Biologically active food supplement

Abstract

FIELD: food industry.SUBSTANCE: used in the production of biologically active food supplements (BAS). A composition containing quercetin, luteolin and resveratrol as active ingredients with a ratio of quercetin to luteolin and resveratrol from 3.9:1:3.9 to 4.3:1:4.3 is used as a dietary supplement to food, helping to improve spatial memory, concentration, reduce neuroinflammation, improve motor coordination, and stabilize the emotional background.EFFECT: obtaining a dietary supplement that has a positive effect on the nervous system with neuroprotective properties.2 cl, 5 dwg, 1 ex

Description

[0001] The invention relates to the food industry and can be used in the production of biologically active food supplements (BAS) that have a positive effect on the nervous system with neuroprotective properties.

[0002] Patients with diseases directly or indirectly associated with neuroinflammation often do not consume sufficient amounts of natural substances with neuroprotective properties in their diets. Based on scientific evidence, supplementation with biologically active substances (in addition to dietary interventions) can reduce the severity of neuroinflammation in patients with neurodegenerative diseases / mental disorders / endocrine disorders that negatively affect the nervous system, as well as in patients at risk for developing such diseases due to increased neuroinflammation (patients with unhealthy habits, chronic stress, autoimmune diseases, etc.).

[0003] The Russian market offers either single-component dietary supplements containing quercetin, luteolin, or resveratrol, or supplements with two of these three components. Furthermore, these well-known products do not claim neuroprotective properties.

[0004] The closest solution to the claimed invention is a biologically active food supplement containing grape seed extract in an amount of 33.3% by weight, dipeptide carnosine in an amount of 49.9993% by weight, quercetin in an amount of 6% by weight, coenzyme Q10 in an amount of 4% by weight, organic selenium in an amount of 0.0007% by weight and gum arabic (fibergum) in an amount of 6.7% by weight (patent RU No. 2364258, published on 20.08.2009). The known supplement allows to obtain a final product that provides an integrated preventive and healing effect on the human body.

[0005] The disadvantage of the known solution is the lack of pronounced neuroprotective properties.

[0006] The technical problem of the invention is the creation of an effective biologically active supplement that provides patients suffering from diseases that are directly or indirectly associated with neuroinflammation with a sufficient amount of natural neuroprotectors.

[0007] The technical result is the manifestation of neuroprotective properties, including protection against memory and concentration impairment, neuroinflammation, decreased cognitive function, deterioration of coordination, and changes in emotional background.

[0008] The technical result is achieved by using a composition containing quercetin, luteolin and resveratrol as active components at a ratio of quercetin to luteolin and resveratrol from 3.9:1:3.9 to 4.3:1:4.3, as a biologically active food supplement that helps improve spatial memory, concentration, reduce neuroinflammation, improve coordination of movements, and stabilize the emotional background.

[0009] Japanese Sophora extract can be used as quercetin.

[0010] Example of obtaining dietary supplements

[0011] The capsule contents (dried Japanese Sophora extract (98% quercetin), 98% resveratrol, 95% luteolin, microcrystalline cellulose) are mixed. They are then encapsulated into hard gelatin capsules using an automatic or semi-automatic machine. Capsules are packaged in quantities of 20-200 pieces in jars with hermetically sealed polyethylene lids with a capacity of 60-250 cm3. 3 A jar is a unit of consumer packaging.

[0012] Capsules may be packaged in 10-20-piece blisters made of polyvinyl chloride film and printed lacquered aluminum foil, which are then placed in packs of 1-20 blisters. Packs constitute a unit of consumer packaging. Consumer packaging labeling complies with the Technical Regulations of the Customs Union "Food Products in Terms of Labeling" (TR CU 022 / 2011).

[0013] The claimed combination of components for the production of a dietary supplement with neuroprotective properties exhibits a synergistic effect, which is not a simple sum of the properties exhibited by each component, allowing to stop or prevent undesirable factors affecting the nervous system and allowing to preserve memory and concentration, prevent neuroinflammation, prevent a decline in cognitive function, improve motor coordination, and stabilize the emotional background.

[0014] The super-synthetic effect of a combination of quercetin, luteolin, and resveratrol has been confirmed by studies. Below are the test results confirming the unexpected effect of this combination of active ingredients.

[0015] The results of the conducted research are reflected in the images, which show:

[0016] Fig. 1 - water maze test results;

[0017] Fig. 2 - TNF-α activity in hippocampal tissues;

[0018] Fig. 3 - discriminant analysis of metabolite content in AD-CON and AD-LR groups;

[0019] Fig. 4 - discriminant analysis of metabolite content in AD-CON and AD-LQR groups;

[0020] Fig. 5 - Analysis of metabolite levels in hippocampal tissue. PPPA - phenylpyruvic acid.

[0021] Male Sprague Dawley rats with an average weight of 202±11 g were used in the study. All rats were raised in individual stainless steel cages in a controlled environment (23°C, 12-hour light / dark cycle) and had unlimited access to food and water. All study procedures complied with GOST 33215-2014 "Guide for the Care and Maintenance of Laboratory Animals. Rules for Equipment of Facilities and Organization of Procedures." Rats underwent a 7-day acclimatization in the vivarium.

[0022] On day 10, rats were anesthetized with an intraperitoneal injection of a mixture of ketamine and xylazine (100 and 10 mg / kg, respectively) and placed in a stereotaxic device. Cannulas were inserted into bilateral CA1 subregions of the hippocampus. The cannula was connected to a 22-gauge tube filled with amyloid-β(25-35) for the four Alzheimer's disease (AD) model groups or amyloid-β(35-25) for the Non-AD-CON group. Amyloid-β(35-25) was used in the Non-AD-CON group as a normal control, since it has the reverse sequence of amyloid-β(25-35) and does not aggregate in the brain. Both types of amyloid-β were dissolved in sterile saline and infused into bilateral CA1 subregions using an osmotic pump (Alzet Osmotic Pump Company; Cupertino, CA, USA) at a rate of 3.6 nmol / day for 14 days. The prescribed diet was provided from day 7 and was continuously provided until day 51.

[0023] Preparing for intervention.

[0024] The doses of flavonoids (luteolin, quercetin, resveratrol) required to evaluate efficacy and interactions were estimated based on our preliminary cell culture study: treatment with luteolin (0.05-0.5 μg / mL) and resveratrol (0.1-1 μg / mL) reduced cell death and tumor necrosis factor-α (TNF-α) expression in PC12 cells treated with amyloid-β (25-35). The doses of luteolin and resveratrol were adjusted according to the corresponding human doses. Based on the preliminary study and typical human doses, the dosage for the animal study was calculated using the animal conversion factor.

[0025] Eighty rats with AD were divided into four treatment groups of 20 rats each, and rats in each group were given test ingredients included in a high-fat diet. The rats with AD were divided into four treatment groups as follows:

[0026] 1. AD model rats that were given dextrin as a control (AD-CON),

[0027] 2. AD model rats given a 1:1 mixture of luteolin and quercetin (50 mg / kg body weight; AD-LQ),

[0028] 3. AD model rats given a mixture of luteolin and resveratrol (50 mg / kg body weight; AD-LR)

[0029] 4. AD model rats were given a mixture of luteolin, quercetin and resveratrol (50 mg luteolin / kg body weight; 200.5 mg quercetin / kg body weight; 208 mg resveratrol / kg body weight; AD-LQR).

[0030] Twenty non-AD rats were given dextrin as normal control (Non-AD-CON).

[0031] All rats had free access to a modified high-fat semi-purified AIN-93 diet without polyphenols, which induces obesity, systemic inflammation, and insulin resistance. The diets of the AD-LQ, AD-LR, and AD-LQR groups contained 0.1% luteolin plus 0.1% quercetin; 0.1% luteolin plus 0.1% resveratrol; 0.1% luteolin plus 0.4% quercetin plus 0.4% resveratrol, respectively. The diets of the AD-CON and Non-AD-CON groups contained 0.2% dextrin instead of flavonoids.

[0032] The diet was prepared weekly using a semi-purified modified AIN-93 diet. Luteolin, quercetin, and resveratrol were uniformly mixed with a vitamin-mineral mixture and sugar, and then the mixture was sifted to break up lumps. Each mixture was mixed with the appropriate amount of starch, casein, and lard, sifted again, and stored at 4°C. Fresh diet was provided by tightly pressing the weighed powder into a food container every other day, and the remaining feed was weighed and discarded. The amount of each supplement consumed (dosage) was calculated based on feed intake each week. The diet composition contained 35% carbohydrates (corn starch and sucrose), 22% protein (casein), and 43% fat (lard).

[0033] Study of manifestations of neuroinflammation.

[0034] Passive avoidance test.

[0035] On day 47 of the study, rats were tested for memory deficits using a passive avoidance apparatus consisting of a dual-chamber, dark / light shuttle box. In the experimental field study, an electric shock (75 V, 0.2 mA, 50 Hz) was delivered for 5 s immediately after the rats entered the dark chamber. Five seconds later, the rat was removed from the dark chamber and returned to its home cage. After 24 h, the retention latency to enter the dark chamber was measured in the same manner as in the field test, but the foot shock was not delivered, and the latency was recorded to a maximum of 600 s. Shorter latencies indicate memory deficits compared with significantly longer latencies.

[0036] Water maze test. Spatial memory function was assessed using the Morris water maze test, as described previously, on day 49. The Morris water maze tests hippocampus-dependent learning, including spatial memory acquisition.

[0038] Locomotor activity.

[0039] At the end of the experiment, locomotive activity was measured using a Linton AM1053 activity monitor, consisting of a three-dimensional array of infrared beams placed around transparent Plexiglas cages with AmLogger software (Linton Instruments, UK). The sum of measurements of rearing, mobility, and activity was considered total locomotive activity. Activity was assessed for 1 hour during the dark phase of the light / dark cycle after 30 minutes of adaptation to the transparent Plexiglas cage.

[0040] Quantitative biochemical analysis.

[0041] The levels of the studied metabolites were measured using real-time polymerase chain reaction (RT-PCR).

[0042] Hippocampal tissues were collected from 3 randomly selected rats from each group. Total RNA was isolated from the tissues using a monophasic solution of phenol and guanidine isothiocyanate. Coding DNA (cDNA) was obtained using a mixture of total RNA, superscript III reverse transcriptase, and high-fidelity Taq DNA polymerase (1:1:1, v:v:v) by polymerase chain reaction (PCR). cDNA was mixed with primers of the genes of interest (TNF-α, IL-1β, and β-actin) and SYBR Green mix, and the expression of the genes of interest was analyzed using a real-time PCR machine. Gene expression levels in unknown samples were quantified using the comparative cycle threshold method.

[0043] Immunoblot

[0044] The hippocampi of four rats from each group were dissected as described previously. The hippocampi were lysed with RIPA buffer supplemented with protease inhibitors, and their protein content was measured using a Bio-Rad protein assay kit (Hercules, CA, USA). Lysates with equivalent amounts of protein (30-50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then analyzed by immunoblotting with antibodies as follows: protein kinase B (PKB or Akt), phosphorylated PKB Ser473 , GSK-3β, phosphorylated GSK-3β ser9 , Forkhead Box O-1 (FOXO-1) protein, phosphorylated FOXO-1 thr24 , cAMP response element binding factor (CREB), phosphorylated CREB ser129 , phosphorylated tau ser396 and β-actin. Protein expression intensity was determined using Imagequant TL.

[0045] Analysis of metabolites in the hippocampus.

[0046] The hippocampus was homogenized with a methanol:water mixture (4:1). The mixture was vortexed for 30 seconds and placed in a sonicator for 30 minutes at room temperature. It was then placed on ice for 30 minutes and centrifuged at 12,000g at 4°C for 10 minutes. The supernatant (aqueous layer) was separated and concentrated using a vacuum centrifuge. Terfenadine as a standard compound was added to the dried samples, and the mixture was dissolved in 20% methanol. Hippocampal metabolites were analyzed using an ultra-throughput LC-quadrupole-TOF (UPLC-Q-TOF) MS system. Samples were injected onto an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm; Waters) and equilibrated with water containing 0.1% formic acid. The samples were eluted with a gradient of ACN containing 0.1% formic acid at a flow rate of 0.35 mL / min for 15 min. Eluted metabolites were analyzed by Q-TOF MS (Waters) with positive ESI.The capillary and sampling cone voltages were set to 2.78 kV and 26 V, respectively. The source and desolvation temperatures were set to 110°C and 300°C, respectively, and the desolvation flow rate was 700 L / h. TOF MS data were collected in the m / z range of 100–000 with a scan time of 0.2 s and an interscan delay time of 0.02 s. Lock spray with leucine enkephalin (0.556 kDa in positive ESI mode) was used at a flow rate of 3 μL / min and a frequency of 10 s to ensure accuracy and reproducibility. A mixture of all samples was injected after every 10 samples for quality control. MS / MS spectra of metabolites were collected in the m / z range of 50–1000 using a collision energy ramp from 10 to 30 eV. Retention time, m / z and ion intensity from mass spectral analysis were extracted using MarkerLynx software (Waters).MarkerLynx software was used for data acquisition, alignment, and normalization of UPLC-Q-TOF MS analyses of serum metabolites. Data were aligned with a mass tolerance of 0.04 Da and a retention time window of 0.15 s. All mass spectra were normalized using an external standard. Metabolites were identified using online metabolite databases (Metlin and HMDB), the ChemSpider chemical structure database, literature references, and purified standards. Norepinephrine and normetanephrine content in hippocampal lysates were determined using high-performance liquid chromatography and electrochemical detection, as described previously.

[0047] Euglycemic hyperinsulinemic "lock" and hyperglycemic "lock" procedures.

[0048] For clamp procedures, the right carotid artery and left jugular vein of each rat were catheterized at the seventh week, and after 5-6 days, each treatment group was divided into two subgroups. In one subgroup (n = 8), a euglycemic hyperinsulinemic clamp was performed on fasted conscious rats, and in the other (n = 9), a hyperglycemic clamp was performed as follows: the plasma glucose concentration is sharply increased to 125 mg / dL above the basal level by continuous glucose infusion. This hyperglycemic plateau is maintained by adjusting the variable glucose infusion based on the rate of insulin secretion and glucose metabolism. Since the plasma glucose concentration is maintained constant, the glucose infusion rate is an index of insulin secretion and glucose metabolism. Hyperglycemic clamps are often used to evaluate insulin secretory capacity.A continuous infusion of regular human insulin (Humulin, Eli Lilly; Indianapolis, IN, USA) was then started at 20 pmol / kg / min to increase plasma insulin concentrations to approximately 1100 pM by 210–240 min. Blood samples were collected at 10-min intervals, and 25% glucose was infused as needed to clamp glucose levels at approximately 6 mM. Whole-body glucose uptake and basal glucose turnover rates, as well as hepatic glucose production rates at baseline and in the hyperinsulinemic clamp state, were measured as described previously. During the hyperglycemic clamp procedure, glucose infusions were used to raise and maintain serum glucose levels 5.5 mM above baseline for 120 min; serum insulin levels were measured at 0, 2, 5, 10, 60, 90, and 120 min.Two days after the clamping procedure, food-deprived rats were injected with human regular insulin (5 U / kg) via the inferior vena cava, and tissues were collected 10 min later.

[0049] The results of the research conducted are shown below.

[0050] Body weight and food intake, luteolin and L-theanine

[0051] Body weight at week 7 did not differ significantly between groups. However, weight gain during the experimental periods was significantly lower in the AD-CON group than in the Non-AD-CON group, and weight gain in the AD-LR and AD-LQR groups was similar to that in the Non-AD-CON group. However, epididymal fat and retroperitoneal fat mass, which represents visceral fat, were significantly higher in the AD-CON group than in the Non-AD-CON group, while visceral fat mass was significantly lower in the AD-LR and AD-LQR groups than in the AD-CON groups. Food intake did not differ significantly between all groups. Luteolin intake in the AD-LQ and AD-LQR groups was approximately 500 and 250 mg / kg, respectively, while resveratrol intake in the AD-LR and AD-LQR groups was approximately 1000 and 500 mg / kg, respectively. No synergistic effect of the combination of luteolin and resveratrol on energy metabolism was observed.

[0052] Memory impairment.

[0053] In the passive avoidance test, the AD-CON group showed a shorter retention time to enter the illuminated room than the Non-AD-CON group, even though the rats had received an electric shock upon entering the illuminated room 12 hours earlier. This finding suggests that the AD-CON group had a greater degree of memory impairment than the Non-AD-CON group. Retention times significantly decreased across groups in the order AD-LQR > AD-LQ > AD-LR > AD-CON.

[0054] In the water maze test, the Non-AD-CON group found the area where the platform was located (area 5) faster and stayed in area 5 longer than the AD-CON group, indicating that the AD-CON group had spatial memory impairment. The spatial memory impairments of the groups were significantly improved in the ascending order of AD-CON < AD-LR < AD-LQ < AD-LQR; in particular, treatment with luteolin in combination with quercetin and resveratrol almost completely prevented the memory deficit (Fig. 1).

[0055] The AD-LQR group's scores were similar to those of the Non-AD-CON group. Thus, the combination of luteolin, quercetin, and resveratrol had a synergistic effect in protecting against memory impairment.

[0056] Insulin signaling and neuroinflammation in the hippocampus.

[0057] The phosphorylation rates of Akt, GSK-3β, and FOXO-1, which are downstream of insulin receptors, were attenuated in the hippocampus of the AD-CON group compared with the Non-AD-CON group (P < 0.05; Fig. 2). In addition, CREB phosphorylation was significantly attenuated in the AD-Con group compared with the Non-AD-CON group. Tau phosphorylation was enhanced in the AD-CON group (Fig. 2). This may be due to the attenuation of insulin signaling in the hippocampus. The administration of luteolin, resveratrol, and quercetin in combination significantly increased the phosphorylation rates of Akt, GSK-3β, and FOXO-1 compared with the AD-CON group, and the pairwise combinations of the ingredients did not cause statistically significant changes (P < 0.05; Fig. 2). CREB phosphorylation also significantly increased in the order AD-CON < AD-LR < AD-LQ < AD-LQR. This finding suggests a synergistic effect of flavonoids against hyperglycemia-induced neuroinflammation.

[0058] The expressions of TNF-α and IL-1β in the hippocampus increased by 2.1 and 1.8 times, respectively, in the AD-CON group compared with the Non-AD-CON group, and their expressions decreased in the order of AD-CON ≈ AD-LQ ≈ AD-LR ≈ AD-LQ > AD-LQR > Non-AD-CON (P < 0.05; Fig. 2). The serum TNF-α level in the AD-CON group was much higher than that in the Non-AD-CON group, however, only the combination of all three bioactive plant compounds significantly reduced the levels of serum TNF-α and IL-1β, an indicator of systemic inflammation, in the brain tissue, thereby exerting a positive effect on neuroinflammation (P < 0.05; Fig. 2).

[0059] Metabolomic analysis of hippocampal tissue.

[0060] Hippocampal metabolites were examined in the AD-CON, AD-LR, AD-LQ, and AD-LQR groups and further analyzed using partial least squares discriminant analysis (PLS-DA). The hippocampal metabolites of AD-LQ, AD-LR, and AD-LQR were separately compared with AD-CON. Compared with AD-CON, AD-LR showed proper R2X (0.509), Q2X (0.592), and Q2 value (0.0338) values, but they were not significantly different (P = 0.144) (Fig. 3), whereas the comparison between AD-LQR and AD-CON showed higher P values ​​(Fig. 4). The model quality parameters for fit and predictability (R2X = 0.509, Q2X = 0.734, Q2 = 0.516) were significantly different only in AD-LQR compared to AD-CON (P=0.038).

[0061] We investigated the significantly different metabolites between AD-CON and AD-LQR by performing the PLS-DA loading test with a two-sample t-test. Five metabolites (proline, nicotinamide, hypoxanthine, phenylpyruvic acid, normetanephrine) showed significant differences between the AD-CON and AD-LQR groups. Compared with AD-CON, the hippocampal hypoxanthine levels significantly increased in AD-LQR, and the levels of proline, nicotinamide, phenylpyruvic acid, and normetanephrine significantly decreased in AD-LQR (Fig. 5). Similar changes were not observed in the AD-R and AD-LQ groups.

Claims

1. The use of a composition containing quercetin, luteolin and resveratrol as active components in a ratio of quercetin to luteolin and resveratrol from 3.9:1:3.9 to 4.3:1:4.3, as a biologically active food supplement that helps improve spatial memory, concentration, reduce neuroinflammation, improve motor coordination, and stabilize emotional background.

2. Use of a composition according to claim 1, wherein the extract of Japanese pagoda tree is used as quercetin.

Images