Standardized plant extracts of biomass from in vitro cultures, methods of preparation and uses thereof

Abstract

The present invention relates to a standardized plant extract of biomass from an in vitro culture of Brassica granatum, containing biologically active compounds and their primary and secondary metabolites, comprising, in% by weight: 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, wherein the content of major mannose glycosides in the polyphenol fraction is 70-96%, making up 18%-35% of the total extract, and to compositions containing the standardized extract and glycerol and to methods for preparing standardized plant extracts. According to the method of the invention, along with the optimally selected steps, specific conditions, parameters such as temperature, duration, agitation, light, growth factors and the like, maximum volume productivity of the target substance and mannoside and stable productivity of in vitro plant cultures are achieved, and the method is a reliable and effective 24 / 7 continuous system for the production of NP.

Description

[0001] This application is a divisional application of the application number 202080097832.3 filed on April 15, 2020 with the title “Standardized plant extracts from biomass of in vitro cultures, methods of their preparation and uses”. TECHNICAL FIELD

[0002] The present invention relates to standardized plant extracts, in particular extracts from biomass of in vitro cultures, wherein the plant extracts comprise valuable bioactive compounds (BACs) and secondary metabolites, and can be used for the preparation of reagents for the pharmaceutical, cosmetic or food industry, as well as to methods of preparation of such extracts. BACKGROUND

[0003] Plants in nature contain a wide variety of BACs and their origin, growth conditions, time of harvest, extraction techniques, etc. affect the quality of the product.

[0004] Cultivated medicinal plants have many advantages over wild plants. Their growth can be monitored and they can be harvested at the most favorable time, while the approach to unsuitable plant species can be avoided.

[0005] Haberlea is a monotypic genus, Haberlea rhodopensis Friv. (Rhodopean Silivryak), or simply HR, is its only member. The plant is a relic of the Tertiary period, distributed in central and southern Bulgaria, found in the Balkan Mountains and the Rhodope Mountains, as well as in the northeastern Greek-Rhodope Mountains, the Mongolian Mountains and the Falakro Mountains. For centuries, the Balkan giant has been traditionally used in ethnopharmacology and local traditional medicine (1).

[0006] Plant extracts are known to be isolated from HR leaves. Biochemical compounds isolated from plant extracts are sources of essential natural ingredients. In addition to fruits and vegetables, polyphenols, glycosides, sugars, etc. are found in plants and due to their physiological functions, they play an important role in human health. Ethanol extract of HR is known to be obtained by spray drying the whole plant, followed by 3 hours of ethanol extraction at room temperature under stirring, filtration, separation of insoluble, followed by vacuum concentration and freeze-drying to obtain the extract. The same source also provides information on aqueous HR extract, in which the whole plant is spray dried, extracted with hot water at 120°C for 20 minutes in an autoclave, then the insoluble is separated by filtration at high temperature, and freeze-dried. The resulting extract has anti-aging, antioxidant, skin lightening and immune stimulating effects (5).

[0007] There are reports of obtaining HR extracts by hydroalcoholic extraction (ethanol / water, no specified method of preparation) and purifying them by gel filtration chromatography on Sephadex LH-20, which contain isolated fractions particularly rich in phenylethanoid glycosides - myconosides - responsible for the biological activity of the extracts of Picraena excelsa. In the same source, the effectiveness of the extracts was observed, and it was reported that myconosides stimulate the antioxidant protection of the skin, to improve its elasticity by stimulating the synthesis of extracellular structures, and have cytoprotective and UV-protective effects, protecting it from oxidation as an anti-aging agent for the skin, increasing its elasticity, enhancing its luster and having potential cosmetic applications (3).

[0008] It is also known that the phytochemical composition of the 70% ethanol extract of HR leaves contains a large group of major bioactive compounds, including secondary metabolites such as: phenolic acids, flavonoids, fatty acids, phytosterols, carotenoids, soluble lipids, oligo- and polysaccharides, free sugars, polyols, organic acids, including myconosides, with proven antioxidant and hepatoprotective effects (1).

[0009] Data on the phytochemical profile of the methanolic extract of HR leaves prepared by the combination of liquid / liquid extraction, preparation and semi-preparative HPLC using two phenolic glycosides: myconoside and pauciflooside, can also be used. The fraction rich in myconoside (caffeoyloxyphenylethanoid glycoside) has a potential role in plant survival and has antioxidant activity due to the presence of a caffeoyloxy group and two free hydroxyl groups in its benzene ring. The main BAC group and secondary metabolites found in various HR extracts during normal growth and during the drying period of plant development are organic acids, fatty acids, amino acids, phenolic acids and sugars (4).

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

[0011] On the other hand, as a particularly valuable and rare endemic species, HR belongs to the plant species prohibited from collection in nature. In recent years, for example, in vitro systems for HR regeneration and micropropagation have been established with the aim of protecting the natural population of this rare plant species. In vitro banks of HR plants from different locations have been established. The same source also describes an effective method of regeneration and micropropagation, in which the seeds of the plant species are sterilized with 70% EtOH for 1 minute, treated with hypochlorite for 6-10 minutes, treated with 0.1% HgCl2for 3-5 minutes and washed three times with distilled water, and the resulting aseptic HR seeds are placed on classic hormone-free MS medium (as well as MS B5, WPM), germinated for 3-4 months and obtained seedlings. The plant clusters are subcultured for 1-1.5 months to obtain an in vitro system of fully developed plants, which are prepared for transformation in non-aseptic conditions in a controlled greenhouse environment in a 6-7 month process (2).

[0012] At the same time, plant biotechnology, especially in vitro cultivation of plant cells, has become a promising tool for the sustainable and continuous production of BACs from plants. The principles of plant cell cultivation and plant cell propagation in solid and liquid nutrient media, as well as general procedures related to the establishment and cultivation of plant cells, have been described for other plant species than HR.

[0013] According to (6), isolated extracts from plant cell cultures of Rosa genus have been obtained, which contain valuable natural products (NPs) for cosmetic skin and hair care, prevention of signs of aging. According to (7), plant cell biomass is used for the production of phenylpropanoids from Ajuga repens, and of verbascosides from Olea europea, Syringa vulgaris or Appia citobara, as well as for the production of verbascoside-standardized extracts from Syringa cell cultures, which have proven antioxidant activity, effective treatment of acne and prevention of hair loss.

[0014] According to (8), preparations in the form of in vitro cultured extracts of Argania spinosa are also prepared, which are intended 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, which contains BACs with proven anti-radical activity and for cosmetic applications for skin protection and regeneration, wherein in vitro propagated undifferentiated cells of D. ruyschiana grown in semi-solid or liquid medium have been obtained in the dark or under a photoperiod of 14-16 hours, the cell biomass is isolated, the biomass is subsequently extracted with ethanol or ethanol, glycerol and water in a ratio of 20:20:60-50:50:0 (v / v), the biomass is dried and further extracted with the same extractant.

[0016] To date, there has been no report of the extraction of BACs and secondary metabolites from the biomass of HR in vitro cultures.

[0017] Although in fact, the general principles for the cultivation of plant cells do not apply to large-scale cell proliferation for production purposes, and the development of specific technological processes is required. In order to maintain in vitro cultures of plants (differentiated or undifferentiated) under in vitro culture conditions, it is important to determine the optimal strict specific ratio of growth regulators (auxins, cytokinins and gibberellins) and to establish it for each cell line (for each plant species). Changes in cell culture conditions, as well as the use of elicitors, precursors and absorption substrates, are crucial for the production of specific bioactive compounds / metabolites and are specific for the plant species used and the target compounds required.

[0018] However, the synthesis of valuable BACs and metabolic ingredients in in vitro cultures is a complex process with many unknown parameters. The accumulation of natural ingredients and secondary metabolites in the cell biomass is caused by a dynamic balance between biosynthesis, biotransformation and biodegradation. It is important to select suitable and optimized conditions - nutrient media and stimulating factors for each ingredient inherent to the whole plant.

[0019] In general, the homogeneity of natural products obtained from traditional extracts is insufficient and the amount of the target therapeutic ingredients tends to vary seasonally and regionally when compared to those obtained from in vitro cultures. It is also necessary to work with standardized raw materials in order to obtain in vitro biomass extracts and incorporate them into products of the cosmetics, pharmaceutical or food industry.

[0020] The problem of the present invention is to obtain a standardized extract, in particular from HR in vitro cultures (seedlings, shoot cultures, root cultures (normal roots, adventitious roots and hairy roots), somatic embryos, callus cultures, cell suspension cultures), with a guaranteed content of phenylethanoid glycosides-mannosides, with unchanging physicochemical properties and the accompanying composition of BACs / components, obtained by biotechnological methods with maximum efficiency design. SUMMARY

[0021] The problem according to the present application is solved by a standardized plant extract, in particular from biomass of in vitro cultures (seedlings, shoot cultures, root cultures (normal roots, adventitious roots and hairy roots), somatic embryos, callus cultures, cell suspension cultures) of HR, containing BAC, including their secondary and primary plant metabolites - fatty acids, sterols, organic acids, amino acids, free phenolic acids and sugars. The amount of BAC in the extract (expressed in wt.%) is: 0.5-1.5 of fatty acids, 0.5-1.0 of sterols, 4.0-6.0 of organic acids, 8.0-12.0 of amino acids, 3.0-6.0 of free phenols, 45.0-55.0 of sugars and 25.0-35.0% of polyphenolic compounds, containing 70-96% of phenylethanoid glycosides - mannose glycosides of the polyphenol fraction. The extract obtained from the biomass of in vitro cultures of HR is rich in mannose glycosides and standardized, the amount of which is 18-35% of the total extract. Depending on the needs of the relevant industry - pharmaceutical, food or cosmetic, the dissolution of the standardized extract in glycerol produces a product with a controlled content of 0.01-15.00% of mannose glycosides in its composition.

[0022] The said standardized extract from in vitro cultures according to the present application is prepared by a method comprising the following essential steps:

[0023] 1) initiation of in vitro cultures of HR, wherein:

[0024] - the explants are selected from individual parts or organs of the plant, in particular from leaves, stems, hypocotyls, roots, seeds, anthers, ovaries, sepals, seedlings,

[0025] - the selected explants are surface sterilized 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 for 10-60 minutes with or without the addition of a detergent, washing with sterile distilled water and drying for 1-20 minutes;

[0026] - inoculate aseptic explants on semi-solid or liquid medium with or without addition of growth regulators and cultivate at 18-32°C in the dark or under photoperiod - for each of them, light / dark 8-16 hours and pH of the medium 5.0-6.2 - to obtain 2-5 weeks of 85-100% differentiated or undifferentiated in vitro cultures (shoots, branch cultures, root cultures, somatic embryos, callus cultures, cell suspension cultures). Transfer the resulting inoculated in vitro cultures to grow independently on semi-solid nutrient medium with or without addition of growth regulators and with or without addition of reducing and / or antioxidant agents. Cultivate at 18-32°C under the same photoperiod for 15-45 days to select from the total number of in vitro cultures produced 5-30% of in vitro lines morphologically stable and high-yielding in terms of accumulation of mannosides. Maintain the selected in vitro lines by regular subculturing every 20-35 days on fresh semi-solid medium with or without addition of growth regulators.

[0027] 2) Biomass production:

[0028] - transfer the selected high-yielding lines for cultivation in sterile liquid nutrient medium supplemented with carbon source, growth regulators, antioxidants to obtain 70-100% of in vitro lines or so-called inocula adapted to submerged cultivation conditions;

[0029] - inoculate the resulting inocula in liquid medium and cultivate it at 18-32°C under the same photoperiod in flasks, bioreactors or temporary immersion systems (immersion period 1-30 minutes and exposure period 1-12 hours) for 1-6 weeks until the control content of mannosides in the biomass is not less than 80 mg / g of dry biomass, followed by stimulation of the production of secondary metabolites by addition of factors selected from the group consisting of elicitors, feeding with fresh nutrient medium, addition of precursors, introduction of a second phase in the cultivation system to recover secreted secondary metabolites, or a combination thereof, for 3-15 days to obtain biomass enriched in mannosides (not less than 100 mg / g of dry biomass);

[0030] - isolate the biomass enriched in mannosides from the culture broth and dry or freeze-dry at 20-80°C, with a dry biomass yield not less than 10-15 g / l, and optionally dry the resulting culture broth by vacuum evaporation or freeze-drying at 30-70°C, with a dry matter yield not less than 15-30 g / l;

[0031] 3) Preparation of HR biomass extract by in vitro cultures:

[0032] - optionally mix the resulting dry biomass with the culture broth in a homogenizer and homogenize;

[0033] -Immerse the dried mixture in 30-80% ethanol at 18-45°C with or without ultrasound for 16-72 hours;

[0034] - Filter the resulting mixture and separate the precipitate. Collect the filtrate and dry it under vacuum at 30-70°C to obtain a viscous concentrate (extract) containing 10-30% water and a mannoside content (not less than 15 g / kg); and

[0035] - The standardized extract is dissolved by adding glycerol and stirring until completely homogenized. Depending on the requirements of the relevant industry—pharmaceutical, food, or cosmetic—the resulting solution has a controlled mannoside content of 0.01%–15.00%.

[0036] Suitable nutrient media are selected from the following standard semi-solid and liquid variants: MS (Murashige and Skoog), WP (McCown Woody Plant), LS (Linsmaier and Skoog), Gamborg B5, Heller, Nitsch, Schenk, and White, or with the addition of modified macrosalts compositions, microsalts, and vitamins. Depending on the method requirements, the medium may be further modified by adding the following: carbon sources, such as sucrose and / or glucose (1%-9%); activated charcoal 0-5%; reducing agents, such as 2-mercaptoethanol and / or dithiothreitol at a concentration of 0-10 mg / L; antioxidants, such as ascorbic acid and / or citric acid at a concentration of 0-10 mg / L; and gelling agents, such as agar or gellan gum at a concentration of 0.1%-10%.

[0037] The main plant growth regulators are selected from: phytoalexins (atrazine and α-naphthylacetic acid), cytokinins (kinin and 6-benzylaminopurine), and / or gibberellins at concentrations of 0-20 mg / L. Atrazine and / or α-naphthylacetic acid can be used as phytoalexins, such as cytokinin-kinin and / or 6-benzylaminopurine, and gibberellin 4+7 and / or gibberellin A3. Other possible phytoalexins are: indole-3-acetic acid, indole-3-butyric acid, dicamba, p-chlorophenoxyacetic acid, and β-naphthyloxyacetic acid; cytokinins: 2-iP, 4-CPPU, 6-benzylaminopurine ribonucleoside, dihydrostatin, zeatin, hyoscyamine, and thidiazuron; and gibberellins: gibberellin.

[0038] The stimulants used are selected from: biological agents, such as polysaccharides or chitosan; or non-biological agents, such as methyl jasmonate, jasmonic acid, abscisic acid, or physical factors (penetrating agents, UV light), which, when added at very low concentrations, act as signals to stimulate secondary metabolism in plant cells. Other strategies can also be applied, such as feeding with fresh nutrient media, adding precursors (amino acids and sugars), or introducing a second phase (activated charcoal or absorbent resin) into the culture system to capture secreted secondary metabolites.

[0039] The in vitro cultures of differentiated and undifferentiated HR cells were cultured as follows:

[0040] - In bioreactors with mechanical (stirred vessel) and pneumatic (bubble cap tower) agitation, which are optimized for in vitro culture growth, for immersion culture under controlled conditions and strictly maintained microenvironments;

[0041] - In a temporary immersion system within a semi-automated aseptic system optimized for the growth of differentiated cultures under controlled conditions and a tightly maintained microenvironment, the plant material undergoes short-term controlled contact with the nutrient medium, providing a controlled time period with temporary air agitation, gravity, or mechanical movement of the liquid phase. Systems using products such as PLANTFORM, PLANTIMA, RALM, RITA, SETIS, or similar agents are employed.

[0042] - In a flask on a fixed-track oscillator at 80-150 rpm.

[0043] The extract obtained from biomass produced from in vitro HR cultures contains the target bioactive compounds, is rich in up to 35% polyphenols (composed of 70-96% mannosides), up to 55% sugars and up to 12% amino acids in the mixture, making the extract extremely valuable.

[0044] The standardized content of phenylethanoid-mannoside makes this extract particularly valuable due to its protective effects on human health, its successful applications in pharmaceuticals and cosmetics, and its use in functional nutrition. Mannoside's antioxidant properties, along with its anti-aging, anti-wrinkle, and anti-pigmentation effects, make it suitable for use in cosmetics.

[0045] The development of a method for preparing extracts according to the present invention, with optimized selection of steps, specific conditions, and parameters such as temperature, time, stirring, light, and growth factors, not only achieves maximum volumetric productivity of the target substance and mannoside, but also achieves stable productivity of plant in vitro cultures, and is a reliable and efficient 24 / 7 continuous system for the production of NPs.

[0046] This approach eliminates dependence on natural factors, the limited availability and protection of rare wild plant communities of wild HR (Hemiberlein Species). It also avoids limitations caused by the seasonality and slow growth of HR through the development of renewable and environmentally friendly methods. This method provides an alternative, renewable, and sustainable source of the raw materials necessary to obtain the target extract.

[0047] This ensures batch uniformity of the final NP, as well as stable quality and guaranteed amounts of mannoside in the standardized extract produced by HR in vitro culture.

[0048] Furthermore, the appropriate culture medium used, along with the optimal levels of added growth factors and stimulants, led to the successful formation of in vitro cultures (seedlings, shoot cultures, root cultures, somatic embryos, callus cultures, and cell suspension cultures) and the production of biomass containing the target valuable BAC. 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.

[0049] The type and concentration of stimulants (activators, precursors, and absorption phases) used, as well as the age and stage of development of the in vitro culture at the time of extraction, are particularly important factors that are optimized by the methods used and contribute to higher levels of biosynthesis and accumulation of NPs with particularly complex molecular structures.

[0050] The risk of microbial contamination and contamination from other plant, fungal, microbial, or animal species in the biomass and the resulting extracts is eliminated. Therefore, extracts with naturally occurring chemical compositions and various valuable BAC contents, as well as methods for obtaining maximum biomass yield and extracts, are considered preferred, and are particularly suitable for the standardization of phenylethanoid glycosides for implementation in the food, cosmetic, or pharmaceutical industries. Attached Figure Description

[0051] Figure 1 This is a comparative HPLC chromatogram of mannoside content in biomass (A) from HR in vitro culture, wild plant biomass (B) and biomass extract (C) from HR in vitro seedling culture. Detailed Implementation

[0052] The invention is described below with reference to more detailed embodiments, but these embodiments are not intended to limit the invention:

[0053] Example 1

[0054] 1) Inducing HR in vitro culture:

[0055] 1.1 Washing

[0056] Wash 10-50 HR seeds (0.6mm × 0.1mm) twice with sterile distilled water containing 0.5mg / L gibberellin for 30-60 minutes, treat with 70% ethanol for 100 seconds and with 8% calcium hypochlorite for 50 minutes, then wash twice with sterile distilled water for 5-10 minutes, and dry the resulting sterile seeds on sterile filter paper for 10-15 minutes.

[0057] 1.2 trigger

[0058] Assess the quality and morphology of sterile seeds, remove dead and morphologically altered individuals, and transfer to semi-solid standard MS nutrient medium (pH 6.0) containing 5% sucrose and 5% agar for initiation. Pre-sterilize at 121°C for 30 minutes. Incubate in the dark at 28°C ± 2°C for 2 weeks, monitoring sterility until 95%–100% seedlings are observed.

[0059] 1.3 Independent growth

[0060] The obtained seedling cultures were transferred to semi-solid MS nutrient medium (pH 6.0) supplemented with 5% sucrose and 5% agar for independent growth. They were cultured in a light / dark incubator at 28±2°C for 12 hours for 30–35 days until morphologically stable lines were obtained. Over-producing lines were selected by periodically monitoring the amount of myconazole produced in the cultures and choosing 25% of the high-producing lines from the total number of lines produced in vitro.

[0061] 2) Biomass production:

[0062] 2.1 Maintenance and Adaptation – Subculture of the Selected In Vitro Seedling Line

[0063] Selected lines were maintained by subculturing every 30 days on fresh semi-solid nutrient medium, as in step 1.3, monitoring changes in morphology and stability, and analyzing the amount of mannoside. 10 g of biomass was taken from high-yielding lines and cultured in sterile liquid MS medium with the same additives and pH in 2000 ml flasks on a fixed-track shaker at 140 rpm. Changes in morphology, growth, uniformity, stability, and the amount of mannoside were monitored, and 95–100% of the most adapted lines were used as inoculum for subsequent submersion cultures.

[0064] 2.2 Culture of adapted in vitro seedling systems for biomass production

[0065] The inoculum consisted of 25 g fresh weight / L of 20-day-old (exponential growth phase) liquid culture. Cultures were incubated for 12 hours in light / dark at 28°C for 5 weeks in a temporary submersion system with a 25-minute immersion period and a 6-hour exposure period. The results yielded 180 g of fresh biomass per L and a mannoside content of 105 mg / g dry biomass.

[0066] 2.3 Stimulus to Biomass Production

[0067] Aseptically add jasmonic acid and methyl jasmonic acid (non-biostimulants) at a concentration of 5 mg / L to biomass aged 20-40 days (in the exponential growth phase) and culture for 12 days under the above conditions. At the end of the process, enriched biomass with a mannoside content of 152 mg / g dry biomass is obtained. The biomass is separated from the culture medium by sterile filtration, washed with sterile distilled water, and dried in a ventilated 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 mannoside content and phenolic compounds.

[0068] The culture medium was collected and dried at 60°C in a vacuum evaporator. The yield was 30 g / L dry weight.

[0069] Figure 1 The HPLC chromatograms show a comparison of mannoside content in biomass (A) from HR in vitro culture, wild plant biomass (B) and biomass extract (C) from HR in vitro seedling culture.

[0070] 3) Preparation of mannoside-standardized extracts from in vitro HR seedling cultures:

[0071] The obtained dry biomass and culture medium were mixed and homogenized in a homogenizer. For this purpose, 2 kg of dry biomass and 2.5 kg of dry culture medium (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 a hydromodule of 20 (by weight / volume) for 35 hours, followed by sonication at 40°C for 15 minutes every 4 hours. The resulting precipitate was removed by vacuum filtration, and the filtrate was collected and dried by vacuum evaporation at 40°C to obtain a viscous concentrate containing 12% water.

[0072] One kg of biomass extract from an in vitro seedling culture of HR was obtained, containing 208 g / kg of mannoside.

[0073] The extract was characterized for its phytochemical composition, and the results are shown in Table 1. The amounts of mannosides, phenolic compounds, fatty acids, organic acids, amino acids, sugars, and sterols were monitored by HPLC and GC / MS.

[0074] Table 2 shows the HPLC content of mannosides in the extracts from HR in vitro biomass obtained according to Example 1, and compares them with standard 70% ethanol extracts from HR plants grown in their natural habitat and standard 70% ethanol extracts from HR in vitro seedling cultures.

[0075] 3.1 Dissolution of HR in vitro culture extracts regarding mannoside content

[0076] 759.6 g of glycerol was added to 240.4 g of an extract containing 208 g / kg of mannoside to produce 1 kg of an extract containing 5% mannoside. The resulting mixture was stirred using a vibratory stirrer until the extract was completely homogenized. The resulting solution was packaged in sterile packaging and stored for use in cosmetics, pharmaceuticals, or food supplements. For cosmetic purposes, standardized extracts from HR in vitro cultures are used in products such as creams, emulsions, and gels at amounts of 0.1–15%. Table 3 also provides a comparative analysis of the antioxidant properties of the extracts from HR in vitro cultures obtained according to Example 1 and standard 70% ethanol extracts from wild-grown HR plants. 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, was evaluated.

[0077] Table 1:

[0078] Phytochemical composition of the biomass extract of HR in vitro culture obtained according to Example 1 / GC / MS and HPLC methods. Results are expressed as weight %.

[0079]

[0080] An Agilent Technology Hewlett Packard 7890 A+ / MSD 5975 device (Hewlett Packard, Palo Alto, CA, US) coupled to an Agilent Technology 5975C inert XL EI / CI MSD mass spectrometer (Hewlett Packard, Palo Alto, CA, US). An HP-5MS column (30 m x 250 μm x 0.25 μm) was programmed at 60°C for 2 min, with the temperature ramped up to 260°C at 5°C / min, and exposed at 260°C for 8 min. The injected sample volume was 1 μl with a split ratio of 10:1. The syringe temperature was 250°C, and the carrier gas (helium) flow rate was 1 mL / min. EI / MS spectra were recorded at 70 eV.

[0081] The HPLC system, consisting of a Waters 1525 binary pump (Waters, Milford, MA, USA) and a Waters 2487 dual-λ absorbance detector (Waters, Milford, MA, USA), was operated using Breeze 3.30 software; a Supelco discovery HS C18 column (5 μm, 25 cm x 4.6 mm) was used at 28°C; the mobile phase contained 2% acetic acid and acetonitrile gradients.

[0082] Table 2:

[0083] Comparative analysis of phenethyl glycosides and mannosides using phytochemical HPLC

[0084]

[0085] Example 2

[0086] The method was the same as in Example 1, except that HR leaves were treated instead of seeds, and root cultures were prepared and used as in vitro cultures. Cultures were carried out in bubble column, biosynthesis was enhanced by feeding rather than activation, and the resulting extract contained only accumulated biomass without culture medium.

[0087] 1) In vitro cultures that trigger HR:

[0088] 1.1 Washing

[0089] Wash three to ten young HR leaves (2-5cm) in sterile distilled water with detergent (Tween 80) for 3 minutes, treat with 80% ethanol for 60 seconds and with 6% calcium hypochlorite for 30 minutes, wash three times with sterile distilled water for 3 minutes each, and dry on sterile filter paper for 2 minutes.

[0090] 1.2 trigger

[0091] Aseptic leaves were prepared by removing dead areas. The leaves were then cut into 0.5–1.0 cm segments and initiated on a semi-solid pH 5.5 standard B5 nutrient medium. The medium was pre-sterilized at 121°C for 30 minutes with 4% sucrose, 8 mg / L atrazine, 3% gellan gum, and additionally supplemented with 5 mg / L ascorbic acid and 5 mg / L 2-mercaptoethanol. The medium was incubated in the dark at 24°C ± 2°C for 4 weeks, monitoring for sterility until root cultures formed from leaves at 90% confluence with the explants.

[0092] 1.3 Independent growth

[0093] The resulting in vitro cultures of adventitious roots were cultured separately on the same medium as in step 1.2 of Example 2, with 3 g / L activated carbon added, and cultured in the dark at 24°C for 37 days to obtain morphologically stable lines that overproduce mannoside, of which 15% of the high-yielding lines were selected from the total number of in vitro lines produced.

[0094] 2) Biomass production

[0095] 2.1 Maintenance and Adaptation – Subculturing of the Selected In Vitro Root Culture

[0096] In step 1.3 of Example 2, the selected root culture lines were maintained by regular subculturing every 37 days on a semi-solid fresh medium, monitoring changes in morphology and stability, and determining the amount of mannoside. 15 g of biomass from high-yielding lines was cultured in sterile liquid B5 medium with the same additives as in step 1.3 of Example 2 in a 500 ml flask on a 100 rpm orbital shaker, and 85% of the most adapted lines were used as inoculum for subsequent submersion cultures.

[0097] 2.2 Cultivation of adapted in vitro root cultures for biomass production

[0098] 30-day-old root cultures (30 g fresh weight / L) at the exponential growth stage were cultured for 4 weeks in a bubble column at 24°C ± 2°C in the dark with an air flow rate of 0.3 L / L / min on liquid B5 medium supplemented with the same additives as in step 1.3. 130 g / L of fresh biomass produced dry biomass with a mannoside content of 120 mg / g.

[0099] 2.3 Stimulus to Biomass Production

[0100] Biomass in the late exponential growth stage (35 days old) was aseptically replenished with fresh liquid B5 medium until the bioreactor reached its maximum working volume, and cultured under the above conditions for 10-15 days. The result at the end of the process was enriched biomass containing 170 mg / g of mannosides in dry biomass. The obtained biomass was separated from the culture by filtration, washed, and dried. The yield was 12 g of dry biomass per liter.

[0101] 3) Preparation of mannoside-standardized extracts from HR root cultures via in vitro biomass:

[0102] Three kg of dried biomass from in vitro root cultures was milled and extracted by maceration with an 80% aqueous ethanol mixture at a modulus of 40 (by weight / volume) for the same time period and temperature as in Example 1 (except without sonication) to obtain a viscous concentrate containing 15% water. 500 g of biomass extract from in vitro HR root cultures was obtained, containing 280 mg / g mannoside.

[0103] 3.1 Dissolution of mannoside content in the HR in vitro root culture extract

[0104] Weigh 357.1 g of HR in vitro root culture rich in mannosides into a container. Add 642.9 g of glycerol to the desired weight of 1 kg of extract containing 10% mannosides. Stir the resulting mixture until the extract is completely homogenized by sonication, then package it in aseptic packaging and store it for later use.

[0105] Example 3

[0106] As in Example 1, except that the ovary was treated instead of the seed, and callus and cell suspension cultures were obtained instead of seedling cultures for use as in vitro cultures. Cultures were carried out in conical flasks.

[0107] 1) In vitro callus cultures that induce HR

[0108] 1.1 Washing

[0109] Wash 2-5 newly formed 0.2-0.5 cm HR ovaries in sterile distilled water for 1-2 minutes, treat with 70% ethanol for 90 seconds, treat with 10% sodium hypochlorite for 40 minutes, then wash with sterile distilled water for 1 minute and dry on sterile filter paper for 1 minute.

[0110] 1.2 trigger

[0111] The obtained sterile ovaries were horizontally cut in half and transferred to a semi-solid pH 5 standard WP nutrient medium for initiation. The medium was pre-sterilized at 121°C for 30 minutes, supplemented with 2% sucrose, 1 mg / L 1-naphthacetic acid, 1 mg / L 6-benzylaminopurine and 4% agar, and cultured in the dark at 26°C ± 2°C for 3 weeks. Sterility was monitored until callus formation was achieved in 93% of the explants.

[0112] 1.3 Independent growth

[0113] The obtained in vitro callus cultures were prepared for independent growth in an incubator on the same medium as in step 1.2, with the addition of 3 mg / L citric acid and 1 g / L activated carbon, and cultured in the dark at the same temperature for 27 days to obtain morphologically stable lines that overproduce mannoside, of which 11% of the high-yielding lines were selected from the total number of in vitro generated lines.

[0114] 2. Biomass production

[0115] 2.1 Maintenance, adaptation of selected in vitro cultures and formation of cell suspension cultures

[0116] As before, the selected callus culture lines were maintained by regular subculturing every 27 days on fresh, semi-solid WP nutrient medium, monitoring changes in morphology and stability, and analyzing the amount of mannoside. The same amount of biomass from high-yielding lines as in step 2.2 of Example 2 was cultured in sterile liquid WP medium with the same additives as in the previous steps in 1000 ml flasks on a steady-state shaker at 80 rpm to obtain a cell suspension culture consisting of small and medium-sized aggregates. 75% of the most suitable lines were used as inoculum for subsequent submersion cultures.

[0117] 2.2 Cultivation of adapted cell suspension cultures for biomass production

[0118] A 7-day-old cell suspension culture at 100 g fresh weight / L in the exponential growth phase was cultured for 9 days in a 2000 ml flask at 80 rpm on a fixed-track shaker in the dark at 26°C. This yielded 110 g / L of fresh biomass, of which the mannoside content was 80 mg / g dry biomass.

[0119] 2.3 Stimulus to Biomass Production

[0120] During the late exponential growth stage (6 days old), 1 g of sterile absorbent resin (AmberliteXAD7) was aseptically added to the biomass as a second phase. Culture was continued for 4 days to obtain enriched biomass with a mannoside content of 100 mg / g dry biomass. This was further processed as in Example 1, and the biomass and culture medium were freeze-dried at -40°C. The yield was 9 g dry biomass per liter and 15 g dry culture medium per liter.

[0121] 3) Preparation of mannoside-standardized extracts from HR suspension cultures via in vitro biomass.

[0122] A total of 1 kg of dried biomass and culture medium was homogenized and extracted, precipitated, separated and dried by impregnation with a 30% ethanol-water-ethanol mixture at the same temperature and duration as in Example 1, at a modulus of 10 (by weight / volume) to obtain a viscous concentrate containing 20% ​​water.

[0123] 100g of extract was obtained from biomass from an in vitro cell suspension culture of Balkan giant moss, containing 150mg / g mannoside.

[0124] 3.1 Dissolution of mannoside content in the obtained in vitro HR suspension culture extract

[0125] Weigh 100.0 g of the HR extract rich in mannosides obtained from in vitro cell suspension culture. Add 400.0 g of glycerol to the extract to a desired weight of 500 g containing 3% mannosides. Stir the mixture until the extract is completely homogenized using a rotary or high-pressure homogenizer, and package the resulting solution in a sterile container for storage.

[0126] Table 3:

[0127] Comparative analysis of the antioxidant properties of extracts from in vitro HR cultures prepared according to Example 1 and standard 70% ethanol extracts of HR from nature:

[0128]

[0129] DPPH (2,2-diphenyl-1-picrylhydrazine) / HR extract / 0.1 mM DPPH radical solution / in the dark at 21°C for 15 min / decrease in absorbance at λ=517 nm compared to control sample (with added methanol) / determination of EC 50 (Effective concentration for inhibiting 50% of DPPH free radicals in 0.1 mM DPPH solution).

[0130] Add TEAC / ABTS (2,2'-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical / HR extract to a pre-generated ABTS radical solution / in the dark at 21°C for 15 min / the absorption at λ=734 nm decreased by % compared to the control (with added methanol) / the result was mM Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).

[0131] The following are used to assess the ability to restore the original state:

[0132] CUPRAC / HR extract / Cu(II) ion solution, in the presence of chelating agent new copper reagent / in the dark at 21°C for 15 minutes / Cu(II) reduction to Cu(I) / maximum absorption at λ=450nm / mM Trolox results;

[0133] FRAP-HR extract / Fe(III) ion solution in the presence of TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) / in the dark at 21°C for 15 min / Fe(III) reduction to Fe(II) / Fe-TPTZ complex / maximum absorption at λ=593 nm / mMTrolox results

[0134] References:

[0135] (1) Journal of Ethnopharmacology “The ancient Thracian endemic plantHaberlea rhodopensis Friv. And related species: A rewiew”, 2019, Yordan N.Georgiev;

[0136] (2) Plant Cell, Tissue and Organ Culture (2005) 80: 115–118Djilianov,

[0137] (3) International Journal of Cosmetic Science, 2012, 34, 132–139 “Skin benefits of a myconoside-rich extract from resurrection plant Haberlearhodopensis”, Dell Acqua and Schweiker;

[0138] (4) Natural Product Research: Formerly; Natural Product Letters “Haberlea rhodopensis: pharmaceutical and medical potential as a foodadditive”, 2015; Rumiana Todorova;

[0139] (5) JP2011168560A;

[0140] (6) JP2015503212;

[0141] (7) ER 1736167;

[0142] (8) RU 2,559,579;

[0143] (9) WO2019175829。

Claims

1. A plant extract derived from in vitro cultured biomass, comprising bioactive compounds and their primary and secondary metabolites, wherein, The plant extract was produced from an undifferentiated in vitro culture of *Haberlea rhodopensis* Friv. (HR).

2. The plant extract according to claim 1, wherein, The plant extract was produced from an in vitro culture of differentiated Balkan giant moss (HR).

3. The plant extract according to claim 1 or 2, wherein the bioactive compounds and their primary and secondary metabolites comprise: Organic acids, fatty acids, amino acids, sterols, free phenolic acids, and sugars.

4. The plant extract according to any one of claims 1 to 3, wherein, The plant extract comprises, by weight percent: 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.

5. The plant extract according to any one of claims 1 to 4, wherein, The plant extract includes myconoside.

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

7. The plant extract according to claim 5 or 6, wherein, The plant extract was standardized relative to myconoside.

8. The plant extract according to any one of claims 5 to 7, wherein, The content of myconoside is 18%-35% of the total extract.

9. A composition comprising the extract according to any one of claims 1 to 8, wherein, The composition also contains glycerin.

10. The composition containing the extract according to claim 9, wherein, The plant extract contains 0.01-15.00% myconoside.

11. A method for preparing a plant extract obtained from an in vitro culture of *Haberlea rhodopensis* Friv. (HR), comprising the following steps: Provide HR explants; The explants were treated and the treated material was cultured with or without growth regulators to obtain cell cultures. The cultures were cultured with or without the addition of reducing agents and / or antioxidants; Select in vitro culture lines that produce an excess of myconoside and are morphologically stable; The stable in vitro culture line was proliferated in an aqueous growth medium with or without growth regulators to produce proliferated biomass. The biomass is separated from the culture medium to obtain a mixture; and The obtained mixture is processed to produce plant extracts.

12. The method for preparing plant extracts according to claim 11, comprising: The HR explants were repeatedly washed with sterile distilled water, treated with 40-85% ethanol, and then treated with 2-10% disinfectant with or without detergent. They were then repeatedly washed with sterile distilled water and dried, and incubated at 18-32°C in dark or light / dark photocycles with or without growth regulators at a specific pH. Sterile explants obtained by culturing on semi-solid or liquid sterile nutrient medium of 5.0-6.2 for 8-16 hours were obtained to obtain differentiated or undifferentiated cultures. Subsequently, in vitro cultures were independently grown for 8-16 hours at 18-32°C under the same dark or light / dark cycle on semi-solid sterile nutrient medium with or without growth regulators and with or without reducing agents and / or antioxidants to obtain morphologically stable in vitro cultures with 5-30% overproduction of myconoside. These were maintained on fresh semi-solid medium with or without growth regulators. The resulting high-yielding in vitro cultures were then cultured in sterile liquid nutrient medium supplemented with carbon sources, growth regulators, and / or antioxidants for further adaptation to obtain cultures suitable for immersion. Uncultured cell lines are re-inoculated into liquid culture medium and cultured for another 8-16 hours under the same dark or light / dark photocycle. Then, factors selected from stimulants that enhance the biosynthesis of plant secondary metabolites are added, fresh nutrient medium is replenished, precursors are added, a second phase is included in the culture system, or a combination of the above methods are used to obtain biomass rich in myconoside. The obtained biomass is then separated from the culture medium and dried or freeze-dried. The resulting dried mixture is then homogenized with 30-80% ethanol at 18-45°C. The dried mixture is filtered, the precipitate is separated, and the filtrate is collected and concentrated under vacuum to obtain a concentrate containing 10-30% water. Finally, the concentrate is dissolved to obtain the plant extract.

13. The method for preparing the plant extract according to claim 11 or 12, wherein, The HR explants, after washing and sterilization, were selected from leaves, stems, hypocotyls, roots, seeds, anthers, fruits, ovaries, sepals, and seedlings.

14. The method for preparing plant extracts according to any one of claims 11 to 13, wherein, Differentiated or undifferentiated cultures obtained from sterile explants were selected from: seedlings, meristematic cultures, root cultures (normal roots, adventitious roots, and hairy roots), somatic embryos, callus cultures, and cell suspension cultures.

15. The method according to any one of claims 11 to 14, wherein, The plant extract was standardized relative to myconoside.

16. The method for preparing plant extracts according to claim 15, wherein, Mannosides comprise 18%-35% of the total extract.

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

18. A biomass produced by the method of any one of claims 11 to 16.

19. The biomass according to claim 18, wherein, The biomass contains not less than 80 mg / g of myconoside in the dried biomass.

20. A use of biomass, said biomass being the biomass according to claim 18 or 19, wherein, The biomass is used to prepare products for use in the food, cosmetics, or pharmaceutical industries.

21. A use of biomass, said biomass being the biomass according to claim 18 or 19, wherein, The biomass is used to prepare plant extracts containing myconoside.

22. The use of biomass according to claim 21, wherein, The biomass is used to prepare a standardized plant extract of myconoside.

23. The use of a plant extract, wherein, The plant extract is a plant extract according to any one of claims 1 to 8 or 17, wherein the extract is used to prepare products for use in the food, cosmetic or pharmaceutical industries.

24. A composition comprising biomass prepared by the method of any one of claims 11 to 16, wherein, The plant extract also contains glycerin.

25. The composition according to claim 24, wherein, The composition contains 0.01-15.00% myconoside.

26. A product comprising a plant extract according to any one of claims 1 to 8 or 17.

27. A product comprising biomass according to any one of claims 18 or 19.

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