Method of obtaining pectins with increased content of galactose residues from callus cultures

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the pharmaceutical industry, namely to a method of obtaining pectins from a biomass of cultured tissues of plants Silene vulgaris (M.) G. The method of obtaining pectins with an increased content of galactose residues in side carbon chains from Calusa cultures includes raw material destruction, extraction with water, processing of the biomass with hydrochloric acid, water washing, extraction with ammonium oxalate solution, sedimentation of polysaccharide with ethanol, dialysis and lyophilising, with Caluse cultures being preliminarily grown for 21 hours on an agarised nutrient medium, which contains the ferment 1,4-β-D-galactozyltranspherase in a specified concentration, and as the raw material the biomass of cultured tissues of plants Silene vulgaris is applied.

EFFECT: method makes it possible to obtain physiologically active pectins, which have a specified structure and stable chemical composition.

1 tbl, 4 ex

 

The invention relates to pharmaceutical industry and relates to the receipt of medicines on the basis of pectin polysaccharides.

A method of producing pectin from sea grass (SU 921500 A1, MKI A23L 1/04, SW 37/06), including the grinding of raw material, processing it with hydrochloric acid at pH 4-5, washing with water, extraction with 1% solution of ammonium oxalate, precipitation with ethanol, filtration, crushing, pectin, rinsing with ethanol, treatment with methanol in the presence of acetyl chloride, repeated filtration and successive washing with methanol and ethanol, drying of pectin.

However, the known method is characterized by complexity, the use of toxic substances methanol and allows you to get from biomass cultivated in vitro plant tissue target product with a given structure and stable chemical composition.

A method of producing pectin from grass dog violet Viola canina L. (RU patent 2285536 C1; MCI AC 36/86), including a preliminary extraction with 70% ethyl alcohol, filtering, extraction with hot water, repeated filtration, extraction with 0.5% solution of oxalic acid, filtration, evaporation of the extracts, precipitation and washing of the precipitate with 96% ethanol, drying.

However, the known method does not allow to obtain biomass for cultivated plant tissue target product with engine speed specified�th structure and stable chemical composition.

The closest is a method of producing pectin polysaccharides from biomass tissue cultured plants of Silene vulgaris (M.) G, Tanacetum vulgare L, Lemna minor L. (RU patent 2175843 C1; MKI 7 A23L 1/0524, SW 37/06), including the destruction of raw materials, separation from the liquid fraction, treatment with ethanol and chloroform to remove low molecular weight impurities, extraction with water to remove water-soluble polysaccharides, process the raw material with hydrochloric acid, washing with water, extraction of pectin polysaccharides aqueous solution of ammonium oxalate, the concentration of the extract, precipitation with ethanol, dialysis and freeze drying (prototype).

However, the known method does not allow to obtain from biomass cultivated in vitro plant tissue target product with a given structure.

The technical result of the present invention is to provide a method of obtaining pectin polysaccharides from biomass cultivated plant tissues, which allows to obtain physiologically active pectins with a given structure, with stable chemical composition, in particular modified pectins from biomass tissue culture of Silene vulgaris (M.) G. with increased content of galactose residues in the carbohydrate side chains.

The technical result is achieved in that the callus cultures grown on agar modified PI�atelnoe environment which contains the enzyme 1,4-β-D-galactosyltransferase (EU 2.4.1.22). Next, the raw material ruin, separated from the liquid fraction, extracted with water to remove water-soluble polysaccharides, then treated with a solution of hydrochloric acid at pH 3.7-4.3, then carry out the extraction of pectin polysaccharides aqueous solution of ammonium oxalate, the extract was concentrated, precipitated with ethanol, dialist and freeze dried, used as raw biomass of cultured plant tissues, such as Campion ordinary (Silene vulgaris).

The obtained pectin polysaccharides possess immunomodulatory activity, fine structure of the carbohydrate side chains of pectins (in particular, linear and branched blocks galactan and arabinan) determines the ability to stimulate the activity of white blood cells. On the basis of pectin with developed an extensive region may receive adjuvants for oral immunization of humans and animals.

The method is carried out as follows. For growing callus tissue Silene vulgaris prepare growth medium, using chemically pure reagents (reagent-grade, analytical grade). Pre-prepared concentrate macrosoma on Murashige and Sugu, wherein each of macrosoma dissolved sequentially in a little water, and then the volume is brought to 1 L. Similarly prepared concentrates mi�of rosola on Murashige and Sugu, Fe-chelate, vitamins for Stubbed, 1,4-β-D-galactosyltransferase. Macro - and microscale, Fe-chelate, vitamins in the form of concentrates mixed in a little water. Then to this mixture was added 6-BAP, 2,4-D, sucrose and all carefully mix.The solution was adjusted with distilled water to 1 l and set the pH to 5.6-5.8. In flasks of 250 ml fill in 2 g of agar-agar, poured into the medium in 250 ml, cover with foil and sterilized in autoclave for 20 min at 2-3 ATM. Concentrate 1,4-β-D-galactosyltransferase sterilized using filters with a pore size of 22 μm was then added to the flask after autoclaving the medium. In a sterile room in Petri dishes poured into 30 ml of medium. The cultivation of the callus tissue is held in an incubator at 26±1°C in continuous darkness. After 21 days of growing callus isolated pectic polysaccharides.

Biomass ruin by a single freeze-thawing, separated from the liquid fraction by centrifugation, extracted with water in the ratio of raw material (wet weight): water 1:7-20 at a temperature of 45-70°C for 2-4 h to remove water-soluble polysaccharides, treated with a solution of hydrochloric acid (pH 3.7-4.3; the ratio of raw material:solution 1:4-10) at 45-55°C for 3-4 h, the biomass was washed with water, then carry out the extraction of pectin polysaccharides water 0.3-0.7% Rast�a PR of ammonium oxalate in the ratio of raw material:solution 1:10-20 at 65-75°C for 2-6 h, then the extract was concentrated, precipitated with 96% ethanol in the ratio extract: alcohol 1:2-4 for 2-24 h, dialist and freeze dried, used as raw biomass cultivated plant tissue, for example, Silene vulgaris.

Example 1 (control). For growing callus tissue Silene vulgaris prepare growth medium, using chemically pure reagents (reagent-grade, analytical grade). Pre-prepared concentrate macrosoma, wherein each of macrosoma dissolved sequentially in a little water, and then the volume is brought to 1 L. Similarly prepared concentrates microsales, Fe-chelate, vitamin. Macro - and microscale, Fe-chelate, vitamins in the form of concentrates mixed in a little water. Then to this mixture was added 0.5 mg/l 6-BAP, 1.0 mg/l 2,4-D, 15 g/l sucrose, 15 g/l of glucose and all carefully mix. The solution was adjusted with distilled water to 1 l and install pH 5.6. In flasks of 250 ml fill in 2 g of agar-agar, poured into the medium in 250 ml, cover with foil and sterilized in autoclave for 20 min at 2-3 ATM. Petri dishes are sterilized in hot air oven at 180°C, wrapped in Kraft paper. In a sterile room in Petri dishes poured into 30 ml of medium. The cultivation of the callus tissue is held in an incubator at 26±1°C in continuous darkness. After 21 days of growing callus isolated pectin�new polysaccharides.

Biomass ruin by a single freeze-thawing, separated from the liquid fraction by centrifugation, twice extracted with water in the ratio of raw material (wet weight): water of 1:10 at a temperature of 50°C for 2 h to remove water-soluble polysaccharides, treated with a solution of hydrochloric acid (pH 3.9-4.1; the ratio of raw material:solution 1:10) at 50°C for 3 h, the biomass was washed with water, then carry out a double-extraction of pectin polysaccharides aqueous 0.7% solution of ammonium oxalate in the ratio of raw material: solution 1:10 at 68-70°C for 2 h, after which the extract was concentrated, precipitated with 96% ethanol in the ratio extract:alcohol of 1:2 for 24 h, separated by centrifugation, dissolved in distilled water and cialiswhat against distilled water. The obtained polysaccharide freeze-dried. The yield of the target product amounted to 3.1% of the dry biomass, the content of D-galacturonase acid in selected pectic polysaccharide 65%, the content of galactose residues 2.9%. The test results are presented in table 1.

Examples 2-4. Growing callus tissue Silene vulgaris and a selection from her pectin was carried out analogously to example 1, the difference is the modification of the culture medium comprising 1,4-β-D-galactosyltransferase in a concentration of 0.001, 0.01 and 0.1 mg/ml. the Yield of the target product from dry biomass SOS�evil 3.2, 2.4 and 2.3% respectively. The content of D-galacturonase acid in the selected pectin 70, 69 and 73%, respectively, residues of galactose 4.7, 3.7, and 4.0%, respectively. The test results are 3 modifications of culture media are presented in table 1.

Table 1
Option1,4-β-D-galactosyltransferase, mg/mlD-galacturonic acid, %Galactose, %
1 (control)065.1±6.42.9±0.3
20.00169.8±4.34.7±0.1∗
30.0168.6±1.83.7±0.2∗
40.173.4±3.24.0±0.5∗
* Differences significant at p<0.05.

Thus, the proposed method allows to obtain high yield of modified pectin polysaccharides with increased content of galactose residues in �touch carbohydrate chains from biomass cultivated plant tissue, what is important for pectins with a given structure with certain types of physiological activity.

A method of producing from callus cultures of pectins with increased content of galactose residues in the carbohydrate side chains, including the destruction of raw materials, extraction with water to remove water-soluble polysaccharides, processing biomass with a solution of hydrochloric acid, rinsing with water, extraction with a solution of ammonium oxalate, the precipitation of the polysaccharide with ethanol, dialysis and lyophilization, wherein the pre-callus culture for 21 days grown on agar medium, which contains the enzyme 1,4-β-D-galactosyltransferase in a concentration of 0.001-0.1 mg/ml and used as raw biomass tissue cultured plants of Silene vulgaris.



 

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