RU2623084C2 - Method for simultaneous production of hypericin and pseudohypericin - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: method for producing hypericin and pseudohypericin from raw phytomass material of the plants of the spotted St. John's wort and common St. John's wort species, comprising grinding the plant raw materials, degreasing the raw materials with an organic solvent, separating the plants lipophilic substances extract, drying the raw material, re-extracting the fat-free raw material with aqueous solutions of the organic solvent, filtering the obtained extracts, the concentration of the extracts, the chromatographic separation of the target pigments on a column containing silica gel with using eluents containing an organic solvent and water, the concentration of the obtained eluates fractions, combining the fractions, the crystallization of hypericin and pseudohypericin with the use of solutions of polar organic solvents in a non-polar one, drying the finished pigment, wherein the aboveground mass of the plants Hypericum perforatum L. and Hypericum maculatum Crantz. collected in the phase of mass budding and beginning of flowering is previously degreased with an organic solvent, thrice extracted by macerating with the 40-55% alcohol of the formula C_nH_2nOH, where n=2 or n=3, the process is conducted in a microwave chamber, the extract is separated by filtration or centrifugation and evaporated in vacuum to dryness, thereinafter the extract is subjected to precipitation on silica gel with the functional pores cover of dimethyloctadecyl groups, with the subsequent drying in vacuum, thereinafter the precipitate is placed in a precolumn for flash chromatography over a sorbent, and naphthodianthrones pigments are eluted by aqueous solutions of ethanol; the crystallization of hypericin and pseudohypericin is carried out from a mixture of ethanol-water in the vacuum crystallization mode with the simultaneous concentration of the mother liquor on a rotary evaporator, the hypericin and pseudohypericin drying is carried out in vacuum, under specific conditions.

EFFECT: method is simplified, it increases the variability of the plant raw material extraction, increases the effectiveness of the deep cleaning stage of hypericin and pseudohypericin.

3 dwg, 17 ex, 1 tbl

Description

The invention relates to the pharmaceutical industry and relates to a method for the production of substances of preparations containing naphthodianthrone plant pigments - hypericin and pseudohypericin for medicine, veterinary medicine, biotechnology and scientific research.

In the scientific and patent literature there is known information about the methods of extraction of these pigments from plant materials, methods of separation of these pigments. In this regard, the team of authors considers it necessary to give a comparative analysis of the already known methods of extraction and separation of these pigments.

In the description of the invention there are the following abbreviations and symbols:

- H - hypericin;

- pH - pseudohypericin;

- MAE - microwave activation of the extraction process;

- (v / v / v) - volumetric relations;

- TLC - thin layer chromatography;

- UZV - ultrasonic bath;

- HPLC - high performance liquid chromatography;

- GLC - gas-liquid chromatography;

- TMS derivatives - trimethylsilyl derivatives;

- IR spectra - infrared spectra;

- absorption spectra in the UV (ultraviolet) region.

A known method of extraction of hypericin and pseudo-hypericin in Soxhlet apparatus and chromatographic isolation of pseudo-hypericin [Zotou Α., Loukou Ζ. Determination of Hypericin and Pseudohypericin in Extracts from Hypericum perforatum L. and Pharmaceutical Preparations by Liquid Chromatography-Fluorescence Detection // Chromatographia, 2001. - V. 54. - P. 218-224]. The method is carried out by extracting pigments with a mixture of methanol-acetone (1: 1) for 6 hours, with constant boiling of the extractant, concentrating the pigment extract to a dry residue, dissolving the dry extract in methanol and isolating the pH by preparative liquid chromatography for ten days, concentration of fractions, containing according to HPLC analysis of pH, by freeze drying.

The disadvantages of this method of extraction of pigments and the allocation of pH are: the use of highly toxic solvents: methanol and a mixture of methanol with acetone (1: 1). Long duration of pigment extraction on a Soxhlet apparatus (6 hours) and high labor costs at the stage of pH isolation by liquid chromatography for 10 days.

A known method of extraction of St. John's wort pigments and enrichment of the extract with hypericins [Stasevich OV, Leontiev VN, Kovalenko NA, Supichenko GN Chromatographic analysis and separation of hypericin-containing St. John's wort extract // Transactions of BSTU, 2012. - No. 4 (151). - S. 183-185]. The method is carried out by extraction of pigments in a Soxhlet apparatus with ethanol, preparative separation of the extract by liquid chromatography on a Diaion HP-20 sorbent, elution of pigments with a sorbent with aqueous solutions of alcohol concentration (0, 20, 40, 60, 80, and 100%), as well as a mixture of ethanol and chloroform (1: 1). It was found that in this case, hypericins are concentrated during elution with 80% ethanol in a volume of 304-356 ml, as well as with a mixture of ethanol with chloroform in a volume of 540-564 ml. As a result, the authors were able to increase the concentration of H in the obtained fractions from 0.08% to 0.34%.

The disadvantages of this method of extraction of pigments and the allocation of H are: the use of highly toxic solvents - a mixture of ethanol with chloroform (1: 1). The use of a sorbent for chromatographic enrichment of pigments that does not provide selective separation of H and pH to obtain preparative pure compounds. The long duration of the process of chromatographic separation of pigments on sorbents with a polystyrene matrix, such as "Diaion HP-20".

A known method for producing H and pH is given in patent [US Patent 5047435 Antiviral compositions containing aromatic polycyclic diones and method for treating retrovirus infections / Lavie; David (Rehovot, IL); Meruelo; Daniel (Scarborough, NY); Lavie; Gad (New York, NY); Revel Michel (Rehovot, IL); Vande Velde; Vincent (Boncelles, BE); Rotman; Dalia (Rehovot, IL) Assignee: New York University (New York, NY); Yeda Research and Development Company, Ltd. (Rehovot, IL), Foreign Application Priority Data: Aug 08, 1986. Intern'l Class: A61K 031/015; A61K 045/08; A61L 002/18; A61L 015/00 /], which consists in drying the plant material at 55 ° C, grinding the raw material, extracting 1 kg of St. John's wort grass with acetone (about 5-10 l per kg) in the Soxhlet apparatus until the solvent is discolored in extraction (9-55 h), concentrating the acetone extract by evaporation in vacuo to dryness to obtain a residue of 95 g, chromatographic fractionation of the obtained dry extract on a column filled with silica gel 60 (0.06-0.20 mm). For this, the authors of the patent dissolve 25 g of the obtained dry extract in 500 ml of acetone, add an equal amount of silica gel 60 and evaporate on a rotary evaporator with stirring until a homogeneous and dry mixture is obtained, which is then placed on top of a chromatographic column containing one kg of silica gel 60 and elute chloroform until the solvent reaches the bottom of the column. The column is then washed with a solvent mixture comprising chloroform-acetone-methanol 75:15:10 (v / v / v). After reducing the intensity of the red color of the eluate, the column is washed with a solvent mixture of chloroform-acetone-methanol, 55:15:10 (v / v / v). Fractions of 250 ml are collected. Each fraction was analyzed by TLC. Further purification and separation of H and pH are achieved sequentially by double flash chromatography using silica gel 60 (mesh 0.04-0.06) under pressure. Two main components are obtained: H with a yield of 0.19 g and a pH with a yield of 0.73 g.

The disadvantages of this method of extraction of pigments and the allocation of H and pH are: the use of highly toxic solvents - chloroform, acetone, methanol. The use of a sorbent for chromatographic enrichment of silica gel 60 pigments, which does not provide selective separation of hypericin and pseudohypericin to obtain preparative pure compounds in one flash chromatography step. The long duration of the process of extraction of raw materials with acetone in a Soxhlet apparatus and chromatographic separation of pigments on sorbents such as silica gel 60.

The closest in technical essence and the achieved result is the method of extraction of H and pH from the medicinal phytomass of St. John's wort, described in the article [Punegov V.V., Kostromin V.I., Fomina MG, Zainullin V.G., Yushkova E .A., Belykh D.V., Chukicheva I.Yu., Zaynullin G.G. Extraction of hypericin and pseudohypericin from St. John's wort perforated under microwave activation of the process // Chemistry of plant raw materials, 2014. - No. 1. - S. 125-130] and taken as a prototype in part of the extraction stage of the target pigments. The method of extracting H and pH consists in grinding the medicinal phytomass of St. John's wort perforated to grass flour, passing through a sieve with a 0.5 mm cell, triple extraction of lipophilic substances with chloroform with a hydromodule of thirty, with activation of the mass transfer process by treating the feedstock-chloroform suspension with ultrasound for 30 c, holding the suspension at room temperature for 30 minutes, filtering the chloroform extract of chlorophyll and lipids through a layer of cotton and paper filter, drying the fat-free raw materials in vacuum in a stream of air in order to completely remove chloroform vapors from the feed, triple extraction of the sum of H and pH from non-fat feed for 60 with an aqueous solution of ethyl alcohol or isopropanol with a hydromodule from 20 to 48 under microwave activation of the extraction process (hereinafter referred to as MAE (e )) with heating the suspension in a microwave chamber to 65 ° C at a microwave frequency of 2450 MHz, the specific microwave power under the experimental conditions of 0.0205 W / cm ³ . The resulting extracts are combined and concentrated in vacuo on a rotary evaporator to recover solvents.

The disadvantages of this method of extraction of H and pH are: the use of toxic chloroform as an extractant of lipophilic substances. It takes a long time to completely remove chloroform vapor from nonfat raw materials. In this case, chloroform vapors are not trapped and pollute the atmosphere. In addition, as a rule, technical chloroform contains dioxins in trace amounts, which can cause contamination of plant materials using chloroform.

H and pH are extracted by rapidly heating the alcohol-raw material suspension in the microwave chamber to 65 ° C in 60 s, but at a temperature of more than 60 ° C, the glycosides of flavonoids and starch are intensively extracted from the phytomass into the solution and the pigment extract is polluted with ballast, in this case substances. These substances further reduce the selectivity of the chromatographic separation of H and pH under flash chromatography. In addition, under conditions of constant rapid heating of the suspension, the extractant feedstock in the microwave chamber may cause local overheating, accompanied by partial destruction of the target pigments, and there is also a real danger of a sudden thermal release of the extraction mass from the extractor.

The technical result consists in simplifying and reducing costs, increasing the variability of extraction of plant materials and extraction conditions, increasing the efficiency of the stage of deep purification of hypericin and pseudohypericin by preparative liquid flash chromatography from associated extractive plant substances.

The invention is illustrated by drawings:

- in fig. 1 shows the chromatogram of the HPLC analysis of the obtained pigments. HPLC chromatograms of pure perforated St. John’s wort pigments — hypericin and pseudohypericin — vertically: units of optical density (B (white)), horizontally — time of chromatographic retention of components, min. Milichrom-5 chromatograph, VIS detector, 80 * 2 mm column, Nucleosil C18 sorbent, 5 μm, eluent acetonitrile-ethanol with a linear gradient of increasing concentration of ethanol acidified with acetic acid (1%).

- in fig. Figure 2 shows the IR spectra (IR spectrum of hypericin (a thin film on a KBr disk) identical to the IR spectrum of pseudohypericin. Infralum FT-02 Fourier spectrometer.

- in fig. Figure 3 shows the absorption spectrum in the UV and visible regions of alcohol solutions of the obtained samples of hypericin and pseudohypericin. Shimadzu UV1700 spectrometer (Japan).

Table 1 shows the conditions for the implementation of the method for the simultaneous production of hypericin (H) and pseudohypericin (PH), the composition of the pigments, the practical yield and the degree of extraction from plant materials.

The method is as follows: the raw material for the production of hypericin and pseudohypericin is the chopped medicinal plant phytomass of Hypericum maculatum Crantz and Hypericum peforatum L., harvested in the phase of budding or mass flowering of plants.

The upper part of the foliage stalks of St. John's wort in the phase of budding-mass flowering 20-25 cm long is cut and dried out of direct sunlight. The dried raw material is ground to particles passing through a 0.5 mm sieve. St. John's wort herbal flour is introduced into a 1 L glass flask and the lipophilic substances (chlorophylls, carotenoids, lipids) are extracted with hexane or petroleum ether containing 10-20% (volume fraction) of acetone, with a hydromodule 4.8. The suspension is intensively mixed for 20 minutes and kept in an ultrasonic bath for 5 minutes at a specific power of the ultrasonic emitter of 39 W / dm ³ . The resulting extract was separated by filtration. The raw materials on the filter are additionally washed with a solution of hexane-acetone, preferably at a volume ratio of 8: 2 three times in 0.1 l. Raw materials purified from lipophilic substances are dried in a vacuum of a water-jet pump to remove traces of solvents. The extract of lipophilic substances is concentrated on a rotary evaporator in vacuum at a temperature of 45 ° C to dryness for the recovery of hexane (or petroleum ether) and acetone. The resulting chlorophyll-carotenoid paste is a co-product in the production of hypericin and pseudo-hypericin. The paste is removed from the bottoms and Packed in sealed plastic containers. Fat-free raw materials are introduced into an extractor equipped with a reflux condenser, and 55% ethanol or isopropanol are added in such an amount that the hydromodule is in the range of 40-48. The suspension is held for 30 minutes to swell the raw materials and extracted three times with hypericin and pseudo-hypericin in a microwave chamber with a radiation frequency of 2450 MHz, with a specific power of 41.6 W / dm ³ with a hydraulic module of 40-48 with heating of the suspension of the raw material-extractant to 60-70 ° C ( preferably 66 ° C) for 29-46 min (preferably 42 min), with periodic stirring of the suspension under conditions of pulse-width modulation of microwave radiation with a pulse duty cycle of 1.3, and a duration of microwave pulse packets of 23 s. The total extract obtained after filtration (or centrifugation) in a volume of 14.3 L was concentrated in vacuo while the extract was irradiated in a 10 L distillation flask with a 95 W incandescent lamp on a rotary evaporator at a temperature of 50 ° C to dryness. Get 60 g of a dry extract of purple color. When the extract is irradiated during concentration in vacuo for 2.5 h, the photocyclization of the concomitant compounds protogipericin and protopsevidogipericin occurs to H and PH, respectively. Photocyclization of these substances can increase the practical yield of target pigments. For the purpose of further fractionation by flash chromatography, the total pigment extract is mixed with water (0.25 L), dispersed in an ultrasonic grinder and mixed with 300 g of the Diasorb 130 C16T sorbent. For precipitation, the sorbent with the extract is dried in vacuum with stirring on a rotary evaporator at a temperature of 50 ° C. Dry the precipitate in a vacuum oil pump at a residual pressure of 0.05-0.08 Pa. The precipitate H and pH on Diasorb C16 is a brown-violet powder in an amount of 360 g. Pigment precipitate is added to the front column of the FLASH150 preparative flash chromatography system and pseudohypericin and hypericin are eluted through a chromatographic column with an inner diameter of 160 mm and a layer height sorbent 320 mm, filled with sorbent, fractions 40-64 microns, containing on the surface of the pores of silica gel a functional coating of dimethyl octadecylsilyl groups. The column is equipped with a pneumatic system for radial compression of the sorbent in the column, as well as an inlet and outlet radial distributor of eluent flow along the column. As an eluent, aqueous solutions of ethyl alcohol are used with an increase in concentration from 5% to 60%. To suppress the dissociation of pigments and increase the selectivity of chromatographic separation, eluents acidified with acetic acid are used. Pigments from the column were eluted in a stepwise gradient mode of ethanol concentration. Pseudohypericin is eluted from the column with 20 and 30% ethanol. Hypericin is eluted with 40 and 45% ethanol. The obtained fractions of 5 l each were analyzed by HPLC using the text on a Milichrome 5 analytical chromatograph and the fractions containing pseudo-hypericin and hypericin were combined separately according to the analysis results. Red fractions containing the desired pigments are concentrated in vacuo on a rotary evaporator. The result is a fraction of a concentrate of pseudohypericin and hypericin. The resulting concentrates of pseudohypericin and hypericin are dissolved in 800 ml and 300 ml of 95% ethyl alcohol, respectively, when boiled. The solutions are filtered through a 0.2 micron fluoroplastic membrane filter and concentrated with simultaneous vacuum crystallization on an IR-1M rotary evaporator at a residual pressure of 33-13 kPa (25-10 mmHg) to a residue of 5 and 2 ml, respectively . The residue containing the desired products is filtered through a 0.2 Fm fluoroplastic membrane filter. Received, after drying in vacuum at 60 ° C, on the filter in a crystalline form, 0.06-0.073 g of pseudohypericin and 0.014-0.21 g of hypericin. According to HPLC analysis, the mass fraction of pseudohypericin and hypericin in the obtained samples is 94-98%.

The mother liquors containing ethanol are evaporated to dryness. The solid residue, which is a mixture of minor diantronic compounds of St. John's wort and traces of plant carotenoids, is a co-product of the production of hypericin and pseudo-hypericin. It is removed from the distillation flask and, after drying in vacuum, is Packed in a sealed container. A mixture of acetone-hexane and acetone-petroleum ether is distilled at a distillation unit and after dehydration with synthetic zeolite CaX, it is used repeatedly for the production of experimental batches of St. John's wort pigments for the purpose of biomedical studies of their properties. After extraction of the target pigments, the plant material is placed in the stillage capacity of the laboratory retification unit and, by distillation, ethyl alcohol or isopropanol sorbed by the vegetable pulp are recovered. The plant residue is disposed of.

This method can be successfully used in the pharmaceutical industry, therefore, it meets the criteria of "industrially applicable", "novelty" and "inventive step".

A method for the simultaneous production of hypericin and pseudohypericin is specified by the following examples:

Example 1. Preparation of plant materials. The upper part of the foliage stalks of St. John's wort (Hypericum maculatum Crantz) in the phase of budding-mass flowering 20-25 cm long was cut and dried out of direct sunlight. The raw materials were prepared in the Syktyvdinsky district of the Komi Republic, and the raw materials of perforated Hypericum (Hypericum perforatum L.) were procured from experimental plots of the Botanical Garden of the Institute of Biology of the Komi Scientific Center, Ural Branch of the Russian Academy of Sciences. The dried feed was ground in a mill to particles passing through a 0.5 mm sieve. According to the results of the HPLC analysis, the obtained grass meal from St. John's wort contains 0.095% of the sum of hypericin and pseudo-hypericin, including 0.075% of pseudo-hypericin and 0.020% of hypericin. Herbal flour from Hypericum perforatum contains 0.087% of the sum of hypericin and pseudo-hypericin, including 0.065% of pseudo-hypericin and 0.022% -hypericin.

Example 2. Extraction of concomitant lipophilic substances (chlorophylls and carotenoids, triglycerides). 124 g of air-dried grassy St. John's wort flour was introduced into a 1 L glass flask and 0.6 L of hexane containing 10% (volume fraction) of acetone was added. Hydro module 5. Intensively mixed the suspension on a magnetic stirrer for 20 min and kept in a UZV-2/150-TN (LLC Reltek, Russia) for 5 min with a specific power of the ultrasonic emitter 39 W / dm ³ . The resulting extract was filtered on a suction filter. The filter feed was washed further with a solution of hexane-acetone (9: 1) three times in 0.1 L each. Raw materials purified from lipophilic substances were dried in a vacuum of a water-jet pump to remove traces of solvents. The extract was concentrated on an IR-1 rotary evaporator (Khimlabribribor OJSC, Klin, Russia) in vacuum at a temperature of 45 ° C to dryness for the recovery of hexane and acetone. The resulting chlorophyll-carotenoid paste is a co-product in the production of hypericin and pseudo-hypericin. The paste was removed from the bottoms and Packed in sealed plastic containers.

Example 3. Extraction of hypericin and pseudohypericin. Fat-free raw materials were introduced into a 6 L polypropylene extractor equipped with a reflux condenser and 4960 ml of 55% ethanol were added. The suspension was held for 30 minutes to swell the raw materials and the pigments were extracted three times in a microwave chamber with a radiation frequency of 2450 MHz, with a specific power of 41.6 W / dm ³ with a hydraulic module of 40 with heating of the suspension of the raw material-extractant to 66 ° C for 42 minutes, with periodic stirring suspension under conditions of pulse-width modulation of microwave radiation with a duty cycle of bursts of pulses equal to 1.3, the duration of bursts of microwave pulses is 23 s. The total extract obtained after filtration in a volume of 14.3 L was concentrated in vacuo with simultaneous irradiation of the extract in a 10 L distillation flask with a 95 W incandescent lamp on an IR-10M rotary evaporator (Khimlaborribor OJSC, Klin, Russia) at a temperature of 50 ° C to dryness. Received 60 g of a dry extract of purple color. When the extract is irradiated during concentration in vacuo for 2.5 hours, the photocyclization of the concomitant compounds protogipericin and protopsevidogipericin occurs to H and pH, respectively. Photocyclization of these substances can increase the practical yield of target pigments. For the purpose of further fractionation by flash chromatography, the total pigment extract was mixed with water (0.25 L), dispersed in a UZV and mixed with 300 g of the Diasorb 130 C16T sorbent (CJSC BioChemMak ST, Moscow, Russia), fractions 160-250 microns. For precipitation, the sorbent with the extract was dried in vacuum with stirring on a rotary evaporator IR-1 at a temperature of 50 ° C. Dried in vacuum of the oil pump at a residual pressure of 0.05-0.08 Pa. The pigment precipitate on Diasorb C16 was a brown-violet powder in an amount of 360 g.

Example 4. Fractionation of pigments by flash chromatography. Prepared flash chromatography system “FLASH150”, (Biotage, USA) introduced 360 g of the pigment precipitate obtained in Example 3, and pseudohypericin and hypericin were eluted through a chromatographic column with an inner diameter of 160 mm and a sorbent layer height of 320 mm filled with sorbent , fractions 40-64 microns, containing on the surface of the pores of silica gel a functional coating of dimethyl octadecylsilyl groups. The column is equipped with a pneumatic system for radial compression of the sorbent in the column, as well as an inlet and outlet radial distributor of eluent flow along the column. Aqueous solutions of ethyl alcohol with increasing concentration from 5% to 60% were used as eluent. To suppress the dissociation of pigments and increase the selectivity of chromatographic separation used eluents acidified with acetic acid with a volume fraction of 1%. Pigments from the column were eluted in a stepwise gradient mode of ethanol concentration. The flow rate of the eluent at the column outlet was 850 cm ³ / min at a column inlet pressure of 6 atm (607.95 kPa). In the first fractions eluted with ethanol 5-15%, the target pigments were not detected by HPLC analysis. Mono- and disaccharides, glycosides of flavonoids, mainly rutin, were found in these fractions by GLC analysis of TMS derivatives. Pseudohypericin was eluted from the column with 20 and 30% ethanol. Hypericin was eluted with 40% and 45% ethanol. The obtained 17 fractions of 5 L each were analyzed by HPLC on a Milichrom 5 analytical chromatograph (Medicant LLC, Orel, Russia) and the fractions containing pseudo-hypericin and hypericin were separately combined according to the analysis results. The red fractions containing the target pigments were concentrated in vacuo on an IR-10M rotary evaporator. As a result, a fraction of pseudohypericin concentrate was obtained in an amount of 0.0883 g (practical yield 0.072%) and hypericin 0.0221 g (practical yield 0.018%). According to HPLC analysis, the content of pseudohypericin in the obtained sample was 95%, and hypericin in the second sample was 96% (see table 1).

Example 5. Purification by recrystallization of samples of hypericin and pseudo-hypericin obtained in example 4. 0.0883 g of pseudo-hypericin and 0.0221 g of hypericin were dissolved in 800 ml and 300 ml of 95% ethanol, respectively, while boiling in conical flasks, the resulting solutions were filtered through a 0.2 micron fluoroplastic membrane filter (BioChemMak ST CJSC, Moscow, Russia) and concentrated with simultaneous vacuum crystallization on an IR-1M rotary evaporator (Khimlaborribor OJSC, Klin, Russia) with a residual pressure of 33-13 kPa (25-10 m Hg. v.) to a residue volume of 5 and 2 ml. The residue containing the desired products was filtered through a fluoroplastic membrane filter with 0.2 μm pores. Received, after drying in vacuum at 60 ° C, on the filter in a crystalline form, 0.0879 g of pseudo-hypericin and 0.0218 g of hypericin. According to the HPLC analysis, the mass fraction of pseudohypericin in the obtained sample is 98.6%, hypericin - 99.5% (Table 1). As an impurity, pseudo-hypericin contains 1.4% hypericin, and a sample of hypericin contains 0.5% pseudo-hypericin. The obtained pigments were investigated by physicochemical methods shown in the figures. In fig. 1 shows the chromatogram of the HPLC analysis of the obtained pigments. In fig. 2 shows the IR spectra, and in Fig. 3 absorption spectra in the UV and visible spectral range of the alcohol solutions of the obtained pigments.

According to the results of the chromatography-mass spectrometric analysis of the solutions of the obtained pigments, it was found that in the mass spectrum fixing negative ions, a molecular ion signal with a value of m / z 519 is characteristic for a pseudohypericin sample, a corresponding signal is recorded for a hypericin sample with a m / z value of 503.

Example. 6. Recovery of solvents, utilization of mother liquors and waste raw materials. Stock solutions containing ethanol were evaporated to dryness. The solid residue, which is a mixture of minor diantronic compounds of St. John's wort and traces of plant carotenoids, is a co-product of the production of hypericin and pseudo-hypericin. It was removed from the distillation flask and, after drying in vacuum, was packaged in an airtight container. A mixture of acetone-hexane and acetone-petroleum ether was distilled at a distillation unit and, after dehydration with synthetic zeolite, CaX was used repeatedly for the production of experimental batches of St. John's wort pigments for the purpose of biomedical studies of their properties. After extraction of the target pigments, the plant raw material was placed in the stillage capacity of the laboratory retification unit, and ethylene recovered by distillation was recovered by distillation. The plant residue was disposed of.

Example 7. Obtaining hypericin and pseudohypericin was carried out according to examples 1-6, but for the degreasing of the raw materials used a mixture of acetone and hexane in a volume ratio of 2: 8 with a hydraulic module 5. The practical yield and composition of the pigments are shown in table. one.

Example 8. The preparation of hypericin and pseudohypericin was carried out according to examples 1-6, but the plant material was degreased with an organic solvent of hexane: acetone (8: 2) or petroleum ether: acetone (8: 2) with a hydromodule 5 for 5 minutes. The practical yield and composition of the pigments are shown in table. one.

Example 9. The preparation of hypericin and pseudohypericin was carried out according to examples 1-6, but the plant material was degreased with an organic solvent of hexane: acetone (9: 1) or petroleum ether: acetone (9: 1) with a hydromodule 5 for 5 minutes. The practical yield and composition of the pigments are shown in table. one.

Example 10. Obtaining hypericin and pseudohypericin was carried out according to examples 1-6, but microwave extraction of the pigments was carried out using 55% isopropanol with a hydromodule 40. The practical yield and composition of the pigments are shown in table. one.

Example 11. Obtaining hypericin and pseudohypericin was carried out according to examples 1-6, but the microwave extraction of the pigments was carried out using 55% ethanol, but with a hydraulic module 48. The practical yield and composition of the pigments are shown in table. one.

Example 12. Obtaining hypericin and pseudohypericin was carried out according to examples 1-6, but the microwave extraction of the pigments was carried out using 55% isopropanol with a hydromodule 48. The practical yield and composition of the pigments are shown in table. one.

Example 13. The preparation of hypericin and pseudohypericin was carried out according to examples 1-6, but microwave extraction of the pigments was carried out using 55% ethanol with a hydromodule 40 with heating to 70 ° C for 46 minutes. The practical yield and composition of the pigments are shown in table. one.

Example 14. Obtaining hypericin and pseudohypericin was carried out according to examples 1-6, but the microwave extraction of the pigments was carried out using 55% alcohol with a hydromodule 40 with heating to 60 ° C. The practical yield and composition of the pigments are shown in table. one.

Example 15. Obtaining hypericin and pseudohypericin was carried out according to examples 1-6, but 124 g of spotted hypericum herbal flour (Hypericum perforatum L) was used as raw material. The practical yield and composition of the pigments are shown in table. one.

Example 16. Obtaining hypericin and pseudohypericin is carried out according to examples 1-6, but the microwave extraction of the pigments is carried out in the mode of pulse-width modulation of microwave radiation with a frequency of 2450 MHz with a specific power of 55.5 W / dm ³ three times with a hydraulic module 40 with heating the suspension of raw materials extractant up to 66 ° C in 29 min. The practical yield and composition of the pigments are shown in table. one.

Example 17. Obtaining hypericin and pseudohypericin is carried out according to examples 1-6, but instead of filtering the extract of the target pigments, centrifugation of the suspension of the raw material-extract on a T230 centrifuge for 10 minutes at a rotational speed of the centrifuge rotor up to 10000 rpm is used.

The supernatant is drained from the precipitate, combined to obtain 15 l of the total extract and concentrated in vacuo. The practical yield and composition of the pigments are shown in table. one.

Thus, the proposed object allows to simplify the technology for the production of target substances - hypericin and pseudohypericin due to the rejection of the use of highly toxic chloroform, reduce the duration of all technological stages of the method, reduce the cost of electricity and labor compared with the analogue and prototype, increase the environmental safety of the production of target pigments.

Claims (1)

A method of producing hypericin and pseudohypericin from the raw plant phytomass of St. John's wort and St. John's wort perforated, including grinding of plant materials, degreasing of raw materials with an organic solvent, separation of the extract of lipophilic substances of plants, drying of raw materials, re-extraction of fat-free raw materials with aqueous solutions of an organic solvent, filtering the obtained extracts, concentration of extracts , chromatographic separation of the target pigments on a column containing silica gel using taking eluents containing an organic solvent and water, concentrating the obtained eluate fractions, combining the fractions, crystallizing hypericin and pseudohypericin using solutions of a polar organic solvent in non-polar, drying the finished pigments, characterized in that the aerial mass of plants Hypericum perforatim L. and Hypericum maculatum Crantz. collected in the phase of mass budding and the beginning of flowering, pre-degreased with an organic solvent of hexane and acetone with a hexane: acetone ratio of 8-9: 2-1 or pet Oleina ether and acetone in a ratio of petroleum ether and acetone 8-9: 2-1, respectively, with liquor ratio of 5 for 5 minutes, three times sequentially extracted by infusion of 40-55% alcohol of formula C _n H _2n OH, where n = 2 or n = 3, with a hydraulic module 40-48 for 29-46 minutes, the process is carried out in a microwave chamber under conditions of pulse-width modulation of microwave radiation with a duty cycle of pulse packets of 1.3, a duration of microwave pulse packets of 23 seconds, a radiation frequency of 2450 MHz, with a specific power of 41.6 W / dm ³ with heating the suspension of the raw material-extractant to 66 ° C with periodic by stirring the suspension, the extract is separated by filtration or centrifugation and evaporated in vacuo to dryness at a temperature of 50 ° C with simultaneous irradiation of the extract with a 95 W incandescent lamp, then the extract is subjected to precipitation on silica gel with a functional pore coating of dimethyl octadecylsilyl groups at a dry residue-sorbent ratio of 1: 5 s subsequent drying in a vacuum of 0.05-0.08 Pa, then the precipitate is placed in a flash column for flash chromatography on top of a sorbent with a dispersion of 40-64 microns and naphthodianthrone pigments are eluted with water ethyl alcohol solutions with a gradient of alcohol concentration from 5% to 60%, crystallization of hypericin and pseudohypericin is carried out from ethanol-water mixture in a vacuum crystallization mode with simultaneous concentration of the mother liquor on a rotary evaporator in vacuum with a residual pressure of 33-13 kPa (25-10 mm Hg), drying of hypericin and pseudo-hypericin is carried out in vacuum at a temperature of 60 ° C.

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