Pharmaceutical composition supporting oncology patients during radiotherapy and healthy individuals exposed to ionizing radiation

Abstract

The invention relate to a pharmaceutical composition for supporting oncology patients during radiotherapy and healthy individuals exposed to ionizing radiation, which content of four natural extracts rich in polyphenolic compounds: chokeberry (Aronia melanocarpa L.), elderberry (Sambucus nigra L.), blackcurrant (Ribes nigrum L.), and evening primrose seeds (Oenothera biennis L.).

Description

[0001] PHARMACEUTICAL COMPOSITION SUPPORTING ONCOLOGY PATIENTS DURING RADIOTHERAPY AND HEALTHY INDIVIDUALS EXPOSED TO IONIZING RADIATION

[0002] The invention relate to a pharmaceutical composition for the production of dietary supplements, intended for individuals exposed to the harmful effects of ionizing radiation, including oncology patients undergoing radiotherapy and medical personnel performing radiological examinations.

[0003] Radiotherapy is considered one of the primary tools in the treatment of various types of malignant tumors. The most commonly used types of radiation in radiotherapy are ionizing radiation: photon and gamma (y) radiation. They are characterized by high penetration and energy, which allows them to act both superficially and reach tumors located inside the body, leading to the damage and death of cancer cells. Despite many therapeutic benefits, exposure to radiation also causes damage to surrounding healthy tissues and leads to side effects in patients undergoing radiotherapy. Moreover, radiation-induced mutations and genomic instability result in stochastic effects, which can also occur in healthy individuals not undergoing radiotherapy but exposed to ionizing radiation, such as medical personnel performing radiological examinations.

[0004] The harmful effects of ionizing radiation result from excessive production of free radicals, which damage cellular structures such as proteins, lipids, and nucleic acids. Furthermore, ionizing radiation can lead to direct damage to genetic material through double-strand breaks in DNA, the formation of cross-links, or mutations. The consequences of this include cell death via apoptosis, the release of molecules associated with cell damage, excessive activation of the immune system, and the induction of inflammation in the body.

[0005] Radioprotectors are substances used to limit or eliminate damage in non-cancerous (healthy) cells caused by ionizing radiation, which lacks selectivity exclusively towards cancer cells. Key criteria for an ideal radioprotector include protecting unaltered cells from the harmful effects of ionizing radiation through: neutralization of free radicals, repair of DNA damage, reduction of inflammation, and delaying cell division— aiming to extend the time available for initiating repair processes in the cells. Additionally, it should exhibit low toxicity, high stability, and selectivity of action towards normal cells. Furthermore, it should be easy to administer, have no side effects following administration, and not decrease the radiosensitivity of cancer cells

[0006] In addition to radioprotectors, among the substances administered to patients undergoing radiotherapy, there are also radiomitigators and radiotherapeutics. The difference between them lies in the timing of their administration to patients: radioprotectors are agents used before radiotherapy, while radiomitigators are administered during exposure to ionizing radiation or shortly afterward. Radiotherapeutics are used after clinical symptoms of toxicity have appeared in healthy tissues as a result of previous radiotherapy. According to current knowledge, among the rad io protectors and mitigators approved by the FDA (U.S. Food and Drug Administration) are Amifostine, Filgrastim, Palifermin, Romiplostim, and Sargramostim. Despite the positive effects of radioprotective substances on non- cancerous cells, their use may be associated with side effects in patients undergoing radiotherapy. For example, Amifostine has been associated with side effects such as excessive sweating, nausea, bronchospasm, hypotension, and bradycardia. The administration of Palifermin has been associated with skin complications in some patients, including rash, erythema, edema, and toxicity in the oral cavity, such as dysesthesia, discoloration or thickening of the tongue, and even changes in taste. For this reason, compounds with effective radioprotective action that do not cause accompanying side effects are still being sought. Notable candidates include natural extracts derived from plants that are rich in substances with proven protective effects against ionizing radiation.

[0007] One of the richest sources of antioxidants is the chokeberry (Aronia melanocarpa L.), which is high in anthocyanins, tannins, proanthocyanidins (PAC), flavonols, and hydroxycinnamic acids, particularly chlorogenic acid. Additionally, chokeberries exhibit anti-inflammatory, anti- mutagenic, and immunomodulatory properties that may help mitigate the effects associated with the harmful actions of ionizing radiation. Dufour et al. demonstrated the broad antioxidant activity of chokeberries in in vitro studies on Caco-2 intestinal cells, HepG2 liver cells, and SH-SY5Y neuronal cells, resulting from both the direct inactivation of reactive oxygen species (ROS) and the modulation of the signaling pathway of the erythroid nuclear transcription factor Nrf2. Bushmeleva et al. showed that the cyanidin-3-glucoside (Cy-3-Gal) present in chokeberries significantly reduced the level of malondialdehyde (MDA) in the blood of rats, which is one of the final toxic products of lipid peroxidation. Nikolova et al. proved in vivo that chokeberry extract has protective effects against gamma radiation doses of 10-25 kGy on the gastrointestinal tract in healthy mice. The radioprotective effects of anthocyanins found in chokeberries were also observed in chinchillas and mice exposed to ionizing radiation at a dose of 4 Gy. Administration of chokeberry pigment and concentrated juice decreased the level of superoxide anion radicals and provided protection to splenic cells. Apedbour et al. demonstrated the radioprotective properties of chlorogenic acid concerning male reproductive cells. It was observed that chlorogenic acid (20-80 mg / kg) inhibited oxidative stress in irradiated testes, contributing to increased levels of glutathione (GSH) and total antioxidant capacity (TAG). Additionally, it normalized MDA levels and the Bax / Bcl-2 ratio compared to the irradiated group.

[0008] Black elderberry (Sambucus nigra L.) also exhibits a wide range of biological activities, being rich in active compounds such as polyphenols, including anthocyanins, proanthocyanidins, flavonols, phenolic acids, and tannins, which are also good sources of protein, amino acids, pectins, and vitamins. The high antioxidant potential of black elderberry has been confirmed in numerous studies, associated with the high content of active substances such as cyanidin- 3-glucoside and cyanidin-3-sambubioside. Following the application of black elderberry extract, inhibition of reactions using the DPPH radical was observed at levels of 82.08 - 89.25%. In vitro studies showed that black elderberry extract reduced the generation of reactive oxygen species by 22% and oxidative DNA damage by 46% in human intestinal mucosal cells. In vivo studies in a hypertension model conducted on Wistar rats demonstrated the cardioprotective effect of black elderberry extract, resulting from the reduction of oxidative stress through normalization of reduced glutathione (GSH) levels. Additionally, a decrease in lipid peroxidation was observed. Furthermore, Olejnik et al. demonstrated the anti-inflammatory properties of black elderberry fruit extract in lipopolysaccharide (LPS)-activated murine macrophages of the RAW264.7 cell line, where a reduced level of released pro-inflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-a), and prostaglandin E2 (PGE2), was observed. Ciocoiu et al. found that the action of polyphenols from black elderberry led to a decrease in the level of pro-inflammatory interleukin ip (I L-ip), which is responsible for chronic inflammatory states leading to various complications. Black elderberry is also characterized by a high content of flavonols, primarily quercetin, rutin, and astragalus, which have potential radioprotective properties. Supplementation with quercetin (at a concentration of 20 mg / kg body weight) contributed to the reduction of damage in the kidneys and bladder resulting from X-ray exposure in rats at a dose of 8 Gy. Additionally, inhibition of neutrophil infiltration and a reduction in the level of the pro-inflammatory mediator TNF-a were observed, along with a decrease in the risk of oxidative DNA damage and apoptosis.

[0009] Literature also indicates a range of positive properties of blackcurrant (Ribes nigrum L.) fruit extracts, among which the ability to reduce reactive oxygen species generated as a result of oxidative stress, alleviate inflammatory conditions, support glucose metabolism in tissues, and exhibit antibacterial and antiviral effects are particularly noteworthy. All the positive properties of the extract are attributed to the main bioactive compounds found in blackcurrant fruits, namely cyanidin-3-rutinoside and 3-O-rutinoside delphinidin, which belong to the group of anthocyanins. Sivarajan et al. demonstrated the positive impact of a mixture of blackcurrant and bilberry (Vaccinium myrtillus L.) extracts on the reduction of replication of measles virus, human herpesvirus 1, human herpesvirus 8, and murine cytomegalovirus in in vitro studies. Diaconeasa et al. highlighted the ability of blackcurrant fruit extracts to inhibit the proliferation of cancer cells in HeLa, A2780, and B16F10 cell lines. Benn et al. investigated the effects of blackcurrant extract on anti-inflammatory properties in splenocytes isolated from C57BL / 6J mice with induced obesity. The results indicated a lower level of pro-inflammatory cytokines in the group supplemented with a high-polyphenol blackcurrant extract. Venkatasubramanian et al. studied the effects of blackcurrant extract on toxins produced by Clostridium difficile, a bacterium that overgrows in the colon due to imbalances in the gut flora caused, among other things, by antibiotic use. The study results showed neutralization of C. difficile toxins using blackcurrant extract in a Caco-2 cell model. Yamamoto et al. demonstrated the protective effect of blackcurrant extract in reducing DNA damage induced by oxidative stress in TK6 cells. A decrease in the levels of reactive oxygen species (ROS), which are the direct cause of genotoxicity, was also observed. The extract from various parts of the evening primrose plant (Oenothera biennis L.) is another example of a plant extract used in medicine due to its antioxidant, anti-inflammatory, antibacterial, and antiviral properties. Studies have also confirmed its antidiabetic and anticancer properties, making it a plant with high health potential. The biological activity of evening primrose extract is primarily attributed to polyphenols such as gallic acid, caffeic acid, epicatechin, coumaric acid, ferulic acid, rutin, and rosmarinic acid. Researchers have demonstrated the positive effects of the evening primrose seed extract on the skin in a hairless mouse model. Animals exposed to UV-B radiation three times a week for 14 weeks and supplemented with the extract showed improvements in skin structure and elasticity, along with lower levels of inflammatory markers and collagenases compared to the group exposed to radiation without supplementation. The literature mainly describes the beneficial properties of Oenothera biennis L. seed oil and extracts obtained from the root and aerial parts of the plant. Lee et al. demonstrated the positive effects of the aerial part extract of evening primrose in human keratinocytes subjected to oxidative stress. The studies observed lower levels of reactive oxygen species in cells preincubated with the extract and then exposed to hydrogen peroxide (H2O2), compared to cells incubated only with H2O2. Similar results were obtained in studies assessing DNA damage levels in cells, showing neutralization of the genotoxic effect induced by hydrogen peroxide after applying the extract. Experiments involving C57BL / 6 mice fed a high-fat diet and supplemented with extracts from different parts of evening primrose showed lower expression levels of oxidative stress markers such as heme oxygenase Ho-1, glutathione S-transferase A4 Gsta4, and P40phox, compared to the group without supplementation. Additionally, lower levels of pro-inflammatory interleukins IL-6, TNFa, and monocyte chemoattractant protein MCP-1 were observed in the adipose tissue of the mice. A similar effect was seen in gene expression studies in the liver. Other research indicated the positive effect of evening primrose seed oil on Swiss mice treated with cyclophosphamide. Two weeks of oil supplementation resulted in the neutralization of the genotoxic effect induced by cyclophosphamide, compared to the group of mice that received only the chemotherapeutic agent. Additionally, lower levels of pro-inflammatory interleukins in the liver and lower levels of lipid peroxidation in the pancreas were demonstrated.

[0010] So far, there is no product on the market with proven radioprotective effects that contains standardized extracts from chokeberry and elderberry fruits, as well as blackcurrant and evening primrose seeds. Obtaining a unique combination of these extracts with proper standardization will enable their use with a minimal consumption dose. At the same time, this will allow for achieving an optimal concentration of the desired active compounds found in these extracts, facilitating a potentiated effect that supports the body against ionizing radiation.

[0011] The invention relate to a composition consisting of four natural extracts rich in polyphenolic compounds. The extracts have been isolated from the following plants: chokeberry fruits (Aronia melanocarpa L.), elderberry fruits (Sambucus nigra L.), blackcurrant fruits (Ribes nigrum L.), and evening primrose seeds (Oenothera biennis L.j. The raw materials used to produce the extracts come from Polish and European crops. The extracts used to produce the composition according to the invention are obtained by standard methods known in the state of the art.

[0012] The composition, as a mixture of extracts from elderberry, chokeberry, blackcurrant, and evening primrose seeds, is characterized by a polyphenol content ranging from 35% to 65% and anthocyanin content from 10% to 35%.

[0013] The composition, in which the extract content is as follows: 30-50% elderberry fruit extract, 10-30% chokeberry fruit extract, 5-25% blackcurrant fruit extract, and 20-40% evening primrose seed extract.

[0014] The composition, where the chokeberry fruit extract is characterized by a polyphenol content ranging from 45% to 80% and anthocyanin content from 20% to 35%.

[0015] The composition, where the elderberry fruit extract is characterized by a polyphenol content ranging from 20% to 55% and anthocyanin content from 10% to 35%.

[0016] The composition, where the blackcurrant fruit extract is characterized by a polyphenol content ranging from 20% to 55% and anthocyanin content from 10% to 25%.

[0017] The composition, where the evening primrose seed extract is characterized by a polyphenol content ranging from 35% to 55% and procyanidin content from 10% to 45%.

[0018] The composition, where the extracts are in liquid, semi-solid, or solid form.

[0019] The composition exhibits radioprotective effects, including antioxidant, anti-inflammatory, and cyto protective properties.

[0020] The composition is intended for the production of a dietary supplement.

[0021] The quantitative and qualitative composition was selected based on research conducted on in vitro cellular models and in vivo studies within the OncoAron project, titled "Innovative polyphenol-based preparations derived from berry fruits, used in the prevention and mitigation of side effects of oncological therapies, including chemotherapy and radiotherapy," granted by the Regional Operational Program of the Pomeranian Voivodeship.

[0022] The combination of plant extracts in the aforementioned proportions allows for the creation of a base composition for the preparation, characterized by multifaceted radioprotective effects, including antioxidant, anti-inflammatory, and cytoprotective effects. Figure description:

[0023] Fig.l - shows the effect of the composition (10 pg / mL) on ROS levels in PBMC cells. Positive control: cells stimulated with 250 pM H2O2. Negative control: cells without stimulant. The results are presented as the mean of three independent experiments. Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test. ***p<0.001.

[0024] Fig.2 - shows the effect of the composition (100 mg / kg body weight) on the changes in the expression levels of selected genes related to oxidative stress: Nrf2, Nqol, Nox4, Ho-1, Cat, Gpxl, Sodl, and Sod2, induced by ionizing radiation in mouse kidneys and liver. Normalized relative mRNA expression was determined with respect to a tissue-specific reference gene. Relative quantitative measurement was performed using the 2-AACtmethod. Blue color indicates decreased expression relative to the Control group (+) Pl. Red color indicates increased expression relative to the Control group (+) Pl.

[0025] Fig.3 - shows the effect of the composition (100 mg / kg body weight) on the lipid peroxidation in mouse liver (A) and kidneys (B). Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test. *p<0.05, ***p<0.001.

[0026] Fig.4 - shows:

[0027] (A) A statistically significant increase in NO levels observed using the Griess method in PBMC cell supernatants after a 24-hour incubation with the tested composition at a concentration of 10 pg / mL.

[0028] (B) A statistically significant decrease in IL-2 and IFN-y levels in PBMC cells observed after a 9-hour incubation with the composition at a concentration of 10 pg / mL. The results are presented as the mean of three independent experiments. Statistical analysis was performed using the Student's t-test and one-way ANOVA, with Dunnett's post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0029] Fig.5 - shows the effect of the composition according to the invention (100 mg / kg body weight) on the expression of the genes: Sirt-1, Nf-KB p65, lcam-1, Caspl, 11-16, 11-6, Ifn-y, and Hmgb-1 in the liver, kidneys, lymph nodes, and spleen cells of mice exposed to ionizing radiation. Normalized relative mRNA expression was determined with respect to a tissuespecific reference gene. Relative quantitative measurement was performed using the 2-AACtmethod. Blue color indicates decreased expression relative to the Control group (+) Pl. Red color indicates increased expression relative to the Control group (+) Pl.

[0030] Fig.6 - shows the effect of the composition according to the invention (100 mg / kg body weight) on the expression of the genes Ape-1, Ogg-1, and Atm in mouse liver, kidneys, and spleen cells. Normalized relative mRNA expression was determined with respect to a tissuespecific reference gene. Relative quantitative measurement was performed using the 2-AACtmethod. Blue color indicates decreased expression relative to the Control group (+) Pl. Red color indicates increased expression relative to the Control group (+) Pl. Fig.7 - shows the effect of the composition according to the invention (100 mg / kg body weight) on the expression of the genes Casp3, Bax, and Tgf-Bl in mouse liver, kidneys, and bone marrow cells. Normalized relative mRNA expression was determined with respect to a tissue-specific reference gene. Relative quantitative measurement was performed using the 2~ AActmethod. An increase in gene expression compared to the Control group (+) Pl is indicated in red, while a decrease is indicated in blue.

[0031] Fig.8 - shows the effect of the composition according to the invention (100 mg / kg body weight) on bone marrow cells obtained from mice exposed to ionizing radiation:

[0032] (A) Flow cytometry analysis of leukocyte levels in bone marrow cells.

[0033] (B) Leukocyte levels and (C) platelet levels in blood collected from mice 24 hours after exposure to ionizing radiation. For (A), (B), and (C), the tables present a comparison of the obtained results between negative Control N and positive Control Pl or P1+P2 (combined positive controls, Pl = IR 4 days, P2 = IR 30 days), presented as a percentage of negative Control N (values obtained for the median). The graphs show changes in the Study group Bl or B1+B2 (combined study groups, Bl = composition + IR 4 days, B2 = composition + IR 30 days), where the obtained results represent a percentage of positive Control Pl or P1+P2. Statistical analysis was conducted using the Mann- Whitney test.

[0034] (D) The level of TNFa was determined using the ELISA method in supernatants obtained from bone marrow cell cultures stimulated with PMA and ionomycin for 5 hours. Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0035] Fig.9 - shows:

[0036] (A) Microscopic sections of mouse liver tissues stained using the H&E method. Cell nucleus are visible as blue-purple areas, while the cytoplasm and extracellular structures appear pink-red. DBV - Dilated Blood Vessels; BD - Bile Ducts; HA - Hepatic Artery; PV - Portal Vein; S - Sinusoids. For each mouse, at least 10 images were taken from different microscopic fields at two magnifications (lOx and 40x).

[0037] (B) Microscopic sections of mouse kidney tissues stained using the H&E method. Cell nuclus are visible as blue-purple areas, while the cytoplasm and extracellular structures appear pink-red. G - Glomeruli; AG - Atrophic Glomeruli; BCD - Bellini Collecting Ducts; CD - Collecting Ducts; T - Tubule; BC - Bowman Capsule; V - Vacuolization; EBC - Enlarged Bowman Capsule. For each mouse, at least 10 images were taken from different microscopic fields at two magnifications (lOx and 40x).

[0038] Fig.10 - shows the effect of the composition according to the invention (dose of 100 mg / kg body weight) on the expression of genes related to oxidative stress (A), inflammation (B), and cyto protection (C) in selected tissues of mice 30 days after exposure to ionizing radiation. Normalized relative mRNA expression was determined with respect to a tissue-specific reference gene. Relative quantitative measurement was performed using the 2-AACtmethod. Blue color indicates decreased expression relative to the Control group (+) P2. Red color indicates increased expression relative to the Control group (+) P2.

[0039] The invention is illustrated by the following implementation examples, which do not constitute a limitation of the invention.

[0040] EXAMPLE 1

[0041] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0042] Effect 1.

[0043] ANTIOXIDANT ACTIVITY OF THE COMPOSITION ACCORDING TO THE INVENTION RELATIVE TO MONONUCLEAR BLOOD CELLS (PBMC)

[0044] RESEARCH METHODOLOGY

[0045] Composition

[0046] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 50 mg / mL was prepared using a water:DMSO (4:6 v / v) mixture and then diluted to a concentration of 10 pg / mL in the culture medium.

[0047] Cell models used for experiments

[0048] Peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from leukocyte-platelet layers of healthy blood donors according to a standard procedure (Bulent et al., 2018) using a density gradient with Ficoll (1.077 g / L). The cells were cultured in RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin, 100 pg / mL streptomycin.

[0049] Evaluation of antioxidant activity

[0050] The level of reactive oxygen species (ROS) in PBMCs was determined using the 2', 7'- dichlorodihydrofluorescein diacetate (H2DCFDA). PBMCs were exposed to a ROS inducer (H2O2 at a concentration of 250 pM) and the composition at a concentration of 10 pg / mL. After a 2- hour co-incubation, the level of DCF was measured using a flow cytometer. The level of dichlorofluorescein (DCF) corresponded to the level of reactive oxygen species. The positive control: PBMCs incubated for 2 hours with the ROS inducer - H2O2 at a concentration of 250 pM. The negative control: PBMCs without any stimulant. Results

[0051] Analysis of the obtained results showed a statistically significant increase of 48% [95% Cl from 33 to 64] in ROS levels in PBMCs after exposure to 250 pM H2O2, compared to the negative control. Cells exhibited a 45% [95% Cl from 30 to 60] decrease in ROS level compared to the positive control, when treated simultaneously with the composition and the stimulant.

[0052] EXAMPLE 2

[0053] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0054] Effect 2.

[0055] ANTIOXIDANT ACTIVITY OF THE COMPOSITION ACCORDING TO THE INVENTION IN MICE EXPOSED TO IONIZING RADIATION

[0056] RESEARCH METHODOLOGY

[0057] Composition

[0058] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight.

[0059] In vivo experimental model

[0060] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 1 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N and Pl groups were administered saline intragastrically (up to 100 pL depending on body weight), while mice from the Bl group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7-day supplementation, mice from the Pl and Bl groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for the next 4 days post-irradiation. After this period, mice from the N, Pl, and Bl groups were euthanized, and tissues were collected for further analysis.

[0061] Table 1. Experimental groups used in in vivo studies. Evaluation of antioxidant activity

[0062] Reactive oxygen species (ROS) generated by ionizing radiation lead to increased oxidative stress in the body. In response to stress-inducing factors, there is an activation of gene expression in cells that encode proteins responsible for inducing the enzymes necessary for neutralizing ROS and maintaining redox balance.

[0063] To assess the antioxidant activity of the composition, an analysis of the expression of selected genes related to oxidative stress was performed. RNA from the liver and kidneys of mice was isolated using the Total RNA Mini kit from A&A Biotechnology ©. The isolated RNA was transcribed into cDNA using the RT-PCR method (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems™). The normalized relative expression of each gene was determined using qPCR (PerfeCTa SYBR® Green FastMix Reagent; Quantabio©) with Tbp (TATA-binding protein) as the reference gene for the analyzed tissues. Relative quantification was determined using the 2-AACtmethod. The sequences of primers used in the qPCR are listed in Table 2. The obtained results were compared to the positive control group (Pl).

[0064] Table 2. The sequences of primers used in the qPCR.

[0065] Results

[0066] The obtained results demonstrated the antioxidant effect of the composition through its influence on the expression of genes related to oxidative stress. In the positive control group (Pl), ionizing radiation induced oxidative stress in both the kidneys and liver (Fig. 2). In the liver, activation of the nuclear transcription factor Nrf2 was observed, which in turn led to the induction of the ROS-neutralizing enzyme NAD(P)H-quinone dehydrogenase Nqol. In the experimental group (Bl), lower expression levels of Nrf2 and Nqol genes were observed compared to the control group (Pl), suggesting that the composition exhibits protective properties. The antioxidant effect of the composition in the kidneys was reflected by the reduced expression of the Nox4 gene (NADPH oxidase 4) and reduced expression of genes associated with the enzymatic defense mechanism against reactive oxygen species, such as Ho-1 (heme oxygenase), Cat (catalase), Gpxl (glutathione peroxidase 1), Sodl (superoxide dismutase 1), and Sod2 (superoxide dismutase 2).

[0067] EXAMPLE 3

[0068] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0069] Effect 3.

[0070] ANTIOXIDANT EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION THROUGH INHIBITION OF UNSATURATED FATTY ACID OXIDATION IN MICE EXPOSED TO IONIZING RADIATION

[0071] RESEARCH METHODOLOGY

[0072] Composition

[0073] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight.

[0074] In vivo experimental model

[0075] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 3 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N and Pl groups were administered saline intragastrica I ly (up to 100 pL depending on body weight), while mice from the Bl group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7-day supplementation, mice from the Pl and Bl groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for the next 4 days post-irradiation. After this period, mice from the N, Pl, and Bl groups were euthanized, and tissues were collected for further analysis.

[0076] Table 3. Experimental groups used in in vivo studies. Evaluation of the effect of the composition on lipid peroxidation

[0077] Reactive oxygen species (ROS) generated by ionizing radiation lead to increased oxidative stress in the body. As a result of ROS activity, damage to cellular membrane lipids occurs through the biological oxidation of unsaturated fatty acids, leading to their degradation. The final products formed during lipid peroxidation are highly toxic and exhibit mutagenic and carcinogenic properties, potentially increasing the risk of cancer development.

[0078] To evaluate the effect of the composition according to the invention on lipid peroxidation, the commercially available Lipid Peroxidation (MDA) Assay Kit from Merck was used. This kit measures the concentration of malondialdehyde (MDA), one of the final products of oxidative lipid degradation. Mouse liver and kidney tissues were homogenized in MDA Lysis Buffer. The MDA concentration in the resulting tissue supernatants was determined by conducting a reaction at elevated temperature with thiobarbituric acid (TBA), producing a colored MDA- TBA2 adduct with a maximum absorbance at A. = 532 nm. The obtained results were compared to the positive control group (Pl).

[0079] Results

[0080] The obtained results showed that ionizing radiation contributes to the initiation of lipid peroxidation. In the positive control group (Pl), a statistically significant increase in malondialdehyde (MDA) concentration was observed, with an increase of 33.26% in the liver [95% Cl from -65.39 to -1.14] and 23.47% in the kidneys [95% Cl from -46.58 to -0.36] compared to the negative control (N). In the experimental group (Bl), a 52% reduction in MDA levels was observed in the liver [95% Cl from 21.97 to 82.07, p-value = 0.001], along with a 10.43% decrease in the kidneys compared to the positive control group (Pl).

[0081] EXAMPLE 4

[0082] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0083] Effect 4.

[0084] ANTI-INFLAMMATORY EFFECTS OF THE COMPOSITION ACCORDING TO THE INVENTION ON PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS)

[0085] RESEARCH METHODOLOGY

[0086] Composition

[0087] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 50 mg / mL was prepared using a water:DMSO (4:6 v / v) mixture and then diluted to a concentration of 10 pg / mL in the culture medium. Cell models used for experiments

[0088] Peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from leukocyte-platelet layers of healthy blood donors according to a standard procedure (Bulent et al., 2018) using a density gradient with Ficoll (1.077 g / L). The cells were cultured in RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin, 100 pg / mL streptomycin.

[0089] Evaluation of anti-inflammatory activity

[0090] The levels of interleukin 2 (IL-2) and interferon gamma (IFN-y) were measured in the supernatants collected from the culture of PBMCs. Phytohemagglutinin-L (PHA-L), a lectin with strong agglutinating and mitogenic activity that binds specifically to lymphocyte surface receptors, was used to induce T lymphocytes. The untreated cells served as the control, while the positive control consisted of cells stimulated with PHA-L for 5 hours (5 pg / mL). PBMCs were pre-incubated with the composition for 4 hours, followed by the addition of PHA-L for an additional 5 hours, resulting in a total incubation time of 9 hours with the composition. The concentrations of the cytokines were assessed using commercially available ELISA DuoSet® Development Systems (R&D) for human cytokines IL-2 and IFN-y. The level of nitric oxide (NO) was measured in the supernatants from PBMCs after a 24-hour incubation with the composition using a commercially available kit from Promega (Griess method).

[0091] Results:

[0092] Analysis of the supernatants showed a 5.65-fold increase in nitric oxide (NO) levels compared to the control cells [95% Cl 3.36 - 5.94], The stimulant PHA-L increased the levels of IL-2 in the supernatants of PBMCs by 82.74% [95% Cl 72.54 - 92.94] and IFN-y by 77.23% [95% Cl 59.84 - 94.62], Following pre-incubation of PBMCs with the composition at a concentration of 10 pg / mL, a statistically significant decrease was observed in IL-2 levels by 15.25% [95% Cl 5.05 - 25.45] and IFN-y levels by 22.88% [95% Cl 5.48 - 40.27] compared to PBMCs stimulated with PHA-L.

[0093] EXAMPLE 5

[0094] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0095] Effect 5.

[0096] ANTI-INFLAMMATORY EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION IN

[0097] MICE EXPOSED TO IONIZING RADIATION

[0098] RESEARCH METHODOLOGY

[0099] Composition According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight.

[0100] In vivo experimental model

[0101] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 4 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N and Pl groups were administered saline intragastrica I ly (up to 100 pL depending on body weight), while mice from the Bl group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7-day supplementation, mice from the Pl and Bl groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for the next 4 days post-irradiation. After this period, mice from the N, Pl, and Bl groups were euthanized, and tissues were collected for further analysis.

[0102] Table 4. Experimental groups used in in vivo studies.

[0103] Evaluation of anti-inflammatory activity

[0104] Ionizing radiation used in radiotherapy leads to the generation of reactive oxygen species (ROS) in the body, resulting in the induction of an inflammatory state in tissues. This inflammatory state is characterized by an increased immune response, manifested by the upregulation of genes encoding pro-inflammatory proteins. To assess the potential anti-inflammatory properties of the composition, an analysis of the expression levels of selected genes associated with the inflammatory process was conducted.

[0105] RNA was isolated from mouse tissues, including the liver, kidneys, lymph nodes, and spleen, using the Total RNA Mini kit (A&A Biotechnology©). The isolated RNA was reverse transcribed into cDNA using the RT-PCR method (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems™). The normalized relative expression of the genes was determined using the qPCR method (PerfeCTa SYBR® Green FastMix Reagent; Quantabio©), with Tbp as the reference gene for the kidneys and liver, Actb (P-acti n ) as the reference gene for the spleen, and Hprt-1 (hypoxanthine phosphoribosyltransferase 1) as the reference gene for the lymph nodes. The relative quantitative expression of the genes was determined using the 2-AACtmethod. The sequences of the primers used in the qPCR reaction are listed in Table 5. The obtained results were compared to the positive control group (Pl). Table 5. The sequences of primers used in the qPCR.

[0106] Results

[0107] Gene expression analysis using the qPCR method revealed the induction of inflammation in the positive control group (Pl) compared to the negative control (N), resulting in increased expression of genes related to the immune response in the studied tissues. Depending on the tissue type, increased amplification of genes such as Nf-KB p65, lcam-1, Casp-1, 11-16, 11-6, Ifny, and Hmgb-1 was observed, which encode the following: the p65 subunit of the nuclear factor kappa B (NF-KB), intercellular adhesion molecule 1, caspase 1, interleukin IL-ip, interleukin 6, interferon gamma, and high mobility group protein 1, respectively. In the liver cells of the liver, kidneys, lymph nodes, and spleen in the experimental group (Bl), a lower expression of the aforementioned genes was observed, indicating strong anti-inflammatory properties of the composition according to the invention. In liver cells of the positive control group (Pl), compared to the negative control (N), a decrease in the expression of the Sirtl gene was noted, which encodes sirtuin 1 - a suppressor of the immune response in the body. Conversely, in the experimental group (Bl), increased transcription of this gene was demonstrated.

[0108] EXAMPLE 6

[0109] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0110] Effect 6.

[0111] PROTECTIVE EFFECTS OF THE COMPOSITION ACCORDING TO THE INVENTION ON DNA

[0112] AGAINST DAMAGE CAUSED BY IONIZING RADIATION EXPOSURE IN MICE

[0113] RESEARCH METHODOLOGY Composition

[0114] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight.

[0115] In vivo experimental model

[0116] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 6 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N and Pl groups were administered saline intragastrically (up to 100 pL depending on body weight), while mice from the Bl group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7-day supplementation, mice from the Pl and Bl groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for the next 4 days post-irradiation. After this period, mice from the N, Pl, and Bl groups were euthanized, and tissues were collected for further analysis.

[0117] Table 6. Experimental groups used in in vivo studies.

[0118] Assessment of protective effects on DNA against damage caused by ionizing radiation exposure

[0119] Ionizing radiation used in radiotherapy is a factor that causes DNA damage, such as the formation of single and double-strand breaks in the DNA helix, modifications of nitrogenous bases, generation of apurinic / apyrimidinic sites, and the formation of cross-links between DNA and proteins. In response to these damages, cells activate DNA repair mechanisms, and when the damages are irreversible, mechanisms of cell death, such as apoptosis, are initiated.

[0120] To evaluate the protective effect of the composition against potential DNA damage, an analysis of the expression of selected genes related to DNA repair and cell cycle regulation was conducted. RNA was isolated from mouse liver, kidneys, and spleen using the Total RNA Mini Kit from A&A Biotechnology©. The isolated RNA was subjected to reverse transcription to cDNA using the RT-PCR method (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems™). The normalized relative expression of genes was measured using the qPCR method (PerfeCTa SYBR® Green FastMix Reagent; Quantabio©), with Tbp as the reference gene for kidney and liver cells, and Actb as the reference gene for spleen cells. The relative quantitative measurement of gene expression was determined using the 2-AACtmethod. The sequences of the primers used in the qPCR reaction are included in Table 7. The obtained results were compared to the positive control group (Pl).

[0121] Table 7. The sequences of primers used in the qPCR.

[0122] Results

[0123] Gene expression analysis using the qPCR method revealed the induction of DNA damage in the positive control group (Pl), which was exposed to ionizing radiation, compared to the Control group N. This resulted in an increased expression of genes responsible for DNA damage repair or cell cycle progression arrest in the studied tissues. Depending on the tissue type, increased transcription of genes such as Ape-1, Ogg-1, and Atm was observed, which encode respectively: apurinic / apyrimidinic endonuclease, 8-oxoguanine glycosylase, and serine / threonine kinase involved in DNA repair. In the liver, kidneys, and spleen of the experimental group (Bl), a lower level of expression of these genes was demonstrated, which may indicate the protective properties of the composition against DNA damage.

[0124] EXAMPLE 7

[0125] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0126] Effect 7.

[0127] ANTI-APOPTOTIC EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION IN MICE EXPOSED TO IONIZING RADIATION

[0128] RESEARCH METHODOLOGY

[0129] Composition

[0130] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight. In vivo experimental model

[0131] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 8 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N and Pl groups were administered saline intragastrica I ly (up to 100 pL depending on body weight), while mice from the Bl group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7-day supplementation, mice from the Pl and Bl groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for the next 4 days post-irradiation. After this period, mice from the N, Pl, and Bl groups were euthanized, and tissues were collected for further analysis.

[0132] Table 8. Experimental groups used in in vivo studies.

[0133] Evaluation of the anti-apoptotic effect on cells exposed to damage caused by ionizing radiation

[0134] Reactive oxygen species generated as a result of exposing cells to ionizing radiation used in radiotherapy are factors that lead to the induction of oxidative stress, inflammation, lipid peroxidation, and DNA damage, among other effects. As a result, various repair mechanisms are activated. In the case of irreversible changes, programmed cell death-apoptosis-is induced.

[0135] To assess the potential anti-apoptotic properties of the composition according to the invention in relation to cells exposed to ionizing radiation, an analysis of the expression of selected genes associated with the regulation of apoptotic processes was conducted. RNA was isolated from mouse liver, kidneys, and bone marrow using the Total RNA Mini kit from A&A Biotechnology©. The isolated RNA was reverse transcribed to cDNA using the RT-PCR method (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems™). The normalized relative expression of genes was measured using the qPCR method (PerfeCTa SYBR® Green FastMix Reagent; Quantabio©), with Tbp used as the reference gene for kidney, liver, and bone marrow cells. The relative quantitative determination of gene expression was performed using the 2’ AAct method. The sequences of the primers used in the qPCR reaction are included in Table 9. The obtained results were compared to the positive control group Pl. Table 9. The sequences of primers used in the qPCR.

[0136] Results

[0137] The qPCR analysis revealed the induction of expression of genes associated with the initiation of apoptosis or the inhibition of cell growth and proliferation in the positive control group (Pl), compared to the negative control N. Depending on the tissue type, an increased transcription of genes such as Casp3, Bax, and Tgf-61 was observed, which encode caspase 3, BAX protein, and transforming growth factor beta 1, respectively. In the liver, kidney, and bone marrow cells of the experimental group Bl, a lower level of expression of the aforementioned genes was demonstrated, indicating the potential anti-apoptotic properties of the composition according to the invention.

[0138] EXAMPLE 8

[0139] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0140] Effect 8.

[0141] PROTECTIVE EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION ON BONE MARROW CELLS IN MICE EXPOSED TO IONIZING RADIATION

[0142] RESEARCH METHODOLOGY

[0143] Composition

[0144] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight.

[0145] In vivo experimental model

[0146] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 10 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N, Pl and P2 groups were administered saline intragastrically (up to 100 pL depending on body weight), while mice from the Bl and B2 group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7- day supplementation, mice from the P1,P2 and B1,B2 groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for the next 4 days post-irradiation. After this period, mice from the N, Pl, and Bl groups were euthanized, and tissues were collected for further analysis. Groups P2 and B2 were subjected to an additional 30-day observation. In group B2, daily supplementation with the composition according to the invention was continued. After the observation period, the mice were euthanized, and serum and tissues were collected for further analysis.

[0147] Table 10. Experimental groups used in in vivo studies.

[0148] Assessment of the protective effect on bone marrow cells against damage caused by exposure to ionizing radiation.

[0149] Bone marrow is a tissue responsible for producing blood morphological elements, such as erythrocytes, leukocytes, and platelets, and is one of the most susceptible organs to damage caused by ionizing radiation. The result of bone marrow damage after irradiation is a decrease in the number of individual blood cells, which can consequently lead to conditions such as anemia, difficulties in blood cloting, and increased susceptibility to infections. Examples of measures taken to mitigate the effects of radiation exposure and bone marrow damage include the administration of cytokines, blood transfusions, and bone marrow transplants.

[0150] To evaluate the protective effect of the composition according to the invention, a cytometric analysis of cells isolated from the bone marrow of mice was conducted four days after exposure to ionizing radiation. Cells, at a quantity of 1 million per individual test, were incubated with antibodies (APC / Cyanine7 anti-mouse CD45.2 Antibody, #109824; BioLegend®) for 30 minutes at 4°C, then washed and fixed in 1% paraformaldehyde. The analysis of the samples was performed using a BD FACSCanto™ II cytometer.

[0151] Additionally, a morphological analysis of blood samples collected from mice was performed 24 hours after exposure to ionizing radiation. The levels of leukocytes and platelets were determined using the SCIL VET ABC PLUS+ blood analyzer.

[0152] Furthermore, the level of the pro-inflammatory cytokine TNFa (tumor necrosis factor) was determined in the supernatants from bone marrow cell cultures using the ELISA method (Mouse TNF-alpha DuoSet ELISA, #DY410, R&D Systems - Biotechne®). Initially, bone marrow cells were isolated from mice four days after exposure to ionizing radiation and then transferred to Petri dishes to establish cell cultures at a density of 3 million cells per replicate. After a 24-hour stabilization period, the cells were incubated with PMA (phorbol myristate acetate) and ionomycin [PMA 50 ng / ionomycin 1 pg / ml] for 5 hours, after which the supernatants were collected to measure TNFa levels in the ELISA test. The obtained results were compared to the positive control group Pl.

[0153] Results

[0154] The cytometric analysis of bone marrow cells (Fig. 8A) revealed a decrease in the level of leukocytes in the positive control group (Pl) (median 6.85) compared to the negative control (N) (median 47.8), reaching 14.33% [95% Cl 42.9 - 49.8 for the negative control group (N), 6.1 - 7.2 for control Pl], In the experimental group (Bl), a higher level of leukocytes was observed compared to positive control (Pl), with an increase of 17.15% (value for the median difference).

[0155] The morphological analysis of blood (Fig. 8B) collected 24 hours after the irradiation of mice showed a decrease in the level of leukocytes in the positive control groups (Pl + P2) (Me = 1.1, 95% Cl 1.0-1.4) by 73.17% compared to the negative control group (N) (Me = 4.1, 95% Cl 1.7- 6.1) (decrease determined for the median difference value). The results for the experimental groups (Bl + B2) indicate a higher level of leukocytes compared to positive control groups (Pl + P2), with a difference of 34.04% (value for the median difference).

[0156] Additionally, the morphological analysis of blood (Fig. 8C) indicated a decrease in platelet levels by 75.11% in the positive control groups (Pl + P2) [Me = 673.0; 95% Cl 594.0-751.0] compared to the negative control group (N) [Me = 896; 95% Cl 876.0-1061.0], A higher platelet level was observed in the experimental groups (Bl + B2), with an increase of 22.09% (value for the median difference) compared to positive controls (Pl + P2).

[0157] The analysis of the results obtained by the ELISA method (Fig. 8D) for the supernatants from bone marrow cell cultures stimulated with PMA and ionomycin showed an increase in TNFa cytokine levels in the control group (Pl) by 56.82% (value for the median difference) compared to negative control (N). A lower level of TNFa was observed in the experimental group (Bl), where the median was 48.66% [95% Cl 47.23 - 50.1] compared to positive control group (Pl) [95% Cl 95.74 - 103.7],

[0158] Bone marrow is one of the most susceptible organs to the destructive effects of ionizing radiation, manifested by a decrease in blood cell production, which consequently leads to impaired immune function. Therefore, the conducted morphological blood analyses, along with cytometric analysis, indicate a protective influence of the composition according to the invention on bone marrow. This effect results in a higher level of immune system cells in the group of mice supplemented with the composition compared to the group exposed to ionizing radiation without supplementation. Additionally, the ELISA test results indicate a neutralization of the inflammatory state in the experimental group (Bl) compared to positive control Pl in bone marrow cells, clearly suggesting the protective properties of the composition according to the invention towards these cells.

[0159] EXAMPLE 9

[0160] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0161] Effect 9.

[0162] RADIOPROTECTIVE EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION ON THE MORPHOLOGY OF KIDNEYS AND LIVERS OF MICE EXPOSED TO IONIZING RADIATION

[0163] RESEARCH METHODOLOGY

[0164] Composition

[0165] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight.

[0166] In vivo experimental model

[0167] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 11 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N and Pl groups were administered saline intragastrica I ly (up to 100 pL depending on body weight), while mice from the Bl group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7-day supplementation, mice from the Pl and Bl groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for the next 4 days post-irradiation. After this period, mice from the N, Pl, and Bl groups were euthanized, and tissues were collected for further analysis.

[0168] Table 11. Experimental groups used in in vivo studies. Assessment of radioprotective effect

[0169] Tissue sections from the kidneys and liver, obtained after the completion of the experiment, were subjected to hematoxylin and eosin staining (H&E Staining Kit, Abeam, #ab245880) for detailed morphological analysis and assessment of potential histopathological changes. A procedure was adopted to decolorize two sections from each mouse. Subsequently, for each of these preparations, at least 15 images were taken from different microscopic fields at two magnification levels (lOx and 40x).

[0170] Results

[0171] Histological analysis of liver tissue preparations (Fig. 9A) revealed significant changes in response to ionizing radiation (IR) in all mice. The presence of dilated blood vessels and intense congestion in the liver tissue serves as a clear indicator of the tissue's reaction to radiation exposure. These phenomena were significantly less intense in the experimental group (Bl), suggesting a potential impact of the intervention related to the composition in mitigating these undesirable effects of radiation exposure.

[0172] As a result of the analysis of kidney tissue preparations (Fig. 9 B) after irradiation (positive control group (Pl)), a clear presence of atrophic glomeruli was observed in the majority of mice. It is noteworthy that in many cases, there was also identification of dilatation of Bowman's capsule, i.e., an increase in the space between the glomerular capsule and the renal glomerulus. This is a characteristic indicator of pathological processes that can affect the physiology and function of the glomeruli, as evidenced by literature reports on kidney microscopy subjected to various stressors, including ionizing radiation. It is important to emphasize that the extent of these changes was significantly lower in the group subjected to irradiation in conjunction with the composition (experimental group (Bl)). This indicates a potential reduction in the effects of glomerular atrophy through the application of the composition according to the invention. Furthermore, the observation of kidney tissues in the positive control group (Pl) indicated the presence of vacuolization of the glomeruli and the surrounding blood vessels. Vacuolization, or the formation of vacuolar bubbles within cells, may result from the activation of apoptotic reaction cascades in response to irradiation. Supplementation with the composition in the irradiated mice of the experimental group (Bl) resulted in a reduction of these undesirable effects, which may be a result of the potential protective capacity of the composition. EXAMPLE 10

[0173] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF ARONIA, ELDERBERRY, EVENING PRIMROSE, AND BLACKCURRANT EXTRACTS

[0174] Effect 10.

[0175] ANTIOXIDANT, ANTI-INFLAMMATORY, AND CYTOPROTECTIVE EFFECTS OF THE COMPOSITION ACCORDING TO THE INVENTION ON THE LATE EFFECTS OF IONIZING RADIATION ON MICE

[0176] RESEARCH METHODOLOGY

[0177] Composition

[0178] According to the invention, a composition contains a standardized extract of aronia, elderberry, evening primrose, and blackcurrant. A stock solution of the composition at a concentration of 20 mg / mL was prepared ex tempore using 0.9% saline and was administered to mice at a dose of 100 mg / kg body weight.

[0179] In vivo experimental model

[0180] Female Balb / c mice aged 10-11 weeks were divided into experimental groups according to Table 12 and underwent a 7-day adaptation / acclimatization period. For the next 7 days, mice from the N and P2 groups were administered saline intragastrically (up to 100 pL depending on body weight), while mice from the B2 group received a suspension of the composition at a dose of 100 mg / kg body weight (maximum volume of 100 pL). After the 7-day supplementation, mice from the P2 and B2 groups were exposed to a single dose of 6 Gy of ionizing radiation using a Varian TrueBeam accelerator. Supplementation continued for another 30 days. After this period, the mice were euthanized, and tissues were collected for further analysis.

[0181] Table 12. Experimental groups used in in vivo studies. Evaluation of the long-term effects of the composition

[0182] Radiotherapy can cause long-lasting side effects due to ionizing radiation. The chronic action of reactive oxygen species (ROS) generated as a result of irradiation leads to the development of chronic inflammation. Inflammation is, in turn, one of the mechanisms responsible for the emergence of late side effects of radiotherapy, which can ultimately lead to DNA damage and cell death.

[0183] In order to assess the radioprotective effects of the composition concerning the late effects of irradiation, an analysis of the expression of selected genes related to oxidative stress, inflammation, DNA damage, and cell cycle regulation was conducted. RNA isolated from tissues collected from mice was obtained using the Total RNA Mini Kit; A&A Biotechnology©. The isolated RNA was reverse transcribed into cDNA using the RT-PCR method (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems™). The normalized relative expression of each gene was measured using the qPCR method (PerfeCTa SYBR® Green FastMix Reagent; Quantabio©), referring to the appropriate reference gene for the analyzed tissues. The relative quantitative measurement was determined using the 2-AACtmethod. The sequences of the primers used in the qPCR reaction were included in Table 13. The obtained results were compared to the positive control group (P2).

[0184] Table 13. The sequences of primers used in the qPCR.

[0185] Results

[0186] Analysis of the collected tissues revealed the long-term effects of ionizing radiation on the systemic system. Thirty days after irradiation, the positive control group (P2) still exhibited persistent oxidative stress and inflammation. Additionally, elevated levels of expression were observed for genes encoding proteins associated with DNA damage and cell cycle regulation. As a result of the continued supplementation with the composition according to the invention in the studied group (B2), for an additional 30 days post-irradiation, the following was observed:

[0187] • Lower relative expression of the oxidative stress-related gene Nrf2 in the liver and spleen.

[0188] • Lower relative expression of inflammation-related genes: Caspl and 11-16 in the liver; lcam-1, 11-16, Tgf6-1 in the kidneys; Hmgbl in the lymph nodes; and lcam-1 and stromal-derived factor 1 Cxcll2 in the spleen.

[0189] • Lower relative expression of cell cycle-related genes such as the cyclin-dependent kinase inhibitor Cdknla in the liver and Tgf6-1 in the kidneys, as well as genes associated with DNA damage: Ape-1 in the lymph nodes and Ogg-1 in the spleen.

[0190] The obtained results indicate the long-term protective effect of the composition (100 mg / kg) in terms of its antioxidant, anti-inflammatory, and cyto protective effects.

Claims

Patent claims1. A pharmaceutical composition for supporting oncology patients during radiotherapy and healthy individuals exposed to ionizing radiation, characterized by its content of four natural extracts rich in polyphenolic compounds: chokeberry (Aronia melanocarpa L.), elderberry (Sambucus nigra L.), blackcurrant (Ribes nigrum L.), and evening primrose seeds (Oenothera biennis L.).

2. The composition according to claim 1, characterized by a mixture of extracts from elderberry fruits, chokeberry, blackcurrant, and evening primrose seeds, which contains polyphenols ranging from 35% to 65% and anthocyanins ranging from 10% to 35%.

3. The composition according to claim 1, characterized by containing 30-50% extract from elderberry fruits, 10-30% extract from chokeberry fruits, 5-25% extract from blackcurrant fruits, and 20-40% extract from evening primrose seeds. The composition according to claim 1, characterized by containing polyphenols ranging from 45% to 80% and anthocyanins ranging from 20% to 35% from chokeberry fruits.

4. The composition according to claim 1, characterized by containing polyphenols ranging from 20 to 55% and anthocyanins ranging from 10% to 35% from elderberry fruits.

5. The composition according to claim 1, characterized by containing polyphenols ranging from 20 to 55% and anthocyanins ranging from 10% to 25% from blackcurrent fruits.

6. The composition according to claim 1, characterized by containing polyphenols ranging from 35 to 55% and procyanidins ranging from 10% to 45% from evening primrose seeds.

7. The composition according to claim 1, characterized by the extracts being in liquid, semi-solid, or solid form.

8. The composition according to claim 1, characterized by its radioprotective effect, including antioxidant, anti-inflammatory, and cytoprotective properties.

9. The composition according to claim 1, characterized by its use in the production of a dietary supplement.

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