RU2372080C1 - Method of rapid purification of dihydroquercetin from polymeric forms - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to engineering and purification of vegetable bioflavonoids. A solution of dihydroquercetin is passed through filters with diametre of not less than 10 cm with pore size from 0.05 to 0.45 microns at 75 to 150 Pa pressure with subsequent concentration of the filtered solution and regeneration of the filters by passing 0.1N hydrochloric acid in reverse direction.

EFFECT: invention realises said function.

1 ex, 1 tbl, 3 dwg

Description

The invention relates to a technology for the isolation and purification of plant bioflavonoids, in particular to a technology for the purification of dihydroquercetin (DHA). The method is aimed at the implementation of quick and effective purification of DHA preparations from high molecular weight polymers (VMP) and their aggregates. It is realized through the use of millipore filters with various pore sizes - from 0.05 to 0.45 microns in the final stages of purification. The method is carried out by passing DHA solutions through filters under pressure up to 75-150 pascals. Filter sizes can vary from a few centimeters to several tens of centimeters. Among the filters suitable are those whose material has a minimum sorption of the monomeric form of DHA and the maximum polymer. The method includes passing a DHA solution through a filter at a certain ratio between the concentration of DHA and the filter area according to a given technology, after which the collected solution is concentrated and the filters are regenerated. Filter regeneration is achieved by acid treatment - passing dilute acid solutions through the filters in the opposite direction to the original one. The use of millipore filters in the final stages allows efficient express cleaning of the DHA fraction from its high molecular weight polymer forms. The invention allows to obtain a better target product, free from VMP.

DESCRIPTION OF THE INVENTION

The invention relates to a technology for the isolation and purification of plant bioflavonoids, and in particular to a technology for the purification of dihydroquercetin (DHA). The process of purification of bioflavonoids requires special techniques aimed at removing impurities, polymer forms, preventing spoilage and oxidation processes, as well as the self-polymerization of DHA during purification.

DHA refers to bioflavonoids, found mainly in higher plants, especially in large quantities - in larch. DHA has a very high antioxidant activity. It is not inferior in effectiveness to such powerful antioxidants as α-tocopherol, vitamin E and β-carotene (Teselkin Yu.O., Zhambalova B.A., Babenkova I.V., Klebanov G.I., Tyukavkina N.A. / / Biophysics. 1996. T.41. No. 3. S.620-623). DHA protects plant cells from free radicals and slows down aging. In tree trunks, DHA is in both monomeric and polymerized form.

DHA used in dietary supplements and medicines has a positive effect on a wide range of physiological processes, inhibits the development of allergies, arthritis, atherosclerosis, cancer, etc. (Uminsky A.A., Havsteen B.Kh., Bakaneva V.F. Biochemistry of flavonoids and their significance in medicine. Pushchino: Photon-Century. 2007).

Due to the strong tendency of DHA to the formation of polymer and aggregated forms with much lower biological activity, one of the main problems is the difficulty in obtaining a pure monomeric form, free of polymer forms and impurities. The fraction enriched with DHA is usually isolated from Siberian larch wood (Larix sibirica). Using extraction with alcohol and ether solvents (usually in the presence of acids, at a pH of 3-3.5) and subsequent multi-stage treatment, it is possible to obtain DKG single crystals and achieve a fairly high degree of purification from impurities - up to 96-98% (Savvateev AM Quality assessment and standardization of compositions on the basis of dikvertin. Abstract of the academic title of Candidate of Pharmaceutical Sciences. M., 2007). In industrial production, purification from impurities usually does not exceed 91-95%. In most cases, however, the contribution of the polymer forms of DHA itself remains unknown.

The covalent polymerization of DHA is very active both during the life of the plant cell and during the long-term extraction of DHA and its storage. The presence of polymeric forms of DCG in the resulting preparations is undesirable for two reasons: a) it is excess ballast, b) it is an adsorbent capable of lowering the active concentration of DHA itself (monomeric form) and other active substances (when used as part of dietary supplements and drugs) - due to their sorption on polymers.

Methods for controlling the degree of DKG purification, including chromatography, are laborious and expensive, which leads to a very high cost of DHA preparations. On the Russian market, 1 kg of purified DHA is sold at a price of 20 thousand rubles. up to 100 thousand rubles, depending on the degree of purification.

Methods for controlling the purification of DCG are not sensitive enough, especially when it comes to the detection of micromolar amounts of polymers. Currently, there is an acute need for highly sensitive express methods that can reliably detect and quickly eliminate polymer forms when receiving DHA preparations and their storage.

Recently, the use of two express methods was proposed for the detection of polymer DKG macromolecules (NL Vekshin // Applied Biochemistry and Microbiology. Vol. 44, 2008, in press): UV spectrophotometry (in combination with filtration) and Rayleigh light scattering (with using a spectrofluorimeter with crossed monochromators). It was these methods that, within the framework of the claimed invention, quantitative measurement of DHA polymers was carried out and their presence or absence after filtration during testing was controlled.

The test of the methodology was carried out at a DHA, which, according to the certificate, has 96% purification from impurities (LLC Flavena, Pushchino). The initial concentrated DHA solution (0.5 g / l or 1 g / ml) in distilled water was diluted twice, half of this solution was doubled again, etc. The obtained samples were used to measure optical density and light scattering. All samples, including concentrated ones, were transparent, obscure, i.e. large aggregates "by eye" were not found.

The concentration of DHA was determined by weight and UV photometrically at 328 nm in quartz 1 cm cuvettes in a standard compartment on an M-40 spectrophotometer (Germany). The contributions of the ionized (monomeric) form and the non-ionized monomeric and polymer forms were from the absorption spectra in the range of 250-400 nm.

The intensity of weak Rayleigh light scattering was measured at right angles on a SLM-4800 spectrofluorimeter (USA) with crossed monochromators (slits of 4 nm) installed at a wavelength of 530 nm. This highly sensitive method of measuring scattering was recently used to detect aggregates of denatured insulin (Vekshin NL // Biochemistry. 2008. V.73. No. 4, in press). DHA solutions were placed in standard transparent 1 cm cuvettes at a temperature of 17 ° C. Each point was recorded in a 6-second accumulation mode.

VMP was removed by filtering the DHA solution through millipore filters from various materials (Millex GV, Millex HP, Millex GP, Flow-pore-D, FP, etc.) with four characteristic pore diameters: 0.45 microns, 0.22 microns, 0.1 microns and 0.05 microns.

DHA preparations in aqueous solution had two intense UV absorption bands — with peaks in the region of 290 and 328 nm (FIG. 1). Based on the well-known Bouguer-Baer law of light absorption (Vekshin NL Fluorescence spectroscopy of biopolymers. Pushchino: Photon-Century, 2006), it was determined that the molar extinction coefficient of DHA at 328 nm is 2 × 10 ⁴ M ^-1 cm ^-1 . This band belongs entirely to those DHA molecules where phenolic hydroxyl groups are ionized (Savvateev AM Author. Abstract. Cand. Sc. Candidate of Pharmaceutical Sciences. M., 2007). The band at 290 nm belongs to non-ionized DHA (Savvateev AM, ibid.). Moreover, it should be noted that at 290 nm not only monomeric but also polymeric forms of DHA are absorbed, because polymerization is carried out mainly due to covalent binding through phenolic hydroxyl groups.

The band at 328 nm belongs predominantly to the monomeric form of DHA, and the band at 290 nm belongs to both monomeric and polymeric. Indeed, when filtering a DKG solution through millipore filters with different pores, the band intensity at 290 nm decreases noticeably, while the band intensity at 328 nm does not change or changes little. For example, in the case of filtering a solution of 94 μm DKG through a 0.45-micron Flow-pore-D filter, the band intensity at 290 nm was halved (Fig. 1), and when using a 0.1-micron filter, it was almost tripled (see table ) compared to the original. The filtering process through a 0.45 micron filter takes about 1 minute.

Purification of DHA from VMP using millipore filters with different pore sizes Filter pore diameter (μm) D at 290 nm D at 328 nm Cleaning from VMP (%) Filtration Time (min) before and after before and after 0.05 1.72 0.6 0.98 0.92 97 four 0.1 1.72 0.7 0.98 0.94 89 3 0.22 1.72 0.8 0.98 0.95 80 2 0.45 1.72 0.9 0.98 0.96 72 one

Thus, the use of millipore filters allows efficient express purification of the DHA fraction from its polymer forms.

From the ratio of the areas of the bands at 290 and 328 nm and their changes after filtration, it follows that in the used DKG preparation the amount of polymers is very large contrary to the 96% degree of purification declared in the certificate. The proportion of polymers larger than 0.45 microns is at least 30%, and more than 0.1 microns is another 10% of the total amount of DHA.

The degree of filtration purification of DHA from VMP was found from the data on the change in optical density (D). Filtration through a 0.05- or 0.1-micron filter gives a significantly greater degree of purification from HFMP than through 0.45-micron, or 0.22-micron, but takes a little longer (see table).

It must be emphasized that the size of the PMF is the length of the polymer. The thickness and width of the polymer macromolecule of DHA are negligible (of the order of 10 Å). Indeed, all the solutions were obscure, completely transparent (the spectral lines in Fig. 1 in the region of 380-400 nm, where DHA does not absorb, are close to the abscissa axis), i.e. large quantities of polymer particles and aggregates with an effective diameter of more than half the light wavelength are not detected spectrophotometrically.

However, aggregates in the preparation of DHA are still available (in small quantities). This is evidenced by the fact that when diluting the DHA solution (pre-filtered through a 0.45 micron filter) 2.5 times, the ratio of optical densities at 290 and at 328 nm decreases to a different extent (Fig. 1): the band at 328 nm decreases by 59%, and at 290 nm - only by 41%. Units make up (under given conditions and concentrations) about 5-10% of all DHA molecules. It is difficult to determine the contribution of aggregates with greater accuracy, since the extinction coefficients of DHA in polymer macromolecules and their aggregates are unknown. In such macromolecules and their aggregates, screening hypochromism usually occurs - a decrease in the average extinction coefficient per chromophore due to the competition of stacked chromophores for a photon, covering them with one wave train (Vekshin N.L. Fluorescence spectroscopy of biopolymers. Pushchino: Photon- century, 2006).

By measuring Rayleigh light scattering by a highly sensitive method — on a spectrofluorimeter with crossed monochromators — it is possible to detect polymer and aggregated forms even at very low concentrations. In the region from 3 to 94 μM DHA, there is an almost linear relationship between the concentration of the substance and the light scattering intensity (Fig. 2). It must be emphasized that Rayleigh scattering is absent on the monomeric DKG molecules, because the size of these molecules is many hundreds of times smaller than the wavelength of light. On polymer macromolecules and their aggregates, if they have an effective diameter of more than half the wavelength, Rayleigh light scattering takes place.

It should be noted that the measurements of light scattering on a spectrofluorimeter were not complicated by fluorescence, because DHA practically does not fluoresce (unlike its closest analogue of quercetin). In addition, at 530 nm, the DHA does not have an absorption band at all.

With an increase in the concentration of DHA over 94 μM, a slight decrease in light scattering occurs (Fig. 2). At first glance, this paradoxical fact is explained by the fact that, at high concentrations, the macromolecules of polymer DHA begin to “stick together” in pairs, i.e. the amount of scattering particles decreases.

Figure 2 shows the average values of the light scattering intensity (each point obtained by averaging at least 30 values) of the polymers and aggregates of DHA contained in the solution. In highly sensitive measurements, some values actually “fell out”: they were approximately 1.5–2 times higher or lower than average. Such a fluctuation spread (Fig. 3) is caused by the entry into a narrow light beam passing through the cuvette of too large (although not numerous) polymer aggregates having a size of the order of 0.1-1 mm. The strongest and largest fluctuations were observed in concentrated solutions.

After filtering the DHA solution, the light scattering intensity and fluctuation amplitude decrease sharply. The maximum effect occurs when using a filter with a minimum pore size of 0.05 microns. An example is shown in FIG. 3, where the bottom curve is the light scattering intensity after filtering a 0.94 μM DHA solution through a 0.05 micron filter. The filtering process took about 4 minutes.

Thus, by filtering DHA solutions through millipore filters from certain materials with a fixed pore diameter, it is possible to carry out effective express purification from VMP.

With regard to the extraction and purification of DHA, for example, such copyright certificates and patents are known:

- RF patent 2000797. A method for the isolation of dihydroquercetin / V.A. Babkin, N.A. Tyukavkina, L.A. Ostroukhova and others // BI 1993.37 38.

- RF patent 2034559. Method for the isolation of dihydroquercetin / Babkin V.A., Svyatkin Yu.K., Ostroukhova L.A., Glazyrin V.V., Vorobev V.G., Tyukavkina N.A. - BI. - 1995. - N13.

- RF patent 2158598. A method of producing dihydroquercetin / V.A. Babkin, L. A. Ostroukhova, D. V. Babkin, Yu. A. Malkov // BI 31.2000.

- RF patent 2091076. BI No. 27. 1997. A method of producing dihydroquercetin. / Tyukavkina N.A., Khutyoryansky V.A., Bazhenov B.I.

- RF patent 2180566. BI No. 8. 2002. / Nifantiev E.E., Koroteev M.P., Kaziev G.Z., Uminsky A.A.

Consider in detail patents, which are close analogues:

1) RF patent 2308267. Application 2006113632/15. Publ. 2007.10.20. A61K 31/35 (2006.01) METHOD FOR ISOLATING BIOLOGICALLY ACTIVE DIHYDROQUERCERETINE ISOMERS. Tikhonov V.P., Kolesnik Yu.A., Shmatkov D.A. etc. The crushed larch wood is extracted with 90-95% ethyl alcohol at 55-66 ° C for 1-2 hours, the extract is filtered, propylene glycol is added, the mixture is evaporated, the obtained residue is treated with 5% sodium chloride solution at 20-30 ° C, the resulting resin is separated. The resulting flavonoid complex is eluted from the column with ethyl alcohol, the eluate is evaporated, precipitated with water cooled to + 2 ° C, the precipitated precipitate is crystallized, filtered and dried. The resulting product, which is a bioflavonoid complex, is crushed, followed by dissolution in a mixture of methyl alcohol: water (10:90) and chromatographed in reverse phase, using as a mobile phase a solution of cyclodextrin in a mixture of methyl alcohol: water (10:90) at cooling the column to 0 ° C. The collected fractions of the individual enantiomers were evaporated and dried. The invention improves the degree of purification of the complex and isolate the individual enantiomers of dihydroquercetin. The disadvantages of this method are: a long duration and multi-stage process, the complexity of chromatography, the use of a toxic solvent - methyl alcohol, low productivity, lack of control over the presence of small amounts of VMP in the resulting preparation.

2) RF Patent 2258525. Application 2004101747/15. Application submission date 2004.02.05. Publ. 08/08/20. A61K 35/78 A61K 31/33 A61K 31/343 A61K 31/353 C07D 301/00. METHOD FOR CLEANING DIHYDROQUERCERETINE AND DYHYDROCEMPHEROL FROM BIOPHLAVANOID EXTRACTS OBTAINED IN THE PROCESS OF WOOD PROCESSING. Shilov E.A., Katasonov A.B. The method of producing dihydroquercetin and dihydrocemferol from larch wood extract containing bioflavonoids is characterized in that ethyl acetate and water taken in a specific ratio are dissolved in an organic solvent, freeze-dried extract is applied, then the resulting solution is applied to a column filled with a sorbent - urea, previously balanced with ethyl acetate in a certain ratio of sorbent to sample; the column is eluted with an ester and / or ketone with a boiling point below 120 ° C or ethyl acetate and acetone taken in a specific ratio; the resulting eluate is collected, evaporated before crystallization begins and cooled, the precipitated crystals are washed and dried. The method allows for a one-step process for the separation of dihydroquercetin and dihydrocemferol from impurity resins and other polymer-like compounds, to reduce the cost of the process of producing dihydroquercetin and dihydrocemferol. Although the method allows you to separate the two necessary substances from impurity resins and polymers, the disadvantages of this method are: the duration and the complexity of the column separation, the use of urea and toxic volatile solvents, as well as the lack of monitoring for the presence of VMP of the DHA itself in the resulting preparation.

3) Patent of the Russian Federation 2114631. Application 97111748/14. Application Date 1997.07.22. Publ. 1998.07.10. A61K 35/78 C07D 311/32. METHOD FOR ISOLATING DIHYDROQUERCETICINE. Tyukavkina N.A., Khutyoryansky V.A., Bazhenov B.N. etc. The method is carried out by pre-treating larch wood with boiling water, followed by extraction with an aqueous solution of acetone and purification of the water-acetone extract by liquid chromatography on reversed-phase sorbents of the spherisorb C2-C18 type with a particle size of 5-10 μm using 30 as the mobile phase 50% solution of acetone in a 0.1% aqueous solution of trifluoroacetic acid and the removal of acetone by evaporation. The technical result consists in obtaining dihydroquercetin of the highest degree of purification, more than 99.5%. The disadvantages of this method are: the long duration and multi-stage process, the complexity of chromatography and the high cost of sorbents, the use of toxic trifluoroacetic acid and volatile acetone, the lack of monitoring for the presence of VMP of the DHA itself in the resulting preparation.

4) Patent 2279284. Application 2005106082/15. Application submission date 2005.03.04. Publ. 2006.07.10 A61K 36/15 (2006.01) METHOD FOR INTEGRATED PROCESSING OF LARCH WOOD. Berezhnoy A.G., Savvin A.V., Gavrilov A.B. A method of complex processing of larch wood, characterized in that larch wood with a certain relative humidity is crushed in the presence of ethyl alcohol to a certain particle size, the obtained crushed pulp is extracted with ethyl alcohol under certain conditions, then the solid and liquid phases are separated mechanically, and the solid and liquid phases are separated mechanically from the latter ethyl alcohol with dihydroquercetin dissolved in it, and the liquid phase is filtered, then evaporated, from the resulting concentrate is isolated a mixture of essential oils and fatty organic acids for use in the cosmetic industry, and the remainder of the organic phase is recovered dihydroquercetin. This method allows to increase the yield of dihydroquercetin and to ensure the most complete use of the feedstock, to simplify the process flow diagram. Too simplified scheme of this method leads to not too high purification from impurities, and the method does not allow to achieve real purification from the polymers of the DHA itself. In such preparations, the proportion of its VMP, as was shown in the experiments (see above), is at least 40%.

The closest analogue (prototype) may be the method described in paragraph 5 (5th embodiment of the method) of the RF Patent 2003111011. Application 2003111011/04. Application submission date 2003.04.18. Application publication date 2004.11.20 C09F 1/00 C08B 37/00 C07D 311/40. METHOD FOR COMPREHENSIVE PROCESSING OF LARCH WOOD, METHOD FOR ISOLATING BIOFLAVONOIDS AND METHOD FOR ISOLATING ARABINOGALACTANE OBTAINED IN THE PROCESS OF INTEGRATED PROCESSING. Uminsky A.A., Uminskaya K.A., Pisarev R.P., Beloev T.T. …5. A method of isolating bioflavonoids by treating a hydrocarbon-ether phase obtained by complex processing of larch wood, followed by crystallization of bioflavonoids, characterized in that the aqueous phase of bioflavonoids is used, which is a product of the complex treatment of larch wood according to claim 1, obtained by absorption of hydrocarbon the ethereal liquid phase of the extract with deionized water at a temperature of from 90 to 94 ° C in an inert gas stream, and a baromembrane membrane is successively carried out filtering the aqueous phase of bioflavonoids at a temperature of from 90 to 94 ° C through porous filters with capillary holes with a diameter of 0.005-0.050 μm, purifying the filtrate on a carbon-absorbing porous layer at an excess pressure of 0.5 to 6.0 atm, evaporating and crystallizing bioflavonoids in the form of crystalline hydrate dihydroquercetin, dihydrocampferol and naringenin, followed by recrystallization from alcohol and water phases. A good solution in this method is the use of porous filters with fixed pore sizes, which can significantly reduce the number of VMP in the preparation. The disadvantages of this method are: the duration and multi-stage process, the complexity, the use of high temperature (which can damage DHA and lead to its polymerization, directly during cleaning), the need to work in an inert gas stream (which complicates the work and increases the cost of the drug), obtaining a mixture of three substances, rather than pure DHA, lack of control over the presence of polymers of DHA itself. Filtration by this method has a significant drawback: the lack of quantitative control and the removal of those VMPs that formed at the final stage at high temperature. The share of VMP in the preparation obtained in this way is (according to the applicant) not less than 10%.

The disadvantage of all these methods is to obtain insufficiently purified (and therefore not enough quality) target product containing a noticeable amount of polymer forms.

In contrast to the specified prototype and the above analogues, the technical result of the invention allows to quickly (in 1-4 minutes) to obtain a better target product. Previously, the author of this application used an approach with millipore filters at the final stage of purification of the smallest biological organelles - protomitochondria (Shishmakov D.A., Anisimov R.L., Vekshin N.L. // Biol. Membranes, 2004, v.21, N5 , p. 389-395). A similar approach has now been applied by the applicant in relation to the purification of DHA from VMP, and, as was presented above, two methods for monitoring VMP in trace (micromolar) quantities have been developed.

The method is implemented as follows.

Initially purified by one of the usual methods, a DHA solution in a concentration of about 1 g / l is passed through millipore filters with a diameter of several centimeters to several tens of centimeters with pore sizes from 0.05 to 0.45 microns under pressure up to 75-150 pascals, and then carry out concentration of the collected solution and regeneration of the filters by passing the diluted acid in the opposite direction.

The proposed method allows to obtain the target product - the preparation of DHA with improved properties, almost free of VMP.

Example

Obtaining DHA, almost free of VMP

1) Prepare an aqueous solution of initially purified from impurities of DHA at a concentration of 1 g / l.

2) Put the solution into a large syringe with a volume of 1 liter.

3) Fit a standard Milllex GV millipore filter nozzle with a diameter of at least 10 cm onto the syringe head or on a special adapter of a suitable size.

4) Firmly tighten the filter head.

5) Apply a pressure of up to 75 pascals to the syringe piston and at the same time collect the leaking filtrate in a glass container while cooling.

6) After a gradual clogging of the filter (indicated by the increase in pressure necessary for filtration), stop filtering and, using another syringe filled with the same volume of 0.1 N. hydrochloric acid, rinse the filter head in the opposite direction.

7) Rinse the filter with water (in any direction).

8) Use the regenerated filter repeatedly.

9) Hydrolyze the polymer solution in acid collected by washing with a filter and then reuse it to obtain monomeric DHA.

10) The collected filtrate (containing DHA free of HFMP) is evaporated in a usual manner until dry and stored in the cold.

From 1 g of DHA initially purified from extraneous impurities, 0.5 to 0.95 g of DHA free from VMP can be obtained. In the case of industrial cleaning, where tens and hundreds of kilograms of extract are usually used, it is only necessary to increase the volume of solutions and the area of filter nozzles.

Thus, the proposed method improves the consumer qualities of the target product. This method is fully compatible with traditional technology, becoming its one of the most important final stages.

Claims (1)

A method of producing dihydroquercetin free of high molecular weight polymers, comprising passing a dihydroquercetin solution through filters of at least 10 cm in diameter with pore sizes from 0.05 to 0.45 μm under pressure up to 75-150 Pa followed by concentrated filtered solution and regenerating the filters by passing 0, 1 n hydrochloric acid in the opposite direction.

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