Myconoside-rich extract extracted from in vitro system of plants belonging to genus Haerlea and Ramonda, preparation method and use as chemotherapy, radiotherapy and UV protection agents

Abstract

The present invention relates to an extract containing Myconoside, which is derived from an in vitro system of a plant of the family Gesneraceae, in particular from the genus Haberlea and Ramonda, including species Haberlea rhodopsis Friv., Ramonda helichii (Bois.C. B. Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancicamp, and a method for preparing the extract containing Myconoside. The extract containing Myconoside, which is derived from an in vitro system of a plant of the family Gesneraceae, in particular from the genus Haberlea and Ramonda, includes the species of Haberlea rhodopsis Friv. The invention relates to an extract, in particular to petrovic, Ramonda serbic Panic, Petrovic and Ramonda serbic Panic, as well as hybrids thereof, and a preparation method of the extract. Furthermore, the invention relates to the use of said extract as a chemical, radiation and UV protection agent in human and veterinary medicine, in nutritional supplements and in cosmetics. The plant in-vitro system extract is used independently or in a mixed mode, and comprises a polyphenol component extract 1 mainly containing flavone-C glycoside and phenethyl alcohol compounds (PHE) and a Mycoside component extract 2 mainly containing phenethyl alcohol compounds (particularly Mycoside and Paucifloside). The extract 1 contains 0.1-55.0 wt% of PHE, free phenol, saccharides, organic acid, fatty acid, amino acid, sterol and the like, and the total amount is 100 wt%; the extract 2 contains 55.0 to 99.9 weight percent of PHE (particularly Myconoside and Paucifloside) and phenolic compounds, monosaccharides, disaccharides or residues thereof, and the total amount is 100 weight percent. These compounds, in particular Myconoside, have high biological activity, anti-oxidation, anti-mutation and anti-chromosome-disruption activity, and thus are suitable for use alone, also can be used in combination by metered mixing, or can be conditioned by dilution, for the preparation of natural chemical, radio and ultraviolet protective agents with a controllable degree of protection. The latter is used in humans and veterinary, nutritional supplements and cosmetics to prevent and treat harmful chemical, radiation and UV effects.

Description

Technical Field

[0001] The present invention relates to an extract containing Myconoside, which is derived from the family Gesneriaceae, particularly the genera Haberlea and Ramonda, and also includes Haberlea rhodopensis Friv., Ramonda heldreichii (Boiss.) C.B.Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancic & Petrovic, and Ramonda serbica Pancic, as well as their hybrids and a method for preparing the same. In addition, the present invention also relates to the use of the above extract as a chemotherapeutic, radioprotective, and ultraviolet protective agent in human and veterinary medicine, nutritional supplements, and cosmetics. Background Art

[0002] Wild plants in nature or plants cultivated using traditional agricultural techniques usually produce low levels of the desired bioactive compounds. In addition, some medicinal plants are difficult to cultivate or are rare and endangered species, including species of the genera Haberlea and Ramonda in the family Gesneriaceae, such as Haberlea rhodopensis, Ramonda serbica, and Ramonda nathaliae. This makes it difficult for the industry to obtain their important bioactive components synthesized in nature. The high market demand for natural plant-derived bioactive substances and the limitations of chemical synthesis methods have jointly promoted the demand for obtaining bioactive components through biotechnological means. Plant biotechnology, particularly plant in vitro culture systems, has been proven to be an effective tool for the sustainable production of high-value bioactive components under controlled conditions. Using plant in vitro culture systems can produce various compounds at much higher concentrations than wild plants without damaging natural habitats and destroying biodiversity.

[0003] In recent years, species of Gesneriaceae plants such as Haberlea rhodopensis and Ramonda serbica have attracted much attention from researchers due to their positive effects on human and animal health. In a joint study, Albania and Bulgaria established an in vitro germplasm bank of Ramonda serbica, while Macedonia established an in vitro herbarium of Ramonda nathaliae. Through the polymorphism evaluation of some natural populations, a micropropagation method was selected (E. Daskalova et al., April 2012, Biotechnology & Biotechnology Equipment 26(1): 16 - 25).

[0004] The metabolic changes of Ramonda serbica and Ramonda nathaliae during dehydration and rehydration were studied by 1H-NMR and GC-MS techniques, and the metabolic profiles have been published (Dejan Godeva et al., Phytochemical Analysis, 33(6) / 961-970, August 2022).

[0005] Studies on the traditional ethanol extract of Loxostigma carinatum have shown its unique medicinal properties and pharmaceutical potential, such as antioxidant, radiation protection, anti-chromosomal breakage, chemoprotection, cytoprotection, antibacterial, antimutagenic, immunomodulatory, anticancer and anti-aging effects (Bankova R. et al., Traditional and Modern Veterinary Medicine, 2022, Vol. 7, No.1(12)).

[0006] Existing literature has confirmed that an increase in intracellular reactive oxygen species (ROS) levels, i.e., oxidative stress, can lead to genomic DNA damage and mutations. ROS generated by redox reactions in vivo are highly reactive and can cause significant biological damage, leading to key gene mutations and ultimately inducing cancer, chronic diseases, etc.

[0007] The antioxidant properties of extracts from whole plants, especially the phenolic components of Loxostigma carinatum, have been reported in the literature (Kondeva-Burdina M. et al., Pharmacogn Mag., October 2013, 9(36) and (Mihaylova D. et al., V., 2013, J.F. Biochemistry 37(3)). Multiple studies have shown that modern humans are chronically exposed to environmental factors such as solar radiation (ultraviolet radiation in the UVA and UVB bands).

[0008] In addition, free radicals generated after exposure to gamma radiation are highly reactive and can trigger chain reactions and cause severe biological damage. Anticancer chemotherapeutic drugs can also induce strong oxidative stress, affecting multiple cellular targets and reducing the efficacy of anticancer drugs. Supplementation with antioxidants during chemotherapy can enhance the therapeutic effect (Conklin KA., December 2004, Cancer Comp Ther 3(4)).

[0009] Extracts from wild plants of *Lophostemon chinensis* have radioprotective properties (Georgieva S. et al., Indian J. Exp. Biol., 2013, 51(1); Georgiev Y.N. et al., J. Ethnopharmacology, 2020, 249; Bankova R., Traditional and Modern in Veterinary Medicine, 2022, 7(1)(12); Georgieva S., Gencheva, D., Popov, B., Grozeva, N., and Zhelyazkova, M. 2019, Radioprotective effects of the resurrection plant *Lophostemon chinensis* (Gesneriaceae) and the role of flavonoids and phenolic acids, Bulgarian Journal of Agricultural Science, 25(Suppl 3), 158-168); however, the compounds with this property have not been studied, and it is speculated that it may be due to the presence of phenolic acids and flavonoids in the extract.

[0010] It is known that certain natural glycosylated polyphenols (GPPs, mainly acteoside) have sunscreen properties (US2017 / 0166138).

[0011] Patent EP2319914 discloses an extract from another type of cell culture of plants of the genus *Syringa*, especially *Syringa vulgaris*, and a method for its preparation. The extract contains 20-90 wt% phenylpropanoids (including 5-20% isomartynoside) and 80-10 wt% colorless components, mainly oligosaccharides, polysaccharides, proteins, and lipid molecules, and has antioxidant, procollagen activity, and pigment regulation activity and characteristics.

[0012] WO2021 / 184086 (Innova BM) discloses an extract from in vitro cultures of Haberlea rhodopensis, comprising 25 - 35 wt% polyphenols in the total extract, and the remaining components up to 100 wt% being organic acids, fatty acids, amino acids, sterols, sugars, and free phenols, wherein the phenylethanoid glycoside Myconoside accounts for 18 - 35 wt% of the polyphenol fraction. The extract contains a limited number of polyphenols, namely phenylethanoid glycoside compounds such as Myconoside. The cited literature details the development principles of in vitro plant systems and the propagation of in vitro plant cultures on solid and liquid media. The specific conditions for large-scale cultivation of in vitro plant systems are applicable to specific plant species and are crucial for the production of specific bioactive compounds / metabolites and the targeted compounds of controlled quantity and their use for different purposes. As highly efficient and low-toxic natural chemical, radiation, and UV protectants, the controllable content range of in vitro plant system extracts is very wide, which is particularly important as it enables their use in products for different purposes and fine formulation at different irradiation levels, thus rapidly eliminating the harmful genotoxic effects of irradiation.

[0013] The technical problem to be solved by the present invention is to provide an extract from in vitro culture systems of plants of the genera Haberlea and Ramonda (including their hybrids) of the Gesneriaceae family, which contains a controllable content of rare phenylethanoid glycoside compounds (specifically Myconoside and paucifloside), has high biological utilization value, can be used for the development of new chemical protection, radiation protection, and UV protection products, and ensures the controllability and stability of their protection effects. Summary of the Invention

[0014] According to the present invention, this problem is solved by an extract containing Myconoside, which is derived from an in vitro culture system of plants of the genera Haberlea and Ramonda of the Gesneriaceae family, specifically Haberlea rhodopensis Friv., Ramonda heldreichii (Boiss.) C.B.Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancic & Petrovic, and Ramonda serbica Pancic and their hybrids. The extract includes polyphenol fraction extract 1 mainly containing flavone-C glycosides and phenylethanoids (PHE), and Myconoside fraction extract 2 mainly containing phenylethanoids (especially Myconoside and Paucifloside). Among them, extract 1 contains 0.1-55.0 wt% of PHE and the total amount of free phenols, sugars, organic acids, fatty acids, amino acids, sterols, etc. up to 100 wt%; extract 2 contains 55.0-99.9 wt% of PHE (especially Myconoside and Paucifloside) and phenolic compounds, monosaccharides, disaccharides or their residues, with a total amount up to 100 wt%.

[0015] Another subject of the present invention is a method for preparing the above-mentioned extract, which method comprises the following steps:

[0016] a) Extracting the plant biomass (containing 0.01-25.0% of phenylethanoid glycosides) obtained after the start of in vitro culture (performed according to WO2021184086) with water or hydroalcohol (C1-C3, 40-90%, such as ethanol, isopropanol, methanol) at a ratio of 1:10 to 1:100 w / v, and then performing single or up to three extractions with non-polar solvents applicable to the food, pharmaceutical, and cosmetic industries until the lipophilic components are removed to the greatest extent;

[0017] b) Vacuum concentrating the purified aqueous phase in step a) to 2 / 3 of its volume, removing the solvent at 40°C - 60°C until a crude extract is obtained, and then performing solid-phase extraction with a hydrophobic interaction resin (especially C18 reversed-phase resin), retaining phenolic compounds and PHE, while sugars and polar compounds (mainly organic acids and amino acids) are eluted through the aqueous phase;

[0018] c) Elute the phenolic components on the resin with a mobile phase selected from solvents of different polarities (especially ethyl acetate or isopropanol). The obtained eluate is evaporated to dryness under vacuum at 20 °C - 70 °C to obtain Extract 1, which contains 0.1 to 55.0 wt% of PHE (especially Myconoside and Paucifloside), as well as free phenols, sugars, organic acids, fatty acids, and amino acids, with a total amount up to 100 wt%;

[0019] d) Elute the PHE components on the resin with a 5 to 70% v / v water-alcohol (C1 - C3) mixture. The collected PHE components are concentrated under vacuum at 40 °C to 60 °C until the organic solvent is completely removed, and then freeze-dried or dried to obtain Extract 2, which contains 55.0 to 99.9 wt% of PHE (especially Myconoside and Paucifloside), as well as phenolic compounds, monosaccharides, disaccharides, or their residues, with a total amount up to 100 wt%.

[0020] The obtained Extract 1 and Extract 2 can be used alone or by metered mixing, and are diluted and adjusted to the desired final concentration of phenylethanoid glycosides (Myconoside and Paucifloside) by using solvents safe in the food, cosmetic, and pharmaceutical industries (such as water, glycerol, propylene glycol, or butanediol). In particular, the final concentration of standardized Myconoside is 0.001% to 99.0%. The product thus obtained can be used directly and provides a controllable content of bioactive substances.

[0021] Another subject of the present invention is the use of the above-mentioned Extract 1 and Extract 2 alone or in combination. These extracts are derived from an in vitro culture system of Gesneriaceae plants, especially the in vitro culture system of plants of the genera Haberlea and Ramonda, specifically Haberlea rhodopensis Friv., Ramonda heldreichii (Boiss.) C.B.Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancic & Petrovic, and Ramonda serbica Pancic and their hybrids, directly or after modulation, to obtain new preparations for human and veterinary use, nutraceuticals, and cosmetics, as chemotherapeutic, radioprotective, and ultraviolet protective agents.

[0022] The metered mixing of Extract 1 and Extract 2 ensures feasibility according to specific requirements and purposes of use. When the final concentration of phenylethanoid glycosides (especially Myconoside and Paucifloside) is from 0.01 to 99.9%, the resulting product can be used as a purified extract for in vitro culture of plants of the genera Haberlea and Ramonda, and its biological activity is significantly higher than that of the unpurified crude extract.

[0023] Extract 1 and Extract 2 obtained according to the present invention mainly consist of PHE (especially Myconoside and Paucifloside) within a broad and quantitative range. These compounds (especially Myconoside) have high biological activities, antioxidant, antimutagenic and anti-chromosomal breakage activities, and thus are suitable for use alone or by metered mixing, and by dilution adjustment, for the preparation of natural chemical, radiation and UV protectants with a controllable degree of protection. The latter can be used in human and veterinary medicine, nutraceuticals and cosmetics for the prevention and treatment of harmful chemical, radiation and UV effects.

[0024] Definitions

[0025] According to the method of the present invention, "extract" refers to a crude extract obtained by direct extraction or separation from an in vitro system of plants of the family Gesneriaceae, especially components containing phenylethanol Myconoside obtained from the genera Haberlea and Ramonda, and from their hybrids.

[0026] As used herein, the term "plant" refers to plants of the family Gesneriaceae, especially plants of the genera Haberlea and Ramonda, specifically including Haberlea rhodopensis Friv., Ramonda heldreichii (Boiss.) C.B.Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancic & Petrovic and Ramonda serbica Pancic and their hybrids, and refers to plant cells, whole plants, plant organs, plant tissues, seeds and their progeny.

[0027] As used herein, the term "in vitro culture system" refers to, but is not limited to, in vitro plants cultured from seeds, embryos, meristematic regions, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores, callus, suspension cultures, shoots, meristem (shoot) cultures, root cultures (normal roots, supplementary roots and transformed roots) and in solid or liquid nutrient media or cultivation substrates under controlled in vitro conditions.

[0028] "Myconoside" is a caffeoyl phenylethanol glycoside with the molecular formula C33 H 44 O 19 , 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 ([[(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)). Detailed implementation manners

[0029] Here, exemplary embodiments will be described in detail, and examples thereof are shown in the drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation manners described in the following exemplary embodiments do not represent all implementation manners consistent with the present application. On the contrary, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0030] The present invention is illustrated by the following exemplary embodiments, but is not limited thereto:

[0031] Example 1

[0032] 1. Use a water-alcohol mixture (70% alcohol, such as ethanol, at a ratio of 1:15 w / v) to extract 500 grams of dry biomass (containing 12% phenylethanoid glycosides) from in vitro cultured [[Conandron ramondioides]] for extraction. After filtration, the liquid phase is concentrated under vacuum until ethanol is removed, and then liquid-liquid extraction is performed three times (3×2 L, 1 hour, 21°C) with purified hexane to remove lipophilic components. The purified aqueous phase is concentrated under vacuum at 40°C to 2 / 3 of its volume to remove solvent residues, obtaining a crude extract and performing solid-phase extraction;

[0033] 2. The crude extract (2.0 L) was subjected to solid-phase extraction using C18 reversed-phase resin (600 g). In this way, phenolic compounds and phenyl ethanol glycosides were retained by the resin, while sugars and polar compounds (mainly organic acids and amino acids) were eluted through the aqueous phase;

[0034] 3. The next step was to elute the phenolic fraction with ethyl acetate (4 L) as the mobile phase. Most phenolic compounds, flavonoids, and some PHE (especially Myconoside and Paucifloside) were eluted. The obtained fraction was evaporated to dryness under vacuum at 40 °C to obtain Extract 1, which contained 54.7 wt% of PHE (especially Myconoside and Paucifloside), as well as free phenols, sugars, organic acids, fatty acids, and amino acids, with a total amount up to 100 wt% (the differences in the concentrations of associated metabolites in different extracts are shown in Table 1. In the GC-MS spectra of Haberlea rhodopensis in vitro culture systems: crude extract, Extract 1, and Extract 2. The results were statistically processed and cluster-analyzed by the Euclidean distance method and presented as a hierarchical cluster heat map. Each colored cell in the figure corresponds to the concentration value of the relevant compound.

[0035] 4. The next step was to elute the phenyl ethanol glycoside fraction with 2 L of a water-ethanol mixture (70%, w / w). The collected PHE fraction mainly contained Myconoside and Paucifloside. This fraction was concentrated under vacuum at 50 °C to form a gel (80 g), and then freeze-dried or dried at 40 °C to obtain Extract 2, which contained 99.5 wt% of PHE (especially Myconoside and Paucifloside, see Table 2), as well as monosaccharides, disaccharides, phenolic compounds, flavonoids, or their residues, with a total amount up to 100 wt% (see Table 1).

[0036] Compared with the original crude extract, the yield of Myconoside was 76.1%.

[0037] Figure 1 shows the GC-MS spectra of the extracts from the Haberlea rhodopensis in vitro culture system obtained according to Example 1: crude extract, Extract 1, and Extract 2. The results were statistically processed and cluster-analyzed by the Euclidean distance method and presented as a hierarchical cluster heat map. Each colored cell in the figure corresponds to the concentration value of the relevant compound.

[0038]

[0039] Figure 1

[0040] Table 1 (as follows) shows the phytochemical components of the extracts from the Haberlea plant in vitro culture system obtained according to the method described in Example 1 of the present invention. The results are the average values of 3 parallel tests for each extract variant and are expressed as a percentage of the dry weight.

[0041] Table 1

[0042]

[0043]

[0044] The Agilent Technology Hewlett Packard 7890A+ / MSD 5975 instrument (Hewlett Packard, Palo Alto, CA, USA) was used in combination with the Agilent Technology 5975C inert XL EI / CI MSD mass spectrometer (Hewlett Packard, Palo Alto, CA, USA). HP-5MS chromatographic column (30 m × 250 μm × 0.25 μm), temperature programmed at 60 °C for 2 minutes, then rising to 260 °C at a rate of 5 °C per minute and held at 260 °C for 8 minutes. The injection volume of the sample was 1 μl and the split ratio was 10:1. The syringe temperature was 250 °C and the flow rate of the carrier gas (helium) was 1 ml / min. The EI / MS spectra were recorded at 70 eV.

[0045] High performance liquid chromatography system, Waters 1525 binary pump (Waters, Milford, MA, USA), Waters 2487 Dual λ absorbance detector (Waters, Mifford, MA, USA), operated by Breeze 3.30 software; Supelco Discovery HS C18 chromatographic column (5 μm, 25 cm × 4.6 mm), at 28 °C; mobile phase, gradient of 2% acetic acid and acetonitrile;

[0046] Table 2 shows the NMR data of the structures of Myconoside and paucifloside (in 500 MHz, D2O) in the extract 2 of the present invention

[0047] Table 2

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Example 2

[0055] 1. 100 g of dry biomass (containing 0.5% phenylethanoid glycosides, mainly Myconoside) was extracted from in vitro cultures of Ramonda serbica using distilled water at a ratio of 1:30 w / v. After filtration, the liquid phase was concentrated to 1 / 3 of its volume under vacuum, and then subjected to liquid-liquid extraction three times (3×2 L, 1 hour, 21 °C) with ethyl acetate to remove lipophilic components. The purified aqueous phase was concentrated to 2 / 3 of its volume under vacuum at 50 °C to remove solvent residues, obtaining a crude extract, and solid-phase extraction was carried out according to the procedures described in points 2, 3, and 4 of Example 1 above.

[0056] 2. The resulting phenolic fraction (Extract 1) contains 2.0 wt% PHE (especially Myconoside and Paucifloside), as well as free phenols, sugars, organic acids, fatty acids, and amino acids, with a total amount up to 100 wt%.

[0057] 3. The resulting phenylethanoid glycoside fraction (Extract 2) contains 57.0 wt% PHE (especially Myconoside and Paucifloside), as well as monosaccharides, disaccharides, phenolic compounds, and flavonoids or their residues, with a total amount up to 100 wt%.

[0058] The yield of Myconoside was 82% compared to the original crude extract.

[0059] Example 3

[0060] The operation of this example is the same as that of Example 1, except that the biomass from in vitro cultures of Conandron ramondioides used in step 1 contains 9% phenylethanoid glycosides and is extracted three times with 50% isopropanol at a ratio of 1:20. The purified aqueous phase is concentrated at 50 °C, and in step 4, 50% v / v methanol is used to elute the phenylethanoid glycoside fraction. The resulting Extract 1 contains 29.5% PHE, specifically Myconoside and Paucifloside, and Extract 2 contains 62.0% PHE, specifically Myconoside and Paucifloside.

[0061] The yield of Myconoside was 92% compared to the original crude extract.

[0062] Example 3a

[0063] The operation was the same as in Example 3, except that the dry biomass used was from in vitro cultures of Conandron ramondioides (containing 12% phenylethanoid glycosides, mainly Myconoside). The extract 1 obtained thereby contained 52.0% PHE, specifically Myconoside and Paucifloside, and the extract 2 contained 98.0% PHE, specifically Myconoside and Paucifloside. The yield of Myconoside was 84% compared to the original crude extract.

[0064] Table 3 (below) shows the phytochemical composition of the extracts of the Ramonda serbica in vitro culture system obtained by the method according to Example 3a of the present invention. The results are the average of 3 parallel tests for each extract variant and are expressed as a percentage of dry weight.

[0065] Table 3

[0066] Metabolite Crude extract Extract 1 Extract 2 Myconoside 7.07 2.53 71.19 Phenols and flavonoid-C glycosides 9.14 44.64 8.75 Organic acids and saccharides 71.31 46.21 16.04 Hydrocarbons and fatty alcohols 4.38 2.04 0.99 Amino acids 3.67 0.28 0.10 Others 4.43 3.4 2.93

[0067] The extract 1 and extract 2 obtained according to the above embodiments can be used alone or in combination, in the dry state, or diluted and adjusted to a final concentration of 0.01 to 99.0% of phenylethanoid glycosides (especially Myconoside and Paucifloside) by using solvents safe in the food, cosmetic and pharmaceutical industries (such as water, glycerol, propylene glycol or butanediol, etc.). The product obtained thereby can be used as a purified extract of in vitro cultures of Conandron ramondioides and Ramonda, with significantly improved biological activity.

[0068] Data on application examples and effects of the extracts according to the present invention

[0069] Materials and methods

[0070] In the present invention, the extract 1 and extract 2 containing phenylethanoid glycosides (especially Myconoside and Paucifloside) were isolated from in vitro culture systems of Gesneriaceae plants, especially plants of the genera Haberlea and Ramonda and their hybrids. The dried extracts were stored at -20 °C protected from light and moisture. The solutions used in the experiments were prepared temporarily based on aqueous solutions.

[0071] Example 4

[0072] According to Example 1, the UV-VIS absorption spectra of extract 1 and extract 2 obtained from the in vitro culture system of Conandron ramondioides were studied.

[0073] Prepare an aqueous solution with a final Myconoside concentration of 22 pM using the studied extract, and record its UV-Vis spectrum using a Shimadzu UV / Vis mini 1240 spectrophotometer. The obtained spectrum is shown in Figure 2, indicating that extract 2 has a significantly improved UV spectral absorption ability, suggesting that the purified Myconoside has a stronger UV spectral capture ability compared to extract 1 containing more accompanying compounds such as phenols and flavone-C glycosides.

[0074]

[0075] Figure 2

[0076] The UVA and UVB transmittance (T%) of extract 2 (obtained according to Example 1 of this article) at different dilution levels were studied, 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, respectively. The results are shown in Figure 3.

[0077]

[0078] Figure 3

[0079] The results show that a minimum concentration of 0.011 mg / ml of Myconoside is sufficient to provide 50% inhibition of UV radiation transmission, and a maximum concentration of 0.11 mg / ml can provide 100% protection. These results demonstrate that the phenylethanoid glycoside components (mainly Myconoside and Paucifloside) contained in extract 1 and extract 2 of the present invention have high potential as UV filters in sunscreen products.

[0080] Example 5

[0081] Study the antioxidant potential of extract 1 and extract 2 obtained from the in vitro culture system of Conandron ramondioides in Example 1 when used alone or in combination with a proven UV protectant

[0082] The radical scavenging activity (% inhibition) of Extract 1, Extract 2 (obtained according to Example 1), Oxybenzone, and an aqueous solution (0.5%) of a 1:1 Extract:Oxybenzone combination (w / w) was evaluated by DPPH testing. A DPPH comparative analysis was conducted to compare the antioxidant potential of Extract 1, Extract 2, a commercially available UV protectant (Oxybenzone), and combinations of Extract 1 with Oxybenzone and Extract 2 with Oxybenzone. The results demonstrated that Myconoside in the purified Extract 2 was the carrier of antioxidant activity. Direct EPR spectroscopy was used (Yordanov and Christova, 1997; Karamalakova, 2014). To measure the DPPH radical scavenging ability, a 98% DPPH ethanol solution (80 mM, stock solution) was used at room temperature (22 °C), and Extract 1, Extract 2, Oxybenzone, and the relevant combinations (at a concentration of 0.5%) were mixed and homogenized. The mixture was incubated in the dark, and the DPPH-H / R radicals generated in the system were examined at 1, 5, and 10 minutes. The DPPH solution was used as an internal standard for the EPR signal (Figure 4).

[0083]

[0084] Figure 4

[0085] The research results showed a statistically significant increase in the percentage of DPPH radicals captured in Extract 2 (from 14.9% to 18.2%, 21.3%, p < 0.001), while the UV protectant Oxybenzone showed the lowest radical scavenging activity (from 4.7% to 3.2% and 2.2%, p < 0.001) (Figure 4). Prolonging the incubation time of the solutions studied did not cause a statistically significant change in the percentage of DPPH radicals captured. The results indicated that Oxybenzone exhibited very low antioxidant activity when used alone, but its antioxidant activity increased significantly (p < 0.001) when combined with Extract 1 and Extract 2. Inducing UV-B stress not only did not reduce the inhibitory capacity but also brought about a statistically significant increase. This became evident after 10 minutes in both combinations. In summary, in an in vitro environment, the extracts studied exhibited strong antioxidant activity and persistent radical scavenging ability at different incubation times, both before and after inducing UV-B stress. This effect was most significant in the purified Extract 2 (mainly Myconoside and Paucifloside) and its combination with the commercially available UV protectant Oxybenzone.

[0086] Example 6

[0087] To study the antioxidant potential of extracts 1 and 2 obtained from the in vitro culture system of Haberlea rhodopensis according to Example 1, used alone or in combination with a proven radioprotector

[0088] It has been demonstrated that free radicals generated after exposure to gamma radiation are highly reactive and can trigger chain reactions, leading to significant biological damage and increased oxidative stress. Therefore, so-called radioprotectors have special significance in practical applications. They are substances and compounds of synthetic or natural origin that can enable human cells to withstand higher levels of radiation or can rapidly eliminate the harmful genotoxic effects of such radiation, maintaining the stability of the human genome. To demonstrate that the phenylethanoid glycoside fraction (mainly containing Myconoside and Paucifloside) is the carrier of antioxidant activity and the main component of the radioprotective properties of extracts from in vitro culture systems of Gesneriaceae plants, especially plants of 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 free radical scavenging ability, a 98% DPPH ethanol solution (80 mM, stock solution) was used at room temperature (22 °C), and extract 1, extract 2, amifostine, and the relevant combinations (at a concentration of 0.5%) were mixed and homogenized. The mixture was incubated in the dark, and the DPPH-H / R free radicals generated in the system were examined at the 1st, 5th, and 10th minutes. The DPPH solution was used as an internal standard for the EPR signal, and the DPPH free radical scavenging activities (% inhibition) of aqueous solutions (0.5%) of extract 1, extract 2 (obtained according to Example 1), and amifostine were tested. (Figure 5).

[0089]

[0090] Figure 5

[0091] The research results showed a statistically significant increase in the percentage of DPPH free radicals captured in purified extract 2 (from 59.5% to 72.7%, 85.32%, p < 0.001), while the commercially available radioprotector amifostine showed the lowest free radical scavenging activity (from 5.1% to 4.5% and 3.9%, p < 0.001) (Figure 5). It was found that extending the incubation time did not change the percentage of DPPH free radicals captured.

[0092] The effects of the studied extracts in combination with the commercially available radioprotector amifostine before and after gamma irradiation were also investigated (Figure 6). 2.0 Gy of 60Co gamma radiation (γ-rays) was carried out in a water bath (37 °C) at 24 Gy / min using a Rokus-M irradiator. The DPPH free radical scavenging activity (% inhibition) of aqueous solutions (0.25%) of amifostine (1), extract 1 (2), 1:1 combination of extract 1 and amifostine (w / w) (3), extract 2 (obtained according to Example 1) (4), and 1:1 combination of extract 2 and amifostine (w / w) (5) was measured before and after exposure to 2.0 Gy of gamma radiation.

[0093]

[0094] Figure 6

[0095] The results showed that after Gy irradiation, there were statistically significant increases in the percentage of DPPH free radicals captured in purified extract 2 (mainly containing Myconoside and Paucifloside) (19.7%, p < 0.001) and its combination with amifostine (25.6%, p < 0.001). Prolonging the incubation time (30 minutes) did not change the percentage of DPPH free radicals captured after exposure to 2.0 Gy of radiation. Amifostine alone (0.25%) showed insignificant antioxidant activity (2.5% before radiation and 5.9% after radiation, p < 0.001), but its activity increased significantly when used in combination with purified extract 2 (21.1% before radiation and 25.6% after radiation, p < 0.005).

[0096] Example 7

[0097] The clastogenic activity and anti-clastogenic potential of extracts 1 and 2 obtained from the in vitro culture system of Conandron ramondioides according to Example 1 were studied, either alone or in combination with a clinically proven cytoprotectant (amifostine), directly added to the cell culture and 1 hour after blood irradiation.

[0098] The micronucleus scoring assay was used to study the anti - chromosomal breakage potential of extract 1 and extract 2 (obtained according to Example 1) and the clinically proven cytoprotectant amifostine in human lymphocyte cultures in vitro before and after exposure to ionizing radiation (gamma rays). The micronucleus test of peripheral blood lymphocytes and polychromatic erythrocytes is the most suitable experiment for screening mutagenicity. A compound is considered mutagenic when it increases the spontaneous frequency of micronuclei (MN) in polychromatic erythrocytes or lymphocytes. This test demonstrates the mutagenic effects of compounds with chromosomal breakage or spindle - inhibiting effects. The cytokinesis - blocked micronucleus assay of Fenech, M. and A. Morley (1985) was used to analyze MN in binucleated peripheral blood lymphocytes. The number of MN in 500 binucleated cells in cell cultures prepared from irradiated and non - irradiated peripheral venous blood collected from 5 healthy volunteers was studied. The blood of the donors was exposed to 2.0 Gy of 60Co gamma radiation (γ - rays) using a Rokus - M irradiator at 24 Gy / min in a water bath (37 °C). The irradiation dose was calculated based on the geometric parameters of the irradiator, the distance from the irradiation source, and the power. Figure 7 shows the number of MN found in 500 binucleated cells in cell cultures prepared from non - irradiated blood (A) and irradiated blood (B): control (1); amifostine (2); extract 1 (3); extract 2 (4); 1:1 extract 1 with amifostine (w / w) (5) and 1:1 extract 2 with amifostine (w / w) (6).

[0099]

[0100] Figure 7

[0101] When amifostine, extract 1, and extract 2 (obtained according to Example 1) were added to non - irradiated blood cells, the results showed that the tested substances lacked chromosomal breakage activity, indicating that they are not mutagenic and can be safely used in medicine (Figure 7A). Comparative statistical analysis of the total number of micronuclei in 500 binucleated cells in different groups of cell cultures after gamma - radiation treatment (Figure 7B) showed a statistically significant difference (p > 0.05) between the control group and the cells treated with extract 1, extract 2, and their combinations with amifostine.

[0102] Example 8

[0103] The chromosomal breakage activity and anti - chromosomal breakage potential of extract 1 and extract 2 obtained from the in vitro culture system of Conandron ramondioides were studied, used alone or in combination with a clinically proven cytoprotectant (amifostine), and added to cell cultures 1 hour before in vitro ionizing radiation (γ - rays) irradiation.

[0104] The chromosomal breakage activity and anti-chromosomal breakage potential of extract 1 and extract 2 (obtained according to Example 1) and the clinically proven cytoprotectant amifostine were studied in vitro in human lymphocyte cultures before and after exposure to ionizing radiation (γ-rays) using a test for detecting induced chromosomal aberrations. The method used was able to accurately identify all the major types of structural chromosomal rearrangements induced by ionizing radiation (γ-rays), such as single or double acentric fragments, dicentric chromosomes, and ring chromosomes.

[0105] The study design included a total of 6 groups, including an untreated control group. In each group, 5 blood donors were examined, and 100 metaphase plates obtained from peripheral venous blood lymphocytes of each donor were analyzed for chromosomal aberrations, for a total of 500 metaphase plates analyzed in each group.

[0106] Cell cultures were prepared using the method of Evans H.J. for short-term culturing of peripheral blood lymphocytes and obtaining metaphase plates for chromosomal aberration reading. Chromosome microscope slides were observed on an Olympus BX41 microscope at the most convenient magnification - screening preparations were carried out at 63 magnification, and chromosomal aberrations were analyzed in detail at 1000 magnification. At least 100 metaphases were analyzed for each sample. The donor cell cultures were exposed to 2.0 Gy of 60Co gamma radiation (γ-rays) using a Rokus-M irradiator at 24 Gy / min in a water bath (37°C). The irradiation dose was calculated based on the geometric parameters of the irradiator, the distance from the irradiation source, and the power. Figure 8 shows the frequency of aberrant cells in treated human lymphocytes before (A) and after (B) 2.0 Gy of gamma radiation as follows: control group (1); amifostine (2); extract 1 (3); extract 2 (4); 1:1 extract 1 and amifostine (w / w) (5) and 1:1 extract 2 and amifostine (w / w) (6).

[0107]

[0108] Figure 8

[0109] The addition of amifostine, extract 1, and extract 2 (obtained according to Example 1) to unirradiated blood cells showed that the test substances lacked chromosomal breakage activity, indicating that they were not mutagenic and could be used safely (Figure 8A). The lack of chromosomal breakage activity of the extracts studied at the concentrations used in the experiment provides a basis for the application of extract 1 and extract 2 obtained in the present invention in phytotherapy and practical medicine. The results of the comparative statistical analysis of pre-treating cells with gamma radiation (Figure 8B) showed a significant difference (p>0.01) with statistical significance compared to the control group cells pre-treated with extracts 1 and 2, and their combinations with amifostine (p>0.05).

[0110] The results of Example 7 and Example 8 showed that the extract rich in phenylpropanoid glycosides (mainly Myconoside and Paucifloside) obtained according to Example 1 had a more significant anti-chromosomal breakage potential than amifostine, which is used as a radioprotector in clinical practice but has many side effects. Based on the extracts of the in vitro culture system of Gesneriaceae plants, especially those of the genera Haberlea and Ramonda and their hybrids, which do not have any cytotoxic and chromosomal breakage activities, it can be reasonably assumed that they can be used as radioprotectors in phytotherapy due to their strong anti-chromosomal breakage potential.

[0111] According to Examples 4 - 8, similar parallel tests were also carried out on the Ramonda serbica extract obtained according to Example 3a, and the results were close to the presented results, with a deviation range of ±0.1% to +0.9%.

[0112] Similar parallel tests were carried out on the combination of Extract 1 and Extract 2 of Haberlea rhodopensis Friv. obtained according to Example 1 in a 1:1 ratio, and the obtained results were close to the presented results, with a deviation range of ±0.05% to ±0.09%.

Claims

1. An extract containing Myconoside, derived from an in vitro culture system of plants of the genus Haberlea and Ramonda, characterized in that Extract 1, which alone or in combination contains a component mainly containing flavone-C glycosides and phenyl ethanol compounds, and a component mainly containing phenyl ethanol compounds, in particular Extract 2 containing Myconoside and Paucifloside, wherein the phenyl ethanol compounds in Extract 1 contain 0.1 to 55.0 wt% of Myconoside and Paucifloside, as well as free phenols, sugars, organic acids, fatty acids and amino acids, with a total up to 100 wt%; while Extract 2 contains 55.0 to 99.9 wt% of the phenyl ethanol compounds Myconoside and Paucifloside and phenolic compounds, monosaccharides, disaccharides or their residues, with a total up to 100 wt%.

2. The extract containing Myconoside according to claim 1, characterized in that, The in vitro culture systems of plants of the genus Haberlea and Ramonda are selected from the following species: Haberlea rhodopensis Friv., Ramonda heldreichii (Boiss.) C.B.Clarke, Ramonda myconi (L.) Rchb., Ramonda nathaliae Pancic & Petrovic and Ramonda serbica Pancic, and their hybrids.

3. The extract containing Myconoside according to claim 1, characterized in that, The combination ratio of Extract 1 to Extract 2 is between 1:99 and 99:

1.

4. A composition comprising the Myconoside extract according to claim 1, for use in pharmaceuticals and cosmetics, such as water, glycerol, propylene glycol or butylene glycol, when Extract 1 and Extract 2 are diluted separately or combined in different ratios, the final standardized concentration of Myconoside is 0.001% to 99.0%.

5. The preparation method of the extract containing Myconoside according to claim 1, characterized in that, Comprising the steps in the following order: a) The biomass obtained after the initiation and amplification of the in vitro culture system is extracted with water or a C1-C3 alcohol, a water-alcohol mixture (40 - 90%) in a ratio of 1:10 to 1:100 w / v, and the aqueous phase is extracted with a non-polar solvent once or up to three times to remove lipophilic components; b) The purified aqueous phase in step a) is vacuum concentrated at 40°C to 60°C to remove the solvent until a crude extract is obtained, and solid-phase extraction is carried out through a hydrophobic interaction resin to retain phenolic compounds and phenyl ethanol compounds, and polar compounds such as sugars, organic acids and amino acids are eluted with the aqueous phase; c) The phenolic components on the resin are eluted with solvents of different polarities (especially ethyl acetate or isopropanol). The resulting eluate is vacuum evaporated to dryness at 20°C to 70°C and freeze-dried or dried to obtain Extract 1, which contains 0.1 to 55.0 wt% of phenyl ethanol compounds (especially Myconoside and Paucifloside). d) Elute the phenylethanoid glycoside fraction on the resin with an aqueous-alcoholic mixture of C1-C3 alcohols and 5 to 70% v / v distilled water. The collected phenylethanoid glycoside fraction is concentrated in vacuo at 40°C to 60°C until the organic solvent is completely removed, and then freeze-dried or dried to obtain Extract 2, which contains 55.0 to 99.9 wt% of phenylethanoid glycosides (especially Myconoside and Paucifloside).

6. The extract containing Myconoside according to claims 1 to 5 for the preparation of a product having chemical, radiological and UV protective effects.

7. The extract containing Myconoside according to claims 1 to 6 for use in human and veterinary medicine, the pharmaceutical industry, cosmetics and the food industry.

8. The extract containing Myconoside according to claims 1 to 7 for the preparation of a dermatological preparation having antioxidant, antimutagenic and anti-chromosomal breakage activities.

9. The extract containing Myconoside according to claims 1 to 7 for the preparation of a food supplement having antioxidant, antimutagenic and anti-chromosomal breakage activities.

10. The extract containing Myconoside according to claims 1 to 7 for the preparation of a drug having antioxidant, antimutagenic and anti-chromosomal breakage activities.

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