EA022691B1 - Method for producing a hepatoprotective preparation based on balsam poplar (populus balsamifera l.) bud pinostrobin - Google Patents

Abstract

The invention relates to the field of bioorganic chemistry, pharmaceutical chemistry and technology of drugs and concerns methods for producing preparations of vegetable raw material, which possess hepatoprotective activity. Technical result of the claimed invention is achieved by that the ecologically safe barothermal method is used for producing extract balsam poplar (Populus balsamifera L.) buds with pinostrobin flavonoid content of 0.9% with reference to air-dry raw material by extraction of raw material at a temperature of 150-160°C, pressure of 1.9 atm., for 60 min, separation and purification of thick extract by method of column chromatography with elution with petroleum ether and mixture petroleum ether-ethylacetate 49:1, recrystallisation of isolated technical pinostrobin with further synthesis of pinostrobin oxime (substance for pharmaceutical dosage form). Pinostrobin oxime substance is not toxic, does not cause acute toxic reactions in tests in mice and rats and demonstrates the high hepatoprotective activity in dose of 100 mg/kg of weight, and in the dose of 50 mg/kg eliminates manifestations of cytolysis, cholestasis and mesenchymal inflammation syndromes. Pinostrobin oxime relates to "short-living" products. In rat organism the product is revealed within 2 hours, irrespective of the administered dose and method of administration, penetrates well mainly in vascularised organs (kidneys, liver, lungs), and absolute bioavailability of pinostrobin oxide is 11%. Antioxidant activity of pinostrobin oxime is 20 times as high as that of pinostrobin. Pinostrobin oxime does not produce any toxic effect on the liver, reduces the lipoperoxidation intensity, the triacylglyceride content by 26% in it and increases phospholipid level by 21%, inhibits lipid peroxidation processes in hepatocytes, reducing diene conjugate content by 35%, increasing the activity of antiperoxide protection enzymes - glutathionperoxidase and glutathion reductase up to 54%.

Description

The invention relates to the field of bioorganic chemistry, pharmaceutical chemistry and drug technology and relates to methods for producing preparations from plant materials with hepatoprotective activity.

The objective of the invention is to develop a method for producing based on the natural flavonoid pinostrobin (1) and its oxime (2), which has hepatoprotective activity.

Known methods for the isolation of pinostrobin.

Method 1. The balsamic poplar buds (Рор1и8 акакатГета б.) Are infused with 65-96% ethyl alcohol under conditions of fractional maceration, combining infusion at room temperature for 24 hours, followed by extraction of the target substances with heating not higher than 80 ° С for 1 hour and combining the first and second extracts. Get the sum of flavonoids and phenylpropanoids with a content of 2.7% (RF patent No. 2135201 from 08/27/1998 Braslavsky V. B., Kurkin V. A., Zapesochnaya G. G., Zhdanov I. P. The method of obtaining tincture of balsam poplar for the treatment of purulent-inflammatory diseases of soft tissues).

Method 2. The balsamic poplar buds (Рор1и8 акакатГета б.) Are extracted with 96% ethyl with a ratio of dry raw materials: extractant = 1: 20, the ethanol extract is evaporated under vacuum, the residue is dissolved in hot water, the flavonoids are extracted with ethyl acetate, at a temperature of -70 ° С, 5 hours. The content of pinostrobin in the extract is 0.17% in terms of a unit of absolutely dry raw materials (Lozhkina G.A., Isaeva E.V., Ryazanova T.V. Influence of various factors on the extraction process of balsamic poplar buds. Chemistry of plant raw materials . - 2007. - No. 2. - S. 51-54).

Method 3. The walnut pericarp (1id1ap8 ged1a b.) Is extracted with 96% ethyl alcohol, the resulting amount of substances is separated on a silica gel column with a mixture of petroleum ether-ethyl acetate (15: 1), the eluates are combined and recrystallization from ethyl acetate gives pinostrobin and betasitosterol with a purity of 98 % (Eigoreap Ra1ep1 ΟΝ 101475459 A; 07/08/2009, Ke Wiiap, Viapysh ш, ΖΙπριπ Ζ1ι; πι§ MeYuu! Gog e \ 1gasypd rzozyoosh ap! Аe1a-8YO81ets Ggot yyukotu pirk! The disadvantages of the above methods are:

1) a low yield of the target substance pinostrobin (0.17% in terms of air-dry raw materials);

2) the use of toxic and flammable solvents, the use of which in pharmaceutical production is prohibited according to the standards of good manufacturing practice (CMP);

3) the time duration of the processes in the above methods for the isolation and purification of pinostrobin;

4) one of the natural sources of pinostrobin - walnut - has a narrow habitat (Zailiysky Alatau, Karatau, Western Tien Shan).

As plant raw materials in the method we offer, they use the balsamic poplar buds (Рор1и8 ЛаНатГета б.), Which is widely distributed in the territory of Northern Kazakhstan. The operational reserves of poplar buds in the North Kazakhstan region are 400 kg per 2.5 ha.

The technical result of the claimed invention is achieved by using an environmentally safe barothermal method for producing balsamic poplar kidney extract (Pori1i8 ba1zatTega b.) With a content of flavonoid pinostrobin 0.9% calculated on air-dry raw materials, extraction of raw materials at a temperature of 150-160 ° C, a pressure of 1.9 atm, for 60 min, separation and purification of the condensed extract by column chromatography, eluting with petroleum ether and a mixture of petroleum ether-ethyl acetate (49: 1), recrystallization technical pinostrobin followed by synthesis of pinostrobin oxime (substance for the dosage form).

The flavonoid pinostrobin from the balsamic poplar buds (Porillaria bapanifacea L.) is isolated in the following way.

Method A. The extraction of balsamic poplar buds is carried out in a barothermic manner. 6 kg of balsamic poplar buds are placed in the reactor and heated at a temperature of 150-160 ° C, a pressure of 1.9 atm for 60 minutes until the oil is completely extracted, a thick extract is obtained (with a yield of 35% by weight of the raw material) and balsamic poplar essential oil ( yield 0.9% by weight of raw materials).

A thick extract weighing 0.25 kg is mixed with silica gel (0.5 kg) in a ratio of 1: 2 (0.5 kg) and mixed thoroughly until a homogeneous mass is obtained. Condensed extract mixed with silica gel is laid out on a flat surface (stainless steel pan or glass) with a thin layer and placed under exhaust ventilation. Dry extract with silica gel is crushed, weighed, its weight is 0.7 kg.

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The chromatographic column is filled with dry silica gel. The mass of silica gel to be poured was 1.0 kg, the volume of petroleum ether was 5.0 l. The column is eluted first with petroleum ether, and then with a mixture of petroleum ether-ethyl acetate (49: 1).

Fractions are evaporated on a rotary evaporator IR-10M. The dry residue of the fractions was monitored by TLC and combined with ethyl acetate.

Fractions containing pinostrobin are washed with petroleum ether and dried under traction. Get 22 ± 0.5 g of pinostrobin technical.

25.0 g of technical pinostrobin are recrystallized. An evaporation flask with technical pinostrobin is installed in a water bath at a temperature of 60-70 ° C, dissolved in ethyl acetate and left for 12 hours. The precipitated crystals are washed with petroleum ether.

After crystallization, the pinostrobins are first dried on a filter under exhaust ventilation for 2 hours, then transferred to a porcelain cup and dried in a vacuum oven at a temperature of 40 ° C and a pressure of (-0.08) - (- 0.09) mPa to constant mass. Pinostrobin has the appearance of a fine crystalline powder, odorless white.

From 6 kg of balsamic poplar kidneys, 22 ± 0.5 g of the active substance pinostrobin is obtained.

The melting point is determined on a device for determining the melting temperature of Niib HUSAUAG (Germany), which is 96-99 ° C (from ethyl acetate).

The purity and authenticity of the obtained pinostrobin (1) purified is determined by HPLC. The purity of the purified pinostrobin is at least 99.5%.

Elemental analysis: found%: C 70.6 (71.57; 71.11; 70.64); H 4.9 (5.18; 5.30; 4.79);

Calculated%: C 71.0; H 5.09.

Mass spectrum (t / ζ), I (%): 270 / M / + (100.0), 252 (6.1), 193 (35.8), 166 (62.4), 138 (29.6), 114 (6.9), 110 ( 11.9), 77 (9.6), 51 (5.9), 39 (4.5).

Found (t / ζ): 270.08932; calculated (t / ζ) for 6ι ₆ Ηι ₄ Ο ₄ (t / ζ) 270.08920.

UV spectrum: (λ, nm, 1 ° C in UNN): 212 (4.31); 289 (4.29); 334 (3.53).

IR spectrum (KBr), ν / cm ^-1 : 3010 (OH, aromatic) 2972, 2935, 1646 (С = О), 1618, 1579 (С = С), 1445, 1381, 1339, 1259, 1209, 1158 , 1092, 998, 960, 916, 887, 840, 800, 766, 742, 717.

¹ H NMR (SES1 ₃ , 300 MHz, δ (ppm)): 5.41 (H-2, dd), 3.08 (H-3 a, dd), 2.83 (H-3b, d.d.), 6.45 (H-6, d.), 6.63 (H-8, d.), 7.44 (H-2 ', m.), 7.39 (H-3', m.), 7.24 ( H-4 ', m.), 7.39 (H-5', m.), 7.44 (H-6 ', m.), 12.15 (OH, s), 3.79 (OCH3, s.).

¹³ C NMR (SES1 ₃ , 300 MHz, δ (ppm)): 79.10 (S-2, d), 43.26 (S-3, bp), 167.00 (S-4, s .), 162.66 (S-5, s), 95.02 (S-6, d.), 195.63 (S-7, d.), 94.14 (S-8, d.), 164, 02 (C-9, s), 103.02 (C-10, s), 138.25 (C-1 ', s), 126.01 (C2', d.), 128.75 (C-3 ', d.), 126.01 (S-4, d.), 128.75 (S-5, d.), 126.01 (S-6, d.), 55.22 (OCH ₃ , q.).

Method B. 6 kg of poplar buds in a bag, cross-linked from calico, placed in a container for extraction of raw materials, then pour pre-prepared extractant - ethyl alcohol 95% in the amount of 33.064 kg (40 l), extracted at a temperature of 60 ° C. After cooling, the liquid extract is drained and the extraction is repeated 2 more times under the same conditions.

After the time of extraction, the liquid extract is poured into a rotary evaporator. In a rotary evaporator, the process of thickening the liquid extract is carried out at a temperature of 60 ° C. Obtain 250 g of a thick extract of balsamic poplar buds.

The purified thick extract with a mass of 0.25 kg from the evaporation flask is poured into an evaporation porcelain cup, the remaining extract is washed off the walls of the flask with a small amount of ethyl alcohol (0.2 kg). The rinse is combined with the thick extract in an evaporation cup and mixed thoroughly until a homogeneous mass of sufficiently liquid consistency is obtained. Silica gel is added to the dissolved extract in a ratio of 1: 2 (0.5 kg) and mixed thoroughly until a homogeneous gruel-like mass is obtained. Condensed extract mixed with silica gel is laid out on a flat surface (stainless steel baking sheet or glass) with a thin layer for the rapid evaporation of ethyl alcohol under exhaust ventilation. Dry extract with silica gel is crushed, weighed, its weight is 0.7 kg.

The chromatographic column is filled with dry silica gel. The mass of silica gel to be poured was 1.0 kg, the volume of petroleum ether was 5.0 l. The column is eluted first with petroleum ether and then with a 98: 2 petroleum ether-ethyl acetate mixture.

Fractions are evaporated on a rotary evaporator IR-10M. The dry residue of the fractions was monitored by TLC and combined with ethyl acetate.

Fractions containing pinostrobin are washed with petroleum ether and dried under traction. Get 12 ± 0.5 g of technical pinostrobin.

g technical pinostrobin is recrystallized in the following way: an industrial flask with a technical pinostrobin is installed in a water bath at a temperature of 60-70 ° C, dissolved in ethyl acetate and left for 12 hours. The precipitated crystals are washed with petroleum ether.

After crystallization, the pinostrobins are first dried on the filter under exhaust ventilation for 2 hours, then transferred to a porcelain cup and dried in a vacuum oven at a temperature of 40 ° C and a pressure of (-0.08) - (- 0.08 09) MPa to constant weight. Pinostrobin has the appearance of a fine crystalline powder, odorless white.

From 6 kg of balsamic poplar kidneys, 12 ± 0.5 g of the target substance pinostrobin is obtained. The melting point is determined on a device for determining the melting temperature of Niib \ Usa1 / 1ag (Germany), which is 96-99 ° C (from ethyl acetate).

The purity and authenticity of the obtained pinostrobin (1) purified is determined by HPLC.

The purity of the purified pinostrobin is at least 98.0%.

From the table. 1 shows that the barothermal method allows to obtain an extract with a higher content of pinostrobin. And the barothermic method of obtaining a thick extract of poplar buds is relatively simple fast and economical than extraction. The thick extract of poplar buds obtained by the barothermic method is cheaper and more environmentally friendly in comparison with the chemical extraction method.

Table 1

Pinostrobin content in thick extracts of poplar buds obtained by barothermic and extraction methods

Ways Weight kidney poplar trees kg Weight thick extract kg Content pinostrobin in extract,% The content of pinostrobin in the extract, g Extraction ny 6 1.8 8.8 12.5 6 1.8 7.8 12.3 6 1.8 8.3 12,4 Barothermic cue 6 1,5 10.3 22.5 6 1,5 9.9 22.4 6 1,5 9.6 22.3

Obtaining pinostrobin oxime (2).

g of pinostrobin and 15 g of hydroxylamine hydrochloride are dissolved at 50-60 ° C in 0.5 l of ethyl alcohol. To an alcohol solution of pinostrobin and hydroxylamine hydrochloride add 15.0 g of sodium bicarbonate.

The reaction mixture was stirred at 50-60 ° C. for 48 hours. The progress of the reaction was monitored using TLC.

A solution of hydrochloric acid 1% is added to the reaction mixture until sodium bicarbonate is completely dissolved and a neutral pH of 6-7 is obtained (control with universal indicator paper).

The reaction mixture is poured into a 1 L separatory funnel. Pinostrobin oxime was recovered from the reaction mixture by extraction with ethyl acetate (3x100 ml). Ethyl acetate extracts were collected in a 500 ml flat-bottomed flask. Calcined sodium sulfate was added to the flask with ethyl acetate extracts and left for 2 hours. Then the solution was separated from the desiccant by filtration through a paper filter.

An ethyl acetate solution is poured into a flask of a rotary evaporator ΙΚΑ KU 05 La81C. In a rotary evaporator, the solvent is evaporated and technical pinostrobin oxime is obtained. The process is carried out at a temperature of 60 ° C.

Pinostrobin technical oxime is dissolved by heating to 50-60 ° C in ethyl alcohol 96%, left for 12 hours in a cold place for crystallization. Then the solvent is separated from the crystals by decantation.

After crystallization, the pinostrobin oxime is first dried on the filter under exhaust ventilation for 2 hours, then transferred to a porcelain cup and dried in a vacuum oven at a temperature of 40 ° C and a pressure of (-0.08) - (- 0.09) MPa to constant weight. Oxime is a crystalline powder of a white color with a yellowish tint, odorless.

35 ± 0.5 g of pinostrobin oxime are obtained from 30 g of pinostrobin.

The melting point is determined on a device for determining the melting temperature of Niib \ Usa1 / 1ag (Germany), which is 182-184 ° C (from ethyl acetate).

The purity and authenticity of the obtained pinostrobin oxime purified is determined by HPLC. The purity of the purified pinostrobin oxime is at least 99.0%.

Elemental analysis: found%: C 67.01 (67.60; 66.52; 66.91); H 5.03 (5.26; 4.90; 4.93); N, 4.68 (4.98; 4.49; 4.57);

Calculated%: C 67.37; H 5.26; N, 4.92.

Mass spectrum (t / ζ), I (%): 285 / M / + (100.0), 268 (16.1), 266 (19.3), 208 (46.4), 179 (9.5), 164 (8.8), 136 ( 5.3), 105 (13.3), 104 (25.9), 91 (12.4.), 91 (12.4), 77 (23.3), 69 (12.1), 39 (5.7). Found (t / ζ): 285.10144;

- 3 022691 calculated (t / ζ) for ί '. ’ΚΗι. · ΝΟ. · Ι (t / ζ) 285.10010.

UV spectrum: (λ, nm, 1 ° C in UN): 205 (4.37); 251 (3.61); 279 (4.13).

IR spectrum (KBr), ν / cm ^-1 : 3430 (Ν-ОН), 3008 (ОН, aromatic), 2918, 2848, 1647 (Ο = Ν), 1617, 1578 (С = С), 1515, 1458 , 1389, 1276, 1227, 1209, 1192, 1149, 1094, 1068, 1054, 1029, 990, 964, 948, 922, 887, 866, 820, 777, 750, 734, 704.

¹ H NMR ίΠ / ΟΙλ + ΙΑΙδΟ-ίΙ. , 300 MHz, δ (ppm)): 5.12 (H-2, dd), 3.3 (H-3a, dd), 2.74 (H-3b, dd), 6.01 (H-6, s), 6.01 (H-8, s), 7.42 (H-2 ', m.), 7.37 (H-3', m.), 7.34 (H-4 ', m.), 7.37 (H-5 ', m.), 7.42 (H-6', m.), 11.24 (OH, s), 3.7 (OCH ₃ , s).

¹³ C NMR (ΌΜδΟ-ά ₆ , 300 MHz, δ (ppm)): 79.10 (C-2, d), 43.26 (C-3, bp), 152.8 (C-4 , s.), 159.2 (S-5, s), 95.55 (S-6, d.), 162.24 (S-7, d.), 94.17 (S-8, d.), 157.93 (C-9, s), 98.62 (C-10, s), 139.83 (C-1 ', s), 126.68 (C-2', d.), 128.65 (S-3 ', d.), 126.68 (S-4', d.), 128.65 (S-5, 'd.), 126.68 (S-6', d.), 55.55 (OCH ₃ , to.).

Study of acute toxicity of pinostrobin oxime (2).

In the experiments 2 types of animals are used: outbred white mice and white rats. Each group consists of 5 animals.

To study the acute toxicity of pinostrobin oxime, its solutions in dimethyl sulfoxide (DMSO) emulsified with sunflower oil are used. The maximum technically achievable dose of pinostrobin oxime is determined by its solubility in DMSO (0.56 g / ml) and a safe dose of DMSO, which was taken as a dose of 1.8 ml / kg. For enteral administration of the drug, feed pellets are prepared by mixing equal parts by weight of pinostrobin oxime and a raw egg. For the formation of granules, bread crumbs are added to the mixture. Feed pellets contain 0.1 g of pinostrobin oxime. Fodder granules are given to animals in separate cages in the morning from half an hour to 3 hours. The dose of pinostrobin oxime received by animals as part of the feed granules is 4.17 g / kg for mice and 7.38 g / kg for rats .

Enteral administration is carried out in two ways - intragastrically (using a probe) and as part of feed pellets; parenteral - intraperitoneally.

During the two-week period, the animals were monitored daily and weighed three times. Fallen animals are opened, organs with marked changes are selected for histological analysis. At the end of the observation period, surviving animals are killed by decapitation, opened, organs are weighed and tissue samples are taken for histological examination. The organs taken for histological examination are fixed in 10% neutral formalin and poured into paraffin wax. Sections 5-7 μm thick are stained with hematoxylin and eosin. All histologic preparations are examined on an MBI-15 microscope in all magnification modes (from 70 to 1000 times).

After receiving pinostrobin oxime in the composition of feed pellets (on average 4.17 g / kg) in the behavior and appearance of mice there are no signs of toxic effects of the drug.

Rats received the highest dose of 8 and 10 g / kg (an average of 7.38 g / kg). No abnormalities were observed in animals.

A macroscopic examination of both mice and rats that received the drug with feed pellets revealed a moderately pronounced plethora of internal organs and brain matter.

In animals that received pinostrobin oxime in a DMSO solution at a dose of 1.1 g / kg, the following were determined: brain - plethora of the vessels of the pia mater; heart - moderate dilation of the heart cavities; liver - moderately pronounced plethora; kidneys - without macroscopic features; spleen - moderately full-blooded, normal sizes; testes and ovaries - without features. Histologically: moderately expressed dystrophic changes in parenchymal and stromal cellular elements of the internal organs and brain were determined.

Thus, the morphological study in animals treated with pinostrobin oxime intragastrically at the highest technically achievable doses does not reveal toxic effects.

After intraperitoneal administration of pinostrobin oxime, a decrease in the motor activity of mice, impaired coordination of movements, are noted. Mice die from 6-18 hours after administration of the drug until the end of the observation period of 14 days.

An autopsy of dead animals revealed macroscopically: the brain is full-blooded soft membranes, the substance of the brain is jelly-like, in the ventricles there is a small amount of clear fluid; heart - cavities dilated, filled with liquid blood, flaccid myocardium; the liver is brownish-gray, sharply increased in size, when touched with tweezers, it is easily destroyed; the kidneys are swollen, dark brown in color, the cortical layer differentiates clearly in the section; spleen - enlarged, full-blooded; gonads are full-blooded.

According to the results of a probit analysis of the dependence of the death of young mice of an intraperitoneally administered drug, an average - lethal dose (LD ₅₀ ) = 0.493 g / kg was determined.

In the table. 2 presents the results of a study of the effect of pinostrobin oxime on the viability of rats after a single intraperitoneal administration.

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table 2

Pinostrobin oxime toxicity measurements for rats with a single intraperitoneal administration

Dose (g / kg) Number of animals Death toll The proportion of dead animals males Females males females 1,1 5 5 one 0 0.1 10 one 0.8 5 1 5 s | about 0.3 10 3 0.5 5 | 5 1 1 2 0.3 10 3 0.2 5 1 5 0 1 1 0.1 10 one 0.1 5 1 5 about | about 0,0 10 0

From the table. 2 shows that the results of the study in rats are similar to the results of experiments with mice. Using the same approach as in the analysis of the mortality of mice, a group of young rats was isolated (males weighing 160-220 g and females 140-160 g, 21 animals in total).

According to the results of a probit analysis, the dependence of the death of rats from this cohort on the dose was determined LD ₅₀ = 0.758 g / kg

In the table. Figure 3 presents the results of a study of the effect of pinostrobin oxime on the viability of rats after a single intraperitoneal administration.

Table 3

Pinostrobin oxime toxicity measurements for rats with a single intraperitoneal administration

Dose (g / kg) Number of animals Death toll Death toll ANIMALS males Females males females 1,1 5 5 one 0 0.1 10 one 0.8 5 5 3 0 0.3 10 3 0.5 5 5 one 2 0.3 10 3 0.2 5 5 ⁰ 1 ¹ 0.1 10 one 0.1 5 5 0 0 0,0 10 0

The table shows that the results of the study in rats are similar to the results of experiments with mice. Using the same approach as in the analysis of mortality in mice, a group of young rats was isolated (males weighing 160-220 g and females 140-160 g, 21 animals in total).

According to the results of a probit analysis of the dependence of death of rats from this cohort on the dose of the drug, LD ₅₀ = 0.758 g / kg was determined.

A single maximum technically achievable dose of pinostrobin oxime solution in DMSO (1.1 g / kg) when administered orally does not cause rat death.

The consumption of pinostrobin oxime with food in a dose of 4.17 g / kg by mice and 7.38 g / kg by rats does not cause death of animals. The average lethal doses with intraperitoneal administration are 0.493 g / kg, (0.128-0.858 g / kg) for mice (males weighing 16-18 g, females 14-16 g) and 0.758 g / kg (0.545-0.972 g / kg) for rats (males weighing 160-220 g and females - 140-160 g).

Pinostrobin oxime belongs to the category of low toxic substances.

Determination of the bioavailability of pinostrobin oxime.

The study of the absolute bioavailability of pinostrobin oxime was carried out by the content of the test substance in the blood plasma of rats after its intragastric and intraperitoneal administration in the same dose of 500 mg / kg. Pinostrobin oxime is rapidly absorbed from the abdominal cavity of rats at a rate of 3.393 h ^-1 with a maximum concentration (Stax) of 4.553 μg / ml. The time to reach the maximum concentration (T _max ) in the blood plasma of pinotrobin oxime is 0.33 hours.

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The drug also quickly eliminates from the body after its intraperitoneal administration. This is evidenced by the value of the parameter ίι / _2β ι - 0.37 hours.

The value of LISo-g and LiS of _{0- /} pinostrobin oxime after its intraperitoneal administration was calculated, which made it possible to determine the absolute bioavailability of the drug, equal to 11%.

Tissue availability of pinostrobin oxime.

The tissue availability of pinostrobin oxime in the liver, kidneys, lungs, mesentery, brain, heart was studied.

Pinostrobin oxime is recorded in all studied organs and tissues. The substance is determined in blood plasma and organs within 2 hours.

Analysis of the absolute values of tissue accessibility of pinostrobin oxime showed that the test compound is most intensively distributed in well-vascularized organs (kidneys, liver, lungs), and the least in moderately vascularized organs (heart, brain, mesentery).

A validated method has been developed for the quantitative determination of pinostrobin oxime in biological materials based on high performance liquid chromatography (HPLC) with tandem mass spectrometric and spectrophotometric detection. Using HPLC and mass spectrometry after intragastric administration of pinostrobin oxime to rats at a dose of 50 mg / kg in their daily urine, it was found that pinostrobin oxime is excreted in the urine mainly unchanged.

The study of the pharmacokinetics of pinostrobin oxime in the blood plasma of rats was carried out after its oral administration at doses of 50.0 and 500 mg / kg.

After intragastric administration of pinostrobin oxime (50 and 500 mg / kg) to animals, the substance is rapidly absorbed from the gastrointestinal tract (GIT) at a rate of 1.917-2.188 h ^-1 (C _max / AIS0 ^ yes) and is determined in blood plasma for 2 h of the study (in the range of 4-12 hours after administration of the drug, the test substance was not registered even in trace amounts). Considering that the half elimination period did not exceed 0.23-0.28 h, pinostrobin oxime can be attributed to the group of short-lived drugs.

Antioxidant activity in the ABT8 oxidation test (TEAC).

The antioxidant activity of pinostrobin oxime is investigated using the AiyohShai reagent kit! A88au Κίΐ (Zfta). The method is based on the detection of the radical cation 2,2'-azino-bis- (3-ethylbenzothiazolin-6-sulfonic acid) (ABT§ * ⁺ ), formed in the reaction of ABT§ with ferryl myoglobin. Antioxidants inhibit the production of radical cation, and antioxidant activity is evaluated in comparison with the activity of the water-soluble analogue of vitamin E - Trolox. Using Trolox makes it possible to evaluate the effectiveness of the anti-radical action through the so-called trolox equivalent of antioxidant effectiveness (Tgo1oh Esstsuaksh ΛηΙίοχίάαηΙ Sarajeiu, TEAC). TEAC values indicate how much trolox in mg / ml inhibits ABT§ * ⁺ with the same efficacy as 1 mg / ml of the analyte.

The antioxidant effect of pinostrobin in terms of TEAC values is minimal and amounts to 0.011 mg / ml, while the antioxidant activity of pinostrobin oxime in comparison with pinostrobin is more than 20 times higher and amounts to 0.265 mg / ml.

Model of acute CC1 ₄ hepatitis.

Acute CC1 ₄ -hepatitis is reproduced by introducing reg 08 using a probe 3 times every other day with a 50% solution of CC14 in liquid paraffin in a dose of 0.15 ml / 100 g of animal body weight. Pinostrobin oxime is studied in the dose range of 25, 50 and 100 mg / kg when administered orally in the form of a suspension in 1% starch mucus according to the treatment-and-prophylactic regimen: within 7 days to CC1 ₄ , and then against the background of model reproduction (5 days). Pinostrobin oxime is administered daily at the same time before feeding the animals 1 hour before hepatotoxin administration. Comparison drug Carsil is studied at a dose of 100 mg / kg.

Chronic alcoholization is carried out by oral administration of 40% ethyl alcohol at a dose of 14 ml / kg for 60 days. The therapeutic effect of pinostrobin oxime is studied at doses of 50 and 100 mg / kg in comparison with Carsil at a dose of 100 mg / kg. A day after a 60-day alcoholization, the test substances begin to be administered within 21 days against the background of ethanol intake in the above doses. Then carry out the slaughter of animals of the experimental and corresponding control groups. The control is animals that continue to receive only 40% ethanol at a dose of 14 ml / kg for 21 days.

Paracetamol hepatitis model.

Acute hepatitis is caused by the introduction of intragastric paracetamol at a dose of 2500 mg per 1 kg of body weight in the form of a suspension in 1% starch mucus for 2 days. From the third day of the experiment, the test substances were injected into rats for 5 days. One day after the last injection, rats are decapitated under mild ether anesthesia, and biochemical parameters (total protein, ALT, AST, bilirubin, glucose, cholesterol), as well as absolute and relative liver masses are determined in blood serum.

Statistical processing of the results is carried out using the software package §1aY8Yea 6.0. The normality of the distribution of indicators is estimated using the criterion 81ι; φίΐΌ- \ νί11 <'5 \ ν-Κ5ΐ.

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Intergroup differences were evaluated by the non-parametric criterion Mapp-HUNPpsu and-1sM.

Evaluation of the hepatoprotective effect and determination of the effective therapeutic dose of pinostrobin oxime in a model of acute tetrachloromethane hepatitis.

Tetrachloromethane is the best known hepatotoxin used to model liver diseases; acute toxic hepatitis develops in the liver when tetrachloromethane is administered to rats.

The therapeutic and prophylactic use of pinostrobin oxime in doses from 25 to 100 mg / kg against the background of acute tetrachloromethane hepatitis to varying degrees influenced the restoration of biochemical parameters of the main pathological syndromes of liver damage with carbon tetrachloride, which is reflected in Table. 4-6.

Table 4

The effect of pinostrobin oxime on the absolute and relative mass of rat liver in acute tetrachloromethane hepatitis

Groups of animals The mass of animals, g The absolute mass of the liver, g The relative mass of the liver,% Intact rats, n = 6 178.7 ± 3.6 7.9 ± 0.3 4,5 ± 0,2 Control (without treatment), n = 6 184.3 ± 6.1 9.8 ± 0.6 * 5.3 ± 0.3 * Comparison group ("Carsil"), n = 6 190.4 ± 5.5 10.4 ± 0.3 * 5.5 ± 0.2 * pinostrobin oxime at a dose of 25 mg / kg, n = 5 176.5 ± 4.6 10.0 ± 0.3 * 5.7 ± 0.3 * pinostrobin oxime at a dose of 50 mg / kg, n = 8 173.0 ± 5.0 9.5 ± 0.6 * 5.5 ± 0.3 * pinostrobin oxime at a dose of 100 mg / kg, n = 7 179.4 ± 6.6 9.6 ± 0.5 * 5.3 ± 0.2 * Note: * - p <0.05 compared with values in intact animals; ⁺ - p <0.05 compared with the values in animals of the control group.

From the table. Figure 4 shows that in acute toxic hepatitis, the absolute and relative mass of the liver increases compared with intact animals. At the same time, the therapeutic and prophylactic use of pinostrobin oxime in doses of 25, 50 and 100 mg / kg does not prevent the growth of liver mass.

From the table. Figure 5 shows that in acute toxic hepatitis, the glucose content decreases, which indicates a violation in the liver of processes associated with carbohydrate metabolism. The liver provides a constant level of glucose in the blood due to the dynamic balance of the reactions of glycogenesis, gluconeogenesis, glycolysis and glycogenolysis. Violation of carbohydrate metabolism in liver pathology consists in a decrease in glycogen synthesis, a violation of its breakdown and the formation of glucose from non-carbohydrate substances (gluconeogenesis occurs only in the liver), which leads to the development of hepatogenic hypoglycemia. When the parenchyma is damaged, the liver loses its ability to absorb all substrates of glycogen synthesis from the bloodstream, which leads to disruption of glycogen synthesis. Pinostrobin oxime inhibits the development of hepatogenic hypoglycemia.

An increase in the concentration of urea in the blood serum indicates hepatic cell failure. Karsil and pinostrobin oxime prevent an increase in the concentration of urea in blood serum (Table 5).

In addition, pinostrobin oxime stimulates protein-synthetic processes in liver cells, as evidenced by an increase in the concentration of total protein in blood serum (Table 5).

Pinostrobin oxime at doses of 25 and 50 mg / kg helps to reduce the concentration of total bilirubin in the blood serum of experimental animals, at a dose of 50 mg / kg it reduces ALT activity, as a result of which the de Ritis coefficient increases slightly. The use of pinostrobin oxime in doses of 50 and 100 mg / kg also helps to reduce the turbidity units in the thymol sample.

Thus, pinostrobin oxime at a dose of 50 mg / kg has the best effect on eliminating the manifestation of cytolysis, cholestasis and mesenchymal inflammation syndromes. Those. the optimal therapeutic dose of pinostrobin oxime in the acute tetrachloromethane hepatitis model is a dose of 50 mg / kg.

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Table 5

The effect of oxime pinostrobin on biochemical parameters of rat blood serum in acute tetrachloromethane hepatitis

Groups of animals Hemoglobin, g / l Total protein, g / l Glucose, mg / dl Urea, mmol / l Intact rats, n = 6 123.0 ± 5.8 65.7 ± 2.7 66.0 ± 1.1 5.05 ± 0.45 Control (without treatment), n = 6 121.6 ± 5.3 69.8 ± 3.5 55.0 ± 3.3 * 6.49 ± 0.41 * Comparison group ("Carsil"), n = 6 121.8 ± 8.5 68.2 ± 2.7 57.6 ± 5.0 4.61 ± 0.53 * pinostrobin oxime at a dose of 25 mg / kg, n = 5 ’- 66.7 ± 3.1 73.5 ± 4.0 * 5.74 ± 0.88 pinostrobin oxime at a dose of 50 mg / kg, n = 8 134.4 ± 8.4 74.9 ± 2.6 * 60.9 ± 3.4 * 5.34 ± 0.49 * pinostrobin oxime at a dose of 100 mg / kg, n = 7 117.5 ± 4.6 74.0 ± 2.7 * 58.3 ± 2.6 5.91 ± 0.57 Note: * - p <0.05 compared with values in intact animals; * - p <0.05 compared with the values in animals of the control group.

Along with this, the administration of pinostrobin oxime significantly reduces the content of primary products of lipid peroxidation (LPO) -diene conjugates in terms of phospholipids in the liver of experimental animals by 58%, which indicates a pronounced antioxidant effect of pinostrobin oxime ίη νίνο.

The course administration of pinostrobin oxime leads to a decrease in the content of triacylglycerides in the liver by 29% and an increase in the level of phospholipids by 18%. At the same time, the ratio of phospholipids to triacylglycerides (TAG) increases to 8.7 times, which indicates an improvement in liver lipid metabolism.

The introduction of pinostrobin oxime does not lead to a significant change in the cholesterol content in the liver of experimental animals.

Thus, the course administration of pinostrobin oxime (25 mg / kg per day) does not have a toxic effect on the liver, reduces the rate of lipid peroxidation in it, the content of triacylglycerides and increases the level of phospholipids.

The course introduction of pinostrobin oxime against the background of toxic hepatitis induced by carbon tetrachloride significantly reduces the yield of marker enzymes alanine aminotransferase (ALT) and aspartamenotransferase (AST) in blood serum by 44 and 20%, respectively.

The mechanism of the hepatoprotective effect of pinostrobin oxime ίη νίνο lies in its ability to inhibit LPO processes in hepatocytes, as evidenced by a significant decrease in the content of diene conjugates by 35%. LPO inhibition protects hepatocyte membrane phospholipids from oxidative degradation. The activity of antioxidant enzymes of glutathione peroxidase and glutathione reductase is significantly increased up to 54%.

The administration of pinostrobin oxime against the background of toxic liver damage reduces the TAG content in it by 26% and increases the level of phospholipids by 21%, while the ratio of PL / TAG increases to 0.9.

Thus, on the model of toxic hepatitis induced by carbon tetrachloride, the hepatoprotective effect of pinostrobin oxime was established. The hepatoprotective effect of the test compound is based on the ability of pinostrobin oxime to increase the level of reduced glutathione and to induce the activity of glutathione-dependent antioxidant enzymes, glutathione peroxidase and glutathione reductase.

Another model for determining the hepatoprotective effect of the studied compound is alcoholic hepatosis associated with oxidative degradation of ethanol in the liver with the participation of alcohol dehydrogenase and the microsomal monooxygenase system.

In chronic alcoholic hepatosis in the untreated group, the protein-synthetic function of the liver decreases, as evidenced by a decrease in the concentration of total protein in the blood serum. Karsil and pinostrobin oxime at a dose of 100 mg / kg restore protein-synthetic liver function.

All other biochemical parameters remain within the normal range (table. 6).

A study of the hepatoprotective effect of pinostrobin oxime on a model of acute tetrachloromethane hepatitis showed that the dose of pinostrobin at a dose of 50 mg / kg has the best effect on eliminating the manifestations of cytolysis syndrome, cholestasis and mesenchymal inflammation.

A study of the hepatoprotective effect of pinostrobin oxime on a model of chronic alcoholic hepatosis showed that pinostrobin oxime at a dose of 100 mg / kg restores the protein-synthetic function of the liver.

The effect of pinostrobin oxime in comparison with the effect of the reference drug Karsil on liver metabolism in acute hepatitis caused by paracetamol in rats was studied.

For 5 days, rats were injected with pinostrobin oxime inside a dose of 100 mg / kg; Karsil - in

- 8 022691 dose of 100 mg / kg; control animals receive an equivolume amount of starch mucus. A day after the last injection, rats are decapitated under mild ether anesthesia and biochemical parameters (total protein, ALT, AST, glucose, cholesterol) are determined in blood serum, de Ritis coefficient, and relative liver mass are calculated.

The percentage of surviving animals at the end of the experiment is: 100% for intact animals; 20% for control animals; 80% - for animals receiving the comparison drug Karsil as a treatment; 67% - for animals receiving pinostrobin oxime as a treatment.

Table 6

The effect of pinostrobin oxime on the restoration of biochemical parameters of the main pathological syndromes of liver damage in rat blood serum in acute tetrachloromethane hepatitis

Groups animals Bilirubin, μmol / l ALT μmol / (s "l) AST μmol / (s’l) De Ritis coefficient, AST / ALT Alkaline phosphatase nmol / (s’l) Thymol try, turbidity 8-N Intact rats, n = 6 10.39 + 1.13 0.074 + 0.038 0.151 + 0.014 1,595 + 0,261 5702 + 651 0.84 + 0.16 Control (without treatment), n = 6 35.78 + 11.29 * 1.402 + 0.118 * 0.427 + 0.036 * 0.308 + 0.018 * 7551 + 1016 1.43 + 0.35 Comparison Group (Carsil), n = 6 44.62 + 14.84 * 1,545 + 0,046 * 0.440 + 0.035 * 0.285 + 0.022 * 9734 + 1346 1.38 + 0.35 OD at a dose of 25 mg / kg, n = 5 21.98 + 6.01 * 1.514 + 0.051 * 0.504 + 0.023 * 0.333 + 0.012 * .... 1.76 + 0.67 OD at a dose of 50 mg / kg, n = 8 19.94 + 2.80 * 1,203 + 0,103 * 0.407 + 0.029 * 0.346 + 0.021 * 7947 + 985 1.30 + 0.32 OD at a dose of 100 mg / kg, n = 7 38.25 + 6.20 * 1.290 + 0.152 * 0.426 + 0.029 * 0.359 + 0.043 * 7808 + 666 1.13 + 0.24

Note: * - p <0.05 compared with values in intact animals; * - p <0.05 compared with the values in animals of the control group.

From the table. 7 shows that with acute paracetamol hepatitis, the relative mass of the liver increases compared with intact animals. At the same time, the therapeutic and prophylactic use of pinostrobin oxime, as well as the comparison drug Karsil, does not interfere with the growth of liver mass.

Table 7

The effect of pinostrobin oxime on the relative mass of rat liver in acute paracetamol hepatitis

Groups of animals Weight animals, g Relative liver% weight Intact rats, n = 5 186 + 10 3.88 + 0.12 Control (without treatment), p-5 197 + 11 4.80 + 0.21 * Comparison Group ("Carsil"), 198 + 12 4.80 + 0.30 * n = 5 pinostrobin oxime in a dose of 100 188 + 6 4.92 + 0.25 * mg / kg, n = 6 Note: * - p <0.05 compared with values in intact animals.

From the table. Figure 8 shows that in acute paracetamol hepatitis, the main biochemical parameters of rat blood serum remain within normal limits.

From the table. Figure 8 shows that in acute paracetamol hepatitis, the level of alanine aminotransferase increases by 2.3 times, and the de Ritis coefficient decreases by 46% compared with intact animals. In this case, treatment with the drug Karsil helps to reduce the level of alanine aminotransferase by 46% and increase the de Ritis coefficient by 57% compared with this indicator in the control group of animals. Pinostrobin treatment with oxime helps to reduce the level of alanine aminotransferase by 34% and increase the de Ritis coefficient by 24% compared with this indicator in the control group of animals.

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Table 8

The effect of pinostrobin oxime on the biochemical parameters of rat blood serum in acute hepatitis paracetamol

Groups of animals General protein, g / l Cholesterol, mg / dl Glucose, mg / dl Intact rats, n = 9 72.18 ± 1.45 60.41 ± 2.36 71.31 ± 1.18 Control (without treatment), n = b 73.17 ± 0.89 67.08 ± 6.41 75.63 ± 4.49 Comparison group ("Carsil"), n = 4 76.05 ± 5.43 68.17 ± 11.14 75.67 ± 6.45 pinostrobin oxime in a dose 100 mg / kg n = 4 67.85 ± 3.33 65.25 ± 5.22 81.53 ± 2.67

From the table. Figure 8 shows that in acute paracetamol hepatitis, the level of alanine aminotransferase increases by 2.3 times, and the de Ritis coefficient decreases by 46% compared with intact animals. In this case, treatment with the drug Karsil helps to reduce the level of alanine aminotransferase by 46% and increase the de Ritis coefficient by 57% compared with this indicator in the control group of animals.

Treatment with pinostrobin oxime helps to reduce alanine aminotransferase by 34% and increase the de Ritis coefficient by 24% compared with this indicator in the control group of animals.

Table 9

The effect of pinostrobin oxime on biochemical markers of liver pathology in rat blood serum in acute paracetamol hepatitis

Groups of animals ALT, E / L AST, E / L Coefficient de Ritis, AST / ALT Intact rats, n = 9 0.61 ± 0.09 0.81 ± 0.04 1.39 ± 0.30 Control (without treatment), n = 6 1.40 ± 0.36 * 0.87 ± 0.03 0.75 ± 0.11 * Comparison Group (Carsil), n = 4 0.75 ± 0.10 0.83 ± 0.05 1.18 ± 0.22 ⁺ OD at a dose of 100 mg / kg, n = 4 0.93 ± 0.15 0.80 ± 0.04 0.93 ± 0.14 Note: * - p <0.05 compared with values in intact animals; ⁺ - p <0.05 compared with the values in animals of the control group.

Thus, the use of pinostrobin oxime, as well as the comparison drug Karsil, prevents the development of cytolysis syndrome in acute paracetamol hepatitis.

The advantages of the method for producing hepatoprotective agents based on pinostrobin from balsamic poplar buds (Рор1и8 акакатёга б.) Are as follows.

1. The availability of plant materials of balsamic poplar buds (Poricon baapiGsga b.) In the territory of Northern Kazakhstan, the total operational stock of poplar buds is 400 kg per 2.5 ha.

2. The barothermal method for producing hepatoprotective substance of pinostrobin oxime from balsamic poplar buds (Poriconium papillon L.) is environmentally friendly, since it eliminates the use of toxic solvents in the production process and is characterized by high productivity, a significant reduction in the duration of the process and provides a quantitative yield of the active substance pinostrobin with the yield 10.3% based on the thick extract.

3. The substance of pinostrobin oxime is not toxic, does not cause acute toxic reactions when tested in mice and rats and exhibits high hepatoprotective activity at a dose of 100 mg / kg body weight, and at a dose of 50 mg / kg eliminates the manifestations of cytolysis, cholestasis and mesenchymal inflammation.

4. Pinostrobin oxime refers to short-lived drugs. In rats, the drug is determined for 2 hours, regardless of the dose and the method of administration.

5. Pinostrobin oxime penetrates well mainly into vascular organs (kidneys, liver, lungs). The absolute bioavailability of pinostrobin oxime is 11%.

6. The antioxidant activity of pinostrobin oxime is 20 times higher compared to pinostrobin 1022691 nom.

7. Pinostrobin oxime has no toxic effect on the liver, reduces the rate of lipid peroxidation in it, the content of triacylglycerides by 26% and increases the level of phospholipids by 21%, inhibits the processes of lipid peroxidation in hepatocytes, reduces the content of diene conjugates by 35%, increasing the activity of antiperoxidizing enzymes protection - glutathione peroxidase and glutathione reductase up to 54%.

Claims (1)

CLAIM A method of obtaining a hepatoprotective agent based on pinostrobin from balsamic poplar buds (Rory1i8 bakashtGega b.), Including the extraction of plant materials, its processing, characterized in that the extraction is carried out in a barothermic manner at a temperature of 150-160 ° C, a pressure of 1.9 atm, for 60 min, chromatographic separation and purification of the thick extract when the column was eluted with petroleum ether and a mixture of petroleum ether-ethyl acetate 49: 1, recrystallization of the isolated technical pinostrobin, followed by synthesis on e based on oxime pinostrobina (substance for formulation).