WO2013095175A1

WO2013095175A1 - Method of obtaining stepharine sulfate in the cell culture of stephania glabra tissue, and method of quantitative determination of stepharine sulfate

Info

Publication number: WO2013095175A1
Application number: PCT/RU2011/001005
Authority: WO
Inventors: Andrey Anatolievich ALIMOV; Vladimir Aleksandrovich VLADIMIROV; Ludmila Vasilievna VORONCHIKHINA; Elena Borisovna KOVALEVA; Valentina Nikolaevna KRUCHININA; Olga Mikhailovna MIKHAILOVA; Andrey Germanovich MOSHKIN; Vladimir Vasilievich Turkin; Lyubov Zakharovna TSIMBALENKO; Ekaterina Ivanovna LAPSHOVA
Original assignee: Zakrytoe Aktsionernoe Obshchestvo "Biogentechnologies"
Priority date: 2011-12-20
Filing date: 2011-12-20
Publication date: 2013-06-27

Abstract

The invention relates to biotechnological industry, specifically to obtaining the substance of medicinal preparation stepharine sulfate out of the cell culture of Stephania glabra plant, and a method of quantitative determination of stepharine sulfate in biomass by means of thin layer chromatography of a prepared sample and Nastyukov color test for stepharine identification.

Description

Method of obtaining stepharme sulfate in the cell culture of Stephania glabra tissue, and method of quantitative determination of stepharine sulfate.

FIELD OF THE INVENTION

The invention relates to biotechnological industry, specifically to obtaining the substance for medical uses, stepharine sulfate from the cell culture of Stephania glabra plant and a method of stepharine sulfate concentration determination in biomass.

BACKGROUND OF THE INVENTION

Stephania glabra [Stephania glabra (Roxb.) Miers, of the family Menispermaceae] is a dioecious perennial climber with a large sphere-like stem tuber (caudex) with a diameter of up to 50 cm and mass of up to 30-50 kg located near the soil surface. Caulis reach 15 m and longer; they are curly and become lignescent at base with age. Leaves are alternate, round-shaped, pointed, and smooth; up to 15-20 cm long. Flowers yellow and green, in umbrella-shaped pendulous inflorescence. Grows in sub-tropical and tropical regions of the South-Eastern Asia: southern China, India, the Himalayan foothills, and Japan. Was introduced into the Transcaucasian Subtropics: in Adjara on the Black Sea coast of the Caucasus since 1958.

Stephania glabra is a plant with one of the highest alkaloid contents on the globe. Its caudex contains up to 6-8% of alkaloids: 2% of hyndarine (and derivatives thereof: hyndarinine and hyndaridine), stephaglabrine (or stepharine), cycleanine, rotundine etc. Tubers and green mass are used in both official and folk medicine. Pharmacological activity of Stephania glabra depends on the content of hyndarine and stephaglabrine alkaloids. Stephaglabrine possesses anticholinesterase action inhibiting cholinesterase. Hyndarine possesses sedative and spasmolytic action.

From a pharmaceutical point of view the most valuable is alkaloid stepharine

Stepharine sulfate is a sulfate of isoquinoline proaporphine alkaloid stepharine extracted out of Stephania glabra. Stepharine sulfate has the chemical formula (C|₈Hi₉N0₃)₂ · ¾S0₄ and is white crystal powder with the melting point in the range of (247-252) °C, with the subsequent decomposition; soluble in water and aqueous alcohol. It is the substance of drug called stephaglabrine sulfate (Stephaglabrini sulfas).

For medical application, the use of stephaglabrine sulfate (0.25% water solution thereof) as an anti-cholinesterase agent (USSR Inventor's Certificate of Authorship Nb. 315388, 1963) is known. It is known that stephaglabrine sulfate has specific connective tissue growth inhibiting properties, preventing the development of a scar in a nerve injury, and may be used as a medicine for treating accident or surgery related damage of the peripheral nervous system (USSR patent No. 1713151, 1985).

Globally, many pharmaceuticals drags, aromatic substances, nutrient additives and other valuable substances are obtained out of natural, vegetable raw materials. About 30% of all medicines used contain products of specialized plant metabolism - secondary metabolites.

They are usually obtained by extraction from plants, the majority of which belong to exotic or vanishing species that grow in remote areas in unregulated climatic, environmental and even political conditions that do not guarantee sustainable supply and quality of raw materials. Apparently, plantation-based growing does not eliminate certain adverse natural conditions, at the same time requiring large areas of land with special utilization regimes combined, probably, with alternative uses. As a result, that method often is not cost-effective and the raw material produced contains, as a rule, a number of harmful impurities, i.e. herbicides, pesticides, radionuclides and other contaminators.

Thus, a method is known of extracting stephaglabrine from plant raw material that includes processing of Stephania glabra roots with xylene in alkaline solution, further processing with sulphuric acid solution, chloroform and final precipitation; dissolution of the precipitate in alcohol, addition of hydrochloric acid, extraction of the target product by chloroforme, further addition of sulphuric acid and crystallization (USSR Inventor's Certificate of Authorship No. 315387, 1977) One major restriction of the method is the shortage of input raw material.

Disadvantages of known methods of obtaining biologically active compounds by extraction from plant raw materials are overcome by extraction performed out of plant cell biomass which could be obtained from plant cell cultures.

Cells from different parts of the plant (the root, stem, caulis, peduncle etc.) are introduced in the culture. The ability of plant cell cultures to synthesize substances inherent in intact plants is proven, with some plant cells producing them in quantities even greater than the parent plant. That is why plant cell cultures are of interest as sources of obtaining a variety of substances for pharmaceutical, perfumes and cosmetics, food and chemical industries. It is an alternative method of obtaining valuable substances of vegetable origin with the following advantages:

- guaranteed production of plant cell biomass in required quantities with specified properties irrespective of the season, climatic or weather conditions;

- virtually absolute environmental cleanliness of cell culture biomass production and freedom thereof from pollutants; - shorter time of biomass accumulation which amounts to several weeks in culture, while being about 10 years in nature, with the content of target substances in cell culture not lower than in a plant;

- possibility in the cell culture to synthesize new substances not generated in plants under natural conditions;

- possibility to use commercial biotechnological methods and equipment to produce plant cell biomass.

A method of producing stepharine sulfate is known which provides for submerged cultivation of cells with the use of Stephania glabra plant cell culture (RF patent No. 2089610, 1997).

According to the aforesaid method, cells are grown in a nutrient medium in dark conditions, at the temperature of 26°C, in conical or flat-bottom Erlenmeyer flasks on a shaker with the following technical specifications: ellipsoid trajectory of the platform movement, vibration amplitude: 5-7 mm, rotations per minute: 100.

Cultivation duration is 14 days. Within that term, which marks the beginning of a stationary phase, the cell biomass produced is separated from culture liquid by filtration and dried, and then stepharine is extracted from the cell biomass.

However, the known method is inapplicable on an industrial scale due to difficulties related to upscaling of cell culture growing process.

A method is known which provides for obtaining stepharine sulfate from Stephania glabra plant cell culture, comprising in-depth cultivation of cells in a nutrient medium in a bioreactor under stirring and aeration of the culture suspension, and accumulation of the target product (RF patent No.2399665, 2009).

However, this method does not provide a concentration determination of stepharine sulfate for the cultivation stages and in the target product.

It is worth noting that one of the main difficulties during plant cells cultivation is the problem of monitoring the target product at different cultivation stages, since the product content in plant cells is not constant and varies in a wide range in the process of cell growing. At the same time, the extraction of the target product is one of the most complicated stages of the whole process, and the most energy- and labor-intensive one. For example, with the stepharine content below 0.05% per one gram of dry biomass, the extraction of the same becomes, technically and economically, hardly feasible. That is why it is particularly important to control stepharine concentration variations in cultivation time. The authors have not discovered any information sources describing the stage of quantitative stepharine determination during industrial-scale cultivation.

Besides, as already noted earlier, root and tubers of the Stephania glabra (Roxb.) Miers. plant synthesize several types of alkaloids: proaporphine (stephaglabrine, pronuciferine); bisbenzylisoquinoline (cycleanine); tetrahydroprotoberberine (hyndarine) etc., which makes analysis more difficult.

Therefore, there is an urgent need to control stepharine concentration during fermentation process and in the target product.

However, the majority of available stepharine quantitative analysis methods are known to be used for chemo-toxicological analysis of biological fluids. Apparently for that reason, none of the authors' multiple attempts to adapt the above-mentioned methods to analyze extracts from the Stephania glabra (Roxb.) Miers. tissue culture succeeded. The presence in the extract of a large number of closely chemically related substances distorts analysis results and renders them unreliable.

A method is known of quantitative determination of stepharine in the cell culture of Stephania glabra tissue suggested by V.N.Davidenkov and co-authors, which has been comparatively recently slightly modified by N.S.Tsibulko (V.N.Davidenkov, N.V.Tareeva, A.A.Kiryanova, and L.T.Bondarenko. Quantitative determination of stepharine in the cell culture of Stephania glabra tissue.//Khim.-Farm. Zhurnal No.3, 1988, p.326-328.; N.S.Tsibulko. In "Issues of Biological, Medical and Pharmaceutical Chemistry", 2010, No.10, p.10-12). The method includes extraction of the sum of alkaloids from dried biomass by chlorophorm during 3 hours.

However, the utilization of chlorophorm in production is associated with quite a few restrictions, primarily due to special requirements to the equipment and materials used in the process. Therefore, the authors opted for the most efficient and affordable extraction with toluene.

In Davidenkov's studies, chromatographic separation of stepharine from the accompanying alkaloids and ballast components is performed on Silufol UV-254 plates in the chamber by the ascending technique in a mixture composed of chlorophorm : benzene : ethyl alcohol: aqua ammonia 25% (50:80: 100:0.2 by volume).

Silufol UV-254 plate are silica gel LS 5-40 fixed by starch on aluminum foil sheets. The plates demonstrate high elution velocity but at the same time low separation capability. In Davidenkov's studies to perform elution, the plate section with the marked zone is completely cut out with scissors and used for elution, with subsequent filtration. The individual character of the zone containing stepharine is proven by comparing the UV range of the alcohol eluate of the tissue culture extract and the UV range of the stepharine reference sample zone. To prove the individual character of the stepharine zone removed from the plate, repeated chromatography is performed on Silufol UV-254 plates in a mixture composed of: ethyl acetate: chlorophorm : methanol : aqua ammonia 25% (100:200:300:0.2). This method of zone individuality confirmation is labor-intensive, requires great amount of materials and does not provide a hundred percent guarantee.

Hereinafter, optical density is determined using a spectrophotometer, while quantitative stepharine concentration is calculated according to the Beer-Lambert law of light absorption. The main downside of the known method is its labor intensiveness and uncertainty of measurements.

Subsequent research of the present authors has shown the possibility to use color test to identify stepharine.

Several color tests have been described in the literature for the identification of stepharine, in particular: Marquis reagent test, diazotized sulphanilic acid reaction, reaction with 2,4-Dinitrochlorobenzene and reaction with 2,4-Dinitrophenylhydrazine. The chemical nature of those reactions has not been sufficiently studied.

The Marquis reagent, a mixture of formaldehyde and sulfuric acid (1 : 1), is used in toxicological chemistry to identify nitrogenous bases, including alkaloids. According to pharmacopeial monograph FS 42-2438-86, 0.01 g of stephaglabrine is dissolved in 2 mL of water, and then 2 drops of the Marquis reagent is added, producing a stable ruby-red coloring (FS 42 24-38-86. Stephaglabrine sulfate.-1989).

The reaction with diazotized sulphanilic acid is rarely used and is hardly compatible with thin layer chromatography (TLC analysis). Stephaglabrine forms compounds with diazotized sulphanilic acid that are colored yellow and orange (V.F.Kramarenko. Toxicological Chemistry - Kiev: Higher School, 1989.- 448 p.).

The reaction with 2,4-Dinitrochlorobenzene was suggested by Schreiner with co-authors in 1983. Essentially, the reaction consists of 1 percent ether solution of 2,4-Dinitrochlorobenzene being added to dry stephaglabrine-containing precipitate. As an isoquinoline derivative, stephaglabrine condenses with 2,4-Dinitrochlorobenzene with the formation of a yellow-colored addition compound (F.Feigl. Drop Method of Organic Substances Analysis. - Moscow.: Goskhimisdat 1962.- 836 p.). The reaction has a low susceptibility threshold and is almost incompatible with TLC analysis.

Multiple studies by the present authors have shown that, to determine stepharine in the adsorbent layer of the zone of interest, it is possible to use the modified Nastyukov color test for aarroommaattiicc hhyyddrrooccaarrbboonnss ((AA..MM..NNaassttyyuukkoovv.. JJoouurrnnaall ooff RRuussssiiaann SSoocciieettyy ooff PPhhyyssiiccaall CChheemmiissttrryy uunnddeerr tthhee aauussppiicceess ooff LLeenniinnggrraadd UUnniivveerrssiittyy.. CChheemmiiccaall sseeccttiioonn.. VV..ll .. --LL..,, ((GGllaavvnnaauukkaa)).. --SSttaattee PPuubbll.. HHoouussee.. 11886699--11993300 vv..3355—— 11990033))

IInn ccoonnttrraasstt ttoo tthhee k knnoowwnn mmeetthhoodd,, iirroonn ssuullffaattee iiss iinnttrroodduucceedd iinnttoo tthhee rreeaaccttiioonn mmiixxttuurree aass ccaattaallyysstt ttoo oobbttaaiinn tthhee ddeessiirreedd rreessuulltt.. TThhee uussee ooff tthhiiss rreeaaccttiioonn aalllloowweedd iinnccrreeaassiinngg ssuusscceeppttiibbiilliittyy aanndd aavvooiiddiinngg tthhee ssttaaggee ooff eelluuttiioonn ooff tthhee bbaassee ffrroomm tthhee aaddssoorrbbeenntt llaayyeerr,, wwhhiicchh mmaaddee tthhee pprroocceessss mmuucchh ssiimmpplleerr..

SSUUMMMMAARRYY OOFF TTHHEE IINNVVEENNTTIIOONN

IInn aaccccoorrddaannccee wwiitthh tthhiiss iinnvveennttiioonn,, aa mmeetthhoodd ooff oobbttaaiinniinngg sstteepphhaarriinnee ssuullffaattee iinn tthhee cceellll ccuullttuurree ooff SStteepphhaanniiaa ggllaabbrraa ttiissssuuee iiss ssuuggggeesstteedd,, wwhhiicchh iinncclluuddeess tthhee oobbttaaiinniinngg ooff sseeeedd mmaatteerriiaall,, iinn--ddeepptthh ccuullttiivvaattiioonn ooff SStteepphhaanniiaa ggllaabbrraa cceellllss iinn aa nnuuttrriieenntt mmeeddiiuumm uunnddeerr ssttiirrrriinngg aanndd aaeerraattiioonn bbyy aaiirr ttiillll tthhee pprroodduuccttiioonn ooff bbiioommaassss,, ffiillttrraattiioonn aanndd ddrryyiinngg ooff tthhee ssaammee,, aanndd sseeppaarraattiioonn ooff tthhee ttaarrggeett pprroodduucctt,, wwhheerreeiinn qquuaannttiittaattiivvee ddeetteerrmmiinnaattiioonn ooff sstteepphhaarriinnee ssuullffaattee bbyy tthhiinn llaayyeerr cchhrroommaattooggrraapphhyy ooff aa pprreeppaarreedd ssaammppllee aanndd ccoolloorr tteesstt ffoorr sstteepphhaarriinnee iiddeennttiifificcaattiioonn aarree ppeerrffoorrmmeedd..

IInn aaccccoorrddaannccee wwiitthh tthhiiss iinnvveennttiioonn,, qquuaannttiittaattiivvee ddeetteerrmmiinnaattiioonn ooff sstteepphhaarriinnee ssuullffaattee mmaayy aallssoo bbee ppeerrffoorrmmeedd iinn tthhee bbiioommaassss pprroodduucceedd ppririoorr ttoo ttaarrggeett pprroodduucctt sseeppaarraattiioonn..

IItt iiss pprreeffeerraabbllee tthhaatt,, ttoo pprreeppaarree aa bbiioommaassss ssaammppllee,, iitt iiss eexxttrraacctteedd wwiitthh ttoolluueennee iinn tthhee rraattiioo ooff 11 :: 1100,, ffiilltteerreedd,, eevvaappoorraatteedd iinn vvaaccuuuumm aanndd rreessiidduuee ddiissssoollvveedd iinn cchhlloorroopphhoorrmm..

IItt iiss pprreeffeerraabbllee tthhaatt tthhiinn llaayyeerr cchhrroommaattooggrraapphhyy ooff aa pprreeppaarreedd ssaammppllee iiss ppeerrffoorrmmeedd oonn SSiilliiccaa ggeell 6600 FF225544 ppllaatteess ooff MMeerrcckk mmaannuuffaaccttuurree..

IItt iiss pprreeffeerraabbllee tthhaatt,, aass aa ccoolloorr tteesstt ffoorr sstteepphhaarriinnee iiddeennttiifificcaattiioonn,, NNaassttyyuukkoovv ccoolloorr tteesstt iiss uusseedd wwiitthh iirroonn ssuullffaattee uusseedd aass ccaattaallyysstt..

IItt iiss pprreeffeerraabbllee tthhaatt,, pprriioorr ttoo ccuullttiivvaattiioonn,, tthhee aavvaaiillaabbiilliittyy aanndd ccoonncceennttrraattiioonn ooff sstteepphhaarriinnee ssuullffaattee iinn sseeeedd mmaatteerriiaall iiss aaddddiittiioonnaallllyy cchheecckkeedd bbyy mmeeaannss ooff tthhiinn llaayyeerr cchhrroommaattooggrraapphhyy..

IItt iiss pprreeffeerraabbllee tthhaatt,, dduurriinngg tthhee ccuullttiivvaattiioonn pprroocceessss,, qquuaannttiittaattiivvee ddeetteerrmmiinnaattiioonn ooff sstteepphhaarriinnee ssuullffaattee iinn ccuullttiivvaatteedd cceellllss iiss aaddddiittiioonnaallllyy ppeerrffoorrmmeedd bbyy mmeeaannss ooff tthhiinn llaayyeerr

A method of quantitative determination of stepharine sulfate in a sample during Stephania glabra tissue cells cultivation by the performance of thin layer chromatography of a prepared sample and color test for stepharine identification has also been suggested.

It is preferable that, to prepare a biomass sample, it is extracted with toluene in the ratio of 1 : 10, filtered, evaporated in vacuum and residue dissolved in chlorophorm. It is preferable that thin layer chromatography of a prepared sample is performed on Silica gel 60 F254 plates of Merck manufacture.

It is preferable that, as a color test for stepharine identification, Nastyukov color test is used with iron (III) sulfate used as catalyst.

DETAIL DESCRIPTION OF PREFERRED EMBODIMENTS

A method of obtaining stepharine sulfate in the cell culture of Stephania glabra tissue, including a method of quantitative determination of the same, is performed as follows:

Seed material is grown in 750-mL (total volume) flasks containing 200 mL of the medium, at the temperature of (26±1)°C, in darkness, under continuous stirring on a shaker (150 rpm) for the period of 2 weeks.

In-depth cultivation of Stephania glabra cells is performed in a liquid nutrient medium of the following composition:

Nutrient medium component ncentration, mg/1

Ammonium nitrate 1700

Potassium nitrate 1800

Potassium monophosphate 234

Potassium chloride 1 64

Magnesium sulfate heptahydrate 850

Potassium iodide 0.83

Cobalt dichloride sulfate 6-hydrate 0.025

Boric acid 6.2

Manganese sulfate 4-hydrate 22.3

Cuprum sulfate 5 -hydrate 0.025

Sodium molybdate 2-hydrate 0.25

Zinc sulfate heptahydrate 8.5

Ferrum sulfate heptahydrate ^Τ 27.8

Sodium ethylenediamine tetraacetate 37.3

Alpha-naphthylacetic acid __L _ 0.5

2,4- dichlorophenoxyacetic acid 0.1

Calcium pantothenate 2

Thiamine chloride 2

Nutrient sugar 50x10³

The mediums are sterilized in steam sterilizers at (1 17±1)° C for a period of 15 minutes. Seed material is grown in 750-mL (total volume) flasks containing 200 mL of the medium, at the temperature of (26±1)°C, in darkness, under continuous stirring on a shaker at the rate of 150 rpm for the period of 2 weeks.

To determine stepharine concentration in the seed material, a reference flask is used. The content of the flask is filtered, dried at 50±1°C, and homogenized in a porcelain mortar down to the particle size of 0.2-0.4 mm. One gram (precise weight) is used to perform toluene extraction with the ratio of raw material to solvent being equal to 1 : 10. The total amount of toluene obtained is filtered with a plaited paper filter and evaporated in vacuum at a temperature not exceeding 56±2°C. The dry residue thus obtained is dissolved in chlorophorm, and an aliquot amount is used for analysis on Silica gel 60 F254 plates of Merck (Germany) manufacture. Concurrently with the sample under study, 50 μΐ, of each of the stepharine reference solutions containing 50 and 100 g of the substance, respectively, are applied on the above-mentioned plate.

Thin layer chromatography is performed in an air-tight closed chamber saturated in advance with vapors of a mixture of solvents composed of chlorophorm: benzene: ethyl alcohol: aqua ammonia 25% (100:80:50:0.2 by volume). After the mobile phase migrates as high as 1 1 cm from the starting line, the plate is withdrawn from the chamber and dried in the air. Approximate stepharine concentration is determined by sight, wherein the plate is viewed under UV light and the marked zone of the sample component is compared against the reference sample. If need be, the exact concentration is determined by means of a color reaction with subsequent spectrophotometric measurement of optical density.

The evaluated seed material is used to obtain inoculum to be transferred to the fermenter. For this purpose, the cells grown in conical flasks under stirring on a shaker are put together, and to those is added an equal volume of fresh medium. The end volume of inoculum is 15L of cell suspension, which corresponds to a seed dose of 30 g/L of raw biomass.

In-depth cultivation of Stephania glabra cells is carried out in an industrial plant cell cultivation bioreactor in the above-described liquid nutrient medium under stirring and aeration by air till the obtaining of biomass. For this purpose, the inoculum is added in a sterile manner into the sterile reactor containing sterile medium (55L) under the pre-set temperature of (26±1)°C, with subsequent stirring and aeration, which marks the beginning of the cultivation process.

During cultivation that is carried out for two weeks, the air flow fed through the bubbler is gradually increased from 0.06 to 1.2 m /hour. If need be, for the reason that the product content in plant cells is not constant during cultivation but fluctuates in a wide range, additional control of stepharine content in cultivated cells is conducted according to the procedure described for seed material.

The cultivation process ends between days 1 1 and 14. The obtained biomass is filtered and dried at the temperature of 50±1°C in the drying chamber. The air-dry biomass is crushed in a mill (to particle size 0.2-1.0 mm) and quantitative determination of the target product, stepharine, performed. For that purpose, two averaged samples are prepared of one gram of milled biomass each. An analysis of each sample is performed according to the scheme below.

The sum of alkaloids of the cell culture from one gram of the sample is extracted with toluene with the ratio of raw material to solvent equal to 1 :10. The extraction is carried out for four hours at a room temperature with four portions of toluene. The extracts are put together and filtered with a plaited paper filter into a 100-mL flask. The whole volume of filtered liquid is quantitatively transferred to a flat-bottom 100-mL flask and evaporated in vacuum at a temperature not exceeding 56±2°C. The obtained dry residue is dissolved in 1 L of chlorophorm, and an aliquot portion is used for thin layer chromatography analysis.

In a general case, thin layer chromatography (TLC) is a kind of chromatography based on the difference of velocity with which components of a mixture migrate in a flat thin layer (0.1- 0.5 mm thick) of adsorbent while moving in the flow of a mobile phase (eluent). Depending on the choice of the chromatographic system (composition of stationary and mobile phases), main roles in the separation of substances may be played by the processes of adsorption, extraction, ion exchange, or complexation. In practice, several separation mechanisms often take place at the same time. Depending on the plate position and the direction of eluent flow, one differentiates among the ascending, descending and horizontal TLC techniques.

In the case under study, the present authors use ascending TLC on Silica gel 60 F254 plates of Merck (Germany) manufacture, featuring a silica gel stationary layer with a standardized particle size of 10 to 12 micrometers and a fixed pore size of 60 A. The method of affixing particles to the glass base serves to increase the plate resolution.

The chlorophorm solutions under analysis, 50 mcL in volume, are applied in the form of a streak by a standard calibrated capillary on the starting line located at a distance of 15mm from the lower and side edges of the plate. Along with the samples under analysis, 50 mcL of each of the reference stepharine solutions containing from 20 to 50 meg of the substance are applied onto the plate. Thin layer chromatography is performed in a closed air-tight chamber saturated in advance with vapors of a solvent mixture composed of chloroform: benzene: ethyl alcohol: aqua ammonia 25% (100:80:50:0.2 by volume). The plate is placed in the chamber vertically, immersed into the solvent 5 mm deep. After the mobile phase migrates as high as 1 1 cm from the starting line, the plate is withdrawn from the chamber and dried in the air. The marking of the sample component zone with the R_f value coinciding with that of the reference alkaloid sample is carried out under UV light (wavelength of 254 nm).

To perform a color (qualitative) reaction, the adsorbent layer of the zone of interest is removed from the base with a spatula and put into a 10-mL beaker. To the sample thus formed are added 1.8 parts of formaldehyde solution in glacial acetic acid (1 :99) and 1 part of ferric sulfate solution in concentrated sulfuric acid, the latter solution being prepared prior to use out of a 5% iron (III) sulfate solution in 10% sulfuric acid by means of the addition of 19 parts of sulfuric acid to 1 part of ferric sulfate. The mixture is thoroughly stirred and left for 12-16 hours at a room temperature till the full development of color. Where stepharine sulfate is present, the mixture will assume crimson color.

The reaction mixture is centrifuged for 5 to 10 minutes at 3,000-5,000 rpm. The optical density of the resulting transparent supernatant is measured with a photoelectrocolorometer (PhEC) at a wavelength of 490 nm in a cuvet with a 0.3-cm thick layer, using the zero sample as a reference solution. Stepharine content is calculated with the use of a calibration curve plotted according to the measurement results.

The extraction of the target product is carried out in an extraction unit equipped with an agitator. The Stephania glabra biomass wetted in a 5% sodium hydroxide water solution is put into the extractor, extraction is carried out under vigorous stirring with the agitator, and liquid (toluene extract) is drained from the extractor. The extraction cake is unloaded from the extractor and dried in the air, and recovery rate is evaluated by means of TLC analysis.

For that purpose, the air-dry extraction cake is homogenized in a porcelain mortar down to the particle size of 0.2-1.0 mm. Ten grams (precise weight) of homogenized cake is mixed with 5 mL of aqua ammonia 25% and kept at a room temperature for 10 minutes. Thereafter, the extraction of toluene and TLC determination of residual stepharine concentration are performed.

The toluene extract produced from biomass is processed with a 2.5% sulfuric acid solution for obtaining extract. The effectiveness of stepharine transfer from toluene extract to 2,5% sulfuric acid solution is determined by TLC analysis, using as a sample an aliquot portion of toluene extract evaporated to dryness in vacuum.

The sulfuric acid solution are alkalized with aqua ammonia 25% and processed with toluene to obtain secondary toluene extracts, which are, in turn, evaporated to dryness in a rotary evaporator at the temperature of 56±2°C and vacuum of 0.081±0.001 MPa. The dry residue is dissolved in ethyl alcohol and titrated with a 20% sulfuric acid alcohol solution to pH 5 in order to obtain technical-grade stephaglabrine sulfate. The technical-grade stephaglabrine sulfate was additionally purified by recrystallization in 80% ethyl alcohol.

The technical stephaglabrine sulfate obtained as a result of the above process is evaluated for recovery. For that purpose, an aliquot portion of alcohol solution is applied to the TLC plate and analyzed as described above.

The following examples illustrate the invention, without limiting the same in any way.

EXAMPLES.

Example 1.

To obtain seed material, Stephania glabra plant cells were cultivated in 750-mL flasks stirred on an orbital shaker at 150 rpm, each containing 200 mL of the nutrient medium seeded with 20 mL of the cell suspension from the previous passage. On the 12^th day of growth, the reference flask was used for the determination of stepharine concentration. The flask content was filtered, dried at the temperature of 50±1°C, and homogenized in a porcelain mortar down to the particle size of 0.2-0.4 mm. One gram (precise weight) was mixed with 1,500 μί of a 5% base solution (caustic soda) and kept at a room temperature for 10 minutes. The mixture was extracted with toluene with the raw material to solvent ration of 1 : 10. The extraction was carried out for four hours at a room temperature with four portions of toluene. The total amount of toluene was 100 mL.

The whole volume of toluene extract was quantitatively transferred to a flat-bottom flask and evaporated in vacuum at the temperature of 56±2°C. The obtained dry residue was dissolved in 700 of chlorophorm. An aliquot portion (60 μϋ) was applied to the starting line of a Silica gel 60 F254 plate of Merck (Germany) manufacture. In parallel with the sample under analysis, reference 50-μί stepharine solutions containing 50 and 100 μg of the substance were applied to the plate. TLC was performed in an air-tight closed chamber saturated in advance with the vapors of a solvent mixture composed of chlorophorm: benzene: ethyl alcohol: aqua ammonia 25% (100:80:50:0.2 by volume). After the mobile phase had migrated as high as 1 1 cm from the starting line, the plate was withdrawn from the chamber and dried in the air.

The marking of the sample component zone with the R_f value coinciding with that of the reference alkaloid sample was carried out under UV light. To obtain a zero sample, a zone was determined and marked on the plate free from any separation products. The adsorbent layer of the zone of interest of the sample, as well as the reference sample zones and the zero sample zone, were removed from the base with a spatula and placed into 10-mL beakers. To the four samples thus obtained were added 0.9 mL of iron (III) sulfate solution and 1.62 mL of formaldehyde working solution, thoroughly stirred and left for 16 hours at a room temperature till the full development of color. The reaction mixture was centrifuged for 10 minutes at 5,000 rpm. The optical density of the resulting transparent supernatant was measured with a PhEC at a wavelength of 490 nm in a cuvet with a 0.3-cm thick layer, using the zero sample as a reference solution. A calibration curve was plotted according to the measurement results, and stepharine content in 1 gram of the air-dry biomass from the flasks determined. The alkaloid content in the dried culture of Stephania glabra tissue was not less than 0.12 %. The flasks of that lot were used to obtain inoculum for the subsequent cultivation in a 100-L bioreactor designed for the industrial-scale plant cell cultivation.

Inoculum - 15 L of cell suspension (in equal proportions with a fresh nutrient medium) - was added to the sterile reactor with a sterile medium (55 L) of the above composition, the reactor having been set at the target temperature regime of (26±1)° C. The seed dose was 30 g/L of raw biomass.

After that, the least required air feed was switched on and cultivation process started, with periodic sample collection for the purpose of determining the current cell concentration. As such concentration grew, air feed was increased. With the total period of cell cultivation of up to 14 days, air flow was increased practically in a linear fashion from 60 L/hour to 1.3 m³/hour at the end of the cycle.

Cell output by raw biomass amounted to 300 g/L, and by dry biomass to 20 g/L, the proportion of living cells reaching 90%. Stepharine content in the target product was determined according to the procedure described above for the seed material. The target product content in the bioreactor was equal to 0.11%.

Thus, continuous control of quantitative stepharine content in the process, under cultivation conditions as close to those of flask cultivation as possible, makes it possible to obtain biomass with the target product content at the level of that of the seed material, and eliminate the inefficient use of biomass for the subsequent product extraction.

Example 2.

Stephania glabra plant cell suspension was cultivated in a bioreactor designed for the industrial-scale plant cell cultivation, as was shown in Example 1.

Alkaloid content of the seed dose was 0.15%. During cultivation, control samples were collected and stepharine concentration determined on the 7^th, 9^th and 1 1^th days of cultivation according to the technique described in Example 1. Alkaloid content in the cultivated cells was 0.12% irrespective of the day of cultivation.

Example 3. Stephania glabra plant cell suspension was cultivated in a bioreactor designed for the industrial-scale plant cell cultivation, as was shown in Example 1. 10 consecutive processes were conducted.

During cultivation, control samples were collected and stepharine content determined on the 9^th and 1 1^th days of cultivation according to the technique described in Example 1. Alkaloid contents in the seed dose and biomass cells are given in table 1.

Based on the data shown in the table, a conclusion may be drawn that in two cases out of ten, stepharinen concentration on days 9 and 1 1 proved to be below the desirable level, which requires termination of the cultivation process and makes the use of the given biomass impossible for subsequent extraction. Therefore, it is reasonably to omit the extraction of the target product, which is one of the most complicated stages and the most energy and labor intensive stage of the whole technological process, and thus increase the overall cost effectiveness of the process.

Example 4

Stephania glabra plant cell suspension was cultivated in a bioreactor designed for the industrial-scale plant cell cultivation, as above shown. The average stepharine concentration in the air-dry biomass during ten fermentations was 0.1%.

The extraction of the target product was performed with the help of an extractor equipped with an agitator.

Stephania glabra biomass wetted with a 5% sodium hydroxide water solution was loaded into the extractor and extracted with toluene to obtain toluene extract. The extraction cake was unloaded from the apparatus and dried in the air; after that, the extraction rate was evaluated by a developed TLC analysis technique. For that purpose, the air-dry extraction cake was homogenized in a porcelain mortar down to the particle size of 0.2-1.0 mm. Ten grams (precise weight) of the homogenized cake was mixed with 5 mL of aqua ammonia 25% and held at a room temperature for a period of 10 minutes. After that, extraction with toluene was performed and residual stepharine content determined by the TLC technique described above. In ten consecutive extractions, no meaningful stepharine quantities were detected in the extraction cake.

The toluene extract obtained from biomass was processed with a 2.5% sulfuric acid solution for the purpose of obtaining extract. The completeness of stepharine transfer from toluene extract to 2,5% sulfuric acid solution was controlled by means of TLC analysis, using 1/100 of the total toluene extract as a sample, which was evaporated in vacuum. In the course of ten consecutive extractions, no meaningful stepharine quantities were detected in the processed extract.

The sulfuric acid solution were alkalized with aqua ammonia 25% and processed with toluene to obtain secondary toluene extracts, which were, in turn, evaporated to dryness in a rotary evaporator at the temperature of 56±2°C and vacuum of 0.081 ±0.001 MPa. The dry residue was dissolved in ethyl alcohol and titrated with a 20% sulfuric acid alcohol solution to pH 5 in order to obtain technical-grade stephaglabrine sulfate. The alcohol solution obtained as a result of the above process was evaluated for recovery. For that purpose, 40-50 μL· of alcohol solution was applied to the TLC plate and analyzed as described above. The recovery from the alcohol solution fluctuated in the range between 50% and 70%; therefore all the alcohol solutions were re-cycled to the sulfuric acid solution stage for a repeat process. After such repeat processing the share of unrecovered stepharine was 10-15%.

The technical stephaglabrine sulfate obtained was recrystallized with 80% ethyl alcohol. The processed alcohol solution was evaluated by TLC as above described. The share of unrecovered stepharine fluctutated within 1% to 3%. All the alcohol solutions were re-cycled to the sulfuric acid solution stage for a repeat process.

Table 1.

Stepharine content in air-dry biomass, %

Claims

1. Method of obtaining stepharine sulfate in the cell culture of Stephania glabra tissue, including the obtaining of seed material, in-depth cultivation of Stephania glabra cells in a liquid nutrient medium under stirring and aeration by air till the production of biomass, filtration and drying of the same, and separation of the target product, wherein concentration determination of stepharine sulfate by thin layer chromatography of a prepared sample and color test for stepharine identification are performed.

2. Method of obtaining stepharine sulfate according to claim 1, wherein quantitative determination of stepharine sulfate is performed in the biomass produced prior to target product separation.

3. Method of obtaining stepharine sulfate according to claims 1 and 2, wherein to prepare a biomass sample, it is extracted with toluene in the ratio of 1 : 10, filtered, evaporated in vacuum and residue dissolved in chlorophorm.

4. Method of obtaining stepharine sulfate according to claims 1, 2 and 3, wherein thin layer chromatography of a prepared sample is performed on Silica gel 60 F254 plates of Merck manufacture.

5. Method of obtaining stepharine sulfate according to claim 1 , wherein, as a color test for stepharine identification, Nastyukov color test is used with iron (III) sulfate used as catalyst.

6. Method of obtaining stepharine sulfate according to claim 1 , wherein, prior to cultivation, the availability and concentration of stepharine sulfate in seed material is additionally checked by means of thin layer chromatography.

7. Method of obtaining stepharine sulfate according to claim 1, wherein, during the cultivation process, quantitative determination of stepharine sulfate in cultivated cells is additionally performed by means of thin layer chromatography and subsequent spectrophotometry.

8. Method of quantitative determination of stepharine sulfate in a sample during Stephania glabra tissue cells cultivation by means of thin layer chromatography of a prepared sample and color test for stepharine identification.

9. Method of quantitative determination of stepharine sulfate according to claim 8, wherein to prepare a biomass sample, it is extracted with toluene in the ratio of 1 : 10, filtered, evaporated in vacuum and residue dissolved in chlorophorm.

10. Method of quantitative determination of stepharine sulfate according to claims 8 and 9, wherein thin layer chromatography of a prepared sample is performed on Silica gel 60 F254 plates of Merck manufacture.

1 1. Method of quantitative determination of stepharine sulfate according to claim 8, wherein, as a color test for stepharine identification, Nastyukov color test is used with iron (III) sulfate used as catalyst.