RU2770470C1 - Geroprotective feed additive - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to a composition for preventing age-related diseases and increasing the healthy life span of unproductive and farm animals, namely, to a geroprotective feed additive. The additive contains a mixture of substances taken in the following ratio of mass parts: resveratrol - from 1 to 20 parts; curcumin - from 4 to 40 parts; epigallocatechin gallate - from 10 to 40 parts; silymarin - from 2 to 40 parts; naringenin - from 2 to 40 parts.EFFECT: use of the invention will make it possible to influence the components of the key signaling pathways of aging and prevent premature aging in animals.1 cl, 6 dwg

Description

The invention relates to food compositions for preventing age-related diseases and increasing the healthy lifespan of non-productive and farm animals using dietary compositions containing biologically active substances that can effectively influence the components of key aging signaling pathways, oxidative stress source markers, apoptosis and insulin resistance markers. A geroprotective feed additive can be added to the basic diet of non-productive and farm animals without its qualitative changes.

A known method includes feeding a companion animal an effective amount of a composition containing at least one omega-3 polyunsaturated fatty acid and various combinations of amino acids, minerals and antioxidants in amounts effective to enhance activity, increase vitality, protect cartilage, maintain muscle mass. , enhancing digestibility and improving the quality of skin and wool in terms of dry matter (RU 2 525 617 C2, 2014.08.20)

Known composition, which is a nutritionally complete diet containing a source of protein, a source of carbohydrates, a vegetable source and a fruit source. The protein source is a combination of chicken, egg white in an amount of 4% to 15% based on the total dry weight of the composition, and corn gluten meal. The source of carbohydrates is a combination of millet, malted rice, and oatmeal. The vegetable source is a combination of carrots, spinach and tomato pomace. The fruit source is citrus pulp (patent RU 2611160, February 21, 2017).

A known composition is a protein hydrolyzate obtained by acid hydrolysis and subsequent neutralization of a peptide-containing animal raw material selected from the following: carcasses of animals or fish, albumin, blood of animals or fish, meat of animals or fish, protein-containing waste in the production of products from animals or fish, filtration to obtain a hydrolyzate and a precipitate, followed by drying of the hydrolyzate, while the finished product contains peptides with a molecular weight above 3000 daltons, not more than 25 wt. %, the optical activity α20D of the finished product is 5-15, the ratio of amino nitrogen: fatty acids: carbohydrates is (10-30): (0.2-2): (0.4-5), and also contains sodium, chromium, nickel, cobalt, selenium, calcium, potassium, sulfur, phosphorus, chlorine, iron, zinc, copper, manganese. (patent RU 2 221456, 20.01.2004)

Known feed composition comprising the following categories of ingredients: antioxidants; anti-glycation agents; agents that reduce body weight or fat; agents that promote high sensitivity to insulin or low levels of insulin or glucose in the blood and anti-inflammatory agents (RU 2449554 C2, 10.05.2012. CN 101298563 A, 05.11.2008.).

Known geroprotective feed additives. For example, according to application US2019350899 - containing resveratrol, genistein, ellagic acid, curcumin and quercetin, according to application CN104543405 - containing plant extracts of artichoke, rosemary, tagete, turmeric extract, citrus extract, preferably grapefruit, nettle, garlic, grape, milk thistle, andrographis, green tea and tomatoes.

Known geroprotective feed additive, including nicotinamide riboside, white grapefruit seed extract containing spermidine, fucus bladder extract containing fucoxanthin (according to patent RU 2738644, selected as a prototype).

The disadvantage of the known additives is the lack of effectiveness associated with the lack of a complex action aimed at regulating key signaling pathways of longevity and aging, including sirtuin proteins, as well as a simultaneous effect on markers of oxidative stress, insulin resistance and apoptosis.

Thus, despite existing inventions, there is a need in the art for additional compositions to correct signaling pathways and markers associated with aging in animals.

The technical objective of the invention is to create a feed composition containing various geroprotectors, including sources of resveratrol, curcumin and epigallocatechin gallate, naringenin and silymarin, which in certain combinations are able to influence the components of key aging signaling pathways, markers-sources of oxidative stress, as well as markers of apoptosis and sensitivity to insulin. The technical result is to increase the efficiency of the geroprotective feed additive due to the complex action.

The technical result is achieved in a geroprotective feed additive containing a mixture of substances taken at the following ratio of mass parts: resveratrol - from 1 to 20 parts; curcumin - from 4 to 40 parts; epigallocatechin gallate - from 10 to 40 parts; silymarin - from 2 to 40 parts; naringenin - from 2 to 40 parts.

The invention is illustrated by drawings:

Fig. 1. Relative amount of SIRT1 protein in the soleus muscle of rats that received a diet supplemented with various combinations of geroprotectors (I - resveratrol and turmeric extract (95% curcuminoids); II - green tea extract (95% polyphenols, 60% catechins, 30% epigalocatechin gallate) , grapefruit seed extract (source of naringenin) and milk thistle extract (silymarin 80%)) and their combinations (III) in comparison with the control group (Control). * - significant differences in relation to the control group (P<0.05).

Fig. Fig. 2. Luminescence intensity (fluorescence) of the marker-source of oxidative stress NOX2 in the soleus muscle of rats that received a diet with the addition of various combinations of geroprotectors (I - resveratrol and turmeric extract (curcuminoids 95%); II - green tea extract (95% polyphenols, 60 % catechins, 30% epigalocatechin gallate), grapefruit seed extract (source of naringenin) and milk thistle extract (silymarin 80%)) and their combinations (III) in comparison with the control group (Control). * - significant differences in relation to the control group (P<0.05).

Fig. Fig. 3. Luminescence intensity (fluorescence) of the glucose transporter GLUT4 in the soleus muscle of rats that received a diet with the addition of various combinations of geroprotectors (I - resveratrol and turmeric extract (95% curcuminoids); II - green tea extract (95% polyphenols, 60% catechins, 30% epigallocatechin gallate), grapefruit seed extract (source of naringenin) and milk thistle extract (silymarin 80%)) and their combinations (III) in comparison with the control group (Control). * - significant differences in relation to the control group (P<0.05).

Fig. Fig. 4. Luminescence intensity (fluorescence) of the proapoptotic factor Bax in the soleus muscle of rats that received a diet with the addition of various combinations of geroprotectors (I - resveratrol and turmeric extract (95% curcuminoids); II - green tea extract (95% polyphenols, 60% catechins, 30% epigallocatechin gallate), grapefruit seed extract (source of naringenin) and milk thistle extract (silymarin 80%)) and their combinations (III) in comparison with the control group (Control). * - significant differences in relation to the control group (P<0.05).

Fig. Fig. 5. Intensity of luminescence (fluorescence) of the anti-apoptotic factor Bcl-2 in the soleus muscle of rats that received a diet with the addition of various combinations of geroprotectors (I - resveratrol and turmeric extract (95% curcuminoids); II - green tea extract (95% polyphenols, 60% catechins, 30% epigalocatechin gallate), grapefruit seed extract (source of naringenin) and milk thistle extract (silymarin 80%)) and their combinations (III) in comparison with the control group (Control). * - significant differences in relation to the control group (P<0.05).

Fig. Fig. 6. The ratio of the proapoptotic factor Bax and the antiapoptotic factor Bcl-2 in the soleus muscle of rats that received a diet with the addition of various combinations of geroprotectors (I - resveratrol and turmeric extract (curcuminoids 95%); II - green tea extract (95% polyphenols, 60% catechins, 30% epigalocatechin gallate), grapefruit seed extract (source of naringenin) and milk thistle extract (silymarin 80%)) and their combinations (III) in comparison with the control group (Control). * - significant differences in relation to the control group (P<0.05).

Sign (*) in the figures shows significant differences in relation to the control group (P<0.05). The values are presented in arbitrary units (c.u.).

Aging includes structural changes at various levels of organization of a living organism: systemic, organ, tissue, cellular and molecular. General signs of aging are primarily manifested at the molecular level, and are also characterized by changes in intracellular and intercellular signaling. In accordance with this, when exposed to age-associated targets, early changes will also be noticeable primarily at the molecular level and in the regulation of signaling pathways (Proshkina et al., 2020). One of the key components of signaling pathways that are currently considered markers of longevity is the family of NAD-dependent proteins - sirtuins (SIRT1-7), among which SIRT1 is the most studied (M. Yu et al., 2021). The role of sirtuin in the suppression of cellular aging may be mediated by delaying the age-related shortening of telomeres, maintaining the integrity of the genome, and also participating in the repair of DNA damage. In addition, SIRT1 may improve the ability to induce cell cycle arrest, mitochondrial biogenesis, and resistance to oxidative stress by inhibiting cell death and the programmed cell death pathway, apoptosis. Age-related features also include changes in the ratio of signaling molecules involved in the activation of apoptotic pathways, for example, Bax, and counteracting apoptosis - Bcl-2: with age, the Bax/Bcl-2 ratio increases (E.Khodapasand et al., 2015; L. Chung et al., 2006). On the other hand, according to the free radical (oxidative) theory of aging, oxidized macromolecules accumulate with age and oxidative stress increases, as a result of which cells lose the ability to function normally (M.T. Lin et al., 2003). One of the critical sources of oxidative stress in cells during aging is various isoforms of NADPH oxidase (NOX) enzymes, in particular, many researchers point to the leading role of the NOX2 isoform (L. Park et al., 2007; L. Geng et al., 2020; L.M. Fan et al., 2017; M.J. Sullivan-Gunn et al., 2013). In addition, age-associated changes may be accompanied by impaired tissue sensitivity to insulin, which can lead to the development of type 2 diabetes mellitus. One of the markers of tissue sensitivity to insulin is the glucose transporter GLUT4, the content of which can be reduced in insulin-dependent body tissues with increasing age (J.A. Houmard et al., 1995; J.M. dos Santos et al., 2012).

Geroprotective feed additive according to the present invention contains a mixture of substances taken in the following ratio of mass parts.

Resveratrol - from 1 to 20 mass parts (otherwise, 50-1000 mg per kg of diet). As a source of resveratrol, an extract of the comb knotweed (Polygonumcuspidatum) can be used.

Curcumin - from 4 to 40 mass parts (otherwise, 200-2000 mg per kg of diet). Turmeric extract (Curcumalonga) can be used as a source of curcumin and curcuminoids.

Epigallocatechin gallate - from 10 to 40 mass parts (otherwise, 500-2000 mg per kg of diet). Green tea extract can be used as a source of epigalocatechin gallate.

Silymarin - from 2 to 40 mass parts (otherwise, 100-2000 mg per kg of diet). Milk thistle extract (Silybummarianum) can be used as a source of silymarin.

Naringenin - from 2 to 40 mass parts (otherwise, 100-2000 mg per kg of diet). Grapefruit seed extract (Citrus paradissi) can be used as a source of naringenin.

Adding concentrations below the specified minimum limit does not provide a geroprotective effect on the animal's body. The use of concentrations above the indicated maximum values reduces the palatability of the product and, as a result, reduces the volume of consumption of the product by animals, or leads to adverse consequences associated with the refusal of food. The concentration can be adjusted individually for each animal species using formulas and interspecies dose transfer coefficients.

The following are the characteristics of each component of geroprotective supplements.

Resveratrol is a plant polyphenol found in a variety of plants, including grape skins, berries, knotweed, peanuts, and more. Resveratrol can affect various intracellular targets associated with longevity, the main one being SIRT1 histone deacytylase. Resveratrol has been shown to prolong lifespan by significantly increasing SIRT1 activity (M. Lagouge et al., 2006; K.T. Howitz et al., 2003). At the same time, many studies report the effect of resveratrol on several types of biological activity, such as antioxidant, anti-inflammatory, antitumor, antidiabetic, neuroprotective and antiplatelet effects, and protection of the cardiovascular system (Dan-Dan Zhou et al., 2021).

Curcumin is the main curcuminoid found in the root of the turmeric plant (Curcumalonga). The role of curcumin in slowing down aging has also been considered in many studies (A. Bielak-Zmijewskaet al., 2019; E. Sikorae tal., 2010). In the root of turmeric, the curcumin content is no more than 3.14% (R.F. Tayyem 2006), therefore, to achieve the desired effect, it is desirable to use turmeric extracts, where the total content of curcuminoids can be more than 90%. The addition of curcumin to the diet increased the lifespan of model animals (V.H. Liao et al., 2011; K.S. Lee et al., 2011; L.R. Shen et al., 2013). Curcumin has many molecular targets in the cell, including the regulation of key aging pathways (sirtuins, AMP-activated protein kinase (AMPK)), antitumor activity (A. Bielak-Zmijewska et al., 2010), anti-inflammatory and proapoptotic mechanisms (Y. Heetal., 2015 ; S.Aggarwaletal., 2006), have an antioxidant effect (V.P. Menonetal., 2007) and other effects.

Epigallocatechin gallate (EGCG) is a catechin found in high amounts in green tea. EGCG has shown a geroprotective effect on a number of model organisms, including mammals (S. Abbasetal., 2009; M.K. Brownetal., 2006; A.E. Wagneretal., 2015). Thus, it has been shown that EGCG increased the lifespan of rats by 8-12 weeks (Y. Niuetal., 2013). EGCG is able to have an antioxidant effect (S. Beyazetal., 2021), is involved in the regulation of AMPK, SIRT1 and Foxo signaling pathways (S. Pournourmohammadi et al., 2017; Li-Gui Xiong et al., 2017), associated with an increase in life expectancy and resistance to stress. EGCG supplementation promoted translocation of the glucose transporter GLUT4 in skeletal muscle, improving glucose transport (M. Ueda et al., 2008). EGCG has also been shown to improve the course and prognosis of diseases in animal models of cancer (Kyoung-jinMinetal., 2014), neurodegeneration (N.A. Singhetal., 2016), and metabolic syndrome (Li-GuiXiongetal., 2018).

Naringenin is a natural flavonoid found in citrus fruits, especially grapefruit, and tomatoes. The highest levels of naringenin have been found in young grapefruit tissues, including its pits (P.S. Jourdan et al., 1985). Naringenin is also a potential geroprotector; certain concentrations of naringenin can increase the lifespan of model animals (D. Chattopadhyay et al., 2016). Naringenin is able to have a protective effect on heart function in mice by activating SIRT1, as well as reducing pro-inflammatory cytokines (L.Testaietal., 2020). In a study in an aging mouse model, naringenin improved behavioral function and reduced neurological deficits by increasing antioxidant enzyme levels and counteracting oxidative stress (Y. Zhangetal., 2017). In another study, naringenin reduced the level of inflammatory cytokines and oxidative stress markers in the skin of mice after ultraviolet irradiation (R.M. Martinez et al., 2015).

2014). Силимарин в исследованиях на модельных животных увеличивал среднюю продолжительность жизни до 24,8% по сравнению с контролем (J. Kumar etal., 2015). Силимарин может увеличивать SIRT1, а также демонстрирует антиапоптотическое действие (L-H Li etal., 2007). Также было показано, что силимарин может активировать фермент теломеразу, тем самым способствуя задержке укорочения теломер в нормальных тканях (A. Parzonkoetal., 2010). В опухолевых клетках силимарин, напротив, снижал активность теломеразы (Z. Faezizadeh etal., 2012; E. Yurtcu etal., 2015). Кроме гепатопротекторного действия силимарин обладает нейропротекторным потенциалом (A.Borahetal., 2013), антиканцерогенным действием (Sung-HyunKimetal., 2019), способен влиять на углеводный обмен и предотвращать нарушение толерантности глюкозы (C.Sotoetal., 2010).Silymarin is a flavanone derived from the seeds of milk thistle (Silybummarianum). It is widely used for the treatment of liver diseases in clinical practice (

2014). Silymarin in animal model studies increased life expectancy by up to 24.8% compared to controls (J. Kumar et al., 2015). Silymarin can increase SIRT1 and also shows anti-apoptotic effects (LH Li et al., 2007). It has also been shown that silymarin can activate the telomerase enzyme, thereby contributing to the delay in telomere shortening in normal tissues (A. Parzonkoe tal., 2010). In tumor cells, silymarin, on the contrary, reduced telomerase activity (Z. Faezizadeh et al., 2012; E. Yurtcu et al., 2015). In addition to the hepatoprotective effect, silymarin has a neuroprotective potential (A.Borahetal., 2013), anticarcinogenic effect (Sung-HyunKimetal., 2019), is able to influence carbohydrate metabolism and prevent impaired glucose tolerance (C.Sotoetal., 2010).

The bioactive compounds used in the compositions have been widely studied individually and are described in detail in the book “Potential geroprotectors” (Moskalev A.A. et al., 2016). However, attempts to combine more than three substances from the listed list have not yet been made. These substances in combination have a synergistic effect, affecting various markers associated with aging.

The proposed geroprotective composition can be used as a nutritional supplement to the daily diet, treats, treats, as part of drinking water or separately in the form of tableted or encapsulated forms, both for non-productive animals (for example, cats, dogs, rodents, ornamental birds), and for agricultural animals (e.g. sheep, cows, pigs, goats, birds). The geroprotective feed additive can be used in mature animals, including both young and old animals. Its manufacture consists in simple mixing of components.

An example of the invention.

The experiments were carried out on elderly Wistar rats aged (25-30 months). The studies were carried out in compliance with the requirements for working with experimental animals, set out in the Order of the Ministry of Health and Social Development dated August 23, 2010 No. 708 “On Approval of the Rules of Laboratory Practice”. Animals were randomly divided into 4 groups (n=6 in each group). The control group (Control) received a standard daily diet. Group I and Group II received a standard daily diet supplemented with the following concentrations of potential geroprotectors:

Group Potential geroprotector Quantity, mg/kg diet I Resveratrol 120 Turmeric extract (curcuminoids 95%) 250 II Green tea extract (polyphenols 95%, catechins 60%, epigalocatechin gallate 30%) 1000 grapefruit seed extract 500 Milk thistle extract (silymarin 80%) 500

Group III received the standard daily ration supplemented with a combination of supplements that groups I and II received. After 30 days of feeding, the animals were taken out of the experiment and the quantitative content of SIRT1, NOX2, GLUT4 Bax, Bcl-2 markers in rat skeletal muscles was studied using immunohistochemistry and Western blotting with antibodies against the corresponding proteins. Skeletal muscles were chosen as an experimental model as a type of tissue that is one of the most sensitive to aging and is characterized by the development of age-related sarcopenia (J.Etienne et al., 2020). Statistical processing was carried out using the Kruskal-Wallis method, statistical differences were considered significant at a significance level of P<0.05.

Figure 1 shows the differences in the level of SIRT1 protein in the control and experimental groups. Compositions I and II show a tendency to increase the level of SIRT1, however, significant differences were found only in group III in relation to the animals of the control group, which reflects the additive (synergistic) effect of the components of this composition. Taking into account the role of SIRT1 described above as a key component of the signaling pathways of aging and longevity, it can be concluded that the combination of RCGGS has the greatest geroprotective effect.

Fig. 2 shows the effect of dietary combinations on the oxidative stress marker NOX2. It was found that combination III showed the greatest effect of negative regulation of NOX2 expression in comparison with the control group of aged rats having a higher level of this prooxidant protein. The resulting changes may indicate a protective effect of the RCGGS combination on oxidative stress. Individual compositions I and II showed no significant effect.

In FIG. Figure 3 shows the levels of insulin sensitivity marker - glucose transporter GLUT4 in the control group and groups with different sets of geroprotective supplements. The lowest levels of GLUT4 fluorescence on transverse sections of muscles were found in the control group of animals. In groups with geroprotective compositions (I, II and III), an increase in the intensity of GLUT4 fluorescence was noted, which may indicate a positive effect of dietary supplements on tissue sensitivity to insulin. Significant differences in groups with the addition of geroprotective compositions were not found.

Fig. 4, 5 and 6 show the effect of geroprotective combinations on apoptosis regulation factors: proapoptotic factor Bax, antiapoptotic factor Bcl-2. All combinations demonstrate an effect on the studied regulators, the most significant in this case being the effect on the Bax/Bcl-2 ratio. The effect of a significant decrease in the Bax/Bcl-2 ratio compared to the control group was found in all groups with geroprotective compositions, which may indicate a protective effect on apoptosis and an increase in tissue viability in general.

Claims (2)

Geroprotective feed additive containing a mixture of substances taken at the following ratio of mass parts: resveratrol 1 to 20 pieces curcumin 4 to 40 pieces epigallocatechin gallate 10 to 40 pieces silymarin from 2 to 40 parts naringenin from 2 to 40 parts.

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