Standardized plant extract from biomass of in vitro cultures, method for preparation and use thereof

Abstract

To provide a standardized extract with a guaranteed content of the phenylethanoid glycoside (e.g., myconoside), with invariable physico-chemical characteristics and composition of the concomitant bioactive compounds / components, obtained by biotechnological method with a maximally efficient design.SOLUTION: The invention refers to a standardized plant extract from biomass of in vitro cultures of Haberlea rhodopensis Friv. (HR), containing bioactive compounds and their primary and secondary metabolites, containing in wt.%, as follows: organic acids from 4.0 to 6.0, fatty acids from 0.5 to 1.5, amino acids from 8.0 to 12.0, sterols from 0.5 to 1.0, free phenols from 3.0 to 6.0, sugars from 45 to 55, and polyphenols from 25.0 to 35.0, with a predominant myconoside content of 70% to 96% in the polyphenolic fraction, constituting 18% to 35% of the total extract.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] FIELD OF THEINVENTION The present invention relates to standardized plant extracts, in particular extracts derived from biomass of in vitro cultures and methods for the preparation of such extracts, which contain beneficial biologically active compounds (BACs) and secondary metabolites and can be used for the preparation of agents for the pharmaceutical, cosmetic or food industries.

[0002] BACKGROUND OF THEINVENTION Natural plants contain a variety of BAS, and their origin, growing conditions, harvest time, extraction techniques, etc. affect the quality of the product.

[0003] Cultivated medicinal plants have many advantages over wild plants: their growth can be monitored, they can be harvested at the most favorable time, and access to unsuitable plant species can be avoided.

[0004] Haberlea is a monotypic genus, of which H. rhodopensis Friv. (Rhodopean Silivryak), or (HR) for short, is its only member. The plant is a relic of the Tertiary period and is endemic to central and southern Bulgaria, found in the Balkan and Rhodope Mountains, as well as in the Rhodope Mountains, Pangaion and Phalacro Mountains in northeastern Greece. For centuries, H. rhodopensis has been traditionally used in ethnopharmacology and local traditional medicine (1).

[0005] It is known that plant extracts have been isolated from HR leaves. Bio-compounds isolated from plant extracts are a source of essential natural ingredients. Apart from fruits and vegetables, polyphenols, glycosides, sugars, etc. are also found in plants and because of their physiological functions, they play an important role in human health.

[0006] It is known that the ethanol extract of HR is obtained by spray-drying the whole plant, followed by ethanol extraction for 3 hours with stirring at room temperature, filtration, separation of insoluble matter, and then vacuum concentration and freeze-drying to obtain the extract. The same source also provides information on the water-soluble HR extract, in which the whole plant is spray-dried and extracted with hot water at 120°C in an autoclave for 20 minutes, followed by separation of insoluble matter through filtration at high temperature and freeze-drying. The obtained extract has the effects of anti-aging, antioxidant, skin whitening, and immunostimulation (5).

[0007] There is a report on an extract of HR obtained by hydroalcoholic extraction (ethanol / water, unspecified preparation method) and purified by gel filtration chromatography on Sephadex LH-20, which contains an isolated fraction particularly rich in phenylethanoid glycosides, the myconosides responsible for the biological activity of the Haberlea extract. In the same source, the effectiveness of the extract was observed and it was reported that the myconosides stimulate the antioxidant protection of the skin, improve its elasticity by stimulating the synthesis of extracellular structures, have cytoprotective and UV-protective effects, act as anti-aging agents for the skin that protect the skin from oxidation, increase its elasticity and increase its radiance, and have potential cosmetic applications (3).

[0008] In addition, the phytochemical composition of the 70% ethanol extract of HR leaves contains a large group of major bioactive compounds, including secondary metabolites (phenolic acids, flavonoids, fatty acids, phytosterols, carotenoids, soluble lipids, oligo- and polysaccharides, free sugars, polyols, organic acids, etc.) including myconosides, known to have proven antioxidant and hepatoprotective properties (1).

[0009] Data are also available on the phytochemical profile of methanol extracts of HR leaves containing two phenolic glycosides: myconoside and paucifloside, prepared using a combination of liquid-liquid extraction, preparative and semi-preparative high performance liquid chromatography. The myconoside-rich fraction (caffeoyl phenylethanoid glycosides) has a potential role in plant survival and possesses antioxidant activity due to the presence of a caffeoyl group and two free hydroxyl groups in its phenyl ring. The main BAC groups and secondary metabolites found in the various HR extracts during normal growth and drought periods of plant growth are organic acids, fatty acids, amino acids, phenolic acids, and sugars (4).

[0010] Today, HR extracts obtained directly from naturally growing species are successfully applied in cosmetics, homeopathy and pharmacy due to their proven antibacterial, antiviral, antioxidant, immunomodulatory, cytotoxic, anticancer, chemopreventive, genoprotective and radioprotective properties.

[0011] On the other hand, being a particularly valuable and rare endemic species, HR is among the plant species whose collection from nature is prohibited. For example, in recent years, in vitro systems for the regeneration and mass propagation of HR have been established with the aim of protecting the natural population of this rare plant species. In vitro banks of HR plants from various locations have been set up. The same source also describes an effective method of regeneration and mass propagation in which seeds of plant species are sterilized with 70% EtOH for 1 min, treated with hypochlorite for 6–10 min, treated with 0.1% HgCl2 for 3–5 min, rinsed three times with distilled water, and the resulting sterilized HR seeds are placed on classical hormone-free MS medium (MS B5, similar to WPM) and allowed to germinate for 3–4 months to obtain seedlings. By subculturing the plant clusters for 1–1.5 months, an in vitro system of fully developed plants was obtained, ready to be transformed over the course of 6–7 months under non-sterile conditions in a controlled greenhouse environment (2).

[0012] At the same time, plant biotechnology, specifically the in vitro culture of plant cells, has become a promising tool for the sustainable and continuous production of plant-derived BACs. The principles of plant cell culture and plant cell growth in solid and liquid nutrient media, as well as the general processes related to the establishment and cultivation of plant cells, have been described for other plant species different from HR.

[0013] According to (6), separate extracts have been obtained from plant cell cultures of Rosa sp., containing valuable natural products (NPs) for cosmetic skin and hair care, preventing signs of aging. Plant cell biomass has been used for the production of phenylpropanoids from Ajuga repens, and verbascoside from Olea europea, Syringa vulgaris, or Appia citobara, as well as the production of a standardized extract of verbascoside from Syringa vulgaris cell culture, which according to (7) has proven antioxidant activity and is effective in the treatment of acne and in the prevention of hair loss.

[0014] According to (8), a preparation in the form of an in vitro cultured extract of Argania spinosa has also been produced for the treatment of skin aging and skin inflammation.

[0015] According to (9), a standardized extract obtained from in vitro undifferentiated plant cells of Dracocephalum ruyschiana, containing BACs with proven antiradical activity and cosmetic applications for skin protection and regeneration, grown in semi-solid or liquid media, in vitro grown undifferentiated cells of Dracocephalum ruyschiana in the dark or with a 14-16 h photoperiod, is obtained by isolation of the cell biomass, followed by extraction of the biomass with ethanol or with ethanol, glycerol and water in a ratio of 20:20:60 to 50:50:0 (v / v), drying of the biomass and further extraction with the same extract.

[0016] To date, there have been no reports of extracts derived from the biomass of HR in vitro cultures, containing BACs and secondary metabolites.

[0017] Despite the fact that general principles for the cultivation of plant cells are not applicable to large-scale cell multiplication for production purposes, specific technological processes need to be developed. To maintain plant in vitro cultures (differentiated or non-differentiated) under in vitro culture conditions, it is important to determine the optimal and strictly specific ratio of growth regulators (auxins, cytokinins and gibberellins) and establish it for each cell line (each plant species). The modification of cell culture conditions, as well as the use of elicitors, precursors and absorption matrices, are crucial for the production of certain bioactive compounds / metabolites, specific to the plant species used and the desired target compound.

[0018] However, the synthesis of valuable BACs and metabolic components in in vitro culture is a complex process with many unknown parameters. The accumulation of natural components and secondary metabolites in the cellular biomass results from a dynamic balance between biosynthesis, biotransformation and biodegradation. It is important to select the appropriate and optimal conditions (i.e., nutrient media and stimulating factors) for each component specific to the intact plant.

[0019] Often, natural products obtained from traditional extracts have poor homogeneity and the amount of the target therapeutic components tends to vary seasonally and geographically when compared to those obtained from in vitro cultures. It is also essential to work with standardized raw materials for the purpose of obtaining in vitro biomass extracts and their incorporation into products for the cosmetic, pharmaceutical or food industries.

[0020] The problem of the present invention is to obtain standardized extracts, in particular extracts derived from HR in vitro cultures (seedlings, shoot cultures, root cultures (normal, adventitious and hairy roots), somatic embryos, callus cultures, cell suspension cultures), with a guaranteed content of phenylethanoid glycosides (i.e. myconosides) with unaltered physicochemical properties and constituents of the associated BACs / ingredients, obtained by biotechnological methods with a maximally efficient design.

[0021] Summary of the Invention The problem according to the invention is solved by a standardized plant extract, specifically an extract derived from the biomass of in vitro cultures of HR (seedlings, shoot cultures, root cultures (normal, adventitious and hairy roots), somatic embryos, callus cultures, cell suspension cultures), containing secondary and primary plant metabolites (i.e. fatty acids, sterols, organic acids, amino acids, free phenolic acids, and sugars), BACs. The amount of BACs in the extract is, in % by weight: fatty acids 0.5-1.5, sterols 0.5-1.0, organic acids 4.0-6.0, amino acids 8.0-12.0, free phenols 3.0-6.0, sugars 45.0-55.0, and polyphenolic compounds 25.0-35.0%, including phenylethanoid glycosides (i.e. myconosides) which account for 70-96% of said polyphenolic fraction. The extract obtained from the biomass of HR in vitro cultures is enriched in myconosides and standardized, the amount of which ranges from 18% to 35% of the total extract. Dissolution of the standardized extract in glycerol results in a product with a controlled content of myconosides in its composition, ranging from 0.01 to 15.00%, depending on the needs of the relevant industry (i.e. pharmaceutical, food or cosmetic).

[0022] The standardized extract from the in vitro culture according to the present invention is prepared by a method comprising the following essential steps: 1) Initiation of HR-derived in vitro cultures: - the selection of explants from individual parts or organs of plants, in particular from leaves, stems, hypocotyls, roots, seeds, anthers, ovaries, sepals, seedlings, - surface sterilization of said selected explants by repeated washing with sterile distilled water for 1-240 minutes, treatment with 40-85% ethanol for 10-190 seconds, followed by treatment with 2-10% disinfectant with or without surfactant for 10-60 minutes, washing with sterile distilled water and drying for 1-20 minutes; - initiation of sterile explants on semi-solid or liquid media with or without growth regulators and 2-5 weeks of cultivation at 18-32°C, in a light period of 8-16 hours in the dark or light period, and at a pH of the medium of 5.0-6.2, to obtain 85-100% differentiated or undifferentiated in vitro cultures (seedlings, shoot cultures, root cultures, somatic embryos, callus cultures, cell suspension cultures). Transplantation for independent growth of the obtained initiated in vitro cultures on semi-solid nutrient media with or without growth regulators and with or without reducing agents and / or antioxidants. Cultivation is carried out for 15-45 days at 18-32°C under the same light period to select 5-30% morphologically stable and high-yielding in vitro strains in terms of myconosides accumulation from the total number of in vitro cultures generated. The selected in vitro strains are maintained by regular subculture every 20-35 days on fresh semi-solid medium with or without growth regulators.

[0023] 2) Biomass production: - transplantation for the cultivation of selected high-yielding strains in sterile liquid nutrient media supplemented with carbon sources, growth regulators and antioxidants to obtain in vitro strains or so-called inocula adapted to the conditions of submerged cultivation from 70 to 100%; inoculation of the obtained inoculum into a liquid medium and cultivation in flasks, bioreactors or temporary immersion systems (with an immersion period of 1-30 minutes and an exposure period of 1-12 hours) at 18-32°C and under the same photoperiod for a period of 1-6 weeks until the control content of myconosides in the biomass is 80 mg / g dry biomass or more, then stimulation of the production of secondary metabolites for a period of 3-15 days by addition of factors selected from among elicitors, feeding of fresh nutrient medium, addition of precursors, introduction of a second phase in the cultivation system for recovery of secreted secondary metabolites, or a combination thereof, in order to obtain a myconosides-enriched biomass (100 mg / g dry biomass or more); - separation of myconoside-enriched biomass from the broth and drying or freeze-drying at 20-80 °C (yield of dry biomass is 10-15 g / L or more) and optionally drying of the resulting broth by evaporation under reduced pressure at 30-70 °C or freeze-drying (yield of dry matter is 15-30 g / L or more); 3) Preparation of HR biomass extracts by in vitro culture: - mixing and homogenizing the obtained dry biomass, optionally with the culture medium, in a homogenizer; - Maceration of the dry mixture in 30-80% ethanol for 16-72 hours at 18-45°C with or without sonication; - filtering the mixture obtained, separating the precipitate, and recovering and drying the filtrate under reduced pressure at a temperature of 30-70°C to obtain a viscous concentrate (extract) containing 10-30% moisture and a myconoside content in the extract of not less than 150 g / kg; and - The obtained standardized extract is dissolved by the addition of glycerol and stirred until completely homogenized. The obtained solution has a controlled myconsides content of 0.01% to 15.00%, depending on the needs of the relevant industry (i.e. pharmaceutical, food or cosmetic).

[0024] Suitable nutrient media are standard semi-solid and liquid modifications selected from MS (Murashige and Skoog), WP (McCown Woody Plant), LS (Linsmaier and Skoog), Gamborg B5, Heller, Nitsch, Schenk, and White, or modified macro-salt compositions, micro-salts, and vitamins. For the needs of this method, the medium was further modified by adding a carbon source such as sucrose and / or glucose (1%-9%), 0-5% activated charcoal, a reducing agent such as 2-mercaptoethanol and / or dithiothreitol at a concentration of 0-10 mg / L, an antioxidant such as ascorbic acid and / or citric acid at a concentration of 0-10 mg / L, and a gelling agent agar or Gelrite at a concentration of 0.1%-10%.

[0025] The main growth regulators are selected from among auxins (picloram and α-naphthaleneacetic acid), cytokinins (kinetin and 6-benzylaminopurine), and / or gibberellins, at concentrations of 0-20 mg / L. Picloram and / or α-naphthaleneacetic acid may be used as auxins, such as cytokinins (i.e. kinetin and / or 6-benzylaminopurine), together with gibberellic acid 4+7 and / or gibberellic acid A3. Other possible auxins are indole-3-acetic acid, indole-3-butyric acid, dicamba, p-chlorophenoxyacetic acid and β-naphthoxyacetic acid), cytokinins:2-iP, 4-CPPU, 6-benzylaminopurine riboside, dihydrozeatin, zeatin, meta-tropolin and thidiazuron, and gibberellins:gibberellic acid.

[0026] The elicitors used are chosen among: biotic factors, such as polysaccharides or chitosan; or abiotic factors, such as methyl jasmonate, jasmonic acid, abscisic acid, or physical factors (osmotic agents, UV light); which, when added in extremely low concentrations, act as a signal to stimulate the secondary metabolism of plant cells. Other strategies such as the supply of fresh nutrient medium, or the addition of precursors (amino acids and sugars), or the introduction of a second phase (activated charcoal or absorbent resins) to the culture system to capture the secreted secondary metabolites can also be applied.

[0027] Cultivation of HR differentiated and undifferentiated in vitro cultures is performed as follows.

[0028] - In bioreactors with mechanical (stirred tank) and pneumatic (bubble column) agitation for submerged culture under controlled conditions, and a tightly maintained microenvironment optimal for in vitro culture growth; - in temporary immersion systems in semi-automated sterile systems with controlled conditions and a strictly maintained microenvironment, optimal for the growth of differentiated cultures under in vitro conditions. Short-term controlled contact of the plant material with the nutrient medium is provided for a controlled period of time, accompanied by temporary air agitation, gravitational or mechanical movement of the liquid phase. Systems such as PLANTFORM, PLANTIMA, RALM, RITA, SETIS or their analogues are used; - In a flask on an orbital shaker at 80-150 rpm.

[0029] The obtained extract produced by in vitro HR cultured biomass contains the target bioactive compounds and is enriched in polyphenolic compounds up to 35% of the mixture (consisting of 70-96% myconosides), up to 55% sugars, and up to 12% amino acids, making this extract extremely valuable.

[0030] The standardized content of phenylethanoid glycosides (i.e. myconosides) makes the extract particularly valuable for its protective effect on human health, its successful use for its pharmaceutical and cosmetic effects, and functional nutrition. The antioxidant effect of myconosides makes them suitable for use in cosmetics due to their anti-aging, anti-wrinkle, and anti-pigmentation actions.

[0031] The process developed for preparing the extract according to the present invention uses optimally selected steps, specific conditions, parameters such as temperature, time, agitation, light, growth factors, etc. to obtain not only maximum volumetric productivity of target substances and myconosides but also a stable productivity of plant in vitro cultures as well as a reliable and efficient 24 / 7 continuous system for the production of NPs.

[0032] The dependency on natural factors, limited availability and protection of HR rare wild plant populations are eliminated. The limitations imposed by seasonal and slow HR growth are also circumvented by developing a renewable and environmentally friendly method, which provides an alternative, renewable and sustainable source of the raw materials required to obtain the target extracts.

[0033] Batch uniformity of the final NP is ensured, as well as consistent quality and guaranteed amounts of myconosides in the standardized extracts produced by HR in vitro cultures.

[0034] In addition, the appropriate culture media used, together with optimal levels of growth factors and stimulants added, result in the successful formation of in vitro cultures (seedlings, shoot cultures, root cultures, somatic embryos, callus cultures, cell suspension cultures) and the generation of biomass containing the targeted beneficial BACs. The resulting cultures have significant scale-up potential in industrially relevant bioreactors and temporary immersion systems designed to maximize the yield and content of biosynthesized NPs.

[0035] The type and concentration of stimulants used (elicitors, precursors, and uptake phase), the age and stage in the development of the in vitro culture at the time of extraction are particularly important factors, contributing to a higher level of biosynthesis and accumulation of NPs, optimized and especially those with complex molecular structures, when using the method of the present invention.

[0036] The risk of microbial contamination is eliminated, as well as contamination of the in vitro biomass and the produced extracts with biological materials of other plants, fungi, microorganisms, or animal species. Therefore, the method used to obtain extracts with naturally occurring chemical composition and content of diverse and valuable BACs, as well as maximum biomass yield and extracts, is considered to be favorable and particularly suitable for standardization in phenylethanoid glycosides for implementation in the food, cosmetic or pharmaceutical industries.

[0037] [Example] The present invention will now be described in detail with reference to the following examples, which are not intended to limit the invention: Example 1 1) Initiation of HR in vitro culture: 1.1 Cleaning Ten to fifty HR 0.6 mm × 0.1 mm seeds were washed twice in sterile distilled water containing 0.5 mg / l gibberellin for 30 to 60 min, treated with 70% ethanol for 100 s and 8% calcium hypochlorite for 50 min, followed by washing twice in sterile distilled water for 5 to 10 min, and drying the resulting sterilized seeds on sterile filter paper for 10 to 15 min.

[0038] 1.2.Start Sterilized seeds are evaluated for quality and morphology, and dead and morphologically altered individuals are removed and transplanted onto semi-solid, standard MS nutrient medium (pH 6.0) containing 5% sucrose and 5% agar, pre-sterilized at 121 °C for 30 min, for initiation. Incubate in the dark in a thermostat at 28 °C ± 2 °C for 2 weeks, monitoring sterility until 95-100% seedlings emerge.

[0039] 1.3.Independent growth For independent growth, the resulting seedling in vitro cultures are transplanted onto semi-solid MS nutrient medium (pH 6.0) supplemented with 5% sucrose and 5% agar. Culture is performed for 30-35 days in a thermostat at 28 ± 2 °C in light / dark mode for 12 h, until morphologically stable strains are obtained. Select myconoside overproducing strains by periodically measuring the amount of myconozide produced in the cultures and selecting 25% high-yielding strains from the total number of in vitro strains produced.

[0040] 2).Biomass production: 2.1. Maintenance, adaptation and subculture of selected in vitro seedlings The selected strains are maintained by regular subculturing every 30 days on fresh semi-solid nutrient medium (same as in step 1.3) to monitor changes in morphology and stability and to analyze the amount of myconosides. 10 g of biomass is taken from high-yielding strains and cultivated in sterile liquid MS medium with the same additives and pH in a 2000 ml flask on an orbital shaker at 140 rpm while monitoring changes in morphology, growth, uniformity, stability and amount of myconosides. 95-100% of the most adaptable strains are used as inoculum to continue for the next submerged cultivation.

[0041] 2.2. Cultivation of in vitro seedling systems adapted for biomass production Inoculation is performed with 25 g fresh weight / l of liquid culture at 20 days of age (logarithmic phase of growth). Cultivation is performed in a temporary immersion system with an immersion period of 25 min and an exposure period of 6 h at 28 °C, 12 h light / dark, for 5 weeks. As a result, 180 g fresh biomass per liter is obtained, with a myconoside content of 105 mg per g dry biomass; 2.3. Promoting biomass production A 20-40 day old biomass (in the logarithmic phase of growth) is sterilely supplemented with the abiotic elicitors jasmonic acid and methyl jasmonate at a concentration of 5 mg / l and cultivated under the above conditions for 12 days. At the end of the process, a concentrated biomass is obtained with a myconoside content of 152 mg per gram of dry biomass. The biomass is separated from the culture broth by filtration through a sterile sieve, then washed with sterile distilled water and dried in a ventilated drying oven at 60°C. The yield is 15 g of dry biomass per liter. The quality of the biomass obtained in each batch is monitored for myconoside content and phenolic compounds.

[0042] The culture broth is collected and dried in a vacuum evaporator at 60° C. The yield is 30 g / l dry weight.

[0043] Comparative HPLC profiles for the myconoside content of biomass from HR in vitro cultures (A), wild plant biomass (B), and extracts from biomass from HR in vitro seedling cultures (C) are shown in Fig. 1 .

[0044] 3) Preparation of myconoside standardized extracts from biomass of in vitro HR seedling cultures: The obtained dry biomass and the culture liquid are mixed and homogenized in a homogenizer. For this purpose, 2 kg of dry biomass and 2.5 kg of dry culture liquid (obtained from 200 l of in vitro seedling culture grown under submerged conditions) were used. A water-ethanol mixture of 70% ethanol was added at hydromodule 20 (wt / vol) for 35 hours at 40° C. with 15 minutes of sonication every 4 hours, the resulting precipitate was removed by vacuum filtration, the filtrate was collected and dried by vacuum evaporation at 40° C. to obtain a viscous concentrate containing 12% moisture.

[0045] A biomass extract from 1 kg of HR in vitro seedling culture is obtained containing 208 g / kg of myconosides.

[0046] The extracts were phytochemically characterized and the results are shown in Table 1. The amount of myconosides as well as the contents of phenolic compounds, fatty acids, organic acids, amino acids, sugars and sterols are monitored by HPLC and GC / MS methods.

[0047] The HPLC content of myconosides in extracts from HR in vitro biomass obtained according to Example 1 is shown in Table 2 in comparison with standard 70% ethanol extracts from HR plants growing in their natural habitat and from HR in vitro seedling cultures.

[0048] 3.1. Solubilization of HR in vitro culture extracts with respect to myconside content To 240.4 g of extract containing 208 g / kg of myconosides, 759.6 g of glycerol are added to produce 1 kg of extract containing 5% myconosides. The resulting mixture is stirred with a vibratory stirrer until complete homogenization of the extract. The resulting solution is packaged in sterile packs and stored for use in cosmetics, pharmaceuticals or dietary supplements. For cosmetic purposes, standardized extracts from HR in vitro cultures are suitable in amounts of 0.1-15% for products such as creams, emulsions, gels, etc. Table 3 also shows a comparative analysis of the antioxidant properties of extracts from HR in vitro cultures obtained according to Example 1 compared to a standard 70% ethanol extract from HR plants growing in the wild. The ability of the extracts to scavenge free DPPH and ABTS radicals, as well as their ability to reduce copper(II) and iron(III) ions, were evaluated.

[0049] [Table 1]

[0050] An Agilent Technology Hewlett Packard 7890 A+ / MSD 5975 instrument (Hewlett Packard, Palo Alto, CA, US) was used in conjunction with an Agilent Technology 5975C inert XL EI / CI MSD mass spectrometer (Hewlett Packard, Palo Alto, CA, US). HP-5MS column (30 m x 250 μm x 0.25 μm), temperature program at 60 °C for 2 min (with a temperature ramp to 260 °C at 5 °C / min), exposure at 260 °C for 8 min. The injected sample volume is 1 μl with a split ratio of 10:1. Injector temperature 250 °C with a flow carrier gas (helium) of 1 mL / min. EI / MS spectra are recorded at 70 eV.

[0051] The HPLC system, Waters 1525 binary pump (Waters, Milford, MA, USA), Waters 2487 Dual λ absorbance detector (Waters, Milford, MA, USA) was operated by Breeze 3.30 software; Supelco Discovery HS C18 column (5 μm, 25 cm × 4.6 mm), t 28 °C; mobile phase with a gradient of 2% acetic acid and acetonitrile;

[0052] [Table 2]

[0053] Example 2 The method is similar to that of Example 1, except that HR leaves were treated instead of seeds, and root cultures were prepared and used as in vitro cultures. The cultures were performed in bubble columns, biosynthesis was enhanced by feeding instead of elicitation, and the resulting extracts contained only accumulated biomass without culture medium.

[0054] 1). Initiation of in vitro culture from HR: 1.1.Cleaning Three to ten 2-5 cm young HR leaves were washed for 3 min in sterile distilled water supplemented with detergent (Tween 80), treated with 80% ethanol for 60 s and 6% calcium hypochlorite for 30 min, washed three times for 3 min in sterile distilled water, and dried on sterile filter paper for 2 min.

[0055] 1.2.Start Sterile leaves are processed by cutting off the dead areas. Leaves are then cut into 0.5-1.0 cm sections and initiated on semi-solid, pre-sterilized at 121 °C for 30 min, standard B5 nutrient medium supplemented with 4% sucrose and 8 mg / l picloram, 3% Gelrite, additionally supplemented with 5 mg / l ascorbic acid and 5 mg / l 2-mercaptoethanol, pH 5.5, and incubated in the dark at 24 °C ± 2 °C for 4 weeks while monitoring sterility until root cultures are formed from leaves in 90% of the explants.

[0056] 1.3.Independent growth The resulting in vitro cultures of adventitious roots are individually cultured on the same medium as in step 1.2 of Example 2, plus 3 g / l activated charcoal, in a thermostat at 24° C. in the dark for 37 days to obtain morphologically stable myconoside overproducing strains, where 15% of high-yielding strains are selected from the total number of in vitro strains produced.

[0057] 2) Biomass production 2.1. Maintenance, adaptation and subculture of selected in vitro root cultures The selected root cultures are maintained by regular subculturing every 37 days on semi-solid fresh medium in step 1.3 of Example 2 to monitor changes in morphology and stability and to determine the amount of myconosides. 15 g biomass from high-yielding strains are cultivated in sterile liquid B5 medium with the same additives as in step 1.3 of Example 2 in a 500 ml flask on an orbital shaker at 100 rpm, and 85% of the most adaptable strains are continued as inoculum for the next submerged cultivation.

[0058] 2.2. Cultivation of in vitro root cultures adapted for biomass production 30-day-old root cultures in the logarithmic phase of growth, 30 g fresh weight / l, are cultivated in a bubble column with an air flow rate of 0.3 l / l / min in liquid B5 medium with the same additions as in step 1.3 at 24°C ± 2°C in the dark for 4 weeks. A fresh biomass of 130 g / l gives a dry biomass with a myconoside content of 120 mg / g.

[0059] 2.3. Promoting biomass production The biomass, which is in the late logarithmic phase of growth (35 days old), is sterilely supplemented with fresh liquid B5 medium up to the maximum working volume of the bioreactor and cultivated for 10-15 days under the conditions described above. The result at the end of the process is a concentrated biomass containing 170 mg of myconosides per gram of dry biomass. The obtained biomass is separated from the culture broth by filtration, washed and dried. The yield is 12 g of dry biomass per liter.

[0060] 3) Preparation of myconoside standardized extract from in vitro biomass of HR root cultures: 3 kg of dried biomass from in vitro grown root cultures is ground and subjected to extraction by maceration with 80% aqueous ethanol at a Hydromodule of 40 (wt / vol) for the same time and temperature as in Example 1, except that no ultrasonic treatment was performed, to obtain a viscous concentrate containing 5% moisture. 500 g of biomass extract from in vitro HR root cultures is obtained, containing 280 mg / g of myconosides.

[0061] 3.1. Dissolution of the obtained HR in vitro root culture extracts with respect to myconoside content Weigh 357.1 g of myconoside-rich in vitro root culture of HR in a container. Add 642.9 g of glycerol to the desired weight of 1 kg of extract containing 10% myconoside. Stir the resulting mixture until complete homogenization of the extract by sonication, package in a sterile pack, and store for future use.

[0062] Example 3 It was carried out as in Example 1, except that ovaries were treated instead of seeds, and calli and cell suspension cultures were obtained instead of seedling cultures and used as in vitro cultures. The cultures were carried out in Erlenmeyer flasks.

[0063] 1) Initiation of in vitro callus culture from HR 1.1.Cleaning Two to five newly formed 0.2-0.5 cm HR ovaries were washed in sterile distilled water for 1-2 min, treated with 70% ethanol for 90 s and 10% sodium hypochlorite for 40 min, followed by rinsing in sterile distilled water for 1 min and drying on sterile filter paper for 1 min.

[0064] 1.2.Start The resulting sterile ovaries are cut horizontally in half and transplanted onto semi-solid, pre-sterilized at 121°C for 30 minutes, standard WP nutrient medium (pH 5) supplemented with 2% sucrose, 1 mg / l 1-naphthaleneacetic acid, 1 mg / l 6-benzylaminopurine and 4% agar for initiation, and incubated at 26°C ± 2°C in the dark for 3 weeks, monitoring sterility until callus formation on 93% of the explants.

[0065] 1.3.Independent growth The resulting in vitro callus cultures are ready for independent growth for 27 days in the dark, at the same temperature, in a thermostat, on the same medium as in step 1.2, supplemented with 3 mg / l citric acid and 1 g / l activated charcoal, to obtain morphologically stable myconoside overproducing strains, where 11% high-yielding strains are selected from the total number of in vitro strains produced.

[0066] 2.Biomass production 2.1. Maintenance, adaptation of selected in vitro cultures and formation of cell suspension cultures The selected callus cultures are maintained by regular subculturing every 27 days on fresh semi-solid WP nutrient medium in the previous step, to monitor the changes in morphology and stability, and to analyze the amount of myconosides. The same amount of biomass from the high-yielding strains as in step 2.2 of Example 2 is cultivated in sterile liquid WP medium with the same additives as in the previous step in a 1000 ml flask on an orbital shaker at 80 rpm to obtain a cell suspension culture consisting of small and medium-sized aggregates, and 75% of the most adaptable strains are continued as inoculum for the next submerged cultivation.

[0067] 2.2. Cultivation of cell suspension cultures adapted for biomass production Seven-day-old cell suspension cultures in the logarithmic phase of growth of 100 g fresh weight / l are cultivated in 2000 ml flasks on an orbital shaker at 80 rpm at 26° C. in the dark for 9 days. A fresh biomass of 110 g / l is obtained with a myconoside content of 80 mg / g dry biomass in the resulting biomass.

[0068] 2.3. Promoting biomass production The late logarithmic (6 day old) biomass is aseptically supplemented with 1 g of sterile absorbent resin (Amberlite XAD7) as a second phase. Cultivation is continued for another 4 days to obtain a concentrated biomass with a myconoside content of 100 mg per gram of dry biomass. It is further processed as in Example 1 and the biomass and culture broth are freeze-dried at -40°C. The yield is 9 g of dry biomass per liter and 15 g of dry weight culture broth per liter.

[0069] 3) Preparation of standardized myconid extracts from in vitro biomass of HR suspension cultures A total of 1 kg of dried biomass and culture liquid is homogenized and extracted by maceration with a water-ethanol mixture of 30% ethanol in a hydromodule 10 (wt / vol) at the same temperature and duration as in Example 1, with settling, separation and drying, to obtain a viscous concentrate with a moisture content of 20%.

[0070] 100 g of extract from the biomass of an in vitro cell suspension culture of Haberlea rhodopensis is obtained, containing 150 mg / g of myconosides.

[0071] 3.1. Dissolution of the resulting in vitro HR suspension culture extracts with respect to myconoside content Weigh out 100.0 g of myconoside-rich HR extract obtained from in vitro cell suspension culture. Add 400.0 g of glycerol to the desired weight of 500 g of extract containing 3% myconosides. Stir the mixture using a rotary or high-pressure homogenizer until the extract is completely homogenized, and bottle the resulting solution into a sterile container and store for future use.

[0072] [Table 3]

[0073] DPPH (2,2-diphenyl-1-picrylhydrazyl) / HR extract / 0.1 mM solution of DPPH radical / 15 min at 21°C in the dark / % decrease in absorbance at λ = 517 nm compared to control sample (with methanol) / EC 50 (effective concentration for 50% inhibition of DPPH radical in 0.1 mM DPPH solution) was determined.

[0074] TEAC / ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical / HR extract added to a solution of preformed ABTS radical / 15 min at 21°C in the dark / results as % decrease in absorbance at λ=734 nm compared to the absorbance of the control (methanol added) / mM Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).

[0075] To assess the reducing capacity, the following are used: CUPRAC / HR extract / Solution of Cu(II) ions in the presence of the chelating agent neocuproine / 15 min at 21°C in the dark / Reduction of Cu(II) to Cu(I) / Absorption maximum at λ = 450 nm / mM Trolox result; FRAP-HR extract / Fe(III) ion solution in the presence of TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) / 15 min at 21°C in the dark / reduction of Fe(III) to Fe(II) / Fe-TPTZ complex / absorption maximum at λ = 593 nm / mM Trolox results

[0076] [Table 4]

[0077] References: (1) Journal of Ethnopharmacology “The ancient Thracian endemic plant Haberlea rhodopensis Friv. And related species: A rewiew“, 2019, Yordan N. Georgiev; (2) Plant Cell, Tissue and Organ Culture (2005) 80: 115-118 Djilianov, (3) International Journal of Cosmetic Science, 2012, 34, 132-139 “Skin benefits of a myconoside-rich extract from resurrection plant Haberlea rhodopensis”, Dell Acqua and Schweiker; (4) Natural Product Research: Formerly; Natural Product Letters “Haberlea rhodopensis: pharmaceutical and medical potential as a food additive”, 2015; Rumiana Todorova; (5) JP2011168560A; (6) JP2015503212; (7) EP 1736167; (8) RU 2 559579; (9) WO2019175829.

[0078] [1] A standardized plant extract from in vitro cultured biomass containing bioactive compounds and their primary and secondary metabolites, comprising: The extract is produced by in vitro cultivation of Haberlea rhodopensis Friv (HR) and contains, by weight percent, the following: 4.0-6.0 organic acids, 0.5-1.5 fatty acids, 8.0-12.0 amino acids, 0.5-1.0 sterols, 3.0-6.0 free phenols, 45-55 sugars, and 25.0-35.0 polyphenols; A standardized plant extract, characterized in that the polyphenol fraction contains 70% to 96% of the major myconosides, constituting 18% to 35% of the total extract. [2] A composition comprising the standardized extract according to [1], A composition characterized in that it also contains glycerol and has a myconoside content of 0.01% to 15.00%. [3] A method for preparing the standardized plant extract according to [1] by in vitro culture, comprising: HR explants were repeatedly washed with sterile distilled water for 1–240 min, treated with 40–85% ethanol for 10–190 s, then treated with 2–10% disinfectant with or without surfactant for 10–60 min, repeatedly washed with sterile distilled water, and dried for 1–20 min. The resulting sterile explants are cultured on semi-solid or liquid sterile nutrient medium with or without growth regulators at 18-32°C in the dark or with a light-dark photoperiod of 8-16 hours, at a pH of 5-6.2 for 2-5 weeks to obtain 85-100% differentiated or undifferentiated cultures; The in vitro cultures are then grown independently on semi-solid sterile nutrient media with or without growth regulators and with or without reducing agents and / or antioxidants at 18-32°C under the same photoperiod for 15-45 days to obtain 5-30% of the total number of in vitro strains generated that overproduce the selected myconosides and are morphologically stable in vitro cultures, which are maintained on fresh semi-solid media with or without growth regulators for 20-35 days; The resulting high yield in vitro culture is cultivated in a sterile liquid nutrient medium supplemented with a carbon source, growth regulators and / or antioxidants for further adaptation to obtain 70-100% cell lines adapted to submerged culture, i.e., inoculum, which is re-inoculated into the liquid medium for further cultivation at 18-32°C and the same photoperiod in flasks, bioreactors or temporary submerged systems with a submersion period of 1-30 minutes and an exposure period of 1-12 hours, for 1-6 weeks, until a control mycoside content of 80 mg or more per gram of dry biomass is obtained in the biomass; Then, over a period of 3 to 15 days, factors that enhance the biosynthesis of secondary metabolites in the plant are added, selected from elicitors, supplementation with fresh nutrient medium, addition of precursors, inclusion of a second phase in the culture system, or combinations thereof, to obtain a myconoside-enriched biomass; The biomass obtained is then separated from the culture broth and dried or freeze-dried at 20-80°C to a yield of dry biomass of at least 10-15 g / L, and, if appropriate, the culture broth obtained is evaporated or freeze-dried at 30-70°C to a yield of dry mass of at least 15-30 g / L, after which the resulting dry mixture containing at least 100 mg / g of myconosides is homogenized in a homogenizer and macerated in 30-80% ethanol with or without sonication at 18-45°C for 16-72 hours, The mixture is filtered to separate the precipitate, and the filtrate is collected and concentrated under reduced pressure at 30-70°C to obtain a viscous concentrate (extract) containing at least 150 mg of myconosides per gram of extract and having a moisture content of 10-30%; Finally, the obtained concentrated extract is dissolved by adding glycerol and homogenized until completely dissolved to obtain a final myconsides standardized plant extract from in vitro culture of Haberlea rhodopensis Friv. [4] The method for preparing a plant extract according to [3], characterized in that the HR explants to be washed and sterilized are selected from leaves, stems, hypocotyls, roots, seeds, anthers, fruit ovaries, sepals and seedlings. [5] A method for preparing an extract according to [3], characterized in that the differentiated or undifferentiated culture of the sterile explant obtained at the start is selected from seedlings, meristematic cultures, root cultures (normal roots, adventitious roots and hairy roots), somatic embryos, callus cultures and cell suspension cultures. [6] A biomass obtained by the culture according to [3], The biomass is characterized in that it contains at least 100 mg of myconosides per gram of dry biomass. [7] Use of the biomass described in [6] for the preparation of a standardized plant extract having a myconoside content of 150 mg / g or more. [8] Use of the standardized plant extract according to [1] to [7] for the preparation of a product intended for use in the food, cosmetic or pharmaceutical industries.

Claims

1. 1. A plant extract from in vitro cultured biomass containing bioactive compounds and their primary and secondary metabolites, comprising: The plant extract is obtained from undifferentiated in vitro cultures of Haberlea rhodopensis Friv. (HR).

2. 2. The plant extract of claim 1, wherein the plant extract is obtained from a differentiated in vitro culture of Haberlea rhodopensis Friv. (HR).

3. 3. The plant extract of claim 1 or claim 2, wherein the bioactive compounds and their primary and secondary metabolites include organic acids, fatty acids, amino acids, sterols, free phenolic acids, and sugars.

4. 4. The plant extract of any one of claims 1 to 3, wherein the extract contains, in weight percent, the following: 4.0 to 6.0 organic acids, 0.5 to 1.5 fatty acids, 8.0 to 12.0 amino acids, 0.5 to 1.0 sterols, 3.0 to 6.0 free phenols, 45 to 55 sugars, and 25.0 to 35.0 polyphenols.

5. 5. The plant extract according to claim 1, wherein the plant extract contains myconosides.

6. 6. The plant extract of claim 5, wherein the myconoside content in the extract accounts for 70% to 96% of the polyphenol fraction.

7. 7. The plant extract of claim 5 or 6, wherein the plant extract is standardized for myconosides.

8. 8. A plant extract according to any one of claims 5 to 7, wherein the myconosides are in the range of 18% to 35% of the total extract.

9. A composition containing a plant extract according to any one of claims 1 to 8, A plant extract-containing composition, wherein the plant extract further contains glycerol.

10. 10. The plant extract-containing composition of claim 9, wherein the plant extract contains myconosides in the range of 0.01% to 15.00%.

11. A method for preparing a plant extract obtained from an in vitro culture of Haberlea rhodopensis Friv. (HR), comprising the steps of: - Providing HR explants; - Treating the explants and culturing the treated material with or without growth regulators to obtain cell cultures; - growing the culture with or without reducing agents and / or antioxidants; - selecting myconoside-overproducing and morphologically stable in vitro culture strains; - growing the stable in vitro culture in an aqueous growth medium with or without growth regulators to produce grown biomass; - separating the biomass from the culture medium to obtain a mixture; and - The resulting mixture is processed to produce a plant extract.

12. The HR explants are treated by repeatedly washing with sterile distilled water, treated with 40-85% ethanol, then treated with 2-10% disinfectant with or without surfactant, washed repeatedly with sterile distilled water, dried, and the resulting sterile explants are cultured on semi-solid or liquid sterile nutrient medium with or without growth regulators at 18-32°C in the dark or under a 8-16 hour light / dark photoperiod, at a pH of 5-6.2, to obtain differentiated or undifferentiated cultures; The in vitro cultures are then independently grown on semi-solid sterile nutrient medium with or without growth regulators, and with or without reducing agents and / or antioxidants, at 18-32°C under the same photoperiod as the dark or 8-16 hour light / dark photoperiod to obtain 5-30% selected mycoside-overproducing and morphologically stable in vitro cultures, which are maintained on fresh semi-solid medium with or without growth regulators, and the resulting high-yield in vitro cultures are cultured in sterile liquid nutrient medium with or without growth regulators for further adaptation to obtain cell lines adapted to submerged culture, which are re-inoculated into the liquid medium for further cultivation under the same photoperiod as the dark or 8-16 hour light / dark photoperiod; Next, adding a factor that enhances the biosynthesis of secondary metabolites in the plant, selected from an elicitor, supplementing with fresh nutrient medium, adding a precursor, including a second phase in the culture system, or a combination thereof, to obtain a myconoside-enriched biomass; Next, the obtained biomass is separated from the culture liquid and dried or freeze-dried, the obtained dry mixture is homogenized with 30-80% ethanol at 18-45°C, the obtained dry mixture is filtered to separate the precipitate, and the obtained filtrate is collected and concentrated under reduced pressure to obtain a concentrate having a moisture content of 10-30%; dissolving the final concentrate to obtain the plant extract; 12. A method for preparing the plant extract of claim 11.

13. 13. The method for preparing a plant extract according to claim 11 or claim 12, wherein the HR explants to be washed and sterilized are selected from leaves, stems, hypocotyls, roots, seeds, anthers, fruit ovaries, sepals, and seedlings.

14. 14. A method for preparing a plant extract according to any one of claims 11 to 13, wherein the differentiated or undifferentiated culture obtained at the initiation of a sterile explant is selected from seedlings, meristematic cultures, root cultures (normal roots, adventitious roots and hairy roots), somatic embryos, callus cultures, cell suspension cultures.

15. 15. The method of any one of claims 11 to 14, wherein the plant extract is standardized for myconosides.

16. 16. The method of claim 15, wherein the myconosides range from 18% to 35% of the total extract.

17. A plant extract obtained by the method according to any one of claims 11 to 16.

18. 17. Biomass produced by the method of any one of claims 11 to 16.

19. 19. The biomass of claim 18, wherein the biomass contains 80 mg or more of myconosides per gram of dry biomass.

20. 20. Use of biomass according to claim 18 or 19 for preparing a product intended for use in the food, cosmetic or pharmaceutical industry.

21. 20. Use of biomass according to claim 18 or 19 for preparing a myconoside-containing plant extract.

22. 22. Use of biomass according to claim 21 for preparing a myconoside-standardized plant extract.

23. 18. Use of a plant extract according to any one of claims 1 to 8 or claim 17 for preparing a product intended for use in the food, cosmetic or pharmaceutical industry.

24. 17. A composition containing biomass produced by the method of any one of claims 11 to 16, wherein the plant extract further contains glycerol.

25. 25. The biomass-containing composition of claim 24, wherein the composition contains myconosides in the range of 0.01% to 15.00%.

26. 18. A product containing the plant extract of any one of claims 1 to 8 or claim 17.

27. A product containing the biomass according to claim 18 or 19.

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