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

Abstract

The invention refers to standardized plant extract from biomass of in vitro cultures of Haberlea rhodopensis Friv. (HR), containing bioactive compounds and their primary secondary metabolites, containing in weight %, as follows: organic acid 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, and to a composition containing the standardized extract and glycerol as well as to a method for the preparation of a standardized plant extract. The method according this invention, along with its optimally chosen steps, specific conditions, parameters such as temperature, duration, stirring, light, growth factors, etc. achieves both maximum volumetric productivity of the target substances and myconoside, as well as stable productivity of the plant in vitro cultures and is a reliable efficient 24/7 continuous system for production of NPs. Dependence on natural factors, limited availability and protection of HR rare wild plant populations are eliminated. The limitations posed by seasonality and slow HR growth are also avoided by developing a renewable, ecologically method. The developed method provides alternative, renewable and sustainable sources of raw plant material necessary to obtain the target extract. The resulting extract standardized in myconoside is especially valuable with its protective action on human health and can successfully be used with its pharmacological, cosmetic effects as well as in functional foods.

Claims

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

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

3. The plant extract according to claim 1, wherein the extract contains in weight % 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.

4. The plant extract according to claim 1, wherein the plant extract contains myconoside.

5. The plant extract according to claim 4, wherein the myconoside ranges from 18% to 35% of the total extract.

6. The plant extract according to claim 1, wherein the plant extract is standardized relative to myconoside.

7. A composition containing the plant extract according to claim 1, wherein the plant extract further contains glycerol.

8. A method for the preparation of a plant extract obtained from in vitro cultures of Haberlea rhodopensis Friv. (HR), the method 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 cultures with or without reducing agents and/or antioxidants; selecting myconoside-overproducing and morphologically stable undifferentiated in vitro culture lines; propagating the stable in vitro culture lines in aqueous growth medium with growth regulators to produce propagated biomass; separating the biomass from the medium to obtain a mixture; and treating the obtained mixture to produce plant extract.

9. The method for the preparation of a plant extract according to claim 8, where the HR explants are treated by repeated washing with sterile distilled water, treated with 40-85% ethanol, subsequently treated with 2-10% disinfectant with or without added detergent, washed repeatedly with sterile distilled water and dried, culturing the resultant sterile explants on semi-solid or liquid sterile nutrient media with or without added growth regulators, at 18-32 C. in darkness or light/dark photoperiod for 8-16 hours, pH 5-6.2, to obtain undifferentiated cultures, followed by independent growth of the in vitro cultures on semi-solid sterile nutrient media with or without added growth regulators, and with or without added reducing agents and/or antioxidants, at 18-32 C. under the same photoperiod as the darkness or light/dark photoperiod for 8-16 hours, to obtain 5-30% selected myconoside-overproducing and morphologically stable in vitro culture lines, maintained on fresh semi-solid medium with or without added growth regulators, where-resulting high-yielding in vitro cultures are cultured for further adaptation in a sterile liquid nutrient medium with added: carbon source, growth regulators and/or antioxidants to obtain cell lines adapted to submerged cultivation, which is re-inoculated into the liquid medium for further cultivation under the same photoperiod as the darkness or light/dark photoperiod for 8-16 hours, followed by the addition of factors to enhance the biosynthesis of plant secondary metabolites selected from elicitors, supplementation with fresh nutrient medium, addition of precursors, inclusion of a second phase in the cultivation system or a combination thereof, to obtain myconoside-enriched biomass, followed by separation of the resulting biomass from the culture liquid and drying or freeze drying, the resulting dry mixture is homogenized with 30-80% ethanol at 18-45 C., filtration of the resulting dry mixture, separation of the precipitate, collection and concentration under vacuum of the resulting filtrate to obtain a concentrate with 10-30% moisture, and finally the resulting concentrate is dissolved to obtain the plant extract.

10. The method for the preparation of a plant extract according to claim 8, wherein the HR explants are selected from leaves, stems, hypocotyls, roots, seeds, anthers, fruitsovaries, sepals, seedlings, and wherein the cell cultures comprise of seedlings, meristem cultures, root cultures, somatic embryos, callus cultures, cell suspension cultures.

11. A plant extract obtained by the method of claim 8.

12. Biomass produced by the method of claim 8.

13. The biomass according to claim 12, wherein the biomass contains myconoside of not less than 80 mg/g of dry biomass.

14. A food, cosmetics or pharmaceutical industries product containing the plant extract according to claim 1.

15. A food, cosmetics or pharmaceutical industries product containing the biomass according to claim 12.

16. A plant extract obtained by the method of claim 9.

17. Biomass produced by the method of claim 9.

18. The biomass according to claim 17, wherein the biomass contains myconoside of not less than 80 mg/g of dry biomass.

19. A food, cosmetics or pharmaceutical industries product containing the plant extract according to claim 17.

20. A food, cosmetics or pharmaceutical industries product containing the plant extract according to claim 11.

21. A food, cosmetics or pharmaceutical industries product containing the plant extract according to claim 16.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A is an HPLC profile illustrating the myconoside content in biomass obtained from in vitro cultures of Haberlea rhodopensis Friv.;

(2) FIG. 1B is an HPLC profile illustrating the myconoside content in biomass obtained from wild Haberlea rhodopensis plants; and

(3) FIG. 1C is an HPLC profile illustrating the myconoside content in extract obtained from in vitro seedling culture biomass of Haberlea rhodopensis Friv.

DETAILED DESCRIPTION OF THE INVENTION

Examples

(4) Hereinafter, the present invention is described in more detailed examples, which however are not intended to limit the present invention:

Example 1

(5) 1) Initiation of In Vitro Cultures of HR:

(6) 1.1. Washing

(7) Ten to 50 pieces of 0.6 mm0.1 mm seeds of HR are washed twice, for 30 60 min in sterile distilled water with added gibberellins 0.5 mg/l, treated with 70% ethanol for 100 sec and with 8% calcium hypochlorite for 50 min, followed by washing twice with sterile distilled water for 5-10 min, and drying the resulting sterile seeds on sterile filter paper for 10-15 min.

(8) 1.2. Initiation

(9) The sterile seeds are evaluated for quality and morphology, dead and morphologically altered individuals are removed, and transferred for initiation on semi-solid, pre-sterilized at 121 C. for 30 min standard MS nutrient medium with 5% sucrose and 5% agar, pH 6.0. Cultured in a thermostat at 28 C.2 C. in darkness, for 2 weeks, with asepsis monitored until 95 to 100% of seedlings appear.

(10) 1.3. Independent Growth

(11) The seedling in vitro cultures obtained are transferred for independent growth on semi-solid MS nutrient media with added 5% sucrose and 5% agar-agar, at pH 6.0. Cultivation is in a thermostat at 282 C. in light/dark mode for 12 hours for 30-35 days until morphologically stable lines are obtained. Myconoside overproduction lines are selected by periodic monitoring the amount of myconozide produced in the cultures and selecting 25% of the high-yielding lines from the total number of generated in vitro lines.

(12) 2) Biomass Production:

(13) 2.1. Maintenance, Adaptation-Subcultivation of Selected In Vitro Seedling Lines

(14) The selected lines are maintained by periodic subculturing at every 30 days on fresh semi-solid nutrient medium, the same as in step 1.3, changes in morphology and stability being monitored, and the amount of myconoside analysed. 10 g of biomass are taken from the high-yielding lines 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, with changes in morphology, growth, homogeneity, stability and amount of myconoside being monitored, and the 95-100% of the most adaptive lines continue for subsequent submerged cultivation as inoculum.

(15) 2.2. Cultivation of Adapted In Vitro Seedling Line Systems for Biomass Production

(16) Inoculation is with 25 g of fresh weight/l of liquid culture, at 20 days of age (exponential phase of growth). Cultivation is performed at 28 C. in light/dark for 12 hours for 5 weeks in a temporary immersion system with an immersion period of 25 min and exposure period of 6 hours. As a result, 180 g of fresh biomass per litre is obtained, and the myconoside content is 105 mg/g dry biomass;

(17) 2.3. Stimulation of Biomass Production

(18) To 20- to 40-day-old biomass (at exponential phase of growth), abiotic elicitors jasmonic acid and methyl jasmonate are aseptically added at a concentration of 5 mg/1, and cultivated under the above conditions for 12 days. At the end of the process, enriched biomass with a myconoside content of 152 mg/g dry biomass is obtained. Separation of the biomass from the culture liquid by filtration through a sterile sieve is followed by washing with sterile distilled water and drying in a ventilation oven at 60 C. The yield is 15 g dry biomass per litre. The quality of the resulting biomass in each batch is monitored for myconoside content and phenolic compounds.

(19) The culture fluid is collected and dried in a vacuum evaporator at 60 C. The yield is 30 g/l dry weight.

(20) Comparative HPLC profiles for myconoside content of biomass from HR in vitro cultures (A), wild plant biomass (B), and extract from biomass from HR in vitro seedling cultures (C) are presented in FIGS. 1A, 1B, and 1C.

(21) 3) Preparation of Myconoside-Standardized Extract from Biomass of In Vitro HR Seedling Cultures:

(22) The resulting dry biomass and 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 2001 in vitro seedling culture grown under submerged conditions) are used.

(23) A water-ethanol mixture of 70% ethanol is added at hydromodule 20 (weight to volume) for 35 hours, at 40 C. with sonication for 15 min every 4 hours, the resulting precipitate is removed by vacuum filtration and the filtrate is collected and dried by vacuum evaporation at 40 C. to obtain a viscous concentrate containing 12% moisture.

(24) One kg of biomass extract from in vitro seedling cultures of HR, containing 208 g/kg myconoside is obtained.

(25) The extract is characterized phytochemically, the results are presented in Table 1. The amount of myconoside and the contents of phenolic compounds, fatty acids, organic acids, amino acids, sugars and sterols are monitored by HPLC and GC/MS methods.

(26) HPLC content of myconoside in the extract from HR in vitro biomass obtained according to Example 1 and compared with standard 70% ethanol extract from HR plants growing in their natural habitat, as well as standard 70% ethanol extract from in vitro seedling culture of HR are presented in Table 2.

(27) 3.1. Dissolution of the HR In Vitro Culture Extract Regarding the Myconoside Content

(28) To 240.4 g of the extract, containing 208 g/kg myconoside, 759.6 g of glycerol is added to produce 1 kg of extract, containing 5% myconoside. The resulting mixture is stirred until complete homogenization of the extract using a vibrating stirrer. The resulting solution is packaged in sterile packs and stored for use in cosmetic products, pharmaceuticals or food supplements. For cosmetic purposes, the standardized extract from HR in vitro cultures is suitable in amounts of from 0.1 to 15% for products like creams, emulsions, gels, etc. Table 3 also presents a comparative analysis of the antioxidant properties of the extract from HR in vitro cultures, obtained according to Example 1, compared to the standard 70% ethanol extract from HR plants growing wild. The ability of the extract to capture free DPPH and ABTS radicals, as well as the ability to reduce copper (II) and iron (III) ions, was evaluated.

(29) TABLE-US-00001 TABLE 1 Phytochemical composition of extract from biomass of HR in vitro cultures, obtained according to Example 1/GC/MS, and HPLC methods. Results are expressed in % by weight. Fatty acids 1.10 Sterols 0.93 Organic acids 5.86 Amino acids 9.91 Free phenolic acids 0.47 Sugars 53.50 Polyphenols 28.23

(30) Agilent Technology Hewlett Packard 7890 A+/MSD 5975 apparatus (Hewlett Packard, Palo Alto, CA, US) coupled with an Agilent Technology 5975C inert XL EV/CI MSD Mass Spectrometer (Hewlett Packard, Palo Alto, CA, US). HP-5 MS column (30 m250 m0.25 m) at 60 C. temperature program for 2 min, with a temperature rise to 260 C. with 5 C. per minute, and exposure at 260 C. for 8 min. The volume of the injected sample is 1 l at a split ratio of 10:1. Injector temperature 250 C. with a flow carrying gas (helium) of 1 mL/min. The EI/MS spectrum is recorded at 70 eV.

(31) HPLC system, Waters 1525 Binary pump (Waters, Milford, MA, USA), Waters 2487 Dual A Absorbance Detector (Waters, Milford, MA, USA) operated by Breeze 3.30 software; Supelco Discovery HS C18 column (5 m, 25 cm4.6 mm), t 28 C.; mobile phase with a gradient of 2% acetic acid and acetonitrile;

(32) TABLE-US-00002 TABLE 2 Phytochemical HPLC comparative analysis of phenylethanoid glycoside-myconoside Standard 70% ethanol HR extract extract from in vitro acc. To Standard 70% ethanol biomass from HR Example 1 extract from HR/ acc. To Example 1; mg/100 g mg/100 g fresh weight mg/100 g fresh extract plant from nature biomass Myconoside 120863.80 1249.61 12053.93

Example 2

(33) The method is the same as in Example 1, except that leaves of HR are processed instead of seeds, and root cultures are prepared and used as in vitro culture. Cultivation is carried out in a bubble column, biosynthesis is enhanced by feeding instead of elicitation, and the resulting extract contains only accumulated biomass without culture liquid.

(34) 1) Initiation of In Vitro Cultures from HR:

(35) 1.1. Washing

(36) Three to ten 2-5 cm young HR leaves are washed for 3 min in sterile distilled water with added detergent (Tween 80), treated with 80% ethanol for 60 sec and with 6% calcium hypochlorite for 30 min, triple-washed with sterile distilled water for 3 min and dried on sterile filter paper for 2 min.

(37) 1.2. Initiation

(38) The sterile leaves are processed by excising dead areas. The leaves are then cut into 0.5 to 1.0 cm segments and initiated on semi-solid, pre-sterilized at 121 C. for 30 min standard BS nutrient medium supplemented with 4% sucrose and 8 mg/l picloram, gellrite 3%, and additional supplementation of 5 mg/l ascorbic acid and 5 mg/l 2-Mercaptoethanol, pH 5.5, cultured at 24 C.+2 C. in darkness for 4 weeks, with monitoring for asepsis until root cultures are formed from the leaves in 90% of the explants.

(39) 1.3. Independent Growth

(40) The resultant in vitro cultures of adventitious roots are cultured individually on the same medium as in Example 2, step 1.2, with the addition of 3 g/l activated charcoal, in a thermostat at 24 C. in darkness for 37 days to obtain morphologically stable myconoside overproducing lines, where 15% of high-yielding lines are selected from the total number of in vitro lines generated.

(41) 2) Biomass Production

(42) 2.1. Maintenance, Adaptation-Subcultivation of Selected In Vitro Root Cultures

(43) The selected root culture lines are maintained by periodic subcultivation every 37 days on the semi-solid fresh medium in step 1.3 of Example 2, changes in morphology and stability are monitored, and the amount of myconoside is determined. 15 g of biomass from the high-yielding lines are cultivated in sterile liquid BS medium with the same additions 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 adaptive lines continue for the subsequent submerged cultivation as inoculum.

(44) 2.2. Cultivation of Adapted In Vitro Root Line Cultures for Biomass Production

(45) 30 g fresh weight/l of 30-day-old root culture at the exponential phase of growth are cultured on liquid BS medium with the same additions as in step 1.3, at 24 C.2 in darkness for 4 weeks in a bubble column at air flow rate of 0.3 l/l/min. 130 g/l of fresh biomass yield dry biomass with a myconoside content of 120 mg/g.

(46) 2.3. Stimulation of Biomass Production

(47) The biomass, which is at the late exponential phase of growth (35 days old), is aseptically supplemented with fresh liquid BS medium up to the maximum working volume of the bioreactor and cultivated under the conditions described above for a period of 10 to 15 days. The result at the end of the process is enriched biomass containing myconoside of 170 mg/g dry biomass. The resulting biomass is separated from the culture fluid by filtration, washed and dried. The yield is 12 g of dry biomass per litre.

(48) 3) Preparation of Myconoside-Standardized Extract from In Vitro Biomass of HR Root Culture:

(49) 3 kg of dried biomass from the in vitro cultivated root culture is ground and subjected to extraction by maceration with 80% aqueous ethanol mixture at hydromodule 40 (weight to volume) for the same period of time and at the same temperature as in Example 1, except that it is conducted without sonication to obtain viscous concentrate containing 15% moisture. 500 g of biomass extract from in vitro HR root culture containing 280 mg/g myconoside are obtained.

(50) 3.1. Dissolution of the Resulting In Vitro Root Culture Extract of HR Regarding Myconoside Content

(51) 357.1 g of the myconoside-rich in vitro root culture of HR are weighed in a vessel. 642.9 g of glycerol are added to the desired weight of 1 kg of extract containing 10% myconoside. The resulting mixture is stirred until complete homogenization of the extract with sonication, packaged in sterile packs and stored for future use.

Example 3

(52) Conducted like Example 1, except that instead of seeds, ovaries are processed, and instead of seedling cultures, callus and cell suspension cultures are obtained and used as in vitro cultures. Cultivation is carried out in Erlenmeyer flasks.

(53) 1) Initiation of In Vitro Callus Cultures from HR

(54) 1.1. Washing

(55) Two to five 5 newly-formed 0.2-0.5 cm HR ovaries are washed for 1-2 minutes in sterile distilled water, treated with 70% ethanol for 90 seconds, and 10% sodium hypochlorite for 40 minutes, followed by washing with sterile distilled water for 1 min and dried on sterile filter paper for 1 min.

(56) 1.2. Initiation

(57) The resulting sterile ovaries are cut horizontally in half and transferred for initiation on semi-solid, pre-sterilized at 121 C. for 30 min standard WP nutrient medium supplemented with 2% sucrose, 1 mg/l 1-naphtalene acetic acid, 1 mg/l 6-benzylaminopurine and agar-agar 4%, pH 5, and cultured at 26 C.2 C. in darkness for 3 weeks, with monitoring for asepsis until callus formation in 93% of explants.

(58) 1.3. Independent Growth

(59) The resulting in vitro callus cultures are ready for independent growth in a thermostat on the same medium as in step 1.2 with added citric acid of 3 mg/l and 1 g/l activated charcoal at the same temperature, in darkness for 27 days to obtain morphologically stable myconoside overproducing lines, where 11% of high-yielding lines are selected from the total number of generated in vitro lines.

(60) 2) Biomass Production

(61) 2.1. Maintenance, Adaptation of Selected In Vitro Cultures and Formation of Cell Suspension Cultures

(62) The selected callus culture lines are maintained by periodic subcultivation every 27 days on fresh semi-solid WP nutrient medium as in the previous step, changes in morphology and stability are monitored, and the amount of myconoside is analyzed. The same amount as in Example 2 step.2.2 of biomass from high-yielding lines is cultured in sterile liquid WP medium with the same additions 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, the 75% most adaptable of the lines continue for the next submerged cultivation as inoculum.

(63) 2.2. Cultivation of Adapted Cell Suspension Cultures for Biomass Production

(64) 100 g of fresh weight/t 7-day-old cell suspension culture at the exponential phase of growth are cultured at 26 C. in darkness for 9 days in 2000 ml flasks on an orbital shaker at 80 rpm. 110 g/l fresh biomass with myconoside content in the resulting biomass of 80 mg/g dry biomass is obtained.

(65) 2.3. Stimulation of Biomass Production

(66) The biomass at the late exponential growth phase (6 days old) is aseptically supplemented with 1 g of sterilized absorption resin (Amberlite XAD7) as a second phase. Cultivation continues for a further 4 days to obtain enriched biomass with myconoside content of 100 mg/g dry biomass. It is further treated as in Example 1, and the biomass and culture fluid are freeze dried at 40 C. The yield is 9 g dry biomass per litre and 15 g dry weight culture fluid per litre.

(67) 3) Preparation of Myconoside-Standardized Extract from In Vitro Biomass of HR Suspension Culture

(68) The total of 1 kg of dried biomass and culture fluid are homogenized and extracted by maceration with a 30% ethanol water-ethanol mixture at hydromodule 10 (weight to volume) at the same temperature and for the same duration, with precipitation, separation and drying as in Example 1 to obtain a viscous concentrate containing 20% moisture. 100 g of extract from biomass of Haberlea rhodopensis in vitro cell suspension culture containing 150 mg/g myconoside are obtained.

(69) 3.1. Dissolution of the Resulting In Vitro HR Suspension Culture Extract Regarding Myconoside Content

(70) 100.0 g of the myconoside-rich HR extract obtained from in vitro cell suspension culture are weighed. 400.0 g of glycerol are added thereto to the desired weight of 500 g of extract containing 3% myconoside. The mixture is stirred until the extract is completely homogenized using a rotary or high-pressure homogenizer, and the resulting solution is packed in sterile containers and stored for future use.

(71) TABLE-US-00003 TABLE 3 Comparative analysis of antioxidant properties of extract from in vitro HR cultures prepared according to Example 1 compared to the standard 70% ethanol extract of HR from nature: Total DPPH TEAC,mM FRAP,mM CUPRAC, phenols EC.sub.50 Trolex Trolex mM Trolex mgGAE/g mg/ml Equivalent/g Equivalent/g Equivalent/g Extract from 30.87 0.57 0.041 493.08 0.97 622.87 3.68 1199.91 14.39 PCC of HR Standard 70% 29.44 0.09 0.042 402.69 3.95 564.07 5.15 1037.90 24.44 ethanol extract of HR DPPH (2,2-diphenyl-l-picrylhydrazyl)/HR extract/0.1 mM solution of DPPH radical/in darkness, at 21 C. for 15 minutes/% decrease in absorption at A=517 nm compared to the control sample (with methanol addition)/determination of EC.sub.50 (effective concentration inhibiting 50% of DPPHradial in 0.1 mM DPPH solution). TEAC/ABTS (2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical/HR extract is added to a solution of pre-generated ABTS radical/darkness at 21 C. for 15 minutes/% decrease in absorption at A=734 nm compared to that of a control (with methanol addition)/result as mM Trolox (()-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).

(72) The following are used to evaluate the reducing capacity: CUPRAC/HR extract/solution of Cu (II) ions in the presence of chelating agent neocuproine/darkness at 21 C. for 15 minutes/reduction of Cu (II) to Cu (I)/absorption maximum at =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)/darkness, at 21 C. for 15 mini reduction of Fe (III) to Fe (II)/Fe-TPTZ complex/absorption maximum at A=593 nm/mM Trolox result

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