RU2769981C1 - Composite material based on hydrolyzed lignin and triterpene saponin - Google Patents

Abstract

FIELD: composite materials.SUBSTANCE: invention relates to a method for producing a composite material based on hydrolyzed lignin and saponin and the composite material itself, which can be used in the pharmaceutical industry, in drug manufacturing technology. The method for obtaining the composite material includes bringing the sorbent that is hydrolyzed lignin into contact with a solution of the immobilized substance, triterpene saponin, holding it under stirring at room temperature for 4 hours at a concentration of an aqueous solution of triterpene saponin 0.8-1 g/l; drying of the resulting composite material is carried out in air for 2 days. The sorption process takes place either under dynamic conditions when a triterpene saponin solution is passed through hydrolyzed lignin at the rate of 1 l / g of hydrolyzed lignin at a rate of 1 ml / min, or under static conditions, with the diameter of the sorption column 1-1.5 cm, the height of the sorbent layer 2.5-3.5 cm, at the rate of 1 l/g of hydrolyzed lignin. As a result, a composite material is obtained in which triterpene saponin is immobilized on lignin in an amount of at least 56% of the sorbent mass.EFFECT: expanding the range of composite materials based on hydrolytic lignin with a high sorption capacity for triterpene saponin.2 cl, 3 dwg, 4 ex

Description

The invention relates to biotechnology, in particular to a method for producing a combined dosage form of hydrolytic lignin and saponin, and can be used in the pharmaceutical industry in drug manufacturing technology.

Triterpene saponins isolated from plants make up a large group of medicinal substances: preparations of ginseng, Manchurian aralia, horse chestnut, calendula, etc. are widely known. Pharmacological studies have revealed a wide range of pharmacological actions: antitumor, anti-inflammatory, analgesic, antipyretic and gastroprotective. Other known pharmacological properties are antinematode, antidiabetic, cytotoxic and immunostimulatory activity, as well as protection against cytotoxic damage [SonawaneS. M. AReviewofRecentandCurrentResearchStudiesontheBiologicalandPharmalogicalActivitiesofSapindusMukorossi I S. M. Sonawane, N. Sonawane // IntemationalJoumaloflnterdisciplinaryResearchandInnovations. - 2015. - Vol. 3, no. 4. - P. 85-95].

It is known that many compounds of this class have significant growth-stimulating activity. Triterpene glycosides have a great influence on the permeability of plant cells, which is associated with their surface activity. It has been established that their certain concentrations accelerate the germination of seeds, the growth and development of plants, and concentrated solutions slow down, i.e. their action resembles the action of growth hormones [FaizalA., GeelenD. Saponinsandtheirroleinbiologicalprocessesinplants // Phytochem. Rev. 2013. Vol. 12. P. 877-893].

The molecular structure of saponins can undergo chemical transformations during storage or processing, which, in turn, can change their pharmacological properties. The glycosidic bond and bonds between carbohydrate residues can undergo partial hydrolysis under conditions close to neutral pH, resulting in the formation of aglycones, prosapogenins, carbohydrate chains or monosaccharides.

Immobilization on the surface of sorbents will prevent their destruction with the preservation of functional properties. Recently, the method of clathriding (complex formation) of sorbents with natural substances has gained particular popularity. Medicinal compositions created in this way make it possible to preserve the integrity of the molecular structures of medicinal substances in various body media, while maintaining their pharmacological properties.

Lignin can be used as a matrix carrier for glycosides. Lignin is an organic heterochain oxygen-containing polymer that lacks a single type of bond between monomer units. The structural units of lignin contain various polar groups, including ionizable (acidic) phenolic hydroxyls and a small number of carboxyl groups, as a result of which lignin is a polar polymer exhibiting the properties of a polyelectrolyte. In lignin, due to the presence of a large number of hydroxyl and other polar groups, hydrogen bonds (intra- and intermolecular) are significantly developed. Due to the high degree of branching, lignin macromolecules have a globular shape and such preparations are powders.

Hydrolytic lignin (HL) has a highly developed inner surface and exhibits significant sorption properties, which serves as a prerequisite for obtaining enterosorbents on its basis, as well as using it as a matrix for the immobilization of biologically active substances.

Known composite materials based on lignin and polyacrylonitrile [Patent RU 2621758, composite material based on polyacrylonitrile with lignin, authors - Shtyagina L.M., Lysenko A.A., Sverdlova N.I., Sazanov Yu.N., Vinogradova L. E., Krutov S.M.], based on dimethylhydrazine [M.P. Semushina, K.T. Bogolitsin, A.Yu. Kozhevnikov, D.S. Kosyakov / Sorption properties of technical lignins in relation to 1,1-dimethylhydrazine // Chemistry for Sustainable Development. - 2013. - V. 21 - p. 551-555.], radioactive strontium [Nikiforov A.F., Yurchenko V.V. Sorption of radioactive strontium from aqueous solutions by modified hydrolytic lignin // Sorption and chromatographic processes. 2010. Vol. 10. Issue. 5. - S. 676-684]. The proposed composition of lignin with polyvinylpyrrolidone in the form of tablets [Application 99107824/14 RF, class. A61K 35/78, Appl. 04/12/99, publ. February 20, 2001].

The closest analogue is a composition for the preparation of wet powder or wet granules of lignin with lactulose, used for the prevention and treatment of diseases of the gastrointestinal tract [Patent 2167669 RF, cl. A61K 35/78, 9/14, Appl. 05/11/99, publ. May 27, 2001]. The technical result is to obtain new dosage forms of hydrolytic lignin, allowing to avoid complications caused by the intake of lignin and to treat diseases of the gastrointestinal tract. The mixture of substances is prepared as a wet powder or wet granules. The composition contains 80% wt. hydrolytic lignin and 20% wt. 45% aqueous solution of lactulose. The resulting mixture is dried in a thermostat to a moisture content of 50% and packaged in bags. Also, the second way to obtain a composite form is to mix wet lignin powder (consisting of particles of 0.25-0.55 μm), taken by weight in an amount of 90% and 10% by weight. 45% lactulose solution. The mixture is dried to a moisture content of 70%. The disadvantage of this method is the inability to assess the ratio immobilized on lignin and free forms of lactulose, as well as the low content of lactulose in the composition of lignin. The method of obtaining this composition is based on mechanical mixing of the components in a rotary mixer.

The prototype of the present invention is [Patent 2680600 RF, class. A61K 33/44, C12N 11/06, C12N 11/14, Appl. 12/27/2017, publ. 02/25/2019] a composite material based on ordinary coal activated at a temperature of 900-1000°C, characterized in that it additionally contains triterpene saponin immobilized on coal in an amount of at least 30% by weight of coal.

At the same time, a method for obtaining this composite material is proposed, including bringing the sorbent into contact with a solution of the immobilized substance, drying, where the solution of the immobilized substance is an aqueous solution of saponin with a concentration of 0.1-0.25 g/l, and the sorption process takes place either under dynamic conditions at passing the solution through the sorbent at the rate of 2.5–3 l/g of the sorbent at a rate of 0.25–0.5 ml/min, or under static conditions at the rate of 1–2 l/g of the sorbent, keeping for at least 4 hours at room temperature. The disadvantages of the material include low sorption capacity for saponin.

The objective of the invention is to develop a method for producing a combined preparation based on a saponin-lignin complex.

The technical result consists in expanding the range of composite materials based on hydrolytic lignin with a high sorption capacity for triterpene saponin, at least 56% by weight of the sorbent.

The technical result is achieved by the fact that in the method for obtaining a composite material based on hydrolytic lignin and triterpene saponin, including bringing the sorbent into contact with a solution of the immobilized substance, drying, according to the invention, the sorption process takes place either under dynamic conditions, when the solution of triterpene saponin is passed through hydrolytic lignin from calculation of 1 l / g of hydrolytic lignin at a rate of 1 ml / min, or under static conditions, while the diameter of the sorption column is 1-1.5 cm, the height of the sorbent layer is 2.5-3.5 cm, at the rate of 1 l / g of hydrolytic lignin; the resulting composite material is kept under stirring at room temperature for 4 hours; the concentration of an aqueous solution of triterpene saponin is 0.8-1 g/l; drying of the composite material is carried out in air for 2 days; the sorption capacity of the resulting composite material for saponin is at least 56% by weight of the sorbent.

The use of saponin immobilized on lignin will change the release rate of the glycoside and preserve the integrity of its structure.

In preliminary studies, it was found that the basis of the resulting supramolecular structures containing medicinal compounds and to maintain the basic pharmacological effects should be not covalent interactions, but hydrogen inter- and intramolecular, as well as dispersion bonds (weak interactions). However, in the work [Sorption of radioactive strontium from aqueous solutions by modified hydrolytic lignin/ Nikiforov A.F., Yurchenko V.V. // Sorption and chromatographic processes. 2010. Vol. 10. Issue. 5] found that the surface of hydrolytic lignin particles in the pH range of 1–4 has a positive charge. In the pH range of 4-11, the surface of lignin particles is negatively charged. Therefore, the pH value approximately equal to 4 corresponds to the zero value of the ξ-potential of the surface of lignin particles (isoelectric point). Thus, during sorption from an aqueous solution of saponin (pH-4.5), the lignin surface has no charge and ionic interactions with the saponin molecule are unlikely. The figure 1 shows the pH values of an aqueous solution of saponin in contact with lignin (2). The behavior of the curve similar to that of the aqueous solution (1) indicates the absence of ionic interactions during the sorption absorption of saponin by lignin.

An example of the formation of lignin-based complexes using hydrogen bonds is the "hardwood lignin - hemicellulose" complex described in [Physical chemistry of lignin: monograph / [K.G. Bogolitsyn and others; ed. K.G. Bogolitsyn and V.V. Lunin; Federal Agency for Education, Arkhangelsk State Technical University - Arkhangelsk: Arkhangelsk state. technical university, 2009. - 489 p.] The lignocarbohydrate matrix obtained in the work is a superposition of interpenetrating networks formed by hydrogen bonds. In coniferous wood, lignin consists mainly of units of the coniferyl type, and hemicelluloses of galactose, mannose, and xylose. If we compare the obtained data on the affinity of coniferyl alcohol for cellulose and hemicellulose precursors, then it is much higher for galactose, mannose and xylose than for glucose. The composition of hemicelluloses includes: D-fructose, D-xylose, D-galactose, D-mannose, L-arabinose, L-rhamnose, D-glucose, D-galacturonic and 4-O-methyl-D-glucuronic acids, which are present in the form of side branches.

A similar monosaccharide composition is also present in the saponin molecule. Therefore, it can be assumed that its binding to lignin will be due to Η-bonds between carbohydrate fragments of the saponin molecule and oxygen-containing functional (syringylpropane units) groups of the lignin structure. The high specific surface area of the sorbent (790 m ² /g), as well as the presence of predominantly macropores (500-5000 nm ² ), makes it possible to adsorb saponin molecules both in free form and in the form of close-packed associates of various structures. The result of immobilization is a high sorption capacity for saponin, reaching at least 56%.

IR spectra were taken of samples of lignin in free form and in the form of saponin (Fig. 2a,b). The stretching vibrations at 1608 cm ^-1 assigned to the carboxyl group in the spectrum of lignin in the form of saponin are shifted to 1585 cm ^-1 due to the formation of intermolecular Η-bonds. The aromatic benzene ring, conjugated with double bonds of the carbonyl groups of lignin, absorbs in the region of 1512 cm ^-1 . In the spectrum of lignin in the form of saponin, this band is shifted to 1504 cm ^-1 , probably due to dispersion interactions with the saponin aglycone ring system. The formation of H-bonds is also confirmed by the shift of the band at 1446 cm ^-1 characteristic of the absorption of oxygen-containing groups and the band at 1218 cm ^-1 attributed to the vibrations of the phenol group up to 1425 cm ^-1 and 1215 cm ^-1 , respectively. The presence of a band at 1035 cm ^-1 attributed to skeletal vibrations of the guaiacyl ring, as well as the absence of absorption bands of the syringyl ring, suggest that this lignin is coniferous. The shift of the absorption band from 1035 cm ^-1 to 983 cm ^-1 in the regions assigned to in-plane deformation vibrations of the CH groups of the guaiacyl ring may indicate dispersion interactions between the lignin structure and the saponin aglycone rings.

The formation of weak intermolecular interactions is confirmed by the high degree of sorbent regeneration. Water or a 0.1 M solution of hydrochloric acid can be used as a desorbing agent. At the same time, the degree of regeneration reaches 68 and 63%, respectively. The obtained results on regeneration, i.e. the degree of release of saponin in molecular form, can be used in assessing the biological activity and the maximum therapeutic effect of the resulting dosage forms.

In FIG. 1 shows the dependence of the acidity of the medium (pH) on the concentration of an aqueous solution of saponin, 1 - solution of saponin, 2 - solution of saponin in contact with lignin.

In FIG. 2a shows the IR spectra of 1-lignin, 2-lignin with saponin C=0.05 mg/ml

In FIG. 2b shows IR spectra of 1-lignin, 2-lignin with saponin C=2 mg/ml

In FIG. 3 shows SEM images of a lignin sample with saponin.

Analysis of SEM images of a sample of lignin with adsorbed saponin (Fig. 3) showed the preservation of the fibrous architecture of the surface with dark areas, presumably, the pore space. The surface relief of the enterosorbent is heterogeneous with characteristic bends.

The resulting modified sorbent has a high saponin capacity due to the filling of macropores (500-5000 nm) with saponin molecules and the formation of associates in the sorbent phase. The concentration of glycoside in the pores of lignin leads to dehydration of the latter and a decrease in moisture content from 12 to 5%. The proposed sorption method is simple in execution and does not require preliminary preparation of the sorbent. The optimal ranges of concentrations of working solutions of saponin were determined, which made it possible to reduce the consumption of the substance while maintaining the maximum value of the sorbent capacity.

Example 1. A sample of air-dry lignin weighing 0.5 g is transferred into a glass column with an inner diameter of 1 cm. The height of the sorbent layer was 3.5 cm. 0.5 l of an aqueous solution of triterpene saponin with a concentration of 0.8 g / l. The flow rate is kept constant at 1 ml/min. At the end of the process, the sorbent is removed from the column and dried in air for 2 days. The amount of adsorbed saponin was 0.56 g/g of sorbent. The degree of extraction of saponin from the solution was 70%.

Example 2. A sample of air-dry lignin weighing 0.5 g is transferred into a glass column with an inner diameter of 1.5 cm. The height of the sorbent layer was 2.5 cm. 0.5 l of an aqueous solution of triterpene saponin with a concentration of 1 g is passed through the sorbent layer. / l. The flow rate is kept constant at 1 ml/min. At the end of the process, the sorbent is removed from the column and dried in air for 2 days. The amount of adsorbed saponin was 0.7 g/g of sorbent. The degree of extraction of saponin from the solution was 75%.

Example 3. A sample of air-dry lignin weighing 0.8 g is transferred into a glass column with an inner diameter of 1.5 cm. The height of the sorbent layer was 3.5 cm. 0.8 l of an aqueous solution of triterpene saponin with a concentration of 1 g is passed through the sorbent layer. / l. The flow rate is kept constant at 1 ml/min. At the end of the process, the sorbent is removed from the column and dried in air for 2 days. The amount of adsorbed saponin was 0.78 g/g of sorbent. The degree of extraction of saponin from the solution was 78%.

Example 4. A portion of a sorbent weighing 0.2±0.0002 g in an air-dry state was poured into a conical flask with a ground-in lid with a 200 ml solution of triterpene saponin with a concentration of 1.0 g/L. The contents of the flasks were kept under stirring for 4 hours until equilibrium was established in the system. Then the sorbent was filtered off and dried in air for 2 days. The amount of adsorbed saponin was 0.60 g/g of sorbent. The degree of extraction of saponin from the solution was 60%.

The use of lower concentrations of saponin to achieve the same values of sorption parameters is possible with a significant decrease in the rate of transmission of the solution (under dynamic conditions to 0.2–0.5 ml/min) or an increase in the contact time (under static conditions up to 6–8 hours). An increase in the concentration of saponin makes the process of its extraction from the solution inefficient (the amount of sorbed saponin increases insignificantly).

Claims (2)

1. A method for producing a composite material, including bringing the sorbent into contact with a solution of the immobilized substance, drying, characterized in that hydrolytic lignin is used as the sorbent, and triterpene saponin is used as the immobilized substance, the sorption process takes place either under dynamic conditions by passing a solution of triterpene saponin through hydrolytic lignin at the rate of 1 l/g of hydrolytic lignin at a rate of 1 ml/min, or under static conditions, while the diameter of the sorption column is 1-1.5 cm, the height of the sorbent layer is 2.5-3.5 cm, based on 1 l/g of hydrolytic lignin; the resulting composite material is kept under stirring at room temperature for 4 hours; the concentration of an aqueous solution of triterpene saponin is 0.8-1 g/l; and the resulting composite material is dried in air for 2 days. 2. Composite material obtained by the method according to claim 1, including triterpene saponin, which is immobilized on a sorbent, which is a hydrolytic lignin, in an amount of at least 56% by weight of the sorbent.

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