilicity [26]. However, these glycosides need to be converted into the aglycones before they are absorbed into the blood circulation [27]. Gastric epithelial cells express βglucosidases, which can deconjugate glucose specifically but the deconjugation of rhamnose occurs only in the colon by the rhamnosidases which are secreted by the microflora [28]. The hydrolysis of the sugar moiety is essential prerequisite for the absorption of soy isoflavones; the reduced bioavailability with higher intakes was thought to be because of the substrate concentration exceeding the luminal hydrolytic capacity of the intestine.

There are significant differences between the administration of drugs and the consumption of dietary antioxidants. Drugs are generally administered as concentrated agents in a formulation; thus, they are available at higher concentrations for the enzymes which can metabolize them, but the dietary agents, because of the food matrix or complicated liberation process which should occur in the GIT before absorption, result in low systemic concentrations. These differences imply that drugs can readily saturate the metabolic pathways that rely on the supply of cofactors, but the dietary agents because of their low concentrations at the site of absorption cannot saturate them competently. Consequently, the dietary agents are found in conjugated form in the blood unlike many of the drugs which are found in free form to a major extent [25]. When food polyphenols are administered at pharmacological doses, they are found in the free form in blood [29]. The dose will also determine the primary site of metabolism mainly in case of conjugates. Large doses are metabolized primarily in the liver. Small doses may be metabolized by the intestinal mucosa and microbial flora, with the liver playing a secondary role to further modify the polyphenol conjugates from the small intestine, which implies that the intestine is an important site for metabolism of food-derived polyphenols. Bioavailability of dietary carotenoids in general depends on many factors like heat treatment, homogenization, fiber content, presence of fat and type of fat present in the diet [30]. Literature suggests that mechanical treatment and heating enhances the release of lycopene from the tomato matrix and may explain the improved bioavailability seen with consumption of processed tomato products over fresh tomatoes [31]. The presence of fat in the diet may also favorably affect the absorption of lycopene. All-trans form of lycopene is the major content in natural sources but the cis form of the lycopene is more bioavailable than all-trans form [32,33]. Re et al. performed in vitro incubations with lycopene from commercially available capsules or tomato puree with either a simulated gastric juice or human gastric juice to find out the efficacy of the formulated lycopene. Their results indicated that the percent cis-isomers in both lycopene sources increased after incubation with gastric juices but the conversion in capsules is more than that of puree suggesting a stabilizing effect of food matrix [34]. Another report demonstrated that little isomerization of all-trans lycopene to cis lycopene was observed with thermal processing [35]. These factors need to be taken into consideration when administering lycopene. Genistein which falls in the isoflavones class has good solubility at higher pH values but it tends to precipitate in the gastric environment where the pH is low. Thus, absorption is more in the fasted animals where the pH is slightly higher than the non-fasted animals [36]. A report suggests that the aglycone part is absorbed but not the glucosides of the genistein from the rat stomach [37]. The absorption of the glucosides is delayed when compared with their aglycones, for example, genestin; the glucoside of genistein is converted to the active form in the intestine by hydrolysis before absorption [36]. Many antioxidants that are pharmacologically proven suffer from poor bioavailability and stability. Considering the factors discussed earlier, it would be

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advantageous to formulate these agents into dosages forms that contain the antioxidants in pure form. 5.4. Physicochemical and biopharmaceutical properties Antioxidants which have been used in diet or as formulated agents have some common problems in their efficacy as preventive or therapeutic agents which are related to their physicochemical and biopharmaceutical properties. Many of the antioxidants including some vitamins have poor oral bioavailability, which may be because of their low solubility, permeability, stability and/or drug biotransformation before they reach systemic circulation. Dose, solubility, permeability, stability, bioavailability and pharmacokinetic parameters absorption, distribution, metabolism and excretion are the important properties that affect the fate of drugs through oral route. These parameters govern the design of dosage form for the specific drug candidate. Solubility and permeability of drugs can be correlated to their absorption through gastrointestinal tract. According biopharmaceutical classification these two are the main parameters affecting the oral bioavailability [38]. The drug discovery setting as described by Lipinski et al. [39] illustrates that potency, solubility and permeability comprise the triad which is responsible to get acceptable absorption from this route. The high solubility of a drug molecule compensates for the poor permeability in some cases, examples include azithromicin and some peptidic-like drugs. The metabolic stability also plays a major role in drug development. Metabolic stability can be considered as the rate and extent to which a molecule is

metabolized. A molecule which is rapidly and extensively metabolized is considered to have a low degree of metabolic stability [40]. Apart from metabolic stability, the physical stability of the drug also contributes for the presystemic loss of drug when administered perorally. While designing a dosage form for any drug candidate, it is important to consider the solubility, permeability and presystemic loss as major contributors for the drugs having poor bioavailability could help in successful design of the suitable dosage form or delivery system which can markedly improve the drug's performance through oral route (Fig. 2). 5.4.1. Dose Dose of a compound gives a fair idea of potency. Several factors may influence the potency of a molecule through oral route other than those which influence through intravenous route. The dose is inversely proportional to the bioavailability of a compound. The first and foremost step in a formulation design is the preformulation study, which will provide the factors influencing the bioavailability and in turn potency. This area is incompetent at least in the development of antioxidants. It is evident from the literature that the oral dose is some tens of times more than the intravenous dose for these antioxidants, which will invariably increase the cost of dosage regimen. Above all delivery of antioxidants through intravenous or other routes is not patient friendly. 5.4.2. Solubility Solubility enhancement can be achieved by increasing the available surface area by particle size reduction, complex

Fig. 2. Role of pharmaceutics in development of antioxidants. Possibility of obtaining a successful candidate depends on various steps depicted. The role of pharmaceutical scientist starts from finding out the reasons for the failure of candidate including physicochemical as well as pharmacokinetics-related toxicity issues. Pharmaceutics will lead to the development of a successful candidate using approaches like chemical alterations to obtain desirable properties or implication of the delivery systems and novel approaches in the development of a successful antioxidant.

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formation, micelle formation, etc. Modulating solubility in some cases provides sustained release of the drugs, where the drugs are highly soluble. Solubility of antioxidants is very diverse depending on the class, source and type of conjugation. EGCG is considered to be less soluble compound among green tea catechins. Major lycopene content in natural sources is in all-trans form [41]. Literature points that trans-lycopene has a higher propensity to precipitate and form crystals affecting its solubility, a fact that may possibly decrease its GI absorption relative to the more soluble cis isomers. This may be because of the compact structure of cis form when compared to lengthy trans structure [32]. The poor solubility of ellagic acid, which is below 10 μg/ml, poses challenge in delivery of this molecule [42,43]. Coenzyme Q10 is practically insoluble attributing to its lipophilic 10 carbon chain which can be related to its poor oral bioavailability. Isoflavones like genistein are freely soluble at neutral and basic pH but the solubility is below 5% of the original solubility at gastric pH which results in rapid precipitation of this agent in the gut; however, it was reported that the solubility cannot be directly correlated with the extent of absorption [37]. Comparatively, the glucosides of the isoflavones have higher solubility when compared with their aglycone counterparts, but these need to be converted to the active aglycone before absorption through the gastric mucosa. The medium for the administration of quercetin affects the extent of absorption. A study showed decreasing order of absorption in the solvent systems propylene glycol, water/ propylene glycol and water alone, possibly because of the decreased solubility of quercetin in water than in propylene glycol [36]. The solubility of highly soluble drugs can be modulated to sustain release or prolong the activity; vitamin C is such a type of compound [44]. 5.4.3. Permeability Permeability through the GIT is one of the major factors affecting the performance of drugs administered through oral route. Permeability of the compound mainly depends on the partition coefficient. There are very few reports on the permeability of antioxidants. The apparent permeability value of EGCG is (0.83 ± 0.24) × 10− 7. This could be one of the contributors for the poor bioavailability [45]. Ellagic acid accumulates in the epithelium of the GIT, because of the extensive protein and DNA binding which consecutively hinders its passage into the blood circulation [46,47]. Unfortunately, permeability, the major factor in determining the deliverability of any agent through oral route remained unaddressed in the case of several antioxidants, which are being evaluated for the improvement of human health. 5.4.4. Stability Many of the antioxidants are unstable in aqueous solutions. EGCG and ellagic acid are unstable in alkaline medium. EGCG is highly unstable in sodium phosphate buffer pH 7.4 at room temperature [48] and ellagic acid degrades up to 80% in phosphate buffer pH 7.2 [43]. EGCG is rather stable in the acidic pH from 2.0 to 5.5; however, in neutral pH, it is autooxidized. It was unstable in McCoy's 5A culture media with

half-life less than 30 min [49]. It is very unstable in presence of metal ions like Cu [II], Fe [II] and Fe [6] ions at 2 ppb concentrations [50]. The state of ionization is the most important factor in determining the stability of EGCG. The stability of EGCG is dependent on temperature and solution pH and at higher temperatures the pH effect appeared to be enhanced [51]. Many of these agents are vulnerable to photo degradation; examples include CoQ10 and carotenoids. CoQ10 melts at around 46 °C; however, temperature alone cannot induce the degradation of this agent even at 60 °C. Temperature with irradiation showed a synergistic effect on the degradation of CoQ10 [52]. The instability of carotenoids is because of their unsaturation which makes them vulnerable to oxidation and other factors such as temperature, light or pH can produce qualitative changes in these compounds through isomerization reactions [53]. The food matrix stabilizes the all-trans form of lycopene from the conversion into cis form which is more bioavailable form. While delivering these agents as diet, the stability may be compromised [34]. The storage conditions also have the effect on the activity of the antioxidants. Isoflavones for example have shown reduced scavenging efficacies after storage at 42 °C [54]. 5.4.5. Bioavailability Bioavailability is defined as the rate and extent to which the active ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of drug action. It is mainly dependent on the solubility, permeability as well as the stability in the GIT and biotransformation before reaching the blood through oral route. High solubility can sometimes compensate for the poor permeability of the drugs and may be vice versa, but the presystemic loss due to instability or metabolism independently affects the bioavailability. Bioavailability pertaining to dietary supplements depends on liberation from the food matrix and stability of the active agent during food processing apart from the aforementioned factors. EGCG bioavailability is less than 2% of the oral dose administered in rats [55] and less than 20% in mice [56]. Poor permeability and efflux mechanisms contribute greatly for the poor bioavailability of this agent. Another report suggests that the quicker rate of glucuronide conjugation may contribute to some extent for the poor bioavailability of EGCG [56]. Hepatic metabolism of this agent plays no significant role in the poor bioavailability [57]. The matrix in which lycopene is found in food appears to be an important determinant of its bioavailability, release of lycopene from food matrix is the first step in the absorption process. The process of cooking usually makes lycopene more bioavailable by its release from the matrix to the lipid phase of the food. In vitro and in vivo studies in lymph cannulated ferrets indicate that the cis isomers are more easily incorporated in the bile acid micelles [32,33] and hence are more bioavailable. Most of the carotenoids including lycopene, when administered along with high-fat diet result in better bioavailability [58]. CoQ10's bioavailability is only 10% from meal and oil suspension [59,60], because of the poor solubility and permeability of the drug. Genistein bioavailability was

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calculated to be 18.3% in male Sprague Dawley rats [61]. Its bioavailability showed nonlinear relationship with the dietary intake, it was also evident from the urinary data [62]. The systemic bioavailability as determined by comparing dosenormalized AUCs was found to be greater for the β-glycosides than for the corresponding aglycones [63]. Similar pharmacokinetics has been described for flavonoids. Quercetin bioavailability was shown to be increased by the presence of fat [64]. Vitamin C has good bioavailability when administered in small doses; however the bioavailability decreases with increase in dose. At 200 mg dose, the bioavailability was 100%, but as the dose was increased up to 1250 mg the bioavailability was decreased below 50%. This may be because of the urinary excretion, which is increased with the dose [65]. 5.5. Pharmacokinetic parameters 5.5.1. Absorption Absorption of EGCG was poor from the Caco2 monolayer. The efflux pumps also play some role in the poor absorption of this agent [45]. Chylomicrons play an important role in the absorption of the CoQ10 and other lipophilic antioxidants like carotenoids. Lycopene is taken up by the enterocytes, incorporated into the chylomicrons, and then released into the portal circulation. Chylomicrons are taken up by the liver and lycopene is released into other lipoproteins like LDL, resulting in several hours delay in the time to reach peak (tmax). Gustin et al. suggest that the absorption of this compound is saturable because they have observed non proportional increase in the pharmacokinetic parameters as they increased the dose [66]. The saturation of absorption process is mainly with the lipophilic antioxidants like carotenoids CoQ10 and vitamin E. Poor absorption of ellagic acid may limit its effectiveness due to its inability to obtain high systemic concentrations for extended period of time. CoQ10 is absorbed slowly from the gastrointestinal tract, attributed to its high molecular weight and low water solubility. After oral administration of I3C (250 mg/kg) to mice, the compound was rapidly absorbed and had already reached an apparent peak concentration of 4.1 μg/ml at the earliest sampling time point of 15 min after dose. In other studies, the levels of I3C were not even detected, may be because of the rapid oligomerization of this agent [67]. The type of sugar, but not its position at which it is attached to the quercetin molecule, is the major determinant of the small intestinal absorption of quercetin in rats [68]. Quercetin glucosides are completely hydrolyzed in the small intestine by β-glucosidases before absorption into glycone and aglycone moieties [69]. The aglycone moiety was found to be absorbed up to 65–81% [70]. On the other hand, quercetin rhamnosides are only metabolized to aglycone in the colon by the microbial rhamnosidases [71]. Coadministration of quercetin and catechin results in the reduced absorption of quercetin [72]. Ascorbic acid is well absorbed at lower doses, but absorption decreases as the dose increases. It is transported into the cell by sodiumdependent vitamin C transporters, one or both of which are found in most tissues [73]. The median bioavailabilities after oral doses of 30, 100, 200 and 500 mg were 87%, 80%, 72%

and 63%, respectively. Less than 50% of 1250 mg dose is absorbed and most of the absorbed dose is excreted in the urine [65]. 5.5.2. Distribution, storage and binding EGCG tends to distribute into the peripheral compartments relatively higher than other green tea components. The plasma protein binding of this agent was also reported to be high. At higher doses ranging from 500 to 2000 mg/kg, EGCG saturated the small intestinal and colonic tissues. However, it still continued to cross the intestinal barrier due to the lower concentrations in the plasma and other tissues. Due to higher saturation point of EGCG in these tissues, a linear increase in plasma or tissue concentration with respect to dose has been observed. EGCG is rapidly conjugated in plasma, however, free EGCG was found in tissues [56]. More than 53% of the orally administered ellagic acid was found remaining in the gastrointestinal tract [46] and was found extensively bound to DNA. Sulphate ester, glucuronide and glutathione conjugates of ellagic acid were present in urine, bile and blood [74–76]. I3C was rapidly absorbed, distributed into liver, kidney, lung, heart and brain with liver having the highest amount than plasma and other tissues [67]. The concentration–time curve of genistein after a single dose in healthy post-menopausal Thai women revealed a biphasic curve when administered as soy beverage or soy extract capsules [77]. This is extensively conjugated to glucuronidated in the small intestine and secreted into the intestinal lumen [78]. Most of the exogenous dose of CoQ10 is distributed to the liver and incorporated into very-lowdensity lipoprotein; same is the case with vitamin E and lycopene [58]. Due to high liposolubility of lycopene, it distributes in peripheral tissues extensively and the volume of distribution was found to be in the range of 2.12–18.54 l/kg. Among the isomeric forms of lycopene, lymph contains >75% of cis isomer but the storage tissues contain ∼ 50% cis-lycopene because this mixture is the most stable and represents an equilibrium among the isomers [66]. Vitamin E, like many other lipophilic antioxidants, binds to specific proteins or lipoproteins during absorption, transportation, and distribution. Release of absorbed vitamin E into the circulation occurs via chylomicrons [79]. 5.5.3. Metabolism and excretion EGCG is mainly excreted through the bile; however, urinary excretion also takes place when administered through oral route or parenteral route. The marked differences in the fecal levels of EGCG between oral and intravenous routes suggest that the orally administered drug is not well absorbed [56]. Methylation and sulfation are the main biotransformation pathways of green tea components [45,80]. I3C is rapidly converted into acid condensation products and oxidative metabolites, however some of the metabolites retain the antioxidative properties, but the activity is inferior to the parent compound [67]. The βglucosidases play important role in the metabolism of many polyphenolic glucosides before absorption [69]. The microbial enzymes in the colon also play significant role in the metabolism of glycosides other than glucosides. Metabolism

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by the gut enzyme β-glucosidase is necessary for the separation of genistein (aglycone part) from the genestin (glucoside) before its absorption [37]. Genistein metabolites were found in blood 3 min after oral administration. The major metabolites are glucuronide and sulfate conjugates, but more conjugates were found in the fasted rats than the non-fasted rats [36]. Quercetin administered in free form is significantly converted into the methyl conjugate isorhamnetin, and the concentration of the latter increases with time. This may be because of the preferential excretion of the non-methylated quercetin or because of effective quercetin methylation [36]. The major part of the circulating metabolites of quercetin (91.5%) are glucurono-sulfo conjugates of isorhamnetin and of quercetin the minor part (8.5%) is constituted by glucuronides of quercetin and its methoxylated forms [81]. Quercetin conjugates are formed in the small intestine during absorption of aglycone, which are secreted into the intestinal lumen, which constitutes the elimination pathway in addition to urinary and biliary pathways [82]. Vitamin C is quickly eliminated from the body; approximately 73% of ascorbic acid is removed from the body in less than 24 h. Ascorbic acid is not protein bound, so it is filtered and reabsorbed by the kidneys in healthy subjects but is lost in patients who have been hemodialyzed. Decreased bioavailability and renal excretion keep plasma vitamin C levels at less than 100 μmol/l, even with an oral dose of 1000 mg [65]. 6. Delivery approaches 6.1. Conventional delivery Conventional dosage forms usually consist of tablets, capsules and liquid orals through the most acceptable oral route. These conventional forms are easy to formulate and are relatively less expensive. Antioxidant enzymes are generally administered through intravenous route. These dosage forms are experiencing serious disadvantages in the delivery of antioxidants. At times, these dosage forms lead to loss of efficacy of the active agent which may be due to various reasons like poor bioavailability, first pass effect or instability of the active agent in GI tract. For example, the systemic administration of native exogenous SOD and/or CAT often failed to produce convincing protection against pulmonary oxidative stress in animals. Administration of large doses of CuZnSOD is able to show some protection against hyperoxic rabbits but failed to prevent hyperoxic injury in rats despite significant elevation of SOD activity in plasma. The conventional delivery of these enzymes experiences unfavorable pharmacokinetics as discussed in Section 4. Presently, there are lots of antioxidant products on market which have been formulated into these conventional dosage forms, with vitamins leading the group. Vitamins have been formulated mainly into tablets and capsules. Generally, these agents were found in combinations rather than individual products. Vitamin C especially has been reported to be stable in the tablets at least for 20 weeks. Storage at 25 °C or daily opening of bottles stored at room temperature resulted in 0–2%

loss of ascorbic acid [83]. Vitamin C has relatively less bioavailability problems in comparison to other antioxidants, but the elimination was reported to be very rapid for this agent and absorption is saturable [65,84]. For this reason, sustained release formulations have been developed for vitamin C. Other antioxidants like EGCG, quercetin, lycopene, ellagic acid and coenzyme CoQ10 are difficult to deliver by these conventional dosage forms. Many of these are not formulated in the pure forms; instead the plant extracts of these agents are generally formulated and marketed. These are rarely formulated as liquid orals considering their instability in the solution form. There have been reports on CoQ10 that powder filled capsules and oily suspension filled capsules have poor bioavailability. The poor bioavailability was attributed to low aqueous solubility of CoQ10 and large particle size in the formulations [85]. Similarly lycopene has poor solubility and stability-related issues. Addressing these problems with the help of conventional dosage forms is difficult and help of advanced delivery systems is a must to maximize the potential roles of antioxidants in prophylaxis and therapy. The choice of dosage form is affected by the properties of the molecule, cost of dosage regimen, patient compliance and intended use in prophylaxis or therapy. Prophylaxis desires oral dosage forms, which can act for prolonged periods of time. Different types of strategies were implicated by different groups to achieve successful delivery of these antioxidants. The major approaches employed in the development of antioxidant delivery are discussed in the following sections. 6.2. Inclusion complexes Cyclodextrins (CDs) are cyclic α(1–4) linked glucose oligomers having a torus shape and characteristic dimensions. With the virtue of their geometry and relatively hydrophobic internal cavity, in contrast to the hydrophilic character of the external hydroxyl faces, CD molecules easily form inclusion complexes with a wide variety of molecules and molecular ions. These complexes were employed to increase the aqueous solubility of drugs and the stability of labile drugs resulting in improved bioavailability. Lycopene and CoQ10 are among the major lipophilic antioxidants, solubility of which has been increased using inclusion complexes. Lycopene and β-cyclodextrin complexes are used in the ORAC assay, which showed increased scavenging activity than pure drug indicating the increased solubility [86]. Another study showed the possibility to obtain inclusion complexes at a ratio of 1:1, in solid state and in aqueous solution, between the cavity of β-CD and hesperetin, hesperidin, naringenin and naringin (flavonoids). In a therapeutical formulation, this could improve the dissolution and subsequently the absorption of the drug [87]. 6.3. Chemical modifications Numerous modifications of endogenous as well as exogenous antioxidants have been attempted in order to (i) prolong the half life of these agents in vivo, (ii) get more stable derivatives, (iii) protect these agents from degradation/

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inactivation in GIT and reduce their immunogenicity, and (iv) attain targeting to the tissues or cells. Some of the major chemical modifications of these antioxidants are discussed briefly in this section. Coupling of polyethylene glycol (PEG) to the antioxidant enzymes increases their bioavailability and enhances their protective effect [88]. In particular, the coupling of SOD and CAT amino groups with PEG minimizes their elimination by the reticuloendothelial system and prolongs their circulation by increasing their half-lives from few minutes to several hours in rats and mice. The increase in lifespan and suppression of the immune response are proportional to the number and length of PEGs attached to the molecule, however, excessive modification resulted in reduced enzyme activity. PEG modification facilitates cellular delivery of these enzymes and augments cellular resistance to oxidative stress [13,89,90]. Other coupling strategies for improving the deliverability to cellular targets are discussed in Section 6.5. CoQ10 being a natural lipid antioxidant, extremely insoluble in water and due to this reason the cellular uptake of this agent is very low. One approach is to derive ubiquinone analogs with a reduced number of carbons in the side chain compared with CoQ10. The clinically used synthetic compound idebenone is derived in this process. However, it has been indicated that many short-chain CoQ10 analogs, including idebenone, enhance superoxide formation by respiratory complex I. Thus, identification of more analogues of CoQ10 without these pro-oxidant activities is of importance. The CoQ10 analog decylubiquinone was synthesized in this process and is warranting clinical importance [91]. Chemical modification of ascorbic acid has led to more stable derivatives such as ascorbyl esters with C6 to C18 fatty acids or ascorbyl phosphate salts. Among the lipophilic derivatives, ascorbyl palmitate (AP) is often used in topical preparations as an antioxidant to protect the skin and reduce wrinkles by stimulating collagen lipophilic ingredients in formulations [92]. In case of vitamin E, the acetate and acid succinate esters are commonly used clinically for their high stability. The hydrochloride salt of D-α-tocopheryl N,Ndimethylaminoacetate is another prodrug of vitamin E which has high solubility and stability. Vitamin E succinate was coupled to PEG similarly as enzymatic antioxidants to improve the efficacy [93]. 6.4. Novel drug delivery systems Delivery systems help compounds to be delivered in efficient manner rather than altering their chemical nature or biological activity. Sometimes, chemical modifications may not help for the efficient delivery. For example, shortening the lipophilic chain of the CoQ10 though resulted in compounds with good pharmacokinetic profiles raised certain doubts about the pro-oxidative properties of the derivatives; on the other hand, little or no changes in drug's properties are observed when delivered through the delivery systems. This facilitated the doorway of novel delivery systems in the development of antioxidants. The delivery systems have evolved over a period

of time and are still evolving to improve drug's efficacy, safety and patient compliance. These approaches work in all areas of the delivery. These can be applied to improve the solubility, permeability, stability of the compounds and some can even surpass the first pass metabolism. These delivery systems have been beneficial to the pharmaceutical industries as it is a strategic tool for expanding drug market and patent life. Oral route is the most convenient route and delivery system that makes delivery of antioxidant efficient by this route should be considered. For the drug to have better efficacy, it should reach the site of action, for example drugs should cross blood brain barrier to treat neurodegenerative disorders and target the malignant tumors in treatment of cancer. Novel drug delivery systems would make antioxidant reach site of action and improve the efficacy of therapy, generally by improving the bioavailability. These delivery systems are applicable to overcome various pharmacokinetic problems associated with antioxidants (Fig. 3). 6.4.1. Self-emulsifying drug delivery systems (SEDDS) SEDDS offer the potential for enhancing the absorption of poorly soluble and/or poorly permeable compounds through oral route. SEDDS were shown to improve the delivery of lipophilic compounds such as CoQ10 by the oral route [94]. Following oral administration, SEDDS provided a two-fold increase in the bioavailability compared to a powder formulation. For drugs that are poorly soluble and/or poorly permeable, a significant improvement in reproducibility in performance and bioavailability might be achieved with SEDDS. However, there are some limitations associated with these formulations, including stability, manufacturing methods, interaction of the fill with the gelatin shell and limited solubility of some drugs in lipid solvents. When the product is stored at a lower temperature, there may be some precipitation of the active ingredient and/or the excipients. The precipitated materials should therefore be dissolved again when warmed to room temperature; otherwise, the drug will not be presented in a solution or as a fine emulsion droplet [95]. 6.4.2. Liposomes Liposomes are potential systems for drug delivery because of their size, hydrophilic and hydrophobic character and biocompatibility. Liposomes are studied extensively as drug carriers for variety of molecules such as small molecular weight compounds, proteins and peptides, nucleotides and plasmids. Properties of liposomes are very versatile and vary with lipid composition, size, surface charge and method of preparation. Components of bilayer determine the strength and surface charge of liposomes [96]. Lectin modification of liposomes promotes binding to Peyer's patches in the GIT and facilitates delivery of the agents loaded in the liposomes through oral route [97]. The term ‘antioxidant liposomes’ is generally used to refer liposomes containing lipid-soluble or water-soluble chemical antioxidants, enzymatic antioxidants or combinations of these antioxidants. Antioxidant liposomes hold great promise in the treatment of many diseases in which oxidative stress plays a

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Fig. 3. Comparison of dietary and formulated antioxidants through the most favorable oral route. The administered drug has to go through various phases like liberation, absorption, distribution, metabolism, elimination and response (LADMER). The problems associated with diet supplement at any stage in LADMER can be overcome by implication of NDDS.

significant role [98]. Human antioxidant enzymes and many of the other antioxidants do not easily penetrate the plasma membrane of cells and some have poor stability and short halflife in plasma when administered through conventional delivery modes. In a study, endothelial cells treated with liposomes containing entrapped SOD and CAT have shown to have cellular specific activity of at least 40-fold within 2 h. A major problem associated with conventional liposomes is that they are recognized by the immune system as foreign substances and rapidly removed by phagocytic cells of the reticuloendothelial system. However, with the advent of stealth technologies, which are based on the polymer coating especially PEG onto the liposomal surface, the commercial value and potential therapeutic applications of the liposomes are increasing. The coupling of PEG onto the liposomal surface creates a water shell surrounding these entities and reduces the binding of complement antibodies and immune cells. CuZnSOD-loaded liposomes increased the SOD activity of human lung epithelial cells (A2182) 24 h after treatment. The highest increase of cellular SOD was observed with anionic liposomes when compared with the neutral as well as cationic liposomes. Exposure of untreated cells to oxidative stress increased the cellular glutathione level after 24 h. Cells pre-treated with liposome encapsulated CuZnSOD were protected from oxidative stress which was evident from the unchanged concentration of cellular glutathione [99]. The intratumor administration of liposomes is a highly effective approach for the treatment of local solid tumors.

Liposomes of catechin, epicatechin and EGCG were prepared and shown that the liposomal preparations of catechin and epicatechin are retained for longer durations in the tumor in comparison to aqueous solutions of these agents [100]. Liposomes of vitamins C and E were used in combination or individually and reported that liposomal antioxidants were able to prevent the ischemia and reperfusion where the free forms of antioxidants failed or showed little effect [101]. This could be because of the faster penetration rate of the liposomal antioxidants into the brain cells than the free forms. Similar potential of liposomes for antioxidant delivery was observed, when liposomal α-tocopherol (LAT) was administered intratracheally to the hypoxic rats, where vitamin E was able to produce marked antihypoxic, antioxidant and antiapoptotic effects, suggesting the potential use of LAT for the correction of hypoxic lung injury. These studies were also carried out with liposomes without α-tocopherol, where it was observed that no significant change in the mortality of the hypoxic rats. The antihypoxic and antioxidant action of LAT cannot be explained only by the direct substitution of the deficit of phosphatidylcholine and antioxidant action of α-tocopherol itself [102]. Other antioxidant liposomes which are tested through intratracheal route to treat oxidative stress-related lung diseases include liposomal N-acetyl cysteine (NAC) [103] and liposomes containing both α-tocopherol and glutathione [104] in acute respiratory distress syndrome and phorbol myristoil acetate induced pulmonary oxidant stress in rat model, respectively.

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6.4.3. Microparticles Microparticles have been designed and evaluated as delivery systems for both endogenous as well as exogenous antioxidants. These particles are either matrix type entrapping the active moiety or capsule type encapsulating the drugs. The matrix is generally a polymer which sustains the release of drug. SOD and CAT have been encapsulated in PLGA microparticles and shown to be efficient in releasing drug slowly for extended periods [105]. Similarly, in an attempt to sustain more constant plasma isoflavone concentrations, a new slow-release formulation of a soybean isoflavone extract was prepared by microencapsulation with a mixture of hydroxypropylcellulose and ethylcellulose to alter its dissolution characteristics. In vitro experiments confirmed slow aqueous dissolution of isoflavones from this formulation when compared with the conventional isoflavone extract [106]. Administration of EA microspheres (mcEA) to rats, reduced the severity of dextran sulfate sodium (DSS) induced colitis in a dose-dependent manner, and a significant effect was observed at 10 mg/kg, the ED50 being 2.3 mg/kg. Although EA alone without using microspheres was also effective in reducing the severity of DSS-induced colitis, this effect was much less potent as compared with that of mcEA and the ED50 was about 15 times higher than that of mcEA. This suggests that ellagic acid microspheres can be used to treat ulcerative colitis through oral route effectively [107]. Eudragit microparticles produced through spray drying have been evaluated as encapsulation devices for the delivery of vitamin C via oral route in a view to slow down the release. However, the release rate was not decreased considerably, but these microparticles showed good morphology and size distribution and can be used as supportive therapy in colorectal cancer [108]. Chitosan, a natural polysaccharide, has been gaining increasing importance in the development of microparticulate systems owing to its good biodegradability, biocompatibility and non-toxicity [109]. Chitosan microparticles of vitamin C were also prepared using the same technique using cross-linking agents. The release of vitamin C from these microspheres was sustained and affected by the volume of cross-linking agent added [110]. 6.4.4. Nanoparticles Nanoparticles are colloidal particles varying in size from 10 nm to 1000 nm. Nanoparticles have been explored as drug delivery systems for both small drug molecules and macromolecules. Either direct nanosizing of drug or incorporation into lipidic and polymeric particles can help deliver drugs with poor aqueous solubility and permeability. Nanoparticles have shown to be absorbed in systemic circulation from GIT through Peyer's patches via M cells in lymphatic systems [111]. The lymphatic absorption of drug via the GALT has an advantage over portal administration since it avoids first pass metabolism in liver [112]. After oral administration, these particles also protect the drug from GI degradation [113]. In recent years, the focus is on developing biodegradable polymeric nanoparticles for drug delivery. These particles apart from increasing the bioavailability provide sustained release of

drug. The drug is dissolved, adsorbed, attached or encapsulated in the polymeric matrix of nanometer size. Depending upon the method of preparation nanospheres or nanocapsules are obtained with different release and surface properties [113– 116]. Nanoparticles are also being explored for targeted drug delivery [117–119]. There are reports suggesting those drugs which are encapsulated in high molecular weight polymeric nanoparticles which passively target the tumor tissue through enhanced permeation and retention effect. This property can widely be utilized for delivering antioxidants in tumors cells as many antioxidants discussed above have anti-cancer activity. Antioxidants like EGCG and ellagic acid which have marked anti-cancer activity would benefit in terms of efficacy and patient compliance when delivered by means of nanoparticles. The nanoparticulate delivery can prevent the degradation of these agents in the GIT, which will help in improving the bioavailability of the antioxidants which degrade in the gastric environment. In case of the drugs where efflux mechanisms play major role in poor oral bioavailability, these nanoparticles will help to improve their bioavailability by virtue of the unique absorption mechanism through lymphatic system. Literature also suggests that polysorbate 80 coated nanoparticles can cross blood brain barrier and deliver the drug to brain [120]. There are few reports on lipidic and polymeric nanoparticles for CoQ10. Hsu et al. have reported on preparation of CoQ10 nanoparticles engineered from microemulsion precursors [121]. Kwon et al. have also reported on CoQ10-loaded PMMA nanoparticles. They demonstrated that CoQ10 was more stable within polymeric nanoparticles over dispersion and oil-based formulation against UV and high temperature [122]. Polymeric nanoparticles thus would help in increasing the drug's stability. Ellagic acid is a difficult to deliver molecule and its formulation is also very challenging, because of its insufficient solubility in most of the solvents [43]. A novel method has been developed for the preparation of ellagic acid nanoparticles using a cosolvent [42]. In situ intestinal permeability studies of these prepared nanoparticles using polyvinyl alcohol (PVA), PVA-CS (polyvinyl alcohol-chitosan) blend and DMAB (didodecyl dimethyl ammonium bromide) as stabilizer showed 75%, 73% and 87% permeation respectively and were quite superior to simple EA, which showed only 66% permeation. Increased permeation would lead to increase in bioavailability, clearly indicating the potentials of nanoparticulate systems in oral delivery. Evaluation of EA nanoparticles for ROS scavenging effect in DCFH-DA assay using yeast cells further revealed the fact that nanoparticles were able to penetrate the thick walls of yeast cells and sustain the release of EA [123]. In another study, the gliadins nanoparticles of vitamin E were prepared and shown to sustain the release over a prolonged period of time [124]. Kristl et al. demonstrated the stability of ascorbyl palmitate (AP), an analogue of vitamin C, in microemulsion, liposomes and solid lipid nanoparticles (SLNs). The hydrophilic part of AP is the reactive moiety, and high stability is obtained in systems in which this part is exposed to a less polar environment. This moiety was more deeply immersed in the interface when entrapped in a liquid-state carrier than when applied in gel-state particles. Encapsulation of AP in SLN core

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leads to greater stability. From their study, it is obvious that the location of the sensitive group of the drug-molecule in a carrier system is crucial for its stability [125]. These polymeric nanocarriers (PNC) are attractive vehicles for vascular drug delivery as well, but remained an orphan technology for antioxidant enzymes due to poor loading and inactivation of proteins during formulation. A novel freeze– thaw encapsulation strategy was designed to provide ∼ 20% loading efficiency of an active large antioxidant enzyme, CAT, into PNC of poly(ethylene glycol)-b-poly(lactic-glycolic acid) with size range of 200–300 nm. H2O2 is freely diffusible into PNC which makes these carriers efficient in oxidative stress conditions. PNC-loaded CAT showed 25–30% of H2O2degrading activity after 18 h exposure to proteolytic environment, while free CAT lost activity within 1 h [126]. From this, it is obvious that the PNC can provide stability to the proteins loaded, for extended periods of time. Nanoparticle surface can also be modulated to attain desirable characteristics similarly as liposomes. Coupling of ICAM-1 antibodies creates multivalent ligands which enter cells and addition of CAT to these particles did not change the mechanisms of nanoparticle uptake or trafficking [127]. The ability of nanoparticles in targeting a particular organ has been discussed in Section 6.5. 6.4.5. Gel-based systems An alternative and promising research field deals with particles obtained from hydrogel systems. The hydrogels may be sensitive to environmental stimuli such as pH, ionic strength, electric/magnetic fields, light and temperature depending on the substrate used. Thermoresponsive gels for the controlled delivery of vitamin E had been shown to release the drug in a controlled fashion [128]. Totally transparent solid matrices resulting from the dehydration of new protein gels revealed variable swelling capacities that depend on the solvent used and physicochemical conditions. The protein hydrogels were formed at pH 8 in 50% ethanol solutions. Basically, the dispersion of a β-lactoglobulin pre-gel in an apolar phase produced gelled droplets. These droplets were then washed and dehydrated under vacuum in order to produce particles of 500 μm mean diameter [129]. These gel based systems could help in encapsulating antioxidants for sustained release. 6.4.6. Other modes of antioxidant delivery Injection is the only mode of therapeutic enzyme administration till now. Conventional delivery has several pharmacokinetic disadvantages as described (Section 4). Several strategies have been studied to reduce the frequency of administration of therapeutic enzymes as described in previous sections. A major challenge is to develop noninvasive modes of administration. Special mixed lipid carriers in the form of ultradeformable vesicles, Transfersomes® (Tfs), may arguably deliver drugs transcutaneously into blood circulation. The hydrophobicity of Tfs makes them good candidates for accumulation in the water-rich narrow gaps between the adjoining cells in the skin. This fact associated with the ability of Tfs to deform allows these systems to transiently open the pores through which water normally evaporates between the

cells. The vesicles finally reach the systemic blood circulation through the lymphatic system. The resulting bioavailability reportedly can be rather high and the biodistribution similar to that resulting from a subcutaneous injection. SOD administered transdermally in the form of tranferosomes ameliorated the disease symptoms of arthritis in a rat model. The therapeutic approach showed practical and therapeutic advantages of the non-invasive transdermal delivery of antioxidant enzymes in comparison with invasive administration, contributing to an innovative approach [130]. 6.5. Targeting approaches The delivery of antioxidants to tissue or organ of interest is not possible by the conventional delivery approaches. To attain the delivery of these agents to the sites where they are required many targeting approaches have been adopted. Targeted drug delivery promises a significant improvement over the current therapeutic means and, therefore, has remained the focus of intense research. Several modifications of SOD and CAT have been devised to provide them with greater affinity or specificity for certain organs and tissues. SM-SOD, a derivative synthesized by covalent coupling of a hydrophobic organic anion [poly (styrene-co-maleic acid)] to the cysteinyl residues of CuZnSOD, binds to serum albumin, provides prolonged circulation and renders significant affinity for plasma membrane components when the pH is below the physiological level. These features may facilitate SM-SOD accumulation in ischemic sites. Chimeric protein constructs consisting of SOD and heparinbinding peptides have an affinity for charged components of the endothelial glycocalix [131], similarly putriscine coupled enzymes accumulate in brain [132], and sugar coupled enzymes are used to target hepatic macrophages [133]. Endothelial cells lining the luminal surface of the vasculature represent an important target for delivery of antithrombotic, anti-inflammatory, antioxidant agents and genetic materials [90]. Cell adhesion molecules platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule (ICAM) represent very attractive endothelial determinants for vascular immunotargeting [134]. Thus, SOD and CAT conjugated to antibodies directed against the constitutively expressed endothelial antigens, angiotensin-converting enzyme (ACE) and adhesion molecules (ICAM-1 or PECAM-1) bind to endothelium in intact animals after intravascular administration, accumulate in the pulmonary vasculature, enter endothelial cells and augment their antioxidant defenses [135]. Recent studies revealed that, although endothelial cells do not internalize monomeric antibodies against PECAM and ICAM, one can facilitate intracellular delivery of therapeutic cargoes by controlling size of the anti-PECAM and anti-ICAM immunoconjugates in the nanoscale range [90]. Cationization is a universal approach that can be applied to increase the interaction of compounds with negatively charged biological components. Cationization of CAT greatly increased the amount and rate of hepatic uptake after intravenous injection in mice [136]. The conjugation of drugs with lipoamino acids

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(LAAs) has been proposed as a means of enhancing the lipophilicity of drugs, while giving them membrane-like character which can increase the uptake by penetration through biological membranes and barriers. LAAs are α-amino acids bearing an alkyl side chain, the structure and length of which can be varied to obtain the desired physico-chemical properties. Idebenone–lipoamino acid (IDE-LAA) conjugates have been developed with a view to enhance the penetration into brain, but the inability of IDE-LAA to reach brain was observed and this is because of the extensive enzymatic degradation of IDE-LAA to IDE [137]. The blood–retina barrier and the extra ocular epithelia represent the obstacle in the drug delivery to the choroid, retina, and vitreous. Only a fraction of the drug administered orally or by subcutaneous or intramuscular routes reaches the retina, requiring large doses to be therapeutically effective. A possible approach to improve retinal drug delivery is to facilitate localized delivery to the posterior segment of the eye by using Anopore™ nanoporous filter. Catalase and vitamin C were delivered using these inorganic nanoporous filter, which is made up of aluminium oxide filter with pores of 20 nm size, as a semipermeable membrane to separate two compartments in vitro. The data shown represented the possibility of biocompatible capsules based on nanoporous filters which are able to provide controlled delivery of antioxidant molecules [138]. Another important site to be targeted in oxidative stressrelated diseases is the mitochondria which is the main source for the production of ROS. A range of possibilities are available for the selective delivery of drugs to mitochondria, including targeting based on biophysical properties of mitochondria, e.g., the high negative internal potential, targeting based on the unique mitochondrial localization of enzymes that catalyze the release of drugs from prodrugs, and targeting based on transporter-dependent delivery of prodrugs. Mitochondrially targeted ubiquinone and vitamin E analogs Mito Q and Mito Vit E were synthesized for efficient antioxidant activities [139]. Vitamin E nanospheres using polyethylene glycol (PEG) were prepared and tested against amyloid-beta (Abeta)induced ROS. Unencapsulated vitamin E prevented Abetainduced ROS in cultured SH-SY-5Y human neuroblastoma cells only if present prior to, or applied simultaneously with, Abeta treatment. In contrast, vitamin E in the form of nanospheres was equally effective if administered 1 h after Abeta exposure. These findings suggest that nanospheremediated delivery methods are useful for antioxidant therapy in AD. The antioxidant vitamin E provided limited neuroprotection in AD, which may have derived from its lipophilic nature and resultant inability to quench cytosolic ROS. The authors suggest that PEG-based nanospheres of vitamin E could efficiently scavenge the ROS because of enhanced hydrophilicity [140]. A different strategy called bioreductive delivery systems can be utilized for the targeting of hopoxic tissues in some cancers, rheumatoid arthritis and diabetes. Hypoxic tissue facilitates the bioreductive drug targeting as the oxygen suppresses the release of the drug from the system at other sites [141].

6.6. Novel methods to treat oxidative stress The topics which are beyond the scope of this review but worth notifying are covered in this section. Gene therapy in contrast to the delivery of human recombinant antioxidant enzymes makes the antioxidant therapy free of immune reactions. Experimental results by groups working on gene therapy suggest gene therapy of antioxidants may eventually evolve into potential approaches for the treatment of diseases related to oxidative stress [142–144]. On the other hand, fullerene molecule and its derivatives have been shown to have antioxidant properties among other activities [145,146]. These nanostructures are under extensive research these days and are also promising some insights in the newer treatment strategies for various diseases including diseases inflicted by oxidative stress. 7. Conclusion Increasing understanding of the free radicals role in diseases is opening new area for the antioxidants to manifest in prevention and therapy of the healthcare system, along with promising role as supportive remedies in many regimens of mainline therapy. Nevertheless, epidemiological studies have been suggesting strongly that antioxidants can decrease the incidence of diseases. However, more number of animal and human studies are required to establish the efficacy and safety of these agents in various chronic and or acute oxidative stressrelated diseases. Some reports indicate the potential pro-oxidant activities of these antioxidants [147] including vitamin C [148]. One of the problems which this field is facing is the poor bioavailability of many of these agents, which may be due to poor preformulation research in this area. Pharmaceutical research will help the antioxidants overcome these problems. Pharmaceutical research will help this field grow in various steps of the development (Fig. 3). Although preformulation studies were conducted for some antioxidants, many agents remain untouched. This area is still in its infancy; investigation is needed in this field. The problems associated with the bioavailability and other formulation-related issues of these agents must be overcome, along with strong evidences of their use before these can be approved by the regulatory authorities and marketed as potential chemopreventive agents or as therapeutic agents. The dose ranges implied by different groups are wide spread and there is a need to establish the pharmacological doses of the individual antioxidants. Another important drawback of the antioxidant therapy is the inability of antioxidants to reach the sites of action. This suboptimal delivery can be overcome by different novel delivery strategies which promise the targeted delivery of these agents. It is clear that the pharmaceutical research in the field of antioxidants is not going with the same pace with which the understanding of the role of free radicals in diseases is increasing. Finally, we feel a thorough organized research is still required in this field with an urge for aggressive research in the preformulation, pharmacokinetic, toxicological studies and delivery aspects of the antioxidant agents which are promising health

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