Myconoside-rich extracts from plants belonging to the genera Haberlea and Ramonda from in vitro systems, their preparation method, and use as chemoprotective, radioprotective, and ultraviolet protection agents

Abstract

The present invention relates to myconoside-containing extracts obtained from in vitro systems of plants belonging to the genera Haberlea and Ramonda, including Haberlea rhodopensis Friv, Ramonda heldreichii (Boiss.) C.B. Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancic & Petrovic, and Ramonda serbica Pancic, as well as hybrids thereof, belonging to the family Gesneriaceae, and to methods for their preparation. Furthermore, the present invention relates to the use of said extracts as chemoprotective, radioprotective, and UV-protective agents in human and veterinary medicine, dietary supplements, and cosmetics. The described in vitro plant extracts contain two fractions, Extract 1, a polyphenol fraction containing primarily flavone C-glycosides and phenylethanoids (PHEs), and Extract 2, a fraction containing primarily phenylethanoid myconosides, particularly myconosides and pauciflosides, either alone or in combination. Extract 1 contains 0.1-55.0 wt% PHEs, with free phenols, sugars, organic acids, fatty acids, amino acids, and sterols accounting for up to 100 wt%. Extract 2 contains 55.0-99.9 wt% PHEs (particularly myconosides and pauciflosides), with phenolic compounds, monosaccharides, disaccharides, or their residues accounting for up to 100 wt%. Extracts 1 and 2 obtained according to the present invention consist primarily of broad-spectrum RHEs (particularly myconosides and pauciflosides) within the limits of quantitation. These compounds, especially the myconosides, have the high biological, antioxidant, antimutagenic and anticlastogenic activity demonstrated herein and are therefore suitable for use alone or in combination by dilution and by metered formulation for the preparation of products to be used as natural chemical, radiological and UV protection agents with a controlled degree of protection.The latter are used in human and veterinary medicine, dietary supplements and cosmetics for the prevention and treatment of harmful chemical, radiation and ultraviolet effects.

Description

[Technical Field]

[0001] The present invention relates to myconoside-containing extracts obtained from in vitro systems of plants belonging to the genera Haberlea and Ramonda, including Haberlea rhodopensis Friv, Ramonda heldreichii (Boiss.) CB Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancic & Petrovic, and Ramonda serbica Pancic, as well as hybrids thereof, belonging to the family Gesneriaceae, and to methods for their preparation. Furthermore, the present invention relates to the use of said extracts as chemoprotective, radioprotective, and UV-protective agents in human and veterinary medicine, dietary supplements, and cosmetics. [Background technology]

[0002] Wild plants and plants cultivated using traditional agricultural techniques often produce low yields of desirable bioactive compounds. Furthermore, certain medicinal plants, including species belonging to the genera Haberlea and Ramonda, such as Haberlea rhodopensis, Ramonda serbica, and Ramonda nathaliae, are unsuitable for cultivation or are rare or endangered, making industrial production of important naturally occurring bioactive compounds impossible. The high demand for natural plant-derived bioactive substances and the limitations of chemical synthesis have led to an increasing need to source active ingredients through biotechnological means. Plant biotechnology, particularly in vitro plant systems, has proven to be a promising tool for the sustainable production of valuable bioactive compounds under controlled conditions. In vitro plant manipulation allows the production of a wide variety of compounds at concentrations much higher than those found in wild plants, without damaging their natural habitats or destroying biodiversity.

[0003] In recent years, plants belonging to the Gesneriaceae family and plant species such as Habalea rhodopensis and Ramonda cerbiculatus have attracted the attention of researchers for their beneficial effects on human and animal health.

[0004] In one collaborative study, national in vitro collections of Ramonda serbicae in Albania and Bulgaria and Ramonda nazariae in Macedonia were established, and tissue culture methods were selected after evaluating the polymorphism of several natural populations (E. Daskalova et al., April 2012, Biotechnology & Biotechnological Equipment 26(1):16-25).

[0005] The metabolic changes of Ramonda cerbiculatus and Ramonda nazariae have been investigated during the desiccation and recovery process using 1H=NMR and GC-MS, and the metabolic composition of the plants has been published (Dejan Godeva, et.al, Phytochemical analysis vol 33, issue 6 / p.961-970, Aug. 2022).

[0006] Studies of a typical hydroalcoholic extract of Habalea rhodopensis have revealed unique medical properties and pharmacological potential related to its antioxidant, radioprotective, anticlastogenic, chemoprotective, cytoprotective, antibacterial, antimutagenic, immunological, anticancer and anti-aging effects (Bankova R., et al., Tradition and modernity in veterinary medicine, 2022, vol. 7, No. 1(12)).

[0007] Literature has confirmed that an increase in intracellular free radicals (ROS), known as oxidative stress, is related to genomic DNA damage and the occurrence of mutations. ROS generated from redox reactions in the body are highly reactive and can cause serious biological damage, leading to major gene mutations and ultimately to the development of cancer and chronic diseases.

[0008] The antioxidant properties of phenolic compounds from whole plant extracts, especially from Habalea rhodopensis, are known from the literature (Kondeva-Burdina M, et al., Pharmacogn Mag. 2013 Oct; 9(36)) and (Mihaylova, D. Et al., V., 2013. JFBiochemistry, 37(3)). Many studies have proven that modern humans are exposed to environmental factors such as solar radiation (ultraviolet radiation in the UVA and UVB bands).

[0009] Furthermore, the free radicals generated after gamma radiation exposure are highly reactive and can trigger chain reactions that lead to serious biological damage. Anticancer chemotherapy also induces strong oxidative stress that affects multiple cellular targets, potentially reducing the effectiveness of anticancer drugs. The use of antioxidant supplements during chemotherapy can enhance therapeutic efficacy (Conklin KA. "Chemotherapy-Induced Oxidative Stress: Impact on Chemotherapy Efficacy." Integr Cancer Ther. 2004 Dec;3(4).

[0010] Extracts from the whole Habalea rhodopensis plant have radioprotective properties (Georgieva S, et.al., Indian J. Exp. Biol.2013 51(1), Georgiev, YN et al., J. Ethnopharmacology, 2020, 249; Bankova, R., 2022, Tradition and modernity in veterinary medicine, 2022, vol. 7, No. 1(12); Georgieva, S., Gencheva, D., Popov, B., Grozeva, N., & Zhelyazkova, M. 2019, "Radioprotective activity of regenerated plants of Habalea rhodopensis hriv (Gesneriaceae) and the role of flavonoids and phenolic acids", Bulgarian Journal of Agricultural Science,25(Suppl.3), 158-168,), however, compounds exhibiting such properties have not been investigated, although the presence of phenolic acids and flavonoids in the extract has been suggested.

[0011] It is also known that certain natural glycosylated polyphenols (GPPs), primarily verbascoside, exhibit sunscreen properties (US 2017 / 16613858).

[0012] Another type of cell culture extract, belonging to the Syringa genus, specifically lilac (Syringa vulgaris), is described in EP 2319914, along with its preparation method. The extract described is composed of 20-90 wt% phenylpropanoids (including 5-20% isoverbascoside) and 80-10 wt% other chromophore-free fractions, primarily oligosaccharides, polysaccharides, proteins, and lipid molecules. This cell culture possesses high antioxidant activity and properties, including collagen stimulation and pigmentation control.

[0013] International Publication No. 2021 / 184086 (Innova BM) describes an extract from in vitro cultures of Habalea rhodopensis. The extract contains 25-35 wt% polyphenols of the total extract, with other fractions reaching 100 wt%. It contains sterols, sugars, and free phenols, as well as organic acids, fatty acids, and amino acids, with the phenylethanoid glycoside myconosides accounting for 18-35 wt% of the polyphenol fraction. The described extract contains limited amounts of polyphenols as phenylethanoid glycosides, such as myconosides. The cited publication provides detailed descriptions of the principles of plant growth in in vitro systems and the propagation of plants in in vitro culture on solid and liquid media. The specific conditions used for large-scale plant in vitro culture, suited to specific plant species, are essential for the production of specific bioactive compounds / metabolites, as well as controlled amounts of target compounds, and their utilization for various purposes. The wide range of controlled compositions of extracts from plant in vitro systems used as highly efficient, low-toxicity, natural chemoprotective, radioprotective, and UV-protective agents is particularly important, as it allows their use in products for different applications, microdosing at different irradiation levels, and rapid elimination of the harmful genotoxic effects of irradiation. Summary of the Invention [Problem to be solved by the invention]

[0014] The problem to be solved by the present invention is to find extracts with a controlled content, obtained from in vitro systems of plants belonging to the genera Habalea and Ramonda of the Gesneriaceae family, and their hybrids, which extracts have a high biological value of rare phenylethanoids, in particular myconosides and pauciflosides, and which can be used in novel products with guaranteed and controlled chemoprotective, radioprotective and UV-protective effects. [Means for solving the problem]

[0015] According to the present invention, this problem is solved by a method for producing a plant extract containing, singly or in combination, Extract 1, a polyphenol fraction containing mainly flavone-C-glycosides and phenylethanoids (PHE), and Extract 2, a myconoside-rich fraction, wherein Extract 1 contains 0.1 to 55.0 wt% of PHE (particularly myconosides and pauciflosides) and reaches up to 100 wt% in combination with free phenols, sugars, organic acids, fatty acids, amino acids, and sterols, and Extract 2 contains 55.0 to 99.9 wt% of PHE (particularly myconosides and pauciflosides) and reaches up to 100 wt% in combination with phenolic compounds, monosaccharides, disaccharides, or residues thereof, from plants belonging to the genera Habalea and Ramonda of the Gesneriaceae family, particularly Habalea rhodopensis hriv, Ramonda heldreicki (Boiss.) C.B. Clark; Ramonda myconii (L.). This problem is solved by minocoside-containing extracts obtained from in vitro systems of Rchb.; Ramonda nazariae Pancic & Petrovich, and Ramonda cerbica Pancic, as well as their hybrids.

[0016] Another subject of the present invention is a method for preparing said extract from the above-mentioned plant in vitro system according to the invention, comprising the following steps: a) initiation of in vitro cultivation (carried out in accordance with WO2021184086) and subsequent aqueous or aqueous alcoholic extraction of the biomass obtained after the cultivation (containing 0.01-25.0% phenylethanoid glycosides) (C1-C3, 40-90%, e.g., ethanol, isopropanol, methanol) at a ratio of 1:10-1:100 w / v, followed by one to up to three extraction steps of the aqueous residue with non-polar solvents approved for use in the food, pharmaceutical and cosmetic manufacturing industries until maximum removal of the lipid-soluble fraction; b) the aqueous phase purified in step a) is concentrated under vacuum to 2 / 3 of its volume at a temperature of 40-60°C to remove the solvent until a crude extract is obtained, and then subjected to solid phase extraction with a hydrophobic interaction resin (especially C18 reversed phase), retaining phenolic compounds and PHEs, and washing away sugars and polar compounds (mainly organic acids and amino acids) with an aqueous mobile phase; c) Eluting the phenol fraction from the resin with a mobile phase selected from solvents of different polarities (especially ethyl acetate or isopropyl alcohol). The resulting fraction is evaporated to dryness under vacuum at a temperature of 20°C to 70°C to obtain an extract 1 containing 0.1 to 55.0 wt% of PHEs (especially myconosides and pauciflosides) and up to 100 wt% of free phenols, sugars, organic acids, fatty acids, and amino acids. d) Eluting the PHE fraction from the resin with a 5-70% v / v mixture of aqueous alcohols (C1-C3). The collected PHE fraction is concentrated under vacuum at 40-60°C until the organic solvent is completely removed, and then lyophilized or dried to obtain Extract 2, which contains 55.0-99.9 wt% PHE (especially myconosides and pauciflosides) and up to 100 wt% phenolic compounds, monosaccharides, disaccharides, or their residues.

[0017] The resulting extracts 1 and 2 can be used alone or in combination by metered mixing. They are diluted with solvents (e.g., water, glycerin, propylene glycol, butyl glycol, etc.) that are safe for use in the food, cosmetic, and pharmaceutical manufacturing industries to achieve the desired final concentration of phenylethanoid glycosides (myconosides and pauciflosides). The final concentration of myconosides is specifically standardized to 0.001% to 99.0%. The resulting product is ready to use and has a controlled content of bioactive substances.

[0018] Another object of the present invention is to obtain novel active substances from extracts 1 and 2 obtained from in vitro plant systems of the Gesneriaceae family, in particular the genera Habalea and Ramonda, specifically Habalea rhodopensis hriv, Ramonda heldreicki (Boiss.) C.B. Clark, Ramonda miconii (L.) Rchb., Ramonda nazariae Pancic & Petrovich, and Ramonda cerbica Pancic, and hybrids thereof, which can be used, directly or after preparation, as chemoprotective, radioprotective, and UV-protective agents, separately or in combination, in human and veterinary medicine, dietary supplements, and cosmetics.

[0019] The possibility of mixing Extract 1 and Extract 2 promises feasibility for specific needs and intended uses. When the final concentration of phenylethanoid glycosides (especially myconosides and pauciflosides) is 0.01-99.9%, the resulting product can be used as a purified extract from in vitro cultures of plants of the genera Habalea and Ramonda, with significantly higher biological activity compared to unpurified crude extracts.

[0020] Extracts 1 and 2 obtained according to the present invention are primarily composed of broad-spectrum and quantitatively limited PHEs (especially myconosides and pauciflosides). These compounds (especially myconosides) have demonstrated high biological, antioxidant, antimutagenic, and anticlastogenic activity, making them suitable for use alone, in metered combinations, and by dilution in the preparation of products used as natural chemoprotectants, radioprotectants, and UV protectants with controlled levels of protection. The latter are used in human and veterinary medicine, dietary supplements, and cosmetics for the prevention and treatment of the effects of harmful chemicals, radiation, and UV rays.

[0021] definition According to the method of the present invention, "extract" refers to a fraction containing phenylethanoid myconosides obtained by direct extraction or fractionation of a crude extract from an in vitro system of plants belonging to the Gesneriaceae family, in particular the genera Haberlea and Ramonda, and hybrids thereof.

[0022] As used herein, the term "plant" refers to the family Gesneriaceae, particularly the genera Habalea and Ramonda, specifically Habalea rhodopensis hriv, Ramonda heldreicki (Boiss.) C.B. Clark, Ramonda miconii (L.) Rchb., Ramonda nazariae Pancic & Petrovich, and Ramonda cerbica Pancic, and hybrids thereof, and refers to plant cells, whole plants, plant organs, plant tissues, seeds, and their progeny.

[0023] As used herein, the term "in vitro system" refers, without limitation, to cells obtained from seeds, embryos, meristematic regions, leaves, roots, shoots, generative cells, sporophytes, pollen and microspores, callus tissue, suspension cultures, sprouts, meristem (shoot) cultures, root cultures (normal roots, auxiliary and transformed roots), and in vitro plant cultures cultivated under controlled in vitro conditions in solid or liquid nutrient media or culture substrates.

[0024] "Micanoside" has the molecular formula C 33 H44 O 19 It is a caffeoyl phenylethanoid glycoside with the IUPAC name "[(2R,3R,4R,5R,6R)-4-[(2R,3R,4R)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy-2-[[(2R,3R,4R)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxymethyl]-6-[2-(3,4-dihydroxyphenyl)ethoxy]-5-hydroxyoxan-3-yl]3-(3,4-dihydroxyphenyl)propanoate."

[0025] Exemplary Embodiments of the Invention The present invention is illustrated by, but not limited to, the following exemplary embodiments. [Example]

[0026] Example 1: 1. 500 grams of dried biomass from an in vitro culture of Habarea rhodopensis (containing 12% phenylethanoid glycosides) is extracted with a water-alcohol mixture (70% alcohol, such as ethanol, in a weight ratio of 1:15). After filtration, the liquid phase is concentrated under vacuum until the alcohol is removed, and then subjected to three liquid-liquid extractions with purified hexane (three times with 2 L, 1 hour at 21 °C) to remove the lipid-soluble fraction. The purified aqueous phase is concentrated under vacuum at 40 °C to 2 / 3 of its volume to remove the solvent residues, resulting in a crude extract that is then subjected to solid-phase extraction.

[0027] 2. The crude extract (2.0 L) is passed through solid-phase extraction using conditioned C18 reversed-phase resin (600 g), whereby phenolic compounds and phenylethanoid glycosides are retained on the resin, while sugars and polar compounds (mainly organic acids and amino acids) are washed away with the aqueous mobile phase.

[0028] 3. The next step was to elute the phenolic fraction using ethyl acetate (4 L) as the mobile phase, which eluted most of the phenolic compounds and flavonoids, as well as some PHEs (particularly myconosides and pauciflosides). The resulting fraction was evaporated to dryness under vacuum at 40 °C to obtain Extract 1, which contained 54.7 wt% PHEs (particularly myconosides and pauciflosides) and 100 wt% free phenols, sugars, organic acids, fatty acids, and amino acids. (See Table 1 for the GC-MS profiles of the in vitro extracts of Habalea rhodopensis, namely, the crude extract, Extract 1, and Extract 2. The results were statistically processed, clustered using the Euclidean distance method, and presented as a hierarchical clustering heat map, where each color cell corresponds to the concentration value of the associated compound.)

[0029] 4. The next step is to elute the phenylethanoid glycoside fraction using 2 L of a 70% water-ethanol mixture (w / w). The collected PHE fraction contains mainly myconosides and a small amount of paucifloside. This fraction is concentrated in a vacuum evaporator at 50 °C until a gummy mass (80 g) forms. This is then lyophilized or dried at 40 °C to obtain Extract 2, which contains 99.5 wt% PHE (especially myconosides and pauciflosides - Table 2) and reaches 100 wt% monosaccharides, disaccharides, phenolic compounds, flavonoids, or flavonoid residues (Table 1).

[0030] The yield of myconosides is 76.1% compared to the content in the original crude extract.

[0031] Figure 1 shows the GC-MS profiles of extracts (crude extract, extract 1, and extract 2) from an in vitro system of Habalea rhodopensis obtained according to Example 1. The results were statistically processed, clustered by the Euclidean distance method, and presented as a hierarchical clustering heat map, with each colored cell on the map corresponding to the concentration value of the associated compound. [ka]

[0032] Table 1 (below) shows the phytochemical composition of the extracts from the in vitro system of plants of the genus Habalea obtained by the method described in Example 1 of the present invention. The results are the average of three parallel tests of each extract variant and are presented as % of dry mass. [Table 1] An Agilent Technologies Hewlett-Packard 7890A+ / MSD5975 instrument (Hewlett-Packard, Palo Alto, CA, USA) was coupled to an Agilent Technologies 5975C inert XL EI / CI MSD mass spectrometer (Hewlett-Packard, Palo Alto, CA, USA). The HP-5MS column (30 m x 250 μm x 0.25 μm) was run at 60 °C for 2 min, ramped to 260 °C at 5 °C per min, and exposed for 8 min at 260 °C. The injected sample volume was 1 μl, with a split ratio of 10:1. The injector temperature was 250 °C, and the carrier gas (helium) flow rate was 1 mL / min. EI / MS spectra were recorded at 70 eV. The HPLC system consisted of a Waters 1525 binary pump (Waters, Milford, MA, USA), a Waters 2487 dual λ absorbance detector (Waters, Milford, MA, USA), and a Supelco Discovery HS C18 column (5 μm, 25 cm × 4.6 mm), run at 28°C. The mobile phase consisted of a gradient of 2% acetic acid and acetonitrile.

[0033] Table 2 shows the NMR data (500 MHz in D2O) showing the structures of myconosides and pauciflosides in Extract 2 of the present invention. [Table 2-1] [Table 2-2]

[0034] Example 2: 1. 100 g of dried biomass from an in vitro culture of Ramonda cervica (containing 0.5% phenylethanoid glycosides, mainly myconosides) is extracted with distilled water in a ratio of 1:30 (w / v). After filtration, the liquid phase is concentrated under vacuum to one-third of its volume. This is followed by three liquid-liquid extractions with ethyl acetate (3 x 2 L, 1 hour, 21 °C) to remove the lipophilic fraction. The purified aqueous phase is concentrated under vacuum at 50 °C to two-thirds of its volume to remove solvent residues, and the crude extract thus obtained is subjected to solid-phase extraction according to the procedure described in points 2, 3, and 4 of Example 1 above.

[0035] 2. The obtained phenolic fraction (extract 1) contained 2.0 wt% of PHE (especially myconosides and pauciflosides), and the total amount of free phenols, sugars, organic acids, fatty acids, and amino acids reached 100 wt%.

[0036] 3. The obtained phenylethanoid glycoside fraction (extract 2) contained 57.0 wt% PHE (especially myconosides and pauciflosides), and together with monosaccharides, disaccharides, phenolic compounds, and flavonoids or flavonoid residues, reached 100 wt%.

[0037] The yield of myconosides is 82% compared to the content in the original crude extract.

[0038] Example 3: The procedure was the same as in Example 1, except that the biomass used, derived from an in vitro culture of Habarea rhodopensis, contained 9% phenylethanoid glycosides in Step 1, and the purified aqueous phase obtained by three extractions with 50% isopropanol at a ratio of 1:20 was concentrated at 50°C, and the phenylethanoid glycoside fraction was eluted with 50% v / v methanol in Step 4. Extract 1 thus obtained contained 29.5% PHEs, specifically myconosides and pauciflosides, and Extract 2 contained 62.0% PHEs, specifically myconosides and pauciflosides.

[0039] The yield of myconosides is 92% compared to the content in the original crude extract.

[0040] Example 3.a: The procedure was carried out as in Example 3, except that the dry biomass was obtained from an in vitro culture of Ramonda cervica (containing 12% phenylethanoid glycosides, mainly myconosides). Extract 1 obtained contained 52.0% PHEs, specifically myconosides and pauciflosides, and Extract 2 contained 98.0% PHEs, specifically myconosides and pauciflosides.

[0041] The yield of myconosides is 84% ​​compared to the content in the original crude extract.

[0042] Table 3 (below) shows the phytochemical composition of the extracts from the in vitro system of Ramonda cerbiculatus plants obtained by the method described in Example 3a of the present invention. The results are the average of three parallel tests of each extract variant and are presented as % of the dry mass. [Table 3]

[0043] Extract 1 and Extract 2 obtained according to the above examples can be used alone or in combination, either in a dry state or diluted with a solvent safe for use in food, cosmetics, and pharmaceuticals (e.g., water, glycerin, propylene glycol, butyl glycol, etc.) to adjust the final concentration of phenylethanoid glycosides (especially myconosides and pauciflosides) to 0.01-99.0%. The product thus obtained can be used as a purified extract of in vitro cultures of Haberlea rhodopensis and Ramonda with significantly increased biological activity.

[0044] Examples of use of the extract according to the present invention and data on its effects material and method In the present invention, extracts 1 and 2 containing phenylethanoid glycosides (particularly myconosides and pauciflosides) are isolated from plants belonging to the Gesneriaceae family, particularly the genera Habalea and Ramonda, and their hybrids in vitro. The dried extracts are stored at -20°C, protected from light and moisture. Solutions used in the experiments are prepared extemporaneously based on aqueous solutions.

[0045] Example 4: Study of UV-VIS absorption spectra of extracts 1 and 2 obtained in vitro system of Habarea rhodopensis according to Example 1.

[0046] The investigated extracts were used to prepare aqueous solutions with a final myconoside concentration of 22 μM, and their UV-Vis spectra were recorded on a Shimadzu UV / Vis mini 1240 spectrophotometer. The obtained spectra are presented in Figure 2 and show that extract 2 has a significantly higher ability to absorb light from the UV spectrum, which is evidence that purified myconosides have a more pronounced ability to capture light from the UV spectrum compared to less purified extract 1, which contains more accompanying compounds (such as phenols and flavone C-glycosides). [ka]

[0047] As shown in Figure 3, UVA and UVB light transmittance (T%) was investigated for different dilutions of Extract 2 (obtained according to Example 1 herein) with myconoside concentrations of 0.11 mg / ml, 0.055 mg / ml, 0.011 mg / ml, 0.0055 mg / ml, and 0.00275 mg / ml. [ka]

[0048] The results show that a minimum concentration of 0.011 mg / ml of myconosides is sufficient to inhibit UV penetration by 50%, while a maximum concentration of 0.11 mg / ml provides 100% protection. These results demonstrate the high potential of the phenylethanoid fractions (mainly myconosides and pauciflosides) contained in extracts 1 and 2 of the present invention to be used as UV filters in sunscreen products.

[0049] Example 5: Investigation of the antioxidant potential of an in vitro extract of Habalea rhodopense obtained according to Example 1, alone and in combination with proven UV protection agents

[0050] The radical scavenging activity (% inhibition) of aqueous solutions (0.5%) of Extract 1, Extract 2 (obtained according to Example 1), oxybenzone, and a 1:1 combination (w / w) of the extracts and oxybenzone was evaluated using the DPPH test. Comparative DPPH analysis was performed to compare the antioxidant capacity of Extract 1, Extract 2, a commercial UV protection agent (oxybenzone), and the combinations of Extract 1 and oxybenzone, and Extract 2 and oxybenzone. The results demonstrated that myconosides from purified Extract 2 were responsible for the antioxidant activity. Direct EPR spectroscopy was used (Yordanov and Christova, 1997; Karamalakova, 2014). To measure the DPPH radical scavenging power, Extract 1, Extract 2, oxybenzone, and the relevant combinations (0.5% concentration) were mixed and homogenized in a 98% ethanol solution of DPPH (80 mM, undiluted) at room temperature (22 °C). The mixture was incubated in the dark, and the DPPH-H / R radicals generated in the system were measured at 1, 5, and 10 minutes. DPPH solution was used as an internal standard for the EPR signal (Figure 4). [ka]

[0051] The study results showed that Extract 2 produced a statistically significant increase in the percentage of scavenged DPPH radicals (from 14.9% to 18.2%, then 21.3%, p<0.001), while the UV protection agent oxybenzone exhibited the lowest radical scavenging activity (from 4.7% to 3.2%, then 2.2%, p<0.001) (Figure 4). Prolonged incubation of the investigated solutions did not result in a statistically significant change in the percentage of scavenged DPPH radicals. The results indicate that oxybenzone alone exhibits very low antioxidant activity, but when combined with Extracts 1 and 2, it significantly increases (p<0.001). Inducing UV-B stress not only did not decrease the inhibitory capacity, but also resulted in a rapid and statistically significant increase, which was evident after 10 minutes for both combinations. In conclusion, in an in vitro environment, the studied extracts exhibited potent antioxidant activity and sustained free radical scavenging capacity at different incubation times before and after induced UV-B stress. This effect was most pronounced when purified extract 2 (mainly myconosides and pauciflosides) was combined with oxybenzone, a commercially available UV protection agent.

[0052] Example 6: Investigation of the antioxidant potential of an in vitro extract of Habalea rhodopensis obtained according to Example 1, alone and in combination with proven radioprotectors;

[0053] Free radicals generated after gamma irradiation have been shown to be highly reactive and can trigger chain reactions leading to significant biological damage and increased oxidative stress. Therefore, so-called radioprotectors are of particular interest for practical application. They are synthetic or naturally occurring substances and compounds that enable somatic cells to tolerate higher levels of radiation or rapidly eliminate the harmful genotoxic effects of such radiation, thereby maintaining the stability of the human genome. To demonstrate that the phenylethanoid fraction (mainly myconosides and pauciflosides) is responsible for the antioxidant activity and is also the main fraction responsible for the radioprotective properties of in vitro extracts from plants belonging to the Gesneriaceae family, particularly the genera Haberlea and Ramonda, and their hybrids, we conducted a comparative study of the antioxidant activities of Extract 1, Extract 2, the commercially available radioprotector amifostine, and the combinations of Extract 1 with amifostine and Extract 2 with amifostine by direct EPR spectroscopy. To measure the DPPH radical scavenging ability, a 98% ethanol solution of DPPH (80 mM, undiluted) was mixed and homogenized with Extract 1, Extract 2, amifostine, and the corresponding combination (0.5% concentration) at room temperature (22°C). The mixture was incubated in the dark, and the DPPH-H / R radicals generated in the system were measured at 1, 5, and 10 minutes. Using the DPPH solution as an internal standard for EPR signals, the DPPH radical scavenging activity (% inhibition) of Extract 1, Extract 2 (obtained according to Example 1), and aqueous solutions of amifostine (0.5%) was tested (Figure 5). [ka]

[0054] The results of the study showed that there was a statistically significant increase in the percentage of scavenged DPPH radicals in purified extract 2 (from 59.5% to 72.7%, 85.32%, p<0.001), and that the commercially available radioprotector amifostine had the lowest radical scavenging activity (from 5.1% to 4.5%, 3.9%, p<0.001) (Figure 5). It was found that the percentage of scavenged DPPH radicals did not change even after prolonged incubation of the investigated solutions.

[0055] The effects of the investigated extracts in combination with the commercially available radioprotector amifostine were also investigated before and after gamma irradiation (Figure 6). 2.0 Gy of 60Co gamma radiation (γ-rays) was administered at 24 Gy / min using a Rokus-M irradiator in a water bath (37°C). DPPH radical scavenging activity (% inhibition) was measured for an aqueous solution of amifostine (0.25%) (1), Extract 1 (2), a 1:1 mixture (w / w) of Extract 1 and amifostine (3), Extract 2 (obtained according to Example 1) (4), and a 1:1 mixture (w / w) of Extract 2 and amifostine (5) before and after 2.0 Gy gamma irradiation. [ka]

[0056] The results showed that the combined use of purified extract 2 (mainly containing myconosides and pauciflosides) and amifostine significantly increased the percentage of scavenged DPPH radicals after Gy irradiation (19.7%, p<0.001) and also increased it when combined with amifostine (25.6%, p<0.001). Prolonged incubation (30 min) of the studied solution did not alter the percentage of scavenged DPPH radicals after 2.0 Gy irradiation. Amifostine (0.25%) alone did not significantly increase antioxidant activity (2.5% before gamma irradiation and 5.9% after gamma irradiation, p<0.001), but its combination with purified extract 2 significantly increased it (21.1% before gamma irradiation and 25.6% after gamma irradiation, p<0.005).

[0057] Example 7: Investigation of the clastogenic and anti-clastogenic potential of an in vitro extract of Habalea rhodopensis obtained according to Example 1, alone and in combination with a clinically proven cytoprotectant (amifostine), added directly to cell cultures and 1 hour after irradiation of the blood.

[0058] The anti-clastogenic potential of Extract 1 and Extract 2 (obtained according to Example 1) and the clinically proven cytoprotectant amifostine was investigated individually and in combination by micronucleus scoring assays in in vitro cultures of human lymphocytes before and after exposure to ionizing radiation (gamma rays). The micronucleus test in peripheral blood lymphocytes and polychromatic erythrocytes is the most suitable test for screening for mutagenicity. A compound is considered mutagenic if it increases the spontaneous frequency of micronuclei (MN) in polychromatic erythrocytes or lymphocytes. This test reveals the mutagenic effect of compounds that are clastogens or spindle inhibitors. MN in binucleated peripheral blood lymphocytes was analyzed using the cytokinesis-block micronucleus assay of Fenech, M. and A. Morley (1985). We investigated the number of MNs present in 500 binucleated cells collected from cell cultures prepared with irradiated and non-irradiated peripheral venous blood from five healthy volunteers. Donor blood was irradiated with 2.0 Gy of 60Co gamma rays (γ-rays) at 24 Gy / min in a water bath at 37°C using a Rokus-M irradiator. The irradiation dose was calculated based on the geometric parameters of the irradiator, the distance from the source, and the power output. Figure 7 shows the number of MNs present in 500 binucleated cells in cell cultures prepared with non-irradiated (A) and irradiated (B) blood after the addition of control (1), amifostine (2), Extract 1 (3), Extract 2 (4), a 1:1 mixture of Extract 1 and amifostine (w / w) (5), and a 1:1 mixture of Extract 2 and amifostine (w / w) (6). [ka]

[0059] The addition of amifostine, extract 1 and extract 2 (obtained according to Example 1) to non-irradiated blood cells showed that the test substances had no clastogenic activity, which is evidence that they are non-mutagenic and can be safely used in medicine (Fig. 7A). A comparative statistical analysis of the results obtained for the total number of MN in 500 binucleated cells of different cell culture groups treated after gamma irradiation (Fig. 7B) revealed a statistically significant difference between the control and cells treated with extract 1, extract 2 and their combination with amifostine (p>0.05).

[0060] Example 8: Investigation of the clastogenic and anti-clastogenic potential of the extract from an in vitro system of Habalea rhodopensis obtained according to Example 1, alone and in combination with a clinically proven cytoprotectant (amifostine) added to cell cultures 1 hour before exposure to ionizing radiation (γ-rays) in vitro

[0061] The clastogenic and anti-clastogenic potential of Extract 1 and Extract 2 (obtained according to Example 1) and of the clinically proven cytoprotective agent amifostine, alone and in combination, was investigated in in vitro cultures of human lymphocytes before and after exposure to ionizing radiation (γ-rays) through tests detecting induced chromosomal aberrations. The method used allows accurate identification of all major types of structural chromosomal rearrangements induced by ionizing radiation (γ-rays), such as single or double acentric fragments, dicentric chromosomes, and ring chromosomes.

[0062] The study design included a total of six groups, including untreated controls. In each group, five blood donors were tested, and for each donor, 100 metaphase plates obtained from lymphocytes in peripheral venous blood were analyzed for chromosomal aberrations, for a total of 500 metaphase plates analyzed per group.

[0063] Cell cultures were prepared using the method of Evans HJ for short-term culture of peripheral blood lymphocytes and obtaining metaphase plates for chromosomal abnormality detection. Chromosome microscope slides were observed under an Olympus BX41 microscope at the most suitable magnification: 63x for screening specimens and 1000x for detailed analysis of chromosomal abnormalities. At least 100 metaphase plates were analyzed from each sample. Donor cell cultures were irradiated with 2.0 Gy 60Co gamma rays (γ-rays) in a water bath (37°C) at 24 Gy / min using a Rokus-M irradiator.

[0064] The dose was calculated according to the geometric parameters of the irradiation device, the distance and power of the radiation source. Figure 8 shows the frequency of abnormal cells in human lymphocytes treated with 2.0 Gy of gamma radiation before (A) and after (B) irradiation: control (1), amifostine (2), extract 1 (3), extract 2 (4), a 1:1 mixture of extract 1 and amifostine (w / w) (5), and a 1:1 mixture of extract 2 and amifostine (w / w) (6). [ka]

[0065] Addition of amifostine, extract 1 and extract 2 (obtained according to Example 1) to non-irradiated blood cells showed no clastogenic activity of the tested substances, proving that they are non-mutagenic and safe for use (Figure 8A). The absence of clastogenic activity of the extracts tested at the concentrations shown in the experiments provides evidence for the use of extracts 1 and 2 obtained according to the present invention in phytotherapy and practical medicine. Comparative statistical analysis of the results obtained using cells pretreated before gamma irradiation (Figure 8B) showed statistically significant differences with control cells pretreated with extracts 1 and 2 (p>0.01) and in combination with amifostine (p>0.05).

[0066] The results presented in Examples 7 and 8 show that the extracts rich in phenylethanoids (mainly myconosides and pauciflosides) obtained according to Example 1 have a more pronounced anti-clastogenic potential compared to the synthetic amifostine, which is used as a radioprotectant in clinical practice but has numerous side effects. Based on the absence of any cytotoxic and clastogenic activity in in vitro systems of extracts of plants belonging to the Gesneriaceae family, in particular the genera Habalea and Ramonda, and their hybrids, it can be reasonably argued that they can be used as radioprotectants in phytotherapy due to their strong anti-clastogenic potential.

[0067] According to Examples 4 to 8, similar parallel tests were also carried out on the extract of Ramonda cerbicana obtained according to Example 3a, and the results were similar to those shown above, with deviations ranging from ±0.1% to ±0.9%.

[0068] Similar parallel tests were also carried out on a combination of Extract 1 and Extract 2 from Habalea rhodopensis hriv obtained according to Example 1 in a 1:1 ratio, and the results obtained were close to those presented above, with a deviation of ±0.05% to ±0.09%.

Claims

1. A myconoside-containing extract from an in vitro system of plants belonging to the genera Habalea and Ramonda, The extract is characterized by containing, either singly or in combination, an extract 1 of a fraction containing mainly flavone-C glycosides and phenylethanoids, and an extract 2 of a fraction containing mainly phenylethanoids, particularly myconosides and pauciflosides, The phenylethanoids in Extract 1 contain 0.1 to 55.0 wt% of myconosides and pauciflosides, and together with free phenols, sugars, organic acids, fatty acids and amino acids, the total amount reaches 100 wt%. Extract 2 contains 55.0-99.9 wt% of phenylethanoid myconosides and pauciflosides, and together with phenolic compounds, monosaccharides, disaccharides or their residues, reaches 100 wt%. The myconoside-containing extract.

2. 2. The myconoside-containing extract according to claim 1, characterized in that the in vitro systems of plants belonging to the Habalea and Ramonda genera are selected from Habalea rhodopensis hriv, Ramonda heldreicki (Boiss.) C. B. Clark, Ramonda miconii (L.) Rchb., Ramonda nazariae Pancic & Petrovich, and Ramonda servica Pancic, and hybrids thereof.

3. 2. The myconoside-containing extract according to claim 1, characterized in that the ratio of extract 1 to extract 2 in the combination varies from 1:99 to 99:

1.

4. A composition containing the myconoside extract of claim 1, prepared with a solvent suitable for food, pharmaceutical and cosmetic applications, such as water, glycerin, propylene glycol or butyl glycol, and characterized in that when extracts 1 and 2 are diluted separately or combined in different ratios, the final standardized concentration of myconosides is 0.001% to 99.0%.

5. 2. A method for preparing a myconoside-containing extract according to claim 1, characterized in that it comprises the following steps: a) the biomass obtained after initiation and growth of the in vitro system is subjected to extraction with water or a water-alcohol mixture (40-90%) containing C1-C3 alcohols in a ratio of 1:10 to 1:100 w / v, and the aqueous fraction is subjected to one or up to three extraction steps with a non-polar solvent to remove the lipophilic fraction; b) concentrating the purified aqueous phase from step a) under vacuum at a temperature of 40°C to 60°C to remove the solvent until a crude extract is obtained, which is then subjected to solid phase extraction with a hydrophobic interaction resin to retain phenolic compounds and phenylethanoids, while washing away the sugars and polar compounds such as organic acids and amino acids with an aqueous mobile phase; c) Eluting the phenol fraction from the resin with a mobile phase selected from solvents of different polarities (particularly ethyl acetate or isopropyl alcohol), evaporating the obtained fraction to dryness under vacuum at a temperature of 20°C to 70°C, and then lyophilizing or drying to obtain Extract 1 containing 0.1 to 55.0 wt% of phenylethanoid glycosides (particularly myconosides and pauciflosides); d) The phenylethanoid fraction is eluted from the resin by eluting with a water-alcohol mixture of C1-C3 alcohol and distilled water (5-70% v / v). The collected phenylethanoid fraction is concentrated under vacuum at a temperature of 40-60°C until the organic solvent is completely removed, and then lyophilized or dried to obtain Extract 2 containing 55.0-99.9 wt% of phenylethanoid glycosides (particularly myconosides and pauciflosides).

6. Use of the myconoside-containing extract according to claims 1 to 5 in the preparation of a product with chemoprotective, radioprotective and UV-protective action.

7. Use of the myconoside-containing extracts according to claims 1 to 6 in human and veterinary medicine, pharmaceuticals, cosmetics and food manufacturing.

8. Use of the myconoside-containing extract according to claims 1 to 7 in the preparation of a dermatological preparation having antioxidant, antimutagenic and anticlastogenic activity.

9. Use of the myconoside-containing extract according to claims 1 to 7 in the preparation of a food supplement having antioxidant, antimutagenic and anticlastogenic activity.

10. Use of the myconoside-containing extract according to claims 1 to 7 in the preparation of a drug having antioxidant, antimutagenic and anticlastogenic activity.

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