SU1659053A1 - Method for antiviral preparation producing from yeast - Google Patents

Abstract

The invention relates to a technology for the production of biologically active drugs. The purpose of the invention is to simplify the technological process of obtaining an antiviral drug from yeast and increase the biomass yield of the product. To do this, the cells separated from the culture liquid and dried are homogenized, soluble substances from the homogenate are extracted with 5-6% acetic acid, centrifuged, and the precipitate is discarded. The extract is evaporated in vacuo at 50 ° C and cooled, the desired product from the concentrate is precipitated with two volumes of ethanol, centrifuged, the precipitate is dissolved in water and dried by sublimation. 9 tab.

Description

BACKGROUND OF THE INVENTION The invention relates to a technology for the production of biologically active preparations for crop and livestock production.

The purpose of the invention is to simplify the technological process of obtaining an antiviral drug from yeast and increase the biomass yield of the product.

The goal is achieved in that instead of ribonuclea and phenol, an organic acid, preferably acetic acid, which simultaneously performs the function of the active extractant, is used to purify the preparation. The acid is used in a concentration of 5-6%, which does not reduce the activity of the resulting preparation, but ensures its high yield and good purification from impurities. According to the proposed method, not only yeast biomass grown under laboratory conditions, but also industrial fodder yeast can be used as a raw material.

Separated from the culture fluid and dried cells are homogenized, soluble substances from the homogenate are extracted with 5-6% acetic acid, centrifuged and the precipitate discarded. The extract is evaporated in vacuum at 50 ° C and cooled, the target product from the concentrate is precipitated with two volumes of ethanol, centrifuged, the precipitate is dissolved in warm water and dried by sublimation.

The preparations isolated by this method consist mainly of polysaccharides, are well soluble in water, and possess high antiviral activity.

PRI me R 1. Yeast Candida troplcalls K-41 is grown in fermenters on aerated liquid medium of the following composition: (NH-ikSO / i 3 g / l; NaCI 0.3 g / l; MgSO4 0.4 l;

CN SL CHE iO SL

(l)

People had 1 g / l; K2HPO / jO, 1 g / l; glucose 5 g / l; Biotin 2 μg / l.

The biomass is separated from the culture liquid by centrifugation (3000 rpm, 15 in), dried in a freeze-drying 30 ° C, pressure 10 n / m2 and used as the antiviral drug is produced. Horn cells are homogenized using a ball mill, soluble substances are extracted with 6% acetic acid (2 hours at + 2 ° C), the precipitate is discarded, and the supernatant is brought to neutral pH by adding 5N NaOH and concentrated 1: 5 by evaporation ( 50 ° C) in vacuum. To the cooled concentrate, 2 volumes of 96% ethanol are added and the desired product is precipitated at + 2 ° C. The precipitate is collected by centrifugation and dissolved in warm (50 ° C) distilled water. The solution is centrifuged, the precipitate is discarded, and the preparation is dried in a freeze-drying.

The yield of the purified preparation is 5.7% of the dry weight of the yeast. The drug has the form of an amorphous white powder, is well soluble in water, is thermostable, is not hygroscopic, and can be stored for a long time in a dry place without changing its powdery consistency and high antiviral activity. It contains more than 80% of carbohydrates, the admixture of proteins is no more than 3%, and nucleic acids are traces.

Example 2. Industrial feed dry yeast is used as a starting material. Extraction and purification of antiviral onset is carried out in the same way as in Example 1. Acetic acid in 5% concentration is used for this. The yield of the drug is somewhat lower (4.9%), however, its antiviral activity is higher than with extra- tion with 6% acid. The preparation is an amorphous powder of slightly brown color, it is well soluble in water, is thermostable, not hygroscopic, and can be stored for a long time in a dry place without loss of physical characteristics and antiviral activity. It consists mainly of polysaccharides, protein impurities do not exceed 5%, nucleic acids - 0.4%.

PRI me R 3. The original raw materials - industrial feed yeast. For the extraction and purification of the antiviral drug, acetic acid is used in the optimal concentration (5.5%). The yield of the drug is quite high - 5.2%, its antiviral activity is higher than with the extraction of substances with 6% acetic acid.

Table 1 summarizes the composition and activity of preparations obtained from various sources in relation to the tobacco mosaic virus on dope.

Example 4: Dry yeast cells (40 g) are treated with 6% acetic acid according to the proposed method or with water (prototype). Soluble materials are separated by centrifugation and the carbohydrate content in the obtained extracts (0.2% antronom in concentrated NAZSm) and proteins (according to Bradford micromethod), as well as antiviral activity of extracts (against TMV on datura leaves). The data from this experiment are given in Tables 2, 3, 4.

The target product, extracted with 6% acetic acid, does not significantly differ in its antiviral activity from the product obtained according to the prototype method. However, the carbohydrate content, which is the active principle of the preparation, is 1.5 times higher in this preparation than in the preparation extracted with water. At the same time, a large proportion (31%) of the latter is made up of proteins whose content in the target product extracted with 6% acetic acid does not exceed 2%.

PRI me R 5. 250 mg of Candida tropicalls K-41 yeast cells, freeze-dried, homogenized, and the target product is extracted from them with 3% -, 5% -, 6% -, 8% -, 10% - or 15% acetic acid. Next, the extract is concentrated by evaporation in a vacuum, precipitated with alcohol. Determine

total content of proteins and polysaccharides in preparations and their ability to inhibit the infectivity of TMV on the leaves of the dope.

The data of this experience are given in

Table 5 shows that as the concentration of acetic acid increases from 3 to 10%, the yield of polysaccharides increases, while the yield of proteins, on the contrary, decreases (from 3.4 to 1.4%). A further increase in acid concentration (up to 15%) is accompanied by a decrease in the yield of polysaccharides. Antiviral activity have all the drugs. However, the highest activity observed in drugs, extracted 5-6%

5 acetic acid. Drugs isolated at a higher concentration of acid (8–15%) in relatively low (1 µg / ml) concentrations do not have activity or this activity is not constant. The activity of the drug, extruded with 3% acetic acid, turned out to be much smaller, probably due to the presence of impurities that reduce the activity of inhibitors or stimulate infectivity of the virus.

When testing the effect of various concentrations of acetic acid as an extractant of the target product in this experiment, neutralization with alkali was not deliberately carried out. This made it possible to determine how high concentrations of hydrogen ions can adversely affect the activity of the antiviral agent. Measurements show that the pH values given by a certain acid concentration do not increase significantly during the process of concentrating the extract in a vacuum due to the evaporation of the acid. Thus, at an acid concentration of 5–6%, the pH value of the extract after concentrating the product in vacuum does not exceed 1-2, and the antiviral activity of the target product (compared to a lower concentration of extractant — 3%) did not decrease.

Thus, the optimal concentration of acetic acid, which ensures a sufficiently high yield of the preparation, the absence of essential impurities of associated proteins in it, and a good preservation of the antiviral activity of the target product, is within 5-6%.

Example 6. In the experiment using dry cells of the yeast Candida tropicalis K-41.

 Getting antiviral drug by the proposed method,

40 g of yeast cells are homogenized and the target product is extracted with 400 ml of 6% acetic acid (2 hours at + 20 ° C), the extract is separated from the cell homogenate by centrifugation and evaporated to 100 ml in vacuo. The desired product from the concentrate is precipitated with two volumes of alcohol, the precipitate is dissolved in water and lyophilized. At all stages of preparation, the content of carbohydrates, proteins in the extracts and the antiviral activity of extractable substances are determined.

Getting antiviral drug according to the method prototype.

40 g of yeast cells are homogenized, the target product is extracted with 400 ml of water (2 hours at + 20 ° C), the extract from the cell homogenate is separated by centrifugation, and then treated with RNase at a concentration of 20 µg / ml (2 hours + 25 ° C in 0.001 M NaCI). The extract is concentrated in vacuo to 100 ml, and then emulsified in a mixture of phenol and chloroform (9: 1) for 30 minutes at room temperature, the aqueous phase with the desired product is separated by centrifugation and dialyzed (24 hours) against water. The target product from dialysate is precipitated with 5 volumes of ethyl alcohol, the precipitate is dissolved in water and lyophilized. At all stages of preparation, the content of proteins and carbohydrates in extracts and the antiviral activity of extractable substances are determined.

Comparative evaluation data of the proposed method and its prototype are given in Table 6. The yield of the preparation obtained by the proposed method, under the conditions of this experiment, reaches 2.5%. While using the prototype method, it is possible for half of the drug to be tested only about 0.7% of the drug by weight of the feedstock.

Example. Datura leaf is treated with the preparation (1 mg / ml) 48 hours before or immediately after the infection with the X virus of potato-1 (HVC) by immersing them in a solution of the test preparation. Control leaves are similarly treated with water. The infectious titer of the virus is determined after 5 days by inoculation of excretory tracts from the leaves of the dope of the Gomphrena globosa plants and by counting the local viral lesions formed in the experiment and control.

The data presented in Table 7 testify to a significant prophylactic effect on PVX. A decrease in the virus titer (by 31%) under the influence of the drug was noted.

In the tobacco system - the virus of bronze

5 tomatoes (VBT), the obtained antiviral drug (AVP) is used by spraying the plants three times before (prophylaxis) or after (therapy) inoculating them with infectious juice with an interval of 2-3

0 days between contiguous treatments. The number of local necrosis on inoculated leaves, as well as the number of systemically affected plants are taken into account, and the nature and extent of the external symptoms of the disease are noted simultaneously.

The data of this experience are shown in tables 8 and 9.

As follows from the above results, the obtained WUA has a high

0 prophylactic and essential therapeutic (reduction of plant death by 50% with 100% death in the control) action on tobacco disease caused by PID

five

Claims (1)

The invention The method of obtaining antiviral drug from yeast, including the cultivation of cells and their separation from culture liquid, extraction, purification, and concentration of the extract in vacuum, followed by sedimentation of the target product and its freeze-drying, characterized in that simplifying the process and increasing the biomass yield of the product; extraction and purification are carried out with acetic acid at a concentration of 5-6%. Yield, composition and antiviral activity of drugs allocated their yeast Characterization of the target product, extracted from 40 g of dry yeast in various ways Table 1. table 2 Monosaccharide composition of antiviral drugs obtained using the known and proposed method Note. The content of monosaccharides in the preparations is determined by gas liquid chromatography. Preparations for GLC are obtained by hydrolysis of an antiviral drug in 2N sulfuric acid (2 h, 100 ° C). Effect of hydrolases on the antiviral activity of an antiviral drug isolated from yeast using the inventive method The output and antiviral activity of the target product, extracted from yeast cells with acetic acid of various concentrations Table 3 Table 4 Table 5 Note. For extraction, 250 mg of dry cells are used in each variant. stimulation of infectivity of TMV. Table .6 The comparative efficacy of two methods of obtaining an antiviral drug from Candida tropicalis K-41 yeast cells. Proposed - g Extraction 6% for k- 21.4 eatable succinic acid II Evaporation in vacuum 9.2 III Deposition with alcohol 2 ° 00 I Extraction with water, 378.0 340 botan by RNase (20 µg / ml in 0.001 M Nad) II Evaporation in vacuum 234.0 1700 Treatment with phenol-chloroform mixture, dialysis, alcohol precipitation 13.6 290 Continued table. five 1120 111.4 1.9 1800 1809.2 0.5 1007.2 0.7 1218 31.0 1934 12.1 303.6 4.5 Table 7 Action of antiviral preperga (HDL) from yeast on the accumulation of X-vir sa potatoes in isolated halves of the leaves of dope Incubation time of halves Infectivity of leaf extracts through leaves 5 days after infection (necrosis) leaf Water WUA, 1 mg / ml, Inhibition,% 48 h before inoculation 352431, A about 48 hours after inoculation 3940 + 1.0 +++, 1%; ° P 5%. Table 8 The preventive effect of antiviral drug from yeast on the susceptibility of tobacco varieties Immune 580 IWT. OptionNumber of local plants Number lesions / sheet with systemic symptoms of the disease, Z Water (control) 11, Oil, 4100 WUA, 0.02% 1.7 0.030: # Three-way spraying of plants with an interval of 3 days before inoculation of VBT plants. T And bl and c and 9 Therapeutic antiviral effect of an antiviral drug from yeast in the system of tobacco varieties Immune 580 - WBT - - | -. Option. Kolishestvo plants, Z ... 10 days pi 30 days PI 50 days pi 1ZZZZILI Water (control) 1000000JOOOO100 WUA, 0, 5 62.5 037,562.5 012.5 37.5 50.0  a threefold spraying with an interval of 2 days, started immediately after infection of plants with UBT: 1 — with severe symptoms; 2nd mild symptoms; 2 - the dead.