WO2025144067A1 - Dietary supplement - Google Patents

Abstract

The invention relates to a dietary supplement for reducing the level of oxidative stress in the body and enhancing immune defence. The composition contains a) a source of zinc, b) a source of anthocyanins, c) a source of vitamin C, and d) a source of copper.

Description

BIOLOGICALLY ACTIVE SUPPLEMENT

The invention relates to the field of biologically active additives for reducing the level of oxidative stress in the body and improving the state of immune defense.

PREVIOUS LEVEL OF TECHNOLOGY

Micronutrients are a general term used to describe essential vitamins and minerals obtained from the diet to support virtually all normal cellular and molecular functions [West KP, Stewart CP, Caballero B, Black RE: Nutrition; in Merson MH, Black RE, Mills AJ (eds): Global Health: Diseases, Programs, Systems, and Policies, ed 3. Burlington, Jones and Bartlett Learning, 2012, pp 271-304]. They perform a variety of functions, including enabling the body to produce enzymes, hormones, and other substances needed for normal growth and development. Although the amounts of micronutrients needed are very small, their deficiencies can have a wide range of negative health effects. Micronutrient deficiencies are a common problem worldwide, with over two billion people suffering from inadequate nutrition in micronutrients, particularly iron, zinc, iodine, and selenium [Committee on Micronutrient Deficiencies, Board on International Health, Food and Nutrition Board; Howson CP, Kennedy ET, Horwitz A: Prevention of Micronutrient Deficiencies: Tools for Policymakers and Public Health Workers. Washington, National Academy Press, 1998]. Micronutrient deficiencies can cause dangerous health conditions and can also lead to clinically significant reductions in energy levels, mental clarity, and overall performance. This can lead to decreased educational outcomes, work productivity, and an increased risk of other diseases and dangerous health conditions [https://www.who.int/health-topics/micronutrients#tab=tab_l].

One of the main causes of micronutrient deficiencies is poor and unbalanced diet. Eating habits around the world have changed dramatically in the last few decades. Globalization and urbanization have paved the way for the rise of frozen convenience foods and drinks, junk food, and eating out, with fewer people growing or preparing food from scratch. These cheap, ready-to-eat foods and drinks are often subject to highly processed and high in calories, fat, salt, sugar, and low in protein and dietary fibre. Highly processed foods and drinks typically have a long shelf life compared to the perishable nature of fresh foods, making them attractive to retailers such as supermarkets. Intensive marketing by the industry, particularly to children, has also increased the consumption of these nutritionally unbalanced products. These products are increasingly replacing fresh, nutritious, and minimally processed foods, changing diets and food systems [Moodie, Rob; Stuckler, David; Monteiro, Carlos; Sheron, Nick; Neal, Bruce; Thamarangsi, Thaksaphon; Lincoln, Paul; Casswell, Sally (2013). Profits and pandemics: prevention of harmful effects of tobacco, alcohol, and ultraprocessed food and drink industries. The Lancet, 381(9867), 670–679].

Unhealthy diets and resulting micronutrient deficiencies are associated with a number of non-communicable diseases, including:

- Overweight and obesity

- Cardiovascular disease (heart disease) and stroke.

- Type 2 diabetes and hypertension (high blood pressure).

- Certain types of cancer, including oesophageal cancer; trachea, bronchus and lung cancer; lip and oral cavity cancer; nasopharyngeal cancer; colorectal cancer [Health effects of dietary risks in 195 countries, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. The Lancet, S0140673619300418. doi:10.1016/S0140-6736(19)30041-8].

Diets with limited variety and low nutrient content can also negatively affect a healthy immune system. Malnutrition or a diet deficient in one or more nutrients can impair the production and activity of immune cells. The inflammatory mechanisms that make up innate immunity are highly dependent on nutritional quality, and if this interaction is disrupted, it can have a major impact on disease development.

The inflammatory process can usually be initiated by various stimuli that activate a physiological response in order to restore homeostasis. Interrelated immune response pathways, the innate and adaptive responses, promote each other, protecting the body from pathogens or stress factors that can initiate an immune response [Belikov AV, Schraven B, Simeoni LT. T cells and reactive oxygen species. J Biomed Sci. (2015) 22:85. doi:10.1186/sl2929-015-0194]. Under physiological conditions, the correct function of T cells is guaranteed by the balance between reactive oxygen species (ROS) and antioxidant systems; thus, T cells demonstrate a controlled immune response. However, the accumulation of extracellular ROS and reactive nitrogen species (RNS) can alter immune responses, which can trigger a systemic inflammatory response, mainly induced by oxidative stress (OS) [Colitti M, Stefanon B, Gabai G, Gelain ME, Bonsembiante F. Oxidative stress and nutraceuticals in the modulation of the immune function: current knowledge in animals of veterinary interest. Antioxidants. (2019) 8:28. doi:10.3390/antiox801002]. Oxidative stress occurs when the body has elevated levels of reactive oxygen species (ROS), but normal or low levels of antioxidants [Schieber, Michael; Chandel, Navdeep S. (2014). ROS Function in Redox Signaling and Oxidative Stress. Current Biology, 24(10), R453R462. doi:10.1016/j.cub.2014.03.034]. ROS are by-products of aerobic metabolism, including immune system processes. ROS are chemically reactive toward a variety of biological targets. Too much ROS can cause membrane lipid peroxidation and damage proteins, enzymes, carbohydrates, and DNA. The term oxyinflammation has recently been coined to explain the thread linking chronic and systemic oxidative stress, which negatively affects the immune state, increasing susceptibility to diseases [Valacchi G, Virgili F, Cervellati C, Pecorelli A. Oxinflammation: from subclinical condition to pathological biomarker. Front Physiol. (2018) 9:858. doi:10.3389/fphys.2018.00858].

The human body has certain mechanisms to combat excess free radicals that cause irreversible destructive effects on the body. The body's main line of defense against superoxides are antioxidants. These antioxidants inhibit cellular damage primarily by their ability to scavenge free radicals [Halliwell B. How to characterize an antioxidant- An update. Biochem Soc Symp. 1995; 61:73- 101]. Low molecular weight antioxidants can safely interact with free radicals and stop the chain reaction before vital molecules are damaged. Some of these antioxidants, including glutathione, ubiquinol, and uric acid, are produced during normal metabolism in the body [Shi HL, Noguchi N, Niki N. Comparative study on dynamics of antioxidant action of a-tocopheryl hydroquinone, ubiquinol and a-tocopherol, against lipid peroxidation. Free Radic Biol Med. 1999; 27:334-46]. Other lighter antioxidants are found in the diet. Although there are several enzyme systems in the body that remove free radicals, the main trace element (vitamin) antioxidants are vitamin E (a-tocopherol) and other tocopherols with stronger antioxidant activity, but those that do not have E-vitamin activity: y-tocopherol, 8-tocopherol, vitamin C (ascorbic acid), and 0-carotene [Levine M, Ramsey SC, Daruwara R. Criteria and recommendation for Vitamin C intake. JAMA. 1991; 281:1415-23]. The body cannot produce these substances, so they must be obtained from food. For example, vegetables and fruits, especially if they are not chemically processed, contain a large number of substances with antioxidant properties. Therefore, diets based on vegetables and fruits are able to bring more of these substances into the body than more common ones, such as the so-called "cafeteria diet". The above-mentioned diets can be characterized by the presence of plant products that have lost some of their antioxidant properties and are rich in "empty" calories due to their refined form; Moreover, excess animal products are associated with microbial changes and production of inflammatory metabolites that contribute to an increased risk of obesity and other non-communicable diseases (cardiovascular diseases, diabetes, osteoarthritis, asthma, etc.) [Rios-Hoyo A. etc. Obesity, Metabolic Syndrome, and Dietary Therapeutical Approaches with a Special Focus on Nutraceuticals (Polyphenols): A Mini-Review. Int. J. Vitam. Nutr. Res. 2014; 84:0113-0123. doi: 10.1024/0300-9831/a000198.]. Epidemiological studies show that consumption of healthy foods currently remains too low. Total vegetable intake is 40% below the recommended three servings per day, fruit intake is 60% below the recommended two servings per day, and legumes and nuts intake is 68-74% below the recommended one to two servings. Red and processed meat intake is almost five times higher than the recommendations. Only milk and fish intake are within the recommended limits. In addition, about half of the world's population (48%) consumes too many or too few calories and has an unbalanced weight level [Miller V, Singh GM, Opora J, et al. Global Dietary Database 2017: data availability and gaps on 54 major foods, beverages and nutrients among 5.6 million children and adults from 1220 surveys worldwide. BMJ Glob Health 2021; 6: e003585]. Thus, unbalanced nutrition can lead to a lack of useful and necessary macro- and micronutrients for human health, which entails the development of various pathological conditions, including the accumulation of ROS and RFA in the body and, as a consequence, the development of inflammation and immune disorders. One of the solutions to the problem may be the use of biologically active additives.

Dietary supplements are products that are taken in addition to the normal diet to provide the body with health benefits. food substances. According to the Dietary Supplement Health and Education Act (DSHEA), a dietary supplement is a product that is intended to supplement the diet; contains food ingredients including vitamins, minerals, amino acids, herbal extracts; is intended to be taken orally in tablet, capsule or liquid form; and is labeled as a dietary supplement [Ronis MJ, Pedersen КВ, Watt J (2018) Adverse effects of nutraceuticals and dietary supplements. Annu Rev Pharmacol Toxicol 58:583-601]. According to the National Medicines Agency, dietary supplements are divided into two categories depending on their intended use [Analyses by EUFIC (2009) Dietary guidelines based food in Europe, 2009]:

- Biologically active additives as a food product that complements the normal diet.

- Food products (functional and specialized) for special nutritional use, such as beverages, which, due to their special composition, are intended for specific groups of the population, for example, for maintaining the health of infants or children aged two to five years or for special categories of persons with metabolic disorders or for categories of persons in special physiological conditions.

Supplements and/or functional products can also be differentiated according to their origin (natural or synthetic). They are classified according to the form in which they are available in the following types: a) vitamin and mineral supplements, whether combined in the form of multivitamins or multiminerals or not, b) protein supplements in liquid form, with or without carbohydrates, fats, vitamins and minerals, c) amino acids of any form and composition, d) weight gain supplements, e) meal replacements in the form of powder, wafers or cookies, f) carbohydrate supplements with or without electrolytes and vitamins, g) supplements that have a natural anabolic effect and that are not included in the "list of prohibited substances", h) supplements that activate growth hormones and other hormones, i) polyunsaturated fatty acid supplements, j) food products or food ingredients such as yeast, garlic, algae, royal jelly, k) plant extracts.

Dietary supplements are widely available on the market. Many of them are quite effective, others are moderately effective, and some provide very little benefit. The main reason for their low effectiveness is their production method and the source of their active ingredients [Rovira MA, Grau M, Castaner O, Covas MI, Schroder H, Investigators REGICOR (2013) Dietary supplement use and health-related behaviors in a Mediterranean population. J Nutr Educ Behav 45(5):386–391]. The lack of reliable data on the safety of supplements or adulteration are reasons why supplements may cause serious side effects [Regina Ewa. W. Dietary Supplements - For Whom? The Current State of Knowledge about the Health Effects of Selected Supplement Use. Int. J. Environ. Res. Public Health 2021, 18(17), 8897].

For example, a large number of consumer complaints concerned green tea extract, which could cause liver damage in more than 50 people. For example, Exolise®, a weight loss supplement containing green tea extract, was withdrawn from the market in Spain and France in 2003 [Wharton, S.; Bonder, R.; Jeffery, A.; Christensen, R.A.G. The safety and effectiveness of commonly-marketed natural supplements for weight loss in populations with obesity: A critical review of the literature from 2006 to 2016. Crit. Rev. Food Sci. Nutr. 2019, 60, 1614-1630]. Another example is Dexaprine, which was a multi-ingredient supplement with green tea extract, which was recalled in 2014 in the Netherlands because even half a tablet caused vomiting, anxiety, and tachycardia [Koncic, M.Z. Getting More Than You Paid For: Unauthorized “Natural” Substances in Herbal Food Supplements on EU Market. Planta Med. 2018, 84, 394-406]. Supplements containing garcinia cambogia may also pose a threat to the patient’s life. An example is the supplement Hydroxycut, which led to liver damage, cardiac arrhythmia, and death [Lunsford, K.E.; Bodzin, A.S.; Reino, D.C.; Wang, H.L.; Busuttil, R.W. Dangerous dietary supplements: Garcinia cambogia-associated hepatic failure requiring transplantation. World J. Gastroenter. 2016, 22, 10071-10076] and the supplement Thermatrim, which was suspected of causing toxic leukoencephalopathy [Escamilla-Ocanas, C.E.; Cantu-Martinez, L.; Martinez, H.R.; Camara-Lemarroy, C.R. Acute toxic leukoencephalopathy associated with a non-prescription weight loss supplement: A report of two cases. Neurol. Sci. 2017, 38, 2199- 2201]. Kratom is another example of a dangerous muscle-building dietary supplement, which was removed from the US market in 2018 after it caused the death of a young man [Kennett, G. Time for change: Stepping up the FDA’s regulation of dietary supplements to promote consumer safety and awareness. J. Law Health 2019, 33, 47-78].

Thus, the unbalanced diet of modern man, including a large amount of harmful and unhealthy food and leading to a lack of essential macro- and micronutrients, insufficient consumption of antioxidant-rich foods, as well as a large number of not only ineffective, but also often unsafe supplements found on the market, are important reasons for the need to create effective, accessible and safe for the general public biologically active supplements that can replenish the lack of nutrients, strengthen the body's antioxidant defense and support weakened immunity. The antioxidant activity of foods and beverages has become a widely and actively studied subject in the scientific community due to the growing evidence of the importance of antioxidant systems for human health. Antioxidants have become interesting (from a scientific point of view) compounds due to their numerous benefits, such as anti-aging and anti-inflammatory effects. One of the main functions of antioxidants is to neutralize ROS (and sometimes ROS) in cells [Abuajah CI, Ogbonna AC, Osuji CM. Functional components and medicinal properties of food: a review. J Food Sci Technol. 2015; 52:2522–2529]. ROS include superoxide anion (O2 'X hydrogen peroxide (H2O2) and hydroxyl radicals (OH'), each of which has unique chemical properties that confer reactivity on various biological targets. ROS are often associated with increased levels of oxidative stress, which causes pathological conditions by damaging lipids, proteins, and DNA. The human body has mechanisms that help prevent the initiation of the pathological process of cellular oxidation. One such important mechanism for protecting against oxidative stress is a group of antioxidant enzymes [Sies H. Oxidative stress: Oxidants and antioxidants. Exp Physiol. 1997; 82:291-5]. Thus, superoxide released as a result of processes such as oxidative phosphorylation is first converted to hydrogen peroxide and then reduced to form water. This detoxification pathway is the result of the work of many enzymes: the first step is catalyzed by superoxide dismutase, and then catalases and various peroxidases that remove hydrogen peroxide.

Superoxide dismutase (SOD) is one of the most important enzymes for the implementation of antioxidant protection of the body. SOD is involved in the disproportionation of superoxide free radicals inside and outside the cells, due to which the cell membrane and DNA are protected from damage by oxygen free radicals [Tikhonov, IV; Pliss, E.M.; Borodin, LI; Sen', VD Effect of superoxide dismutase on the oxidation of methyl linoleate in micelles inhibited by nitroxyl radicals. Russ. Chem. Bull. 2016, 65, 2985-2987]. Superoxide dismutase plays an important role in balancing the concentration of biological oxidants, which can protect all aerobic organisms from oxidative damage caused by biological oxidants. SOD is one of the most important antioxidant enzymes, which can catalyze O2»' to form H2O2 to regulate signaling in the body. H2O2 can be used as a second messenger to regulate a variety of signaling processes in inflammation, angiogenesis and other processes that affect the expression of many genes in physiological and pathological conditions. states. In addition, H2O2 diffuses across the cell membrane via aquaporin water channels (AQPs), transferring redox signals from the site where the redox signal is generated to the site of interest [Bienert, GP; Moller, AL; Kristiansen, K.A.; Schulz, A.; Moller, IM; Schjoerring, JK; Jahn, TP Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J. Biol. Chem. 2007, 282, 1183-1192].

The ability to scavenge radicals at very high reaction rates is due to the transition metal ions present in the active site. The SOD free radical scavenging process requires cofactors such as iron, manganese, copper or zinc to exert maximum catalytic activity in the metabolism of toxic intermediates. The metal binding site is located between the two SOD domains and the side chains include aspartate, histamine and histidine. These cofactors tend to donate electrons to O2*' and are regenerated as part of the catalytic mechanism.

Three different SOD isoforms have been identified: SOD1 (CuZn-SOD), localized in the cytoplasm, in the catalytic site of which copper and zinc are present, SOD2 (Mn-SOD), localized in the mitochondria, the cofactor of which is manganese, and, finally, SOD3 (EC-SOD), an extracellular antioxidant enzyme, which, like SOD1, contains copper and zinc in its catalytic center. Thus, it can be said that zinc and copper ions are among the important microelements necessary for the normal functioning of the antioxidant system of the body. Thus, in addition to the fact that zinc is a cofactor of SOD1 and SOD3, it also helps to maintain sulfhydryl groups of proteins [Bray, T.M., Bettger, W.J., 1990. The physiological role of zinc as an antioxidant. Free Radic. Biol. Med. 8, 281-291], protection against vitamin E depletion, stabilization of membrane structure and maintenance of tissue concentrations of metallothioneins, potent free radical scavengers [DiSilvestro, RA, 2000. Zinc in relation to diabetes and oxidative disease. J. Nutr. 130 (5S Suppl.), 1509S-1511S]. It was found that zinc deficiency apparently causes a mutant conformation of superoxide dismutase, which causes chronic endoplasmic reticulum stress [Notta, K.; Fujisawa, T. SOD1 as a molecular switch for initiating the homeostatic ER stress response under zinc deficiency. Mol. Cell. 2013, 52, 75- 86]. Consequently, this leads to inhibition of protein synthesis and induction of the zinc transporter Zip-14. The research also demonstrated that after zinc supplementation, the activity of Zn-dependent enzymes (SOD1 and SOD2) and plasma zinc concentrations were significantly higher than before supplementation. The results were not influenced by age, gender, or geographic area. These data suggest a potential beneficial effect of zinc supplementation on Zn-dependent antioxidant enzymes in healthy older adults. [E. Mariani etc. (2008). Effects of zinc supplementation on antioxidant enzyme activities in healthy old subjects, 43(5), 445-

451. doi:10.1016/j.exger.2007.10.012]. Another mechanism of action of zinc as an antioxidant is through its effect on the expression of glutamate-cysteine ligase, which is the rate-limiting enzyme for de novo glutathione synthesis. This results in a dual effect of free radical scavenging, either directly by glutathione or indirectly via the cofactor glutathione peroxidase [Eide, D.J. The oxidative stress of zinc deficiency. Metallomics 2011, 3, 1124-1129]. It has been previously demonstrated that administration of 100-150 mM zinc to cultured human retinal pigment epithelial ARPE-19 cells increases the mRNA level of glutamate-cysteine ligase via nuclear factor erythroid 2 (NFE2) [На, K.N.; Chen, Y. Increased glutathione synthesis through an ARE-Nrf2 -dependent pathway by zinc in the RPE: Implication for protection against oxidative stress. Investig. Ophthalmol. Vis. Sci. 2006, 47, 2709-2715]. In this regard, it can be said that zinc modulates the total concentration of glutathione in cells [Foster, M.; Samman, S. Zinc and redox signaling: Perturbations associated with cardiovascular disease and diabetes mellitus. Antioxid. Redox. Signal. 2010, 13, 1549-1573]. Thus, zinc supplements can be useful for providing powerful antioxidant protection of the body.

Another important element for maintaining the body's antioxidant system is copper. The body uses copper to produce energy and to maintain healthy bones, skin, nervous system, and cardiovascular system.* Along with zinc, copper is a cofactor for the superoxide dismutase enzymes SOD1 and SOD3. Although copper deficiency is rare, it may result from high zinc intake due to a decreased ability to absorb and utilize copper from the diet [Festa M, Anderson H, Dowdy R, Ellersieck M. Effect of zinc intake on copper excretion and retention in men. Am J Clin Nutr 1985; 41(2):285-29], [Samman S, Roberts D. The effect of zinc supplements on plasma zinc and copper levels and the reported symptoms in healthy volunteers. Med J Aust 1987; 146(5):246-249]. A number of studies have examined the relationship between zinc intake and copper status. These studies analyzed the amount of zinc and the duration of supplementation. Daily supplementation with 60 mg zinc for 10 weeks (well above the upper limit of the recommended daily intake) was associated with decreased copper status [Yadrick M, Kenney W, Winterfeldt E. Iron, copper, and zinc status: response to supplementation with zinc or zinc and iron in adult females. Am J Clin Nutr 1989; 49(1): 145-150].

Additional studies show that copper supplements with zinc help to balance the absorption of both nutrients [August D, Janghorbani M, Young V. Determination of zinc and copper absorption at three dietary Zn-Cu ratios by using stable isotope methods in young adults and elderly subjects. Am J Clin Nutr 1989; 50(6): 1457- 1463]. Thus, adding copper ions to zinc supplements will prevent copper deficiency in the body.

In addition to known micronutrients such as zinc and copper, and vitamins, there are a wide variety of parapharmaceuticals (as opposed to nutraceuticals) that, according to numerous studies, may have a beneficial effect on maintaining healthy functions in the human body. One of these beneficial phytochemicals are flavonoids. Anthocyanins, which are the most important group of flavonoids in plants, are pigments with a flavylium cation structure (AH ⁺ ) that act as an acid. This structure is directly related to their antioxidant activity. Anthocyanins are present in blue, red, and purple vegetables, fruits, and flowers, and fruits with the highest concentrations of anthocyanins include berries, currants, grapes, and some tropical fruits. The main anthocyanins found in the human diet are the glycoside forms of anthocyanidins, which include pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin [H.E. Khoo, A. Azlan, S.T. Tang, S.M. Lim, Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits, Food Nutr. Res. 61 (2017)]. There is an inverse correlation between fruit and vegetable consumption and cardiovascular disease, which is believed to be due in part to their content of polyphenols, such as anthocyanins [A. Scalbert, C. Manach, C. Morand, C. Remesy, L. Jimenez, Dietary polyphenols and the prevention of diseases, Crit. Rev. Food Sei. Nutr. 45 (2005) 287–306.] Anthocyanins were once thought to be poorly bioavailable, but it has been demonstrated that the parent compounds are immediately absorbed and converted to bioactive metabolites that remain in the circulation [C. Czank, et al., Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a 13C-tracer study, Am. J. Clin. Nutr. 97 (2013) 995–1003]. Thus, the bioavailability of anthocyanins is higher than previously thought given their biotransformation. Dietary anthocyanin intake has been associated with a reduction in cardiovascular disease risk factors, as well as a lower risk of coronary heart disease (CHD) and cardiovascular mortality [SS Hassellund, et al., Effects of anthocyanins on cardiovascular risk factors and inflammation in pre-hypertensive men: a doubleblind randomized placebocontrolled crossover study, J. Hum. Hypertens. 27 (2013) 100-106]. It has been shown in the literature that anthocyanins extracted from plants have antioxidant properties. Pelargonidin-3-glucoside, cyanidin-3-glucoside and delphinidin-3-glucoside isolated from the seed coat of Phaseolus vulgaris L. (black bean), as well as their standard aglycones, have strong antioxidant activity in the liposomal system and reduce the formation of malondialdehyde upon irradiation with UVB rays [Tsuda T, Shiga K, Ohshima K, et al. Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L. Biochemical Pharmacol. 1996; 52(7): 1033- 1039]. The study also shows that delphinidin and delphinidin-3-glucoside have the greatest inhibitory effect on lipid peroxidation and increase the activity of O2 scavenging. However, as demonstrated earlier, the consumption of vegetables and fruits as the main anthocyanin-containing products is insufficient in the diet of modern humans, and therefore the addition of anthocyanin-containing dietary supplements to the compositions may be a good strategy for providing the human body with sufficient levels of antioxidants in the blood.

Combining components with different mechanisms of action into one product may be a good strategy for enhancing the body's antioxidant defense. Thus, additional use of various parapharmaceuticals with antioxidant activity can contribute to improvements in antioxidant functions [Zhang, Y.-J., Gan, R.-Y., Li, S., Zhou, Y., Li, A.-N., Xu, D.-P., & Li, H.-B. (2015). Antioxidant Phytochemicals for the Prevention and Treatment of Chronic Diseases. Molecules, 20(12), 21138-21156. doi:10.3390/molecules201219753]. However, there are a number of limitations to the combined use of trace elements such as zinc and copper with polyphenolic compounds. Thus, it has been demonstrated in literary sources that anthocyanins have the ability to bind divalent metals and remove them from the body [Havsteen, B. (1983). Flavonoids, a class of natural products of high pharmacological potency. Biochemical Pharmacology, 32(7), 1141-1148]. Flavonoids, including anthocyanins, have a strong affinity for divalent ions of heavy metals, which also include copper and zinc, since the position of the hydroxyl substituents and the electronic properties of the ring system suggest that flavonoids can be good ligands for the d3 electrons of transition elements of the fourth period. For example, a fairly simple, sensitive, cheap and fast

AND analytical tool for the determination of metals in environmental samples, especially in the case of copper and zinc [Josephine N. Ike et al. Application of Cyanidin in Quantitative Estimation of Metals in Fish Samples. American Journal of Analytical Chemistry. Vol.10 No.12, December 2019]. Also, a number of studies have demonstrated that polyphenolic compounds present in A. melanocarpa fruits may be responsible for the capture of divalent cadmium ions Cd ²⁺ in the lumen of the digestive tract, which in turn reduced its absorption and bioavailability [Ling-Peng Dai et al. Antioxidative and Chelating Properties of Anthocyanins in Azolla imbricata Induced by Cadmium. Pol. J. Environ. Stud. 2012; 21(4):837-844]. Thus, the combination of divalent metal ions with polyphenolic compounds, including anthocyanins, may, on the contrary, worsen the bioavailability of these microelements and prevent their interaction with target targets, which, in turn, will be reflected in the assessment of clinical indicators in patients taking this supplement.

Theoretically, it can be assumed that flavonoids will “capture” and inhibit the absorption of zinc and copper worse if zinc and copper are already bound to amino acids. Bisglycinate forms of minerals are ion-neutral molecules that have a low degree of dissociation and are absorbed in the duodenum through two types of transporters (ionic and peptide - in contrast to standard forms, for example, citrate, lactate, etc., which are absorbed mainly through the ionic transporter).

However, possibly confirming the hypothesis, the authors of the present invention unexpectedly discovered that the present composition, containing bioavailable (chelated) forms of copper and zinc in the form of bisglycinates, elderberry extract standardized for anthocyanin content, and L-ascorbic acid, helps improve the antioxidant profile in the body, as demonstrated by an increase in superoxide dismutase in the blood of most volunteers who tested the supplement for 3 weeks, as well as zinc absorption, which is reflected in an increase in alkaline phosphatase activity as an indirect biomarker of zinc status in the blood (Example 1) [S. Samman; C. Soto; L. Cooke; Z. Ahmad; E. Farmakalidis (1996). Is erythrocyte alkaline phosphatase activity a marker of zinc status in humans? 51 (3), 285-291. doi: 10.1007/bf02784083]. Example 1 also demonstrated a general decrease in the group of such an indicator as circulating immune complexes. Circulating immune complexes (CIC) are a heterogeneous group of immunoreactants formed as a result of a non-covalent union of an antigen and an antibody. The level of CIC is one of the indicators of the immune system and autoimmune symptoms. The study proved that the level of CIC increases consistently with the level of immunoglobulins. This proves the development of a humoral response to antigens and the progression of an autoimmune disease with the development of vascular, pulmonary or renal complications [Gaiduchok, L, Fedorov, Y., Lishchuk-Yakymovych, K., & Pukalyak, R. (2020). Condition of humoral immunity in patients with systemic diseases of connective tissue with activated chronic simple herpetic infection. Immunology and Allergology: Science and Practice, (1), 64-71. https://doi.org/10.37321/immunology.2020.01-09]. An increased level of CIC is due not only to an increase in its production, but also to a violation of its elimination. The phagocytic system is responsible for the neutralization and extraction of immune complexes. With normal functioning of mononuclear and tissue phagocytes, an increased level of CIC does not cause its accumulation in organs and the bloodstream. However, any failure in phagocytosis leads to serious consequences. O2 absorption, glucose consumption and lactic acid accumulation increase during phagocytosis of immune complexes. This is accompanied by oxidative stress and loss of a large amount of energy [Mekhno N, Yaremchuk O (2023) Impact of nitric oxide synthesis modulators on the state of humoral immune system in experimental antiphospholipid syndrome. Pharmacia 70(1): 9-14. https://doi.org/10.3897/pharmacia.70.e94246]. Thus, a decrease in the level of circulating immune complexes may indicate a decrease in the level of oxidative stress in the body.

The biologically active supplement L-OptiZinc from the manufacturer Now Foods (USA) is known. The composition of this supplement includes zinc in the form of zinc monomethionine and chelated copper. The manufacturer assures the normalization of the immune system, help in reducing cholesterol levels, promoting the rapid elimination of inflammation, a beneficial effect on the quality of vision, promoting the saturation of cells with oxygen, improving the digestive process, helping the endocrine system, producing enzymes and hormones as effects on the body. However, the manufacturer does not indicate the antioxidant effect of the system as a potential effect, and the composition does not contain additional components aimed at enhancing the antioxidant effect due to a mechanism of action different from zinc, which can contribute to a synergistic effect in preventing oxidative stress.

The biologically active supplement Zinc-Vitamin C from Evalar (Russia) is known [https://shop.evalar.ru/catalog/vitaminy-i-mineraly/zink-vitamin-c/offer-5220/]. Vitamin C is indicated in the form of ascorbic acid, and zinc in the form of zinc lactate. The manufacturer claims that this product will ensure the smooth operation of the body's defenses throughout the entire period of seasonal colds. However, the manufacturer does not use the most bioavailable form of zinc, as it was It has been established that the most absorbable form of zinc is bisglycinate [J. Osko, W. Pierlejewska, M. Grembecka. Comparison of the Potential Relative Bioaccessibility of Zinc Supplements-In Vitro Studies. Nutrients. 2023 Jun; 15(12): 2813]. Also, this product does not contain any parapharmaceutical components that could enhance the antioxidant activity of zinc and vitamin C, and does not contain copper, which, with long-term use of such a supplement, can disrupt the balance of copper and zinc in the body and cause alimentary diseases associated with copper deficiency.

A well-known biologically active supplement in the form of lozenges for adults with an immunity complex from the company LIVS (Israel) [https://www.eapteka.ru/goods/id515537/]. These lozenges contain sugar, glucose syrup (tapioca syrup), black elderberry fruit extract, water, L-ascorbic acid (vitamin C), pectin, citric acid (acidity regulator), sodium citrate (acidity regulator), natural flavoring (elderberry), black carrot juice concentrate (natural dye), cholecalciferol (vitamin D3), zinc citrate. According to the manufacturer, this product is aimed at maintaining immunity during the cold season. However, the main disadvantage of this product is the content of simple sugars, which, on the contrary, are known to induce the generation of ROS, which negates the effect of the antioxidant components in the product [Prasad, Kailash; Dhar, Indu (2014). Oxidative Stress as a Mechanism of Added Sugar-Induced Cardiovascular Disease. International Journal of Angiology, 23(4), 217-226. doi: 10.1055/s-0034-1387169]. Also, this product does not contain the most bioavailable form of zinc (zinc citrate), and the standardization of polyphenolic components in the elderberry extract is not indicated, which is a significant drawback due to the dependence of the extraction method and the percentage of active molecules in the composition [X.Liu et al. Effects of five extraction methods on total content, composition, and stability of flavonoids in jujube. Food Chem X. 2022 Jun 30; 14: 100287].

A well-known supplement in the form of effervescent tablets with zinc, elderberry and vitamin C from the company Evalar (Russia) [https://shop.evalar.ru/catalog/vitaminy-i-mineraly/elderberry-immunity/offer-869335/]. The main effect indicated by the manufacturer is strengthening the immune system. The composition of this product includes citric acid and sodium bicarbonate (acidity regulators), sorbitol (carrier), maltodextrin (thickener), powdered black elderberry juice, ascorbic acid (vitamin C), beet extract (natural dye), natural flavor Berry mix, dextrose, polyethyleneglycol (carrier), zinc sulfate, sucralose (sweetener), poly dimethyl siloxane (antifoam). However, the disadvantages of this product are use of black elderberry juice instead of standardized extract. Concentrated juices, compared to extracts, have a lower content of biologically active substances, which are present in greater quantities in standardized fruit extracts. The manufacturer also uses inorganic zinc salt (zinc sulfate), which, in addition to low bioavailability, has a high potential for the development of side effects from the gastrointestinal tract in humans. The manufacturer also does not add copper to the composition of the product, which can negatively affect the copper-zinc balance in the body.

A well-known biologically active supplement with elderberry extract, vitamin C and zinc from the OVER company (Russia) [https://www.ozon.ru/product/buzina-immunitet-s-tsinkom-vitaminom-s-i-d3-bad-dlya-immuniteta-60-kapsul-1055479906/]. The manufacturer claims that this supplement will strengthen the immune system, provide antioxidant protection for the body and improve the condition of the skin. The product contains the following active ingredients: elderberry extract, zinc citrate, ascorbic acid (vitamin C), cholecalciferol (Vitamin D3). The main disadvantage of this product is the lack of copper, which can negatively affect the copper-zinc balance in the body. Also, the zinc citrate form is not the most digestible compared to zinc bisglycinate. It is worth noting that the manufacturer did not indicate the standardization of elderberry extract for active substances, which gives reason to doubt the content of effective polyphenolic molecules capable of providing the effects declared by the manufacturer.

All of the above-mentioned disadvantages are eliminated in the present invention by combining into a single composition a small number (four) of components that have fundamentally different mechanisms of antioxidant activity, which allows for a synergistic increase in the effectiveness of the antioxidant action, as well as not causing potential deficiencies in the body of other microelements when taken.

It is also known (Interacting elements - zinc-induced copper deficiency. Prescriber Update, Published 5 March 2020, 41(1):16-17 [https://www.medsafe.govt.nz/profs/PUArticles/March2020/Interacting-elements-zinc-induced-copper-deficiency.html] that zinc can inhibit copper absorption, which can interfere with copper absorption. It is also known (Cheryl Chung et al. Enhanced stability of anthocyanin-based color in model beverage systems through whey protein isolate complexation, Food Research International (2015), 76(Pt 3) doi:10.1016/j.foodres.2015.07.003) that vitamin C accelerates the degradation of anthocyanins. However, the combination of the invention surprisingly showed synergism of a zinc source, an anthocyanin source, a vitamin C source and a copper. ESSENCE OF THE INVENTION

1. A composition for reducing the level of oxidative stress in the body and improving the state of immune defense, containing: a) a source of zinc, b) a source of anthocyanins, c) a source of vitamin C, d) a source of copper.

2. The composition according to claim 1, wherein the zinc source is zinc bisglycinate.

3. The composition according to claim 1, wherein the source of anthocyanins is selected from standardized extracts obtained from plant sources such as elderberries, cranberries, blueberries, raspberries, black currants, cherries, and chokeberries.

4. The composition according to claim 1, wherein vitamin C is L-ascorbic acid.

5. The composition according to claim 1, wherein the copper source is copper bisglycinate.

6. The composition according to item 1, characterized in that it is in the form of a capsule.

7. The composition according to claim 1, containing: a) 20-30 mg zinc bisglycinate, b) 90-110 mg black elderberry extract standardized to 6% anthocyanins, c) 235-250 mg L-ascorbic acid, d) 0.30-0.45 mg copper bisglycinate.

8. The composition according to claim 7, containing: a) 25 mg zinc bisglycinate, b) 100 mg black elderberry extract standardized to 6% anthocyanins, c) 243 mg L-ascorbic acid, d) 0.39 mg copper bisglycinate. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a composition and its use for reducing the level of oxidative stress in the body and improving the state of immune defense. The composition according to the invention contains a combination of a) a zinc source, b) a source of anthocyanins, c) a source of vitamin C, d) a source of copper.

The composition according to the invention in a particular case comprises a zinc source which is zinc bisglycinate, such as having the CAS number 7214-08-6.

The composition according to the invention in a particular case contains a source of anthocyanins selected from standardized extracts obtained from plant sources such as elderberries, cranberries, blueberries, raspberries, black currants, cherries, and chokeberries. Such extracts may be commercially available extracts.

The composition according to the invention in a particular case contains vitamin C, which is L-ascorbic acid.

The composition of the invention in a particular case comprises a copper source which is copper bisglycinate, such as having the CAS number 13479-54-4.

The composition according to the invention may be characterized in that it is in the form of a capsule.

In one embodiment of the invention, the composition contains: a) about 20-30 mg zinc bisglycinate, b) about 90-110 mg black elderberry extract standardized to 6% anthocyanins, c) about 235-250 mg L-ascorbic acid, d) about 0.30-0.45 mg copper bisglycinate.

In one embodiment of the invention, the composition contains: a) 20-30 mg zinc bisglycinate, b) 90-110 mg black elderberry extract standardized to 6% anthocyanins, c) 235-250 mg L-ascorbic acid, d) 0.30-0.45 mg copper bisglycinate.

In one embodiment of the invention, the composition comprises: a) about 25 mg zinc bisglycinate, b) about 100 mg black elderberry extract standardized to 6% anthocyanins, c) about 243 mg L-ascorbic acid, d) about 0.39 mg copper bisglycinate. The zinc bisglycinate content may be 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 mg or any amount in between. The black elderberry extract content standardized to 6% anthocyanins may be 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 119, 120, or 121 mg, or any value between these amounts. The L-ascorbic acid content may be 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268 or 270 mg or any value between the specified. The copper bisglycinate content may be 0.27, 0.30, 0.33, 0.36, 0.39, 0.42, 0.45, 0.48 or 0.51 mg or any value between the specified.

Moreover, the term "about" means a deviation from the specified value by ±10%. That is, when, for example, about 10-20 mg is indicated, this is identical to an indication from about 10 to about 20 mg. At the same time, when, for example, about 10 mg is indicated, this is identical to an indication of 9.9-10.1 mg, which should be easily understood by specialists.

EXAMPLES

Example 1.

A test was conducted on a dietary supplement in the form of a capsule containing 25 mg zinc bisglycinate, which is a concentration of 4.7% of the total weight of the capsule, 0.39 mg copper bisglycinate, which is a concentration of 0.07% of the total weight of the capsule, 243 mg L-ascorbic acid, which is a concentration of 45.4% of the total weight of the capsule, 100 mg black elderberry extract standardized for anthocyanins (6%), which is a concentration of 18.7% of the total weight of the capsule. The auxiliary components used were gum arabic (carrier) at a concentration of 14.7% of the total capsule weight, hydroxypropyl methylcellulose (capsule shell, carrier) at a concentration of 14.2% of the total capsule weight, and a complex food additive (lecithin (emulsifier) and gum arabic (stabilizer)) at a concentration of 2.2% of the total capsule weight.

All components were carefully weighed, mixed and encapsulated before the test. The average age of the test participants was 38.3 years (minimum 25 years, maximum 75 years). The participants used the dietary supplement according to the instructions for use, 1 capsule 2 times a day for three weeks. Before and after the test, blood test data were collected for the following parameters: Superoxide dismutase (SOD) (U/g Hb), Alkaline phosphatase (ALP) (U/L), Circulating immune complexes (U/L). Blood samples were collected in morning time on an empty stomach, were analyzed in one authorized laboratory. The results were processed using the standard functionality for statistical processing of MS Excel (Table 1).

Table 1. Values of antioxidant indicators in the blood before and after testing the dietary supplement

In 67% of participants after 3 weeks of using the dietary supplement, the level of the enzyme superoxide dismutase, which is zinc- and copper-dependent, increased, indicating an increase in the level of zinc and copper and their bioavailability; the level of alkaline phosphatase, which is known to be zinc-dependent enzyme and can serve as a biomarker of zinc content in human blood. The results of the analysis of the level of CIC in the blood demonstrated a decrease in the overall indicator for the group by 15.7%, which indicates an increase in the humoral immune status of the test participants.

Claims

CLAUSE OF INVENTION 1. A composition for reducing the level of oxidative stress in the body and improving the state of immune defense, containing: a) a source of zinc, b) a source of anthocyanins, c) a source of vitamin C, d) a source of copper. 2. The composition according to claim 1, wherein the zinc source is zinc bisglycinate. 3. The composition according to claim 1, wherein the source of anthocyanins is selected from standardized extracts obtained from plant sources such as elderberries, cranberries, blueberries, raspberries, black currants, cherries, and chokeberries. 4. The composition according to claim 1, wherein vitamin C is L-ascorbic acid. 5. The composition according to claim 1, wherein the copper source is copper bisglycinate. 6. The composition according to item 1, characterized in that it is in the form of a capsule. 7. The composition according to claim 1, containing: a) 20-30 mg zinc bisglycinate, b) 90-110 mg black elderberry extract standardized to 6% anthocyanins, c) 235-250 mg L-ascorbic acid, d) 0.30-0.45 mg copper bisglycinate. 8. The composition according to claim 7, containing: a) 25 mg zinc bisglycinate, b) 100 mg black elderberry extract standardized to 6% anthocyanins, c) 243 mg L-ascorbic acid, d) 0.39 mg copper bisglycinate.