RU2846480C1 - Biologically active additive of vegetal origin and its use for prevention of viral, cardiovascular diseases and optimization of metabolism - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to a biologically active additive and use thereof for preventing viral, cardiovascular diseases and optimizing metabolism. Biologically active additive containing the following active components, wt. %: 4.275-4.5 of gallate epigallocatechin, 1.33-1.4 of formononetin, 8.645-9.1 of oleuropein, 16.15-17.0 of methyl-β-cyclodextrin, 64.4-68.0 of quercetin. Using the above biologically active additive for preventing viral, cardiovascular diseases and optimizing metabolism.

EFFECT: group of inventions makes it possible to considerably improve the quality of life of patients.

7 cl, 6 dwg, 3 tbl, 3 ex

Description

The invention relates to medicine, namely to the creation of food additive compositions for therapeutic and dietary use. The present composition of natural plant compounds has a wide range of positive effects, including antioxidant, anti-inflammatory, anti-cancer, antibacterial and antiviral. In addition to these qualities, the main criterion for selecting the components of the composition was their ability to suppress endocytosis and, thus, the transport of various pathogenic factors into the cell.

The transport of nutrients, metabolites, large fragments, signaling molecules, bacteria, and viruses into cells occurs either by direct interaction with the cell membrane or by binding to receptors embedded in the membrane. However, the physical transport of these objects into cells is determined by a mechanism called endocytosis. In endocytosis, the membrane forms folds that surround receptors, large fragments, or droplets of extracellular fluid. These folds eventually form vesicles (endosomes) that are released into the cells.

Thus, endocytosis represents a natural therapeutic target for a wide range of acute and chronic pathological conditions that can be counteracted by transient non-specific inhibition of cell-to-cell communication. Inhibition of endocytosis has been shown to be beneficial in a variety of pathologies, including acute myocardial infarction, stroke, and viral infections, including COVID. The natural compounds in the proposed mixture target various aspects of endocytosis (including phagocytosis, pinocytosis, clathrin, and caveolin-dependent endocytosis) and thus may provide broad protection against most pathogens.

The composition includes the following components of plant origin: Epigallocatechin gallate, EGCG (inhibitor of caveolin-dependent endocytosis), Formononetin (inhibitory effect on dynamin, involved in clathrin-dependent endocytosis), Oleuropein (caveolin-dependent endocytosis and lipid rafts), Methyl-β-cyclodextrin (suppression of endocytosis by reversible extraction of cholesterol from cell membranes) and Quercetin (suppression of liquid phase endocytosis, pinocytosis).

The proposed composition can be used as 1) an adjuvant component of therapies against a wide range of diseases and pathological conditions, including (among other things) infarction, stroke, other forms of acute ischemia, as well as acute conditions requiring protection of cells from internalization of toxic components of necrotic cells and bioactive molecules, 2) a prophylactic agent that allows to significantly reduce the possibility of penetration of viral or bacterial particles into the cell and 3) for the purpose of correcting metabolic syndrome and preventing its consequences.

A large number of different pharmaceutical compositions are known, aimed at the treatment and prevention of the diseases mentioned above.

Thus, a method for treating a patient with cancer is known, which includes: joint administration to said patient of a therapeutically effective amount of quercetin and sodium phenylbutyrate (SBP), wherein each of quercetin and sodium phenylbutyrate is administered intravenously, and wherein the dose of quercetin is from 0.5 to 1.5 g and the dose of sodium phenylbutyrate is from 1 to 10 g. (RU 2644635 C2, published 20.08.2015)

Patent RU 2242984 C2, published 27.12.2004, proposes a means for treating and preventing obesity. The means proposed include a green tea extract with a high content of catechins, which contains, in particular, from 20 to 50 wt.% of catechins, calculated as epigallocatechin gallate. A method for preventing and treating with means based on green tea extracts is also proposed. Catechins present in high concentrations in green tea extracts have an inhibitory effect on catechin-O-methyltransferase, while the concentration of caffeine in green tea extracts according to the claimed means inhibits phosphodiesterase, which leads to an increase in the activity of norepinephrine on thermogenesis.

The objective of the proposed invention is to create a pharmaceutical composition for the prevention and treatment of viral, cardiovascular diseases and optimization of metabolism, which has high efficiency and safety of use.

The technical result consists in achieving the action of the compounds included in the composition, as well as in a significant improvement in the quality of life of patients taking the claimed composition.

The technical result is achieved by the fact that a biologically active pharmaceutical composition of dietary supplements for the prevention and treatment of viral, cardiovascular diseases and optimization of metabolism is proposed, including the following active components:

Epigallocatechin gallate,

Formononetin,

Oleuropein,

Methyl-β-cyclodextrin,

Quercetin.

A biologically active pharmaceutical composition of dietary supplements for the prevention and treatment of viral, cardiovascular diseases and optimization of metabolism according to paragraph 1, characterized in that the composition contains the following active components in wt.%:

Epigallocatechin gallate 4,275-4,725 Formononetin 1.33-1.47 Oleuropein 8,645-9,555 Methyl-β-cyclodextrin 16.15-17.85 Quercetin 64.4-71.4

The use of the said biologically active pharmaceutical composition of dietary supplements in a therapeutically effective amount for the prevention and treatment of viral and cardiovascular diseases and for optimizing metabolism is proposed.

Brief description of the figures:

Fig. 1 - (A) - Diagram of the mechanisms of import of damage signals by endocytosis. (B) - Expected effect of the use of endocytosis inhibitors on the reduction of the volume of damage after ischemia-reperfusion;

Fig. 2 - Endocytic pathways involved in receptor-dependent and -independent import of damage by non-ischemic cells;

Fig. 3 - ROS production in HL-1 cardiomyocytes subjected to anoxia and reoxygenation. A - sample images - MitoSOX; Legend: C - control; A - anoxia; R - anoxia/reoxygenation;

Fig. 4 - ROS production in HL-1 cells incubated in media from hypoxic and reoxygenated cultures. Data are from 4-6 independent replicates and are expressed as mean ± SE. Key: C - control; CMA - conditioned medium from anoxic cultures; CMR - conditioned medium from anoxic/reoxygenated cultures;

Fig. 5 - Transferrin uptake analysis in HL-1 cells subjected to anoxia, reoxygenation, or conditioned medium. Key: C - control; CMA - conditioned medium from anoxia cultures; CMR - conditioned medium from anoxia/reoxygenation cultures;

Fig. 6 - Conditioned medium from reoxygenated cells enhances the cytotoxicity of TNFα and Ang II. Dynasore is also shown to reverse the negative effects of anoxia and anoxia/reoxygenation conditioned medium. Key: C - control; CMA - conditioned medium from anoxia cultures; CMR - conditioned medium from anoxia/reoxygenation cultures.

State of the art

The presence of a wide variety of endocytosis mechanisms allows the cell to capture both bulk particles and cellular fragments, as well as relatively small molecules, either selectively (as part of ligand-receptor complexes) or non-selectively, as a component of the extracellular environment (Fig. 1). Capture of large particles/fragments is carried out in the process of phagocytosis. Capture of the liquid phase by macropinocytosis includes capture of extracellular fluid with substances dissolved in it using membrane folds/protrusions directed into the extracellular space and folding on themselves, forming vesicles, macropinosomes. This mechanism is regulated by a wide variety of growth factors, such as platelet-derived growth factor (PDGF), which stimulates the actin-dependent process of membrane fold formation through activation of Rho GTPases. This is a mechanism of non-selective capture of molecular components of the extracellular environment, which does not depend on the recognition and capture of ligands by receptors.

Clathrin-mediated endocytosis (CME) is the canonical pathway for internalization of cell surface receptors following their binding to the appropriate ligand. Complete, systemic inhibition of clathrin pit formation is lethal. CME is involved in nutrient and ion exchange (i.e., LDL, transferrin, voltage-gated calcium channels), regulation of cell signal transduction (i.e., EGF, insulin, TGFβ, TNFα), and internalization of several other receptors of potential importance for understanding the concept of “import of damage”: beta-adrenergic receptor 2, interleukin-1 receptor (IL1R1), and angiotensin II receptor type 1 (AT1R).

Finally, caveolin-mediated endocytosis refers to the uptake of extracellular components via cell surface membrane microdomains laden with lipids and receptor proteins such as scavenger receptors (CD36, SR-B1) and receptors for folate, urokinase, PDGF, epithelial growth factor (EGF), advanced glycation end-products (AGEs), acetylcholine (M2), tissue factor, catecholamines, bradykinin, endothelin, and oxidized LDL receptor (LOX-1).

Proof-of-concept experimental data using the example of myocardial infarction modeling ( Khaidakov et al., 2014)

Myocardial infarction results from sudden blockage of a coronary artery and interruption of oxygenated blood flow to the irrigated region of the heart muscle. The immediate zone of myocardial infarction contains predominantly necrotic cardiomyocytes, characterized by swelling of cellular organelles, granulation of the cytoplasm, DNA fragmentation, and disruption of the integrity of the plasma, mitochondrial, and lysosomal membranes of the cell. Although ischemic cells with signs of apoptosis (some of which are restored upon reperfusion) are also present in this zone, most of these myocytes are located in the peri-infarction zone, which is much more variable in size.

Stopping blood flow and oxygen supply to the myocardium leads to cessation of aerobic metabolism and, for a short time, increased glycolysis, with subsequent disruption of the main mechanisms of ion exchange, acidification of the cell cytoplasm, accumulation of intracellular Na+ and Cl- and water, and osmotic damage to cell membranes and cellular organelles. Moreover, collapse of the Ca2+ gradient leads to disruption of cardiomyocyte contractility already at the earliest stages of hypoxia.

Resumption of oxygen supply normalizes cytoplasmic pH and leads to partial restoration of ion pump activity. However, disturbances in intracellular Ca2+ metabolism persist or (in the sarcoplasmic reticulum and mitochondria) even increase. Reperfusion causes a sharp increase in the production of active oxygen and massive oxidative damage to cellular components. Ischemia activates hypoxia inducible factor 1 alpha (HIF-1α), which stimulates numerous targets, including genes involved in the control of anaerobic metabolism, angiogenesis, autophagy, and cytokine production. Thus, the products released in the necrotic zone can be divided into products of the early stress response of cardiomyocytes to hypoxia and reoxygenation and molecules and organelles damaged and/or modified as a result of massive oxidative stress developing already during the necrotic phase of infarction.

During hypoxia/reoxygenation, necrotic myocardial cells secrete a complex mixture of soluble factors, including VEGF, TNFα, bFGF, prostaglandins, various pro- and anti-inflammatory interleukins, catecholamines, angiotensin II (Ang II), interferon, and many others. Some of these molecules are released in concentrations sufficient to activate apoptosis in nonischemic cells. For example, one of the immediate consequences of cardiac ischemia is a 100- to 1000-fold increase in catecholamine production, to concentrations that have a direct cytotoxic effect in vitro . Also, in response to hypoxia/reoxygenation, necrotic cells synthesize TNFα in concentrations sufficient to initiate apoptosis in border regions of the heart muscle.

Endocytosis plays a critical role in the internalization of injury signals by non-ischemic cells. From the point of view of effective therapy, the negative effects of hundreds, if not thousands, of signals emanating from the necrotic injury zone cannot be prevented by selecting one or a few elements for neutralization. Evidence accumulated over the past two decades from the results of numerous clinical trials clearly supports this conclusion, since all tested single-target therapies, including ROS inhibitors, calcium channel blockers, TNFα blockers, Ang II receptor blockers, have failed or shown only limited success. Thus, the accumulated experimental and clinical data show that the problem of developing effective approaches to limit the consequences of infarction should be considered in terms of the possibility of preventing the propagation of ALL injury signals.

Suppression of endocytosis as a universal mechanism for import of various damage signals by cardiomyocytes appears to be the simplest and most accessible way to achieve the above goal. Thus, by “closing the door” to all signals coming from outside during ischemia/reperfusion, temporary suppression of endocytosis has the potential to significantly limit the import of damage and, thus, prevent the spread of myocardial infarction beyond the immediate zone of necrosis and significantly improve the long-term cardiac function of post-infarction patients (Figure 2).

In our experiments, we used cultures of HL-1 cardiomyocytes (a cell line widely used in cardiovascular research, derived from mouse atrial tumor myocytes) subjected to 6-hour anoxia followed by reoxygenation (1 hour).

Reoxygenation leads to a massive increase in ROS production . ROS production was investigated using the fluorescent indicator of mitochondrial superoxide MitoSOX® (Invitrogen). Compared to the control, the intensity of the MitoSOX fluorescent signal increased significantly after 6 h of anoxia. Anoxia also led to a redistribution of this signal from localized in the mitochondria to diffusely distributed in the cytoplasm and nucleus of the cell (Fig. 3). The latter was most likely associated with a decrease in the mitochondrial membrane potential, the normal value of which is critical for the selective distribution of MitoSOX® in the cell. Return to normoxia (reoxygenation) led to the restoration of mitochondrial localization of MitoSOX® and an approximately 10-fold increase in the fluorescent signal intensity (Fig. 3).

Anoxic and reoxygenated cells also showed an increase in the expression of several proteins involved in the cellular response to oxidative stress. Thus, the expression of the scavenger receptors CD36 and LOX-1 was increased twofold during both anoxia and reoxygenation (p < 0.05). Under the same conditions, the expression of the p22phox subunit of NADPH oxidase and SOD2 was also increased. In addition, the expression of the autophagy marker beclin-1 was increased (p < 0.05; data not shown).

Damage signals can be transferred to non-ischemic cardiomyocytes by the medium in which hypoxic and reoxygenated cells were cultured. To model the diffusional ability of damage export from the infarct zone to the peri-infarct zone of the heart, we incubated non-ischemic, “healthy” HL-1 cardiomyocytes in medium collected from anoxic and reoxygenated cell cultures. One hour of such exposure resulted in a dramatic increase in mitochondrial ROS generation and stress signals to levels comparable to those achieved with direct exposure to anoxia/reoxygenation (Fig. 4).

Endocytosis is enhanced in cardiomyocytes incubated in the medium in which cells subjected to anoxia/reoxygenation were incubated. Flow cytometric analysis of Alexa-fluor 488 transferrin uptake showed a 2-fold increase in the accumulation of this endocytosis marker in the cytoplasm (Fig. 5) of normal myocytes cultured in medium harvested from anoxic cultures. Normal myocytes cultured in medium harvested from reoxygenated cultures also showed evidence of increased endocytosis, although in this case the increase in Alexa-fluor 488 signal was observed primarily around the cell nucleus.

We next analyzed the activity of receptor-independent fluid-phase endocytosis using Qtracker 655 quantum dots with PEG coating (neutral surface charge) as a marker. In these experiments, normal myocytes cultured in medium collected from anoxic and reoxygenated cultures showed approximately a quarter more pronounced intracellular accumulation of the label than in controls (p < 0.05).

Inhibition of endocytosis suppresses healthy myocyte responses to injury signals In these experiments, we measured the effects of two well-known mediators of the cardiomyocyte stress response (TNFα and angiotensin II) on ischemia. In control cultures, both TNFα (10 and 100 ng/ml) and Ang II (10-7 M) stimulated the metabolic activity of cardiomyocytes (Fig. 6). However, the same concentrations of TNFα and Ang II were toxic (suppressed metabolism) to cells incubated in the medium from reoxygenated cardiomyocyte cultures. Importantly, in these latter experiments, addition of dainosor (an inhibitor of endocytosis) to the medium completely abolished the metabolic toxicity of both substances.

Composition: Epigallocatechin gallate, Formononetin, Oleuropein, Methyl-β-cyclodextrin, Quercetin

Epigallocatechin gallate, EGCG , is the main catechin found in green tea. This polyphenolic compound and some related catechins are also found in white tea, black tea, apples, blackberries, and cocoa products. Effect on endocytosis: In vitro studies, EGCG reduced LPS-induced innate immune responses by inhibiting endocytosis, interfering with Rab5-caveolin-1 interaction, and reducing Rab5 activity. The results indicate that Rab5 and caveolin-1 are molecular targets of EGCG. Thus, EGCG may act as an inhibitor of caveolin-dependent endocytosis (Hagivara and Matsushita, 2014).

Preclinical and clinical data: EGCG and other related catechins act as potent antioxidants that can protect against free radical-induced cellular damage. Research suggests that catechins like EGCG may reduce inflammation and prevent several chronic diseases, including cardiovascular disease, diabetes, metabolic disorders, and certain cancers. EGCG has been shown to inhibit a number of viral infections, including hepatitis B virus (HBV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza A virus (IAV), enterovirus (CVB3, EV71), Chikungunya virus (CHIKV), and coronaviruses (Bimonte et al., 2021). A prospective cohort study of over 8,000 individuals found that daily green tea consumption was associated with a delay in cancer progression, and a subsequent study in breast cancer patients found that patients with stage I and II breast cancer had a lower recurrence rate and longer remission (Fujiki et al., 1999). Furthermore, EGCG delivered in capsule form (200 mg orally) for 12 weeks was reported to be effective in patients with human papillomavirus-associated cervical lesions (Ahn et al., 2003). An epidemiological study found a negative association between green tea consumption and the prevalence of cognitive impairment in older adults over 70 years of age (Molino et al., 2016). In a 5.7-year prospective cohort study of 13,645 elderly Japanese adults aged 65 years and older, frequent green tea consumption was associated with a lower risk of dementia (Tomata et al., 2006).

Formononetin is an isoflavone from the phytoestrogen group found in plants such as red clover, green beans, lima beans, soybeans and many others. It is a major component of many traditional Chinese medicines. Effect on endocytosis. Formononetin slowed the development of atherosclerosis by reducing SRA expression and reducing monocyte adhesion. It is believed that changes in SRA expression are associated with the effect of formononetin on dynamin, which is involved in clathrin-dependent endocytosis (Ma et al., 2020).

Preclinical and clinical data: Phytoestrogens are involved in the suppression of inflammatory cytokines. They increase reverse cholesterol transport and insulin sensitivity. In addition, they have an antioxidant effect by activating antioxidant genes, increasing energy expenditure and inhibiting adipogenesis (Jungbauer and Medjakovic, 2014). Formononetin has been shown to inhibit EV-A71 entry into cells (Li et al., 2017). In animal studies where mice were injected with a lethal dose of EV71, formononetin administration resulted in 75% survival (Dai et al., 2019). Studies have shown that regular isoflavone consumption, typical for the Korean and Japanese diet, significantly reduces the incidence of metabolic syndromes, especially obesity (Jungbauer and Medjakovic, 2014). Similarly, a study in Japanese women found that the group that consumed the highest amount of isoflavones had lower rates of menopause syndrome, migraine, diabetes, and hypertension (Melby, Murashima, & Watanabe, 2004). Compared with Western countries, populations in countries that traditionally consume soy foods high in phytoestrogens have a lower incidence of breast cancer (Korde et al., 2009). Formononetin can effectively inhibit tumor growth in vivo and has been reported to be highly effective against a wide range of malignancies, including breast, bone, multiple myeloma, colorectal, and nasopharyngeal cancers (Aliya et al., 2023).

Oleuropein is a biophenol commonly found in olive leaf, extra virgin olive oil, and some species of the Oleaceae family. Effects on endocytosis: Oleuropein and hydroxytyrosol can inhibit viral fusion with host cell membranes (LeRoy and Wrana, 2005; Alkafaas et al., 2022) by affecting caveolin-dependent endocytosis and lipid rafts.

Preclinical and clinical data: The main pharmacological actions of oleuropein include anticancer, cardioprotective, neuroprotective, gastroprotective, hepatoprotective, antidiabetic, antiobesity and radioprotective effects, which are largely attributed to its putative antioxidant and anti-inflammatory effects (Hassen et al., 2015). Oleuropein has demonstrated antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria, mycoplasma and viruses (Omar 2010) and improved survival in animal models of multidrug-resistant Pseudomonas aeruginosa sepsis (Giamarellos-Bourboulis et al., 2006). In a clinical trial of 141 hospitalized patients with COVID-19, consumption of olive leaf extract (250 or 500 mg every 12 hours for five days) significantly improved oxygen saturation and reduced body temperature and respiratory rate (Ahmadpour et al., 2023). Extra virgin olive oil consumption was associated with a 31% reduction in the risk of all malignancies. The greatest risk reduction was observed for pancreatic, esophageal, urinary tract (54%), breast (33%), and gastrointestinal (23%) cancers (Markellos et al., 2022). People consuming more than 7 g of olive oil per day had a 29% lower risk of mortality from neurodegenerative diseases than those who never or rarely consumed olive oil (Guasch-Ferré et al., 2022).

Methyl-β-cyclodextrin - Cyclodextrins (MβCD) are a type of cyclic oligosaccharide found in potato, maize and cassava. Effect on endocytosis: Extraction of cholesterol with methyl-β-cyclodextrin impairs the formation of clathrin-coated endocytic vesicles (Rodal et al., 1999). MβCD inhibits cholesterol-dependent endocytic processes by reversibly extracting the steroid from the plasma membrane. Extraction of cholesterol with methyl-β-cyclodextrin impairs the formation of clathrin-coated endocytic vesicles. In vitro studies, MβCD potently inhibited the endocytosis of transferrin and EGF. Typical invaginated caveolae were no longer present and invagination of clathrin-coated pits was strongly inhibited.

Preclinical and clinical data: Cholesterol depletion by MβCD significantly reduces the infectivity of influenza A (H1N1 strain) viral particles in vitro (Verma et al., 2018). Sulfonated CDs have been shown to reduce HIV infectivity and halt viral replication in vitro by inhibiting the ability of HIV to fuse with target cells (Weiner et al., 1992). Cholesterol depletion by methylated beta- has been shown to affect the entry of flaviviruses into target cells as well as their ability to replicate inside cells (Lee et al., 2008). Studies in mice have shown that βCD helps dissolve cholesterol deposits in arterial walls and reprogram macrophages to metabolize cholesterol into soluble oxysterols (Zimmer et al., 2016).

Quercetin is a bioflavonoid found mainly in onions, grapes, berries, cherries, broccoli and citrus fruits. Effect on endocytosis: Quercetin inhibits the fluid phase endocytic activity (pinocytosis) of dendritic cells (Huang et al. 2010).

Preclinical and Clinical Data: A study by Ganesan et al. (2012) showed that quercetin treatment inhibited rhinovirus endocytosis in airway epithelial cells. Viruses that commonly respond to flavonoids include adenovirus, herpes simplex virus, Japanese encephalitis virus, and respiratory syncytial virus. Quercetin also exhibits broad antibacterial activity (Anand David et al., 2016). In rats, quercetin (80 mg equivalent dose) suppressed both acute and chronic inflammation and also exhibited significant antiarthritic activity against adjuvant-induced arthritis (Guardia et al., 2001; Mamani-Matsuda et al., 2006). People who consume quercetin and non-alcoholic red wine extract (which contains quercetin) have been found to have lower LDL oxidation (Chopra et al., 2000). In another study, a 6-week course of quercetin at a dose of 150 mg/day reduced systolic blood pressure, plasma oxidation, and LDL levels in overweight subjects (Egert et al., 2009). Quercetin reduced tumor growth and inhibited the proliferation of malignant cells in vivo (Vásquez-Garzón et al, 2009).

Methods of using food additives

Epigallocatechin gallate, EGCG, is highly soluble in water. Take on an empty stomach. One oral dose in the morning after an overnight fast, at least 30 minutes before breakfast, and a second dose in the afternoon 30 minutes before dinner. One group of researchers has suggested that a safe intake level of EGCG as a solid supplement is approximately 300 mg per day.

Formononetin has a good safety profile, as demonstrated by acute and subchronic toxicity studies. The optimal daily dose of formononetin in humans has not been precisely established. However, studies have used doses in the range of 20 to 50 mg/kg orally, i.e. 1400–3500 mg daily (Singh et al., 2011).

In human studies, participants typically took 500–1000 mg of standardized olive leaf extract per day, corresponding to a daily dose of 50–100 mg oleuropein. In a clinical study, patients took 500 mg of olive leaf extract twice daily for 8 weeks. A 12-week crossover study of olive leaf extract was used to evaluate the effects on insulin action and cardiovascular risk factors in men with a BMI of 28 (±2) kg/m2. Oleuropein is highly water-soluble. Liquid oleuropein preparations showed higher peak oleuropein levels in plasma and faster absorption of hydroxytyrosol metabolites (De Bock et al., 2013).

Methyl-β-cyclodextrin is soluble in water (150 mg/mL), forming a clear, colorless solution. Cyclodextrins are widely used in pharmaceutical technology and drug formulations to improve the aqueous solubility, delivery, and bioavailability of insoluble drugs. The maximum level of β-cyclodextrin in food is 5 mg/kg per day. β-Cyclodextrin is recognized as a “natural product” in Japan. All parent cyclodextrins are accepted as food additives and “generally recognized as safe” (GRAS) in the United States. As a food additive, the recommended total daily oral dose is 500 mg/day.

The solubility of quercetin dihydrate ranged from 0.00263 g/L at 25°C to 1.49 g/L at 140°C. The solubility of quercetin in water can be significantly improved (254-fold) by complexation with methyl-β-cyclodextrin (Gulec and Damirel, 2016). Quercetin has been safely used at doses up to 1 gram per day for 12 weeks. Quercetin has most commonly been used in adults at doses of 250-1000 mg orally daily for 12 weeks.

Table 1. Suggested daily dose ranges for the ingredients of the composition:

Ingredient Daily Dose (mg) Daily Dose (%) Epigallocatechin gallate 20-80 4.5 Formononetin 6-24 1.4 Oleuropein 40-160 9.1 Methyl-β-cyclodextrin 75-300 17.0 Quercetin 300-1200 68.0 Total 441-1764 100%

As a special case of the invention implementation, a composition may be presented that includes the following plant components:

Ingredient
(Active connection) Daily Dose (mg) Daily Dose (%) Green Tea Extract (Epigallocatechin gallate) 150-600
(20-80) 10.5 Red Clover Extract (Formononetin) 600-2400
(6-24) 42.1 Olive leaf extract
(Oleuropein) 300-1200
(40-160) 21.1 Methyl-β-cyclodextrin 75-300 5.3 Quercetin dihydrate 300-1200 21.1 Total 1425-5700 100%

In one embodiment, the main ingredients of the composition may be supplemented with aromatic or flavor additives. Honey, berry, fruit, coffee and chocolate food flavors are used.

In another embodiment, the mixtures of ingredients of the composition are taken in the form of a powder for dilution in warm water in combination with aromatic and flavor additives. A single dose, constituting half of the daily effective therapeutic dose, will be contained in tea bags to create a drink in warm water for oral administration in the morning and evening.

Ingredient Dose (mg) Dose (%) Epigallocatechin gallate 40-80 mg 4.5 Formononetin 12-24 mg 1.4 Oleuropein 80-160 mg 9.1 Methyl-β-cyclodextrin 150-300 mg 17.0 Quercetin 600-1200 mg 68.0 Total 882-1764 mg 100%

As a special case of the invention implementation, a composition may be presented that includes the following plant components:

Ingredient Dose (mg) Dose (%) Green tea extract 300-600 mg 10.5 Red Clover Extract 1200-2400 mg 42.1 Olive leaf extract 600-1200 mg 21.1 Methyl-β-cyclodextrin 150-300 mg 5.3 Quercetin dihydrate 600-1200 mg 21.1 Total 2850-5700 mg 100%

- In another embodiment, the mixtures of ingredients of the composition can be taken in the form of a tablet for oral administration. Lactose, sucrose, corn starch, microcrystalline cellulose, povidone polyvinylpyrrolidone and/or modified cellulose will be used as binding components for the production of tablets.

Ingredient Dose (mg) Dose (%) Epigallocatechin gallate 40-80 mg 4.5 Formononetin 12-24 mg 1.4 Oleuropein 80-160 mg 9.1 Methyl-β-cyclodextrin 150-300 mg 17.0 Quercetin 600-1200 mg 68.0 Total 882-1764 mg 100%

As a special case of the invention implementation, a composition may be presented that includes the following plant components:

Ingredient Dose (mg) Dose (%) Green tea extract 300-600 mg 10.5 Red Clover Extract 1200-2400 mg 42.1 Olive leaf extract 600-1200 mg 21.1 Methyl-β-cyclodextrin 150-300 mg 5.3 Quercetin dihydrate 600-1200 mg 21.1 Total 2850-5700 mg 100%

In another embodiment, powder mixtures of the ingredients of the composition can be taken in the form of a gel capsule for oral administration. Gelatin or non-gelatin components that are a product of collagen hydrolysis or based on cellulose will be used as a material for the production of capsules.

Ingredient Dose (mg) Dose (%) Epigallocatechin gallate 40-80 mg 4.5 Formononetin 12-24 mg 1.4 Oleuropein 80-160 mg 9.1 Methyl-β-cyclodextrin 150-300 mg 17.0 Quercetin 600-1200 mg 68.0 Total 882-1764 mg 100%

As a special case of the invention implementation, a composition may be presented that includes the following plant components:

Ingredient Dose (mg) Dose (%) Green tea extract 300-600 mg 10.5 Red Clover Extract 1200-2400 mg 42.1 Olive leaf extract 600-1200 mg 21.1 Methyl-β-cyclodextrin 150-300 mg 5.3 Quercetin dihydrate 600-1200 mg 21.1 Total 2850-5700 mg 100%

In another embodiment, the doses of the nutritional supplements in the composition may be one third of the daily dose on a three times daily basis, given the relatively short half-life in circulation.

In another embodiment, the doses of food additives in the composition may be increased in situations of increased likelihood or signs of the onset of a viral infection.

In another embodiment, the doses of nutritional supplements in the composition may be increased in situations of increased likelihood or signs of onset of acute ischemic conditions such as (for example) myocardial infarction or stroke.

In another embodiment, the doses of food additives in the composition may be decreased or increased depending on height, weight, gender and general condition.

All ingredients can be used in the form of powder extracts or plant parts in dry form or in the form of solutions intended for oral administration or intravenous injection.

Example 1. Prevention and treatment of viral diseases.

One of the fundamental conditions for a successful viral infection is the penetration of the viral particle into the host cells. Viruses are unable to reproduce independently and therefore must use the resources of the host cells to replicate and assemble new viral particles. The formula of the composition is specifically aimed at suppressing the main mechanisms of endocytosis and, thus, complicating the internalization of viral particles. The composition can be used both for the prevention of viral diseases and for a more favorable course of infection, reducing the likelihood of complications, and an earlier recovery.

For the prevention of viral diseases, it is recommended to use the standard daily dose (Table 1) of the composition, dosed three times a day (Table 2).

Table 2. Doses of the composition for prophylactic treatment regimen

Ingredient Dose to take 3 times a day Daily Dose (%) Epigallocatechin gallate 13.3 4.5 Formononetin 4 1.4 Oleuropein 26.7 9.1 Methyl-β-cyclodextrin 50 17.0 Quercetin 200 68.0 Total 294 100%

As a special case of the invention implementation, a composition may be presented that includes the following plant components:

Ingredient Dose to take 3 times a day Daily Dose (%) Green tea extract 100 10.5 Red Clover Extract 400 42.1 Olive leaf extract 200 21.1 Methyl-β-cyclodextrin 50 5.3 Quercetin dihydrate 200 21.1 Total 950 100%

If symptoms of the disease appear, it is recommended to double the dose of the composition over 7-10 days (Table 3).

Table 3. Doses of the composition for the treatment of acute conditions

Ingredient Daily Dose (mg) Dose to take 3 times a day Daily Dose (%) Epigallocatechin gallate 80 mg 26.7 4.5 Formononetin 24 mg 8 1.4 Oleuropein 160 mg 53.3 9.1 Methyl-β-cyclodextrin 300 mg 100 17.0 Quercetin 1200 mg 400 68.0 Total 1764 mg 588 100%

As a special case of the invention implementation, a composition may be presented that includes the following plant components:

Ingredient Daily Dose (mg) Dose to take 3 times a day Daily Dose (%) Green tea extract 600 mg 200 10.5 Red Clover Extract 2400 mg 800 42.1 Olive leaf extract 1200 mg 400 21.1 Methyl-β-cyclodextrin 300 mg 100 5.3 Quercetin dihydrate 1200 mg 400 21.1 Total 5700 mg 1900 100%

Example 2. Prevention and treatment of cardiovascular diseases.

In terms of cardiovascular diseases, the components of the composition have a wide range of action including antioxidant, anti-inflammatory, and anti-atherosclerotic effects.

The development of atherosclerosis is typical for the vast majority of middle-aged and older people. Regardless of the presence or absence of obvious clinical manifestations, atherosclerosis is the leading risk factor and the leading cause of morbidity and mortality in developed countries. Each component of the proposed composition has demonstrated the ability to slow down and partially reverse the age-related development of atherosclerosis. To slow down the development of atherosclerosis, long-term consumption of the composition in preventive doses is recommended in the format of either once a day ( Table 1 ) or 3 times a day ( Table 2 ).

In the case of early signs of development of such acute cardiovascular events as myocardial infarction or stroke, the main goal of formulating therapy and choosing dosages is to prevent the spread of the zone of irreversible damage beyond the ischemic regions. In this context, the most important mechanism of action of the ingredients of the composition is their inhibitory effect on endocytosis.

In addition to the dose that should be increased ( Table 3 ), the onset and duration of use of increased doses are of great importance in order to achieve the maximum therapeutic effect. Given the mechanism of action involved, it is recommended to take the composition immediately after the first symptoms appear and before the onset of reperfusion, followed by continued administration for 8-10 days after the infarction/stroke.

Example 3. Correction of metabolic syndrome.

One of the standard strategies for correcting metabolic syndrome is a diet aimed at reducing the intake of excess nutrients - in terms of the actual biological energy requirement. The transition to a diet is a rather painful and often unsuccessful process, since this method relies mainly on self-discipline required for long-term (often permanent) abstinence from consuming excess food or a certain category of food products. The use of natural compounds aimed at reducing the activity of endocytosis represents an alternative approach that allows achieving the desired correction of metabolism against the background of a significant reduction in diet-related stress, since moderate inhibition of global endocytosis reduces the efficiency of nutrient internalization and, thus, prevents their processing.

To achieve stable correction of metabolism and desired results in maintaining optimal weight, consumption of the composition in prophylactic doses ( Tables 1 and 2 ) is expected to become a daily routine for many years.

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Claims (7)

1. A biologically active supplement containing the following active components, wt.%: 4.275-4.5 epigallocatechin gallate, 1.33-1.4 formononetin, 8.645-9.1 oleeuropein, 16.15-17.0 methyl-β-cyclodextrin, 64.4-68.0 quercetin. 2. A biologically active supplement according to paragraph 1, characterized in that the main components are supplemented with aromatic or flavor additives. 3. A biologically active supplement according to item 1 in the form of a powder for dilution in water. 4. A biologically active supplement according to item 1 in the form of a tablet for oral administration. 5. A biologically active supplement according to item 1 in the form of a gel capsule for oral administration. 6. Use of the biologically active supplement according to paragraph 1 for the prevention of viral and cardiovascular diseases and optimization of metabolism. 7. Use according to item 6, characterized in that the daily dose of the biologically active supplement is: 20-80 mg epigallocatechin gallate, 6-24 mg formononetin, 40-160 mg oleuropein, 75-300 mg methyl-β-cyclodextrin, 300-1200 mg quercetin.