Method for production of siberian cedar seeds liqueur and hepatoprotective preparation produced by such method
FIELD: food industry.
SUBSTANCE: method for production of Siberian cedar seeds liqueur (with hepatoprotective, antioxidant, antihypoxic, hypolipidemic effect) by way of maceration with ethyl alcohol usage; whole Siberian cedar seeds are loaded into the reactor, poured with 70% ethyl alcohol water solution; extraction is performed under preset conditions. The medicinal preparation with hepatoprotective, antioxidant, antihypoxic, hypolipidemic effect contains Siberian cedar seeds liqueur. Usage of the medicinal preparation as a hepatoprotective remedy.
EFFECT: liqueur has pronounced hepatoprotective, antioxidant, antihypoxic and hypolipidemic effect.
6 cl, 3 dwg, 8 tbl
The invention relates to pharmaceutical industry and can be used for obtaining a medicinal product of tincture of seeds, Siberian cedar pine tree and its use as a hepatoprotective funds.
In numerous studies (Lizunov, V. V. Pine nuts as an industrial oilseed raw materials. - Author. Cand. tech. Sciences. - M, - 1969. - 23 p.; Donskaya L. I., Muzalevskaya O. V. / Plant resources. - 1994. - vol.3. - S. 64-66; Outcall V. A., Pimenov, A. I., Makarov V. G., Krasnov, K. A., Strelnikova E. P., Prokhorov L. V., Yevtushenko N. With. To the question about the chemical composition of extracts from the seed coat of the Siberian cedar pine tree // II Intern. Congress "Actual problems of creation of new medicinal preparations of natural origin": Mater. St. Petersburg, 1998. - 214 s.; Strelnikova E. P., L. V. Prokhorov Seeds, Siberian cedar pine tree - component elixirs. In proc. Elixirs edited by Makarov V. G., St. Petersburg. - 1996. Pp. 87-106; P. V. Karetnikov, Dmitrichenko M. M. About the content of some trace elements in pine nuts // food Matters. - 1966. - Vol. 25. - No. 5. - P. 79-80) found that in seeds, Siberian cedar pine tree contains important chemicals such as flavonoids, lignans, nonesterified fatty acids, ubiquinone, amino acids and other valuable biological activities (antioxidant, antigipoksicski�th, anti-inflammatory, reparative, lipid-lowering, etc.). Also contained in the seeds of Siberian pine Siberian minerals and vitamins, which play a critical role in the body (a coenzyme, the regulation of intracellular signaling, metabolism, nerve and muscle activity), further contribute to the pharmacological activity of seeds. This was the basis for the development of a method for producing tinctures and drug, which is a water-alcohol extract of the seeds of the Siberian cedar pine tree.
Known methods for preparing aqueous-alcoholic tincture of cedar nuts and shell pine nuts (patents of the Russian Federation№2471862, №2016891, №2420563), used in alcoholic beverage industry.
Known method of preparing a therapeutic alcohol tinctures according to the patent of Russian Federation №2170097 (the closest analogue) from a mixture of minced raw pine nut and 40-50% alcohol at a ratio of raw material : alcohol 6-10:100. However, the level of development of this method is insufficient for its industrial implementation, and the output of the target product is unpredictable and low.
The object of the invention is to provide a method of obtaining a medicinal tincture of the seeds, Siberian cedar pine tree with high pharmacological activity and obtaining a medicinal product with hepatoprotective properties.
To do this, the method of producing a tincture of the seeds, Siberian cedar pine tree, using ethyl alcohol of whole seeds, Siberian cedar pine tree was charged into the reactor, pour the extractant at a ratio of seeds : extractant 1:5, and the extractant used 70% aqueous ethanol solution, extraction is carried out for 10-15 days at room temperature with stirring daily, get a tincture with an alcohol content not less than 65% and the dry residue is not less than 0.5%, the tincture settle and the precipitate is separated to obtain the finished product.
The technical result is to increase the efficiency of extraction of target components with therapeutic properties.
In the particular case, the ratio of seeds : extractant 1:5 provide taking into account the absorption coefficient of 1.15.
In another particular case, the infusion get with the total content of phenolic compounds in terms of Gallic acid is not less than 0.05%.
In another particular case, the infusion defend at least two days at a temperature of 8-10°C, ensuring no visible suspension.
In another particular case, the precipitate is separated by filtration or decantation.
Drug tincture of the seeds, Siberian cedar pine tree, obtained the claimed method, has hepatoprotective action. In addition, a significant antioxidant activity, anti-hypoxic activity and lipid-lowering effect of the drug.
Graphic material explaining the essence of the invention presented in the figures:
Fig.1 - kinetics of extraction of phenolic compounds (A) and dry residue (B) upon receipt of tincture of seeds, Siberian cedar pine tree.
Fig.2 is the flowchart of the tincture of the seeds, Siberian cedar pine tree. The scheme made the following notation: KtToxToICD- control of technical, chemical and microbiological respectively.
Fig.3A - liver of intact rats. Coloring hematoxylineosin, magnification × 100. Typical ordered structure of the lobules.
Fig.3B - chronic toxic steatohepatitis with marked activity. The liver of rats after 2 months of chronic experiment, treated with CCl4. Coloring hematoxylineosin, magnification × 100. Pronounced sclerosis patriliny paths, the penetration of connective tissue into lobules, globular fatty degeneration, fatty necrosis of hepatocytes, severe inflammatory infiltration of the liver tissue.
Fig.3B - chronic toxic steatohepatitis with minimal activity. The liver of rats after 2 months of chronic experiment, treated with CCl4and a tincture of the seeds of Siberian pine in a dose of 1.4 mg/kg. Coloring hematoxylineosin, magnification × 100. Ill-defined sclerosis and inflammatory infiltration in the portal tracts, isolated necrosis of hepatocytes in the field of view, atomized fatty degeneration in the periportal �areas.
When developing the method of extraction was carried out a preliminary assessment of the content in the product abietic acid, pinocembrin and pinostrobine by high performance liquid chromatography (HPLC). Spectrophotometric method for the reaction products with ninhydrin in the presence of sodium acetate has been evaluated by the content digitisation compounds in terms of arginine (primarily amino acids). Evaluate the content of the core group of active substances of the drug - phenolic compounds - used permanentresidence titration in the presence of indigenously (the methodology of the State Pharmacopoeia XI (GF XI)). The total content of phenolic compounds in terms of Gallic acid was determined spectrophotometrically using the standard sample solution of Gallic acid. The dry residue was determined by the method of GF XI, issue.2, p. 148. To study the transition of different groups of compounds from plant material in the hood as the extractant was investigated 40 and 70% ethyl alcohol. Extraction was carried out by the method of maceration in the ratio raw : extractant 1:5 for 10 days in a dark place with daily stirring. Quantitative data definitions are presented in Table 1.
|The choice of the concentration of the extractant|
|Parameters||40% ethyl alcohol||70% ethyl alcohol|
|The dry residue, %||0,8||0,8|
|The amount of oxidizable substances in terms of tannin, %||0,08||0,085|
|The amount of phenolic compounds in terms of Gallic acid, %||0,76||0,78|
|The amount digitisation compounds in terms of arginine, %||0,23||0,15|
|Abietic acid, mg/l||3,2||14,0|
It is established that the content of abietic acid increases with increasing the concentration of ethanol in the extractant. At the same time, the content of the main studied groups Biol�cally active substances, the alcohol concentration has no significant effect. The choice of the extractant 70% alcohol allows you to extract from the seeds of Siberian pine Siberian complex compounds of medium polarity, which we can assume the presence of terpenes, phenolic compounds, possibly amino acids, vitamins and fatty acids.
For making tinctures can be used by various technologies. The results of analytical studies-brewing with stirring for 3 hours, the stirrer rpm 200 rpm) show that this extraction occurs a slight increase in the amount of content of phenolic and oxidizable substances. The study of the process of remuneratio showed that this technology minor dry residue (table 2). This is probably due to the fact that not as active processes of swelling and diffusion due to the morphological structure of the seed coat of the Siberian cedar pine tree.
The use of maceration for 10 days with daily agitation has led to a simultaneous increase in the content of dry substances with a significant amount of phenolic and oxidizable substances.
|Substantiation of the technology of a pine cedar tincture|
|Remunerate||Maceration with agitation||Maceration|
|The dry residue, %||0,43±0,01||0,76||0,80±0,06|
|The amount of oxidizable substances in terms of tannin, %||0,064±0,002||0,028||0,085|
|The amount of phenolic compounds in terms of Gallic acid, %||0,073±0,002||0,045||0,078±0,006|
|The amount digitisation compounds in terms of arginine, %||0,13±0,01||0,11||0,15±0,01|
To determine the optimal conditions of the extraction process when receiving infusions was studied the kinetics of extraction (Fig.1). Pre-determined the content of extractives from the whole seeds, Siberian cedar pine tree extracted in 70% alcohol, by the method of GF XI, issue.1, p. 295. Found that the extraction process runs for at least 8 days. Extraction efficiency was about 90%.
Thus, based on�the spec of the research was developed by the proposed method of producing tinctures seeds, Siberian cedar pine tree, allowing you to get a drug that meets the requirements of the global Fund XI editions and storage stable for at least 2 years.
As an example of the invention, the following is the technology (Fig.2) obtain the tincture of the seeds, Siberian cedar pine tree, used in the enterprise of the applicant.
BP.1. Preparation of production
At all stages of technological process, the organization of production should be in accordance with the requirements of GMP and ISO 9001:2000.
BP.2.1. Preparation of water
Water to ensure quality indicators according to the norms of FS 42-2619-97 subjected to further purification.
TP.3. Obtaining tinctures
TP.3.1. The preparation of the extractant.
The extractant used 70% ethyl alcohol. Into the reactor using a pump pumped the calculated amount of purified water, then ethyl alcohol 96%. The mixture was stirred for 20 minutes using a vacuum pump, then select a sample to determine the strength of alcohol. The concentration of extractant obtained in control using spycamera. The resulting solution of alcohol poured into the reactor in operation
Maceration is carried out in a reactor with a false bottom and a removable cover. Before you start, check that the name and number� series of raw materials to the packaging data analysis passports. Are entered in the log at the upload date, no series of raw materials, full name of the operator, no analytical passports, No. series of tincture. Manually load the whole seeds, Siberian cedar pine tree. Downloaded raw material distribute evenly. Download data (quantity of raw material, download time) are entered in the logbook. Then pour the prepared solution of the extractant (in the ratio 1:5) taking into account the absorption coefficient of 1.15. Extraction is carried out for 10-15 days at room temperature, stirring daily. After 10 days take a sample for the determination of dry residue and the amount of phenolic compounds. After receiving a positive result, the extract is drained. In the case of low solids or content of active ingredients infusion and continue for several days. After the positive data analysis the tincture is poured into the receiver. Obtained after extraction of the meal is collected in the receiver. Get tincture with an alcohol content not less than 65%, of solids not less than 0.5% and total content of phenolic compounds in terms of Gallic acid is not less than 0.05%.
TP.3.3. Settling and separation of the precipitate
Then, the resulting tincture defend not less than 2 days at a temperature of 8-10°C. after 48 hours of settling, take a sample and visually inspect Otsu�of suspension. In the presence of suspended matter sedimentation continued for 24 hours. After obtaining satisfactory data to proceed to the separation of the precipitate by filtering. After filtering again selected a sample of tincture to control the alcohol content. This infusion is passed further to the stage of packing.
Characterization of specific pharmacological activity of tincture of seeds, Siberian cedar pine tree
Analysis of literature data and the results of previously conducted investigations indicate that drugs derived from the seeds of Siberian pine Siberian (core and shell), contain a rich complex of biologically active substances and possess a wide spectrum of pharmacological activity. So, the elixir Kedrova, the predominant component of which is an aqueous-ethanol extract from the seeds of Siberian pine Siberian, the experimental and clinical study showed a pronounced hepatoprotective, antioxidant, antihypoxic, hypolipidemic and cardiotoxin activity. This determined the main focus of pharmacological research obtained in the manner of a new drug alcoholate of whole seeds of Siberian pine Siberian Pine cedar tincture" (IPS).
As in the draft monograph of the enterprise quantitative standardizat�u "Pine cedar tincture" suggested by the content of total phenolic compounds, in experimental studies, the drug is dosed in mg (total content of phenolic compounds) per kilogram of body weight of the animal.
Study of hepatoprotective activity of tincture of seeds, Siberian cedar pine tree
In seeds, Siberian cedar pine tree contains a complex of biologically active substances, has almost all the mechanisms of action inherent in the known medications. So, in the seeds of Siberian pine Siberian widely represented group of polyphenols (flavonoids, lignans, etc.), and it is known that polyphenolic compounds possess such parties of the action as antioxidant and antihypoxic, which are regarded as basic for the formation of various pharmacological activities requiring energy security and the integrity of cell membranes and organelles (A. Aleksandrova, 1988; L. V. Pastushenkov, E. E. Lesiewska, 1994; Y. V. Medvedev. A. D. Tolstoy, 2000, etc.). These activity are the basis of many types of actions, including anti-inflammatory, reparative, immunomodulating, providing a variety of protective effects, hepatoprotective, cardioprotective, gastroprotective, etc.).
It is possible to believe that the antioxidant effect of polyphenolic compounds seeds, Siberian cedar pine tree is complemented and supported �walking in their composition α-tocopherol, ubiquinone and carotenoids. A certain contribution to the normalization of the functioning of the respiratory function of mitochondria and improve the delivery of oxygen to tissues (antihypoxic effect) can make contained in the seeds of Siberian pine Siberian zinc ions and copper. Inhibition of the process of destruction and the maintenance of reparative-regenerative processes in the liver is supported by the phospholipids. contained in the seeds.
Was used a model of chronic liver injury (vengerovsky A. I., Golovina, E. L., Kovalenko M. Yu., etc. combined use of prednisone and hepatic containing phospholipids in experimental chronic hepatitis // Experim. and the clinical. pharmacology. - 1999. - Vol. 62, No. 2. - Pp. 28-30). The duration of the experiment was 30 days. Animals 3 times a week administered orally 2 ml/kg CCl450% oil solution. Beginning with the 15th day after the beginning of intoxication, rats of the 3rd, 4th and 5th groups, along with CCl4also received per os daily test substance at doses of 0.35 mg/kg, 0.7 mg/kg and 1.4 mg/kg, respectively; the comparison medication zelibor at a dose of 100 mg/kg were injected to animals 6-th group. On the 30th day all animals were sacrificed by decapitation using a guillotine according to the recommendations for euthanasia of experimental animals.
About the severity of the lesion �Iceni judged by the values of the ratio of the mass of the liver, which was calculated as the ratio of liver weight in mg to the weight of animal in grams, histological assessment of sections of the authority and activity in plasma marker enzymes of liver alanine aminotransferase (ALT) and aspartate aminotransferase (ACT).
In histological studies, pieces of liver were fixed for 24 hours in a solution of 10% formalin. In the study of histological preparations that have passed the standard method of wiring using alcohols, embedded in paraffin and stained with hematoxylin-eosin, were used optical microscope BIOLAM. Counting and measuring changes were carried out at magnification × 100 in 10 fields of view.
ALT activity and ACT were determined by optimized UV analysis in accordance with the recommendations of the JFCC (Bergmeyer N. U. et al. // J. Clin. Chem. Clin. Biochem. - 1986. - V. 24. - P. 497). Used reagents company Diasys. Blood sampling was made from the sublingual vein.
Results and discussion
The introduction of carbon tetrachloride to rats 3 times per week for 4 weeks resulted in severe liver damage, as evidenced by the ratios of liver weight and histological and biochemical studies.
The degree of increase of the ratio of the mass of the liver of animals with toxic her defeat is an objective indicator of the severity of the inflammatory process in the body (Alexandrov AE., Vinogradova T. I., Litvinov V. A., etc. Methods of prevention and elimination of hepatotoxicity // for the First time in medicine, St. Petersburg. 1 995. - №2, 3. - P. 8). From Table 3 it can be seen that in untreated animals treated with CCl4the ratio of liver weight compared with intact increased by 52.4% (from 38.9 to 59.3). In animals that were administered the tincture of the seeds, Siberian cedar pine tree in the lowest dose (0.35 mg/kg), the decline was manifested only in the form of trends. At doses of 0.7 mg/kg and 1.4 mg/kg test substance as a drug comparison zelibor, statistically significantly reduced the ratio of liver weight in the absence of visible differences between these groups.
|Changing the ratios of liver weight|
|№ p/p||Terms of experience||The number of animals in the group||The ratio of the mass of the liver|
|3||CCl4+ JMC 0.35 mg/kg||6||5,1±2,7|
|4||CCl4+ JMC 0.7 mg/kg||8||48,7±1,3 p5-2<0,05|
|5||CCl4+ JMC 1,4 mg/kg||7||of 44.7±1.8 p5-2<0,05|
|6||CCl4+ Silimar 100 mg/kg||9||43,4±1,5 p6-2<0,05|
Histological studies showed that the developed experimental model corresponded to histological criteria, characterizing the development of chronic toxic active steatohepatitis. Were evaluated the following characteristics:
fat necrosis of hepatocytes in the lobules, in which the prevalence was characterized as periportal, centrolobular, diffuse as a percentage based on the number of hepatocytes in the lobule (about 300). Accordingly, the percentage of necrotic hepatocytes was evaluated the activity of chronic toxic steatohepatitis: weakly pronounced activity - up to 10%, moderate activity - 11-29%, pronounced activity - 30% or more;
- degeneration as initial et�p damage. In this experimental model of fat intracellular characterized by localization (periportal, centrolobular) and the amount of fatty vacuoles (small, globular);
- fibrosis. Semi-quantitative method was estimated sclerosis in the portal tracts on the severity and the width of the interlayer connective tissue in the portal tracts (mild, moderate, marked);
- inflammation, which was characterized by limfogistiocitarnom leukocyte and neutrophil infiltration of the portal tracts and lobules on the severity (mild - to 50 cells, moderate - from 51 to 100, expressed more than 100).
In the study of drugs in the liver of intact rats in the lobules of the detected radial location of beams in the form of a heavyweight from the Central veins to portal tracts, hepatocytes places different optical density in connection with the degree of accumulation of glycogen in the cytoplasm. Absent degenerative and necrotic changes observed were minor, within the norms of lymphoid infiltration of parenchyma. The portal tracts contain a triad: the artery, vein and bile duct, which were surrounded by the small number of connective-tissue stroma. Sinusoidal capillaries in the lobules is not extended, there was hyperemia of the Central veins (Fig.3A).
Histological preparations of the liver �the lynx, treated with CCl4(group 2) had evidence of chronic active toxic steatohepatitis with marked activity, resembled toxic dystrophy of the liver and consisted of the following: liver damage wore a total character, hepatic lobules with sharply expressed disconnecktie beam structure on the background of the predominance of globular fatty dystrophy of hepatocytes, which were localized in all parts of the lobules. In some lobules degenerative changes prevailed periportal. There was fat necrosis (phaneros) more than 30% of hepatocytes slices in different areas (mainly colportage). In these zones - lymphocytic infiltrate. The inflammation was characterized by expressed and captured the portal tracts and lobules, in addition, the inflammatory infiltrate was composed of histiocytes and neutrophilic leukocytes, a small number of the latter penetrated into the lobule, sinusoids met copperhouse cells. Fibrosis of the portal tracts is expressed ubiquitously, a layer of connective tissue narrow, in single fields of view - wide, was surrounded by slices with a tendency to annularia sclerosis and the formation of CKD cirrhosis. In some segments there was no Central Vienna ("false segments"), which is a diagnostic criterion for the development of cirrhosis of the liver (Fig.3B).
In animals treated with JMC in the dose of 0.7 mg/kg, changes consistent with chronic toxic steatohepatitis with moderate activity and was characterized by: necrosis 10-20% of hepatocytes with localization periportal, degeneration, still prevailed in the form of coarsely�abulnaga obesity hepatocytes scattered nature. Sclerosis of the portal tracts were mild in nature in the form of narrow layers of Mature connective tissue. Inflammatory infiltrate with mononuclear cells (lymphocytes, histiocytes) and pronounced moderately expressed, and neutrophilic leukocytes is ill-defined.
In preparations of rat liver treated JMC at a dose of 1.4 mg/kg (5th group) (Fig.3B), and celebram (6th group), revealed histological signs were the same with minor differences. In these groups the development of chronic toxic steatohepatitis characterized by minimal activity, which was confirmed periportal necrosis up to 10% of hepatocytes or absence of some slices of necrosis at all, mainly periportal atomized fatty degeneration in the liver of rats 5-th group, and in animals treated with zelibor, - the recent localization centrilobular, mild infiltration of the portal tracts and lobules of lymphocytes and histiocytes. Fibrosis of the portal tracts in both groups was mild. It should be noted that the existence of a protein, including balloon, degeneration in this experimental model (rats) were detected.
On the basis of a comparative histological evaluation of the liver of rats of different groups, you can make the following conclusion: all groups showed the development of chronic t�xeonsaga of steatohepatitis with different degree of activity, which manifested itself in a number of hepatocytes undergoing necrosis, the level of inflammatory and degenerative changes in the untreated group of rats treated with CCl4- necrosis of more than 30% of hepatocytes, fibrosis and inflammatory infiltrate pronounced. In the groups receiving drugs: JMC at a dose of 0.35 mg/kg - necrosis of more than 30%, fibrosis and inflammation somewhat less pronounced; JMC in the dose of 0.7 mg/kg - necrosis of 10-20%, fibrosis and mild inflammation; JMC at a dose of 1.4 mg/kg and seliber - less than 10% necrotic hepatocytes, mild fibrosis and inflammation in the portal tracts.
Thus, the marked increase in the hepatoprotective action of the drug with increasing doses. Comparative analysis showed that the ANC at a dose of 1.4 mg/kg and zelibor the most favourable impact on the morphological state of the liver of rats and had a pronounced hepatoprotective effect: necrosis and fibrosis in these groups was similar and minimal. When identifying atomized fatty in liver cells because of its preferential localization in rats receiving ANC at a dose of 1.4 mg/kg - periportal, and in the group of animals treated with cilibia, - centrolobular defeat of hepatocytes (Fig.3B), i.e. in the zone of action of P-450.
An important indicator reflecting the degree of liver damage, is the activity of enzymes of hepatic origin�Oia in the blood (an indicator enzymes cytolysis). It is known that changing their activity may occur much earlier than histologically registered destructive processes in the cells of the parenchyma of the organ (Bluher F. A. the Diagnosis and differential diagnosis of chronic hepatitis and liver cirrhosis / A. F. Bluger, I. N. Novitsky // Klin, med. - 1985. - Vol. 63, No. 2. - P. 134-140).
We determined the activity of serum alanine aminotransferase and aspartate aminotransferase. The results obtained are presented in Table 4, indicate a marked cytolysis of liver cells and the output of indicator enzymes in the blood of untreated animals treated with the hepatotoxin (CCl4). ALT activity they have increased by 150%, a ACT - on 71,1%. The most pronounced hepatoprotective effect, comparable to the action of cilibia, JMC showed at a dose of 1.4 mg/kg, reducing the value of the activities of ALT and ACT 34.3% and 65.1% respectively. Significantly, though less pronounced decreased values of activities aminotransferase and under the influence of the ANC in the dose of 0.7 mg/kg In a dose of 0.35 mg/kg, the decrease in ALT activity was registered only in the form of trends.
Thus, the study of hepatoprotective activity of "Pine cedar tincture" on the model of chronic toxic liver injury by carbon tetrachloride showed that the test substance has expressed, comparable with the effect of known�about the hepatoprotector of cilibia, hepatoprotective activity as evidenced by a significant decrease in the ratio of liver weight, histological and biochemical studies. Morphometric evaluation of the liver revealed some of the benefits of ANC prior to product comparison. If animals treated with cilibia, atomized fatty degeneration remained the most important for the functional activity of the organ centrolobular zone (the zone of action of cytochrome P-450), under the action of a test substance atomized fatty degeneration was observed only periportal.
|Changes in the activity of aminotransferases in the serum of rats|
|№ p/p||Terms of experience||The activity of ALT, units/l||Activity ACT, u/l|
|1||Intact animals (n=6||79,2±6,6||164,0±9,2|
|3||CCl4+ JMC 0.3 to 5 mg/kg n=5||171,3±14,3** (-13,5%)||278,8±35,7** (-41,2%)|
|4||CCl4+ JMC 0.7 mg/kg n=6||145,2±13,4** (-26,8%)||199,1±41,1** (-58%)|
|5||CCl4+ JMC 1.4 mg/kg n=6||130,1±18,3** (-34,3%)||165,5±17,3** (-65,1%)|
|6||CCl4+ zelibor, 100 mg/kg n=6||129,8±17,6** (-34,5%)||169,2±26,3** (-64,3%)|
|* - significantly compared to the 1st group|
|** - significantly compared with the 2nd group|
|In brackets is the reduction in enzyme activity compared with the indicators in the 2nd group (%)|
Study of antioxidant activity of tincture of seeds, Siberian cedar pine tree
For the understanding of the mechanisms of hepatoprotective activity JMC is important to study the antioxidant status of the organism. Study of the antioxidant action of "Pine cedar tincture" was conducted in the experimental model of toxic hepatitis induced with carbon tetrachloride. As the comparison drug was used�van are widely used in medical practice hepatoprotective drug silimar.
The choice of this model was motivated by the fact that the damaging factor of the action of carbon tetrachloride are free-radical products formed during its metabolism by cytochrome P-450 liver: -CCl3·, CHCl2· and other radicals (Burlakova E. B., Khrapova N. G., 1985; Bobylev V. N., 1987; Bobylev V. N. et al., 1994; Dadali V. A., 1999, 'connor, 1990).
Determination of the concentration of products of lipid peroxidation on the test with thiobarbituric acid (TBA) was carried out according to the method (Tian L. Alteration of antioxidant enzymes and oxidative damage to macromol-ecules in different organs of rats during aging // Free Radical. Biol. Med., 1998. - Vol.24, No. 9. - P. 1477-1484). The principle of the method based on the reaction of interaction of malondialdehyde with thiobarbituric acid with the formation of coloured product. In the case of determining peroxidation products in liver tissue (100 mg of tissue homogenized with 3 ml of physiological solution. Measurements were performed at wavelengths of 535 and 580 nm. The concentration of malondialdehyde was calculated by the formula
C is the concentration of malondialdehyde (µmol/l);
ΔE535-580- the difference of the optical densities of the sample at 535 and 580 nm;
1,88·105M-1cm-1the molar extinction coefficient.
Determination of antioxidant activity of blood plasma was performed by the method (Valkonen N., Kuusi T. Sectrophotometric assay for total peroxylradical trap-ping antioxidant potential in human serum // J. Lipid Res., 1997. - Vol.38. - P. 823-833). Principle of method: under the influence of hydroxyl radicals occurs the solvolysis of dibutyrate fluorescein, with the formation of optically active fluorescein having both the absorption (490 nm) and fluorescence (530 nm). Antioxidant activity of blood plasma was calculated by the formula
P - the final plasma dilution;
0,077 M-1cm-1the molar extinction coefficient of fluorescein;
E - the optical density at a wavelength of 490 nm.
Determination of reduced glutathione in liver homogenate was performed by the method (Ellman G. L., 1959). The principle of the method is based on the ability of low molecular weight thiol compounds in interaction with Ellman's reagent to form a colored compound thio-2-nitro-benzoic acid, the aqueous solution of which has a maximum absorption at a wavelength of 412 nm. The portion of the liver tissue (100 mg homogenized with 3 ml of physiological solution. The concentration of SH-groups is calculated by the formula
ΔE is the change in optical density of the solution;
13·103the coefficient of molar extinction;
P - the final dilutions of the homogenate.
Determination of catalase activity was performed by the method (Leff J. A., Oppengard M. A., Curiel, T. J. et al. Progressive increases in serum catalase activity in advancing human immunodeficiency virus infection // Free Radic. Biol. Medicine. - 1992. - Vol.13, No. 2. - P. 143-149). With�orost reduce the concentration of hydrogen peroxide in the incubation medium is recorded spectrophotometrically at a wavelength of 230 nm for 3 minutes.
Determination of the activity of superoxide dismutase reaction using autookislenia of quercetin was performed by the method (Kostyuk V. A., Potapovich A. I., J. V. Kovalev Simple and sensitive method for the determination of superoxide dismutase activity based on the oxidation of quercetin. // Voprosy med. chemistry. - 1990. - Vol. 36. - vol.2. - P. 88-91). The method is based on determining the degree of inhibition of the oxidation of quercetin. As a result of oxidation of quercetin at pH of 10.0 in the presence of tetramethylethylenediamine under aerobic conditions occurs the generation of superoxide anion radical, which, in turn, in the presence of SOD undergoes dismutation. The incubation mixture contained 1.4 µm quercetin, of 0.015 M phosphate buffer (pH 7.8), 0.08 mm EDTA, 0.8 mm of tetramethylethylenediamine, 0.1 ml of biological material in the final volume of 3.5 ml. the Rate of oxidation of quercetin was estimated by the change of absorbance at 406 nm for 20 minutes.
Determination of glutathione peroxidase activity using digestion of tertbutyl method (Gavrilova A. R., Khmara N. F. Determination of glutathione peroxidase activity of erythrocytes at saturating substrate concentrations. // Laboratory work. - 1986, No. 12. - Pp. 721-724). Method principle: glutathione peroxidase, using as a coenzyme reduced glutathione, catalyzes the reduction of hydrop�recise of tertbutyl. On the activity of glutathione peroxidase was judged by the speed of decline in the concentration of reduced glutathione.
Determination of glutathione reductase activity was performed by the method (Beutler E. Red cell metabolism. A manual of biochemical metods. 2-d. Edition. - N. Y., S. Francisco, London: Grune and Stratton. - 1975. - P. 69-71). As the recovery of the oxidized glutathione is consumed equimolar amount of NADPH, the rate of reducing the content of which is determined by decrease in optical density at a wavelength of 340 nm for 10 minutes. Non-specific NADPH-oxidase activity consider using a blank sample containing no glutathione.
The results of the study
With the development of induced carbon tetrachloride hepatotoxicity was observed strengthening of processes of lipid peroxidation, as reflected in increases of 30% of the content of malondialdehyde and a decrease of 22% in the content of glutathione in the liver (table 5).
In parallel there was a significant (9.4%) increase in the concentration of MDA in the serum, while the total antioxidant activity of blood serum (TRAP) was not significantly altered, that, apparently, is connected with sharp increase in the activity of inducible antiradical enzymes (table 6): glutathione peroxidase (544%), catalase (178%), glutathione reductase (71%). In the conditions of this experiment there were not any significant changes�in termination of superoxide dismutase activity, observed only a tendency to decrease.
|Change indicators that reflect the intensity of lipid peroxidation and total antioxidant activity in liver and serum of blood (M±M)|
|№ p/p||Groups of animals, dose||Mdev liver (nmol/g)||Glutathione in liver (nmol/g)||MDV serum (nmol/ml)||Total antiradical activity of blood plasma (nmol/ml)|
|1.||Intact group||451,6±6,5 n=6||1369±25,3 n=6||12,7±0,5 n=6||13,2±0,3 n=6|
|2.||Group CCl4||587,6±13,9 n=6*||1068±31,7 n=7*||13,9±0,3 n=8*||13,9±0,2 n=8|
|3.||The group JMC 0.35 mg/kg||530,3±10,9 n=5**||1495±27,7 n=5**||13,8±0,3 n=6||14,8±,6 n=6|
|4.||The group JMC 0.7 mg/kg||491,1±9,9 n=7**||1499±30,5 n=8**||10,6±0,3 n=8**||13,2±0,2 n=7**|
|5.||The group JMC 1,4 mg/kg||524,4±9,0 n=7**||1394±22,7 n=6**||9,3±0,4 n=7**||13,8±0,2 n=5|
|6.||Group Silimar 100 mg/kg||479,1±10,9 n=9**||1620±36,0 n=9**||10,1±0,4 n=8**||13,5±0,2 n=9|
|* - significantly compared with the first group;|
|** - significantly compared with the second group.|
|Changes in the activity of antioxidant enzymes during the development of toxic liver damage caused by carbon tetrachloride|
|№ p/p||Groups of animals, dose||Catalase activity (µkat/Hb)||The activity of HL�of stateoperated (µkat/Hb)||The activity of glutathione reductase (µkat/Hb)||The activity of superoxide dismutase (.e./Hb)|
|1.||Intact group||49,9±2,8 n=6||3,8±0,7 n=6||277,5±60,5 n=6||29,0±2,2 n=4|
|2.||Group CCl4||139,5±7,9 n=6*||24,5±2,2 n=7*||to 475.6±32,7 n=7*||23,5±2,7 n=8|
|3.||The group JMC 0.35 mg/kg||of 76.6±5,9 n=6**||15,4±1,1 n=6**||490,9±41,6 n=6||25,1±1,4 n=5|
|4.||The group JMC 0.7 mg/kg||62,4±3,7 n=8**||13,6±0.9 n=7**||347,0±14,9 n=8**||33,0±3,1 n=7**|
|5.||The group JMC 1,4 mg/kg||62,0±4,3 n=7**||13,2±1,2 n=7**||339,8±54,7 n=7||32,7±1,4|
|6.||Group Silimar 100 mg/kg||96,0±19,2 n=8**||7,0±0,9||341,2±17,5 n=9||27,8±3.3 n=9|
|* - significantly compared with the first group;|
|** - significantly compared with the second group.|
Increased activity of inducible enzymes, particularly glutathione peroxidase and glutathione reductase, is associated with increased oxidation of reduced glutathione in glutathionylated under the action of free radicals generated by the metabolism of tetrachloroethylene (Connor H. D. et al. Reaction of glutathione with a free radical metabolite of CCl4 // Mol. Pharmacol. - 1990. - No. 37. - P. 443-451). The increase of catalase activity is dependent on enhanced production of hydrogen peroxide.
Thus, under the action of CCl4profound disturbance occur enzyme and links-enzymatic antioxidant system of the liver.
Use as a hepatoprotector "Pine cedar tincture in a dosage of 0.35 mg/kg, 0.7 mg/kg and 1.4 mg/kg and celebra at a dose of 100 mg/kg led to a normalizing changes of processes of lipid peroxidation in the liver. In all groups of experimental animals showed a significant decrease of MDA level respectively 9.8; 16,5; 10.8 and 18.5% and a significant increase ur�VNA glutathione, respectively, at 40; 40,3; 30.5 mm and 51.7%. The most effective should recognize the action of the tincture in the dose of 0.7 mg/kg.
Serum drugs also significantly reduced the intensity of lipid peroxidation. When using AIO installed a dose-dependent effect. With increasing doses of the JMC (0.35 mg/kg, 0.7 mg/kg and 1.4 mg/kg) MDA content in the serum was decreased by 0.7%, 23,8% and 32.8% respectively. The nature of the influence of the studied drugs on the antioxidant enzymes had its own peculiarities. With the introduction of the JMC in doses of 0.7 mg/kg and 1.4 mg/kg, the activity of the inducible enzymes significantly decreased: catalase by 55.3% and 56%; glutathione peroxidase by 44.5% and 54%; glutathione reductase by 27% and 28.6%, respectively, for catalase and glutathione reductase within the range of indicators of intact animals. While there was significant compared with the control group increased activity of superoxide dismutase 40.4% and 39% respectively. The introduction of celebra at a dose of 100 mg/kg was accompanied by a significant decrease in activity induced catalase and at the same time did not lead to increased activity of superoxide dismutase. Changes in activities of glutathione reductase and glutathione peroxidase were similar in the groups of animals receiving ANC at doses of 0.7 mg/kg and 1.4 mg/kg, the activity of glutathione reductase was significantly decreased by 28.3%; deep�of actionprocess - 54%.
Thus, comparable to the severity of the action "Pine cedar tincture" (in doses of 0.7 mg/kg and 1.4 mg/kg) and standard drug celebra (optimal dose) was observed in relation to glutathione and related enzymes, glutathione peroxidase, glutathione reductase, and also in regard to the effect on lipid peroxidation in hepatocytes and total antioxidant activity of blood. Changes antiradical activity and antiperoxidant enzymes were different: the introduction of the JMC was accompanied by a more pronounced response of superoxide dismutase and celebra - catalase.
One can assume that the hepatoprotective action we have developed a new drug "cedar Pine tree tincture" both due to its own antioxidant activity and induction of endogenous antioxidant systems.
Antihypoxic activity of tincture of seeds, Siberian cedar pine tree
Antihypoxic activity of pharmacological substances evaluated on models of different forms of hypoxia: hypoxic, circulatory, himicheskoi and gistologicheskoe. Most often used varieties of hypoxic hypoxia. Our assessment antihypoxic activity of the test substances were conducted on the model of hypoxic hypoxia with hypercapnia. Mice-males (16-20 g) were placed in hermetically closed chamber (e�sekator). To ensure additional sealing between the outside lid of the desiccator and his body laid special sealer. The camera was placed on 3 animals from the control and two experimental groups. AIO was administered orally daily for five days, the mice of one group at a dose of 0.7 mg/kg native liqueurs; other - 1.4 mg/kg dealcoholizing. The last introduction was carried out for 2 hours before placing animals into the chamber. The mice of the control group at the same time and in the same volume of water was injected. The results are presented in Table 7.
|The influence of the "Pine cedar tincture" on the lifespan of mice in a confined space|
|№ p/p||Terms of experience||The duration of life, min||The increase in life expectancy, %|
|1||Control mice (n=6||32,4±0,3||-|
|2||JMC 0.7 mg/kg n=6||43,9±0,4*||35,5|
|3||JMC (d/a) 1.4 mg/kg n=6||45,1±0,4*||39,2|
As the table shows, the test substance showed a pronounced antihypoxic effect in the middle "therapeutic" dose of 0.7 mg/kg, and in most of the tested dose (1.4 mg/kg in dealcoholization), almost equally, statistically significantly, increasing the duration of life of animals in the hermetically closed space (35.5% and 39.2%, respectively). No dozozavisimoe in this experiment, obviously due to the fact that in smaller doses (0.7 mg/kg) was observed synergism of the action of biologically active substances tinctures and ethyl alcohol, included in its composition. It is known that under the influence of alcohol in low doses occurs shallow inhibition of the Central nervous system, conditions are created for an economical expenditure of energy and increases the body's resistance to hypoxia (Botondi A., Chlan L. Effect of alcohol on experimental cerebral hypoxia // Veng. pharmacotherapy. - 1972. - №1-2. - P. 78-81; Kyol Kikuo Effect of hypoxta on the ratio of transport in rat brain slices and the protective action of anaestetic drugs with special reference of ethylcarbamate // J. Na-ra Med. Assoc. - 1974. - Vol.25, No. 1-2. - P. 83-93).
Thus, on the model of hypoxic hypoxia with hypercapnia established the existence of a "Pine cedar tincture" antihypoxic activity.
The study of hipolite�chemical action of tincture of seeds, Siberian cedar pine tree
It is known that the liver plays a key role in the metabolism of lipids and lipoproteins. Violation of metabolism in the liver contributes to the development of dyslipidemia and atherosclerosis (Klimov A. N., Nikulicheva N. G. Lipids, lipoproteins, and atherosclerosis. - St. Petersburg: Piter, 1995. - 298 p.; Brown M. S., Goldstein I. L. A receptor-also been other ideas where pathway for cholesterol homeostasis // Science. - 1986. - Vol.232. - P. 34-47). In the liver, the synthesis of cholesterol (LDL), aobaku, lipoprotein very low density, nascentric, potentially antiatherogenic high density lipoproteins (HDL), and enzyme degradation of cholesterol 7-α-holesterinesterzy. In addition, it is concentrated more than 70% of apob, E receptors, contributing to the capture of the blood of atherogenic lipoproteins with subsequent degradation of cholesterol and formation of bile acids (Brown M. S., Goldstein J. L., 1986). Violation of activity of these homeostatic mechanisms of the liver leads to the development of atherogenic dyslipidemia nature rotatsionnogo type (Gainer J. I. Hypoxia and atherosclerosis: a reevaluation of the old hypothesis // Atherosclerosis. - 1987. - Vol.68, No. 3. - P. 263-266).
Even in normal circumstances, the arterial wall characterized by a significant restriction of blood supply and, under the influence of different risk factors of atherosclerosis revealed a marked predominance of the anaerobic pathway of energy production on aerobic and activation of processes of lipid peroxidation that contributes to the development of edema, increase permeability of blood components, including atherogenic lipoproteins and their deep oxidation (Chernov, Yu. N., Vasin mV, Bathsheva G. A. Pathological changes in cell membranes in ischemic heart disease and possible ways of pharmacological correction // Experim. and the wedge. farmakol. - 1992. - No. 4. - P. 67-72; S. R. Bedwell, Dean T., Jessup W. The action of defined oxygencentered free radicals on human low density lipoprotein // Biochem. J. - 1989. - Vol.262. - P. 707-712; Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Re-view article // Nature. - 1993. - Vol.362. - P. 801-809). There is a certain parallelism between the degree of arterial wall hypoxia (decreased activity of respiratory enzymes), on the one hand, and inhibition of prostacyclin synthesis and its atherosclerotic lesions on the other (G. Schneiderman, T. Goldstick Carbon monoxide induced arterial wall hypoxia and atherosclerosis // Atherosclerosis. - 1978. - Vol.30, No. 1. - P. 1-13).
In the literature there is evidence of positive effects of possessing antihypoxic and antioxidant properties (GHB, olifen, antizol, uninon. α-tocopherol, etc.), with hyperlipidemia and atherosclerosis both in experiment and clinic (Dudayev V. A., Borodkin, V. A. Abboud, etc. the Use of antioxidant Winona in complex treatment of patients with ischemic heart disease // Cardiology. - 1989. - No. 1. - P. 48-52; O. V. Remezov current data on the pathogenesis of atherosclerosis. Message 2. Metabolic, etc�many violations of the arterial wall and liver, contributing to the development of atherosclerosis // Efferent therapy. - 1998. No. 4. Pp. 15-20; Choares M. Y., Perdriset G. M., Lemeberdiere F. A. et al. Alfa-tocoferol: effect on plasma lipoprotein in hypercholesterolemic patients // Israil. J. Med. Sc. - 1987. - Vol.23. - No. 3. - P. 869-872).
The presence of "Pine cedar tincture" hepatoprotective activity and anti-hypoxic and antioxidant activities have provided the background for the study of the hypolipidemic properties. Used rats with chronic toxic Tetrachloromethane lesion of the liver, and the study of hepatoprotective and antioxidant activities.
The study of the hypolipidemic properties of the JMC held in accordance with the guidelines MOH, 2000 (Ryzhenkov V. E., Remezov O. V., Makarov V. G. guidelines for the study of lipid-lowering and anti-atherosclerotic action of pharmacological substances // Guidance on experimental (preclinical) study of new pharmacological substances. - M - 2000. Pp. 224-227). Determination of cholesterol (LDL) in serum was carried out by enzymatic colorimetric method - SAD-RAHR (Schlettler G., Nussler E. // Arbeitsmed Socialmed. Preventivmed, 1975. - No. 10. - S. 25) with the use of a set of reagents firm "Vital Diagnostics" (Germany). Calculating the concentration of HDL was performed according to the formula
the concentration of the standard, where
D - Opticheskie� density (W. Richmond, Nussler E. // Clin. Chem. - 1973. - Vol.19. - P. 1350.).
The content of α-XC is found by using the reagents of the same company at a wavelength of 546 nm on the spectrophotometer SF-26 after a preliminary deposition of β-lipoproteins by heparin in the presence of ions of Mn++(Burstein, M. et al., 1970). The basis for the determination of triglycerides (TG) using reagents firm "Vital Diagnostics" is a cascade of cleavage reactions TG, at the end of which a colored coupling Henneman with maximum absorption at 500 nm, the concentration of which is determined photometrically, is proportional to the concentration of triglycerides in the blood. Determination of hydroperoxides (HP) in apob-containing LP, precipitated by heparin in the presence of ions of Mn++that was performed by the method of El-Saadani, M. et al. (El-Saadani, M. et al. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent // J. Lipid Res. - 1989. - Vol.30, No. 4. - P. 627-630).
The results of these studies are presented in Table 8.
|Hypolipidemic action of the JMC in chronic toxic hepatitis in rats|
|№ p/p||Terms of experience||Total cholesterol, mmol/l||TG, mmol/l||α-LDL, nmol/l||Hydrobar�Kishi in apob-LP, nmol/l|
|3||CCl4+ JMC 0.35 mg/kg n=6||to 2.74±0,05**||0,62±0,08**||0,85±0,03||10,03±0,34|
|4||CCl4+ JMC 0.7 mg/kg n=8||2,67±0,07**||0,81±0,03**||0,93±0,05**||9,28±0,33|
|5||CCl4+ JMC 1.4 mg/kg n=7||2,49±0,04**||0,78±0,04**||0,9±0,02**||of 8.47±0,34*|
|6||CCl4+ silimar 100 mg/kg n=9||to 2.74±0,06**||0,66±0,05**||0,89±0,05**||7,65±0,34*||* - significantly in comparison with the 1st group|
|** - significantly in comparison with the 2nd group|
As might be expected, the development of chronic toxic steatohepatitis in rats was accompanied by a significant rise in serum cholesterol (2,77±0,05 to 3.12±0,07 mmol/l), triglycerides (from 0.65±0.04 to 1,03±0,03 mmol/l) and also by the statistically significant decrease in the level of atherogenic lipoprotein fractions - α-cholesterol (1,04±0,03) to 0.71±0,02 mmol/l). The test substance used in all doses prevented gipolipidemicheskie shifts observed in chronic hepatitis. The level of CL and TG under the influence of "Pine cedar tincture" was reduced to the values of intact rats. Statistically significantly recovered and blood levels of atherogenic lipoproteins.
As already mentioned, the lipid peroxidation of atherogenic low density lipoproteins are much more damaging effect on the arterial wall and is recognized as one of the main factors of atherogenesis. In this regard, we investigated the effects of the JMC on the peroxide oxidation of atherogenic apob-containing lipoproteins in the serum of rats.
As seen from Table 8, in terms of experimental hepatitis and developed hyperlipidemia significantly from�alicias the content of peroxidation products in apob-containing lipoprotein. "Cedar pine tree tincture in a dose of 1.4 mg/kg significantly reduced the content of hydroperoxides in apob-containing lipoproteins. Thus, on the model of chronic toxic liver injury in rats, accompanied by the development of atherogenic dyslipidemia in nature, found a significant hypolipidemic activity of SKN, the normalization of blood levels of antiatherogenic lipoproteins and a decrease in the content of hydroperoxides in apob-containing LP, one of the main factors of atherogenesis.
The main conclusions
1. On the model of chronic toxic hepatitis caused by long-term administration of carbon tetrachloride using a biometric, biochemical and histological methods, "Pine cedar tincture" revealed a well-defined, comparable with the effect of the reference drug celebra, hepatoprotective effect. For some indicators morphometric assessment of liver condition the advantages of the JMC at a dose of 1.4 mg/kg prior to product comparison.
2. Significant antioxidant activity of a test substance, which was manifested as a significant reduction in indicators that indicate activation of the FLOOR when induced liver disease, and the enhancement of endogenous antioxidant activity of tissues.
3. In conditions of hypoxic hypoxia with hypercapnia"cedar Pine tree infusion showed antihypoxic activity, statistically significantly extending the life of mice in an enclosed space.
4. In experiments with induced hyperlipidemia (in liver damage in rats with carbon tetrachloride) of the test substance had a distinct hypolipidemic effect: prevented the increase in blood levels of cholesterol and triglycerides; increases the content antiatherogenic lipoproteins and decreased digestion in atherogenic apob-containing lipoproteins.
The study of General toxic action of tincture of seeds, Siberian cedar pine tree
When conducting Toxicological experiments were guided by the Methodological guidelines for the study obshestvennogo action of pharmacological substances (E. V. Arzamastsev, Guskova T. A., Berezovskaya N. In. . guidelines for the study of General toxic effect of pharmacological substances // Guidance on experimental (preclinical) study of new pharmacological substances. - M - 2000. - P. 18-24) and the guidelines for preclinical study for new drugs developed from natural raw materials (Kukes V. G., Bulaev, V. M., Kolker V. K. et al. guidelines for preclinical study for new drugs developed from natural raw materials // Guidance on experimental (preclinical) study of new pharmacological substances. - M., 2000. Pp. 346-348). Animal�e (white adult mongrel mice and rats) were obtained from the nursery "Rapolano" Leningrad region and detained in conditions of vivarium on a standard diet with free access to water.
In the study of General toxic action of tincture of seeds, Siberian cedar pine tree were carried out:
1. Acute toxicity estimates
2. The chronic toxicity study, including the influence of the "Pine cedar tincture" on the General condition of the animals, the dynamics of body weight and the weights of organs, the influence of the "Pine cedar tincture" on the functional status Central nervous system, study of the effect of "Pine cedar tincture" on the cardiovascular system
3. Study of the influence of "Pine cedar tincture" in the peripheral blood.
4. Study of the effect of "Pine cedar tincture on serum biochemical parameters of blood
5. Study of the influence of "Pine cedar tincture" at the state of the urinary system of rats
6. Morphological evaluation of internal organs of rats at the end of a two-month experiment
7. The study of local irritating action of a Pine cedar tincture"
8. Assessment of the allergenic properties of Pine cedar tincture"
General conclusions according to the results of toxic action of tincture of seeds, Siberian cedar pine tree, the following:
1. "Cedar pine tree tincture" has a low acute"�lichnosti. To determine its LD50 when administered in dealcoholization the form was not possible: greater than 75 ml/kg.
2. If you receive a "chronic" toxicity ANC (two-month introduction) daily observations of the animals did not reveal any abnormalities in the condition and behavior of animals, no significant differences in biometric parameters (body weight, the ratios of masses of bodies of animals of experimental and control groups. Prolonged exposure of the drug did not cause abnormalities in the peripheral blood, liver, cardiovascular, Central nervous and excretory systems. In animals, a long time treated with the test substance, is not revealed no macro-or microscopic abnormalities in the structure of the internal organs from physiological norms.
3. "Cedar pine tree tincture" is not an irritant to the mucous membrane of the stomach, as after a two-month oral administration of the drug to animals with macro and histological study no pathological changes as its mucosa and submucosa.
4. Experiments conducted on Guinea pigs using two tests ("systemic anaphylaxis" and "konjuktivalnyj test") for identifying allergenic action, showed that a two-week introduction issleduemoj� substance did not cause sensitization in animals.
5. The analysis of literary data and the results of our studies suggest that "cedar Pine tree tincture" has no immunotoxicity. This is evidenced by:
- centuries-old experience of the use of seeds, Siberian cedar pine tree in folk medicine and food industry;
- lack of information on allergenic properties and immunotoxicity have used in medical practice the elixir Kedrova, the main component of which are the seeds of SCS (V. G. Makarov, 1999);
- the results of hematological studies obtained in the study "chronic" toxicity of the JMC; no change in the content of lymphocytes, monocytes, eosinophils;
the results of histological evaluation of important immune organ of the spleen in the research "chronic" toxicity; no change of mass and its structure compared to control.
Conclusion and recommendations on the clinical application of tincture of seeds, Siberian cedar pine tree
Experimental (preclinical) studies on the specific activity of "Pine cedar tincture" on two animal species (rats and mice) using biometric, biochemical and histological methods. Revealed distinctly pronounced hepatoprotective effect of the test substance, comparable effektivnosti of hepatoprotector of cilibia.
The analyte detected a pronounced antioxidant activity, which was manifested by a decrease induced by carbon tetrachloride increases the generation of free radicals, the level of TBA-reactive products in the serum and in the liver and increased activity of antioxidant enzymes: glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase.
In conditions of hypoxic hypoxia with hypercapnia (closed space) "cedar Pine tree tincture" had antihypoxic action.
Against the backdrop of induced chronic steatohepatitis hyperlipidemia installed hypocholesterolemic effect of the JMC, and the increase in the reduced content of α-cholesterol, reflecting the normalization of anti-atherogenic lipoprotein fractions. Significantly decreased the content of hydroperoxides in atherogenic (apob-containing) lipoproteins, indicating that reducing their atherogenicity.
In the Toxicological studies set, "cedar Pine tree tincture" has a low acute toxicity - no death of animals when applying the maximum possible (by volume) dose; not marked qualityimage of drug action; in conditions of chronic experiment SKN no irritant action on the mucous membrane of the stomach, does not cause sdhow the peripheral blood functional and morphological disorders of the liver, cardiovascular, excretory and Central nervous systems. The drug has no allergenic activity.
Based on the results of the research, "cedar Pine tree tincture" can be recommended as a hepatoprotector plant origin is a preventive treatment that increases the resistance of liver tissue to various damaging influences.
1. A method of producing tinctures, possessing hepatoprotective, antioxidant, antihypoxic, a hypolipidemic action, from the seeds of Siberian pine Siberian maceration using ethanol, characterized in that the whole seeds, Siberian cedar pine tree was charged into the reactor, pour the extractant at a ratio of seeds: extractant 1:5, and the extractant used 70% aqueous ethanol solution, extraction is carried out for 10-15 days at room temperature with stirring daily, get a tincture with an alcohol content not less than 65% and the dry residue is not less than 0.5%, tincture defend at least two days at a temperature of 8-10°C and the precipitate is separated to obtain the finished product.
2. A method according to claim 1, characterized in that the ratio of seeds: extractant 1:5 provide taking into account the absorption coefficient of 1.15.
3. Ability� according to claim 1, characterized in that get tincture containing amounts of phenolic compounds in terms of Gallic acid is not less than 0.05%.
4. A method according to claim 1, characterized in that the precipitate is separated by filtration or decantation.
5. Drug, possess hepatoprotective, antioxidant, antihypoxic, lipid-lowering effect containing tincture of the seeds, Siberian cedar pine tree, obtained by the method according to claim 1.
6. The use of a medicinal product according to claim 5 as a hepatoprotective funds.
SUBSTANCE: method for producing a pigment complex of bisnaphthazarin for preventing inflammatory diseases, involving demineralising commercial sea urchins' crusts and needles in an organic acid solution, separating organic acid salts and protein, applying pigment solution on a chromatographic column, washing the column with diluted mineral acid and distilled water, eluting the pigment complex, combining fractions containing the pigments, removing ethanol, lyophilising concentrate in the certain environment. The complex of pigments bisnaphthazarins for preventing inflammatory diseases.
EFFECT: complex of pigments prepared by the above method is effective for preventing the inflammatory diseases.
3 cl, 2 dwg, 2 tbl, 4 ex
FIELD: machine building.
SUBSTANCE: for extraction (leaching) of the substances extracted from the plant materials in food, chemical-pharmaceutical and other industries, for output increasing of the substances extracted from the extractable plant materials and for increasing of their concentration in the ready extraction it is suggested to provide the extractor with the extractant recirculation circuit containing devices for solid phase separation, pump, discharge tank, flowmeter, shutdown valves system, and in the extractor bottom part additionally union will be installed for continuous liquid phase supply.
EFFECT: wider possibility of return in the vessel of part of extraction after solid phase separation in specified ratio with fresh extractant, thus improving conditions of mass exchange due to decreasing of the surface tension of the liquid phase.
SUBSTANCE: method for preparing an agent possessing anti-inflammatory, diuretic and antioxidant activity, involving milling Spiraea salicifolia shoots representing a mixture of leaves, blossom and shoots, extracting them three times by gradual maceration, mixing in infusing, filtering, condensing, separating, drying in the certain environment.
EFFECT: agent shoes the pronounced anti-inflammatory, diuretic and antioxidant activity.
2 dwg, 12 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmaceutical and consumer industry in preparing dry herbal extracts used for later colouration of textile fabrics with this extract. A method for preparing the dry herbal extract of St. John's wort involves grinding herbal raw material, extracting in water, filtering, boiling down in a rotary evaporator and drying the residual product to constant weight in a drying chamber in the certain environment.
EFFECT: method described above enables preparing the extract with dry colorant weight 25% of dry herbal raw material weight.
SUBSTANCE: method of obtaining sanguinarine and chelerythrine sulphates includes extraction of milled overground part of macleaya microcarpa and/or macleaya cordata with water aliphatic alcohol, removal of water aliphatic alcohol in vacuum, alkalinisation of water distillation residue with hydrophobic solvent, processing organic phase with sulphuric acid, filtration, washing and drying of target product, with extraction of milled raw material being carried out with water aliphatic alcohol in presence of methanesulphoacid, and solution of alkaloid bases in hydrophobic organic solvent is additionally filtered through layer of hydrophobic solvent, and target product is subjected to boiling in acetone.
EFFECT: method makes it possible to increase quality of finished product, simplify technology of production of sanguinarine and chelerythrine sulphates, and reduce the process duration.
8 cl, 14 ex
SUBSTANCE: method of bee pollen processing consists in the fact, that bee pollen is extracted with CO2 by pumping CO2, obtained fat extract is separated, remaining oil-seed meal is subjected to enzymatic hydrolysis in presence of enzyme Distizym Protacide Extra, obtained fermentolisate is separated into solid and liquid phases, solid phase is dried, liquid phase is filtered, preservative potassium sorbate and sodium benzoate are added into filtered liquid fraction under specified conditions.
EFFECT: method makes it possible to efficiently process bee pollen with obtaining fractions, characterised by microbiological purity, high degree of bioavailability, and a long storage term.
SUBSTANCE: invention relates to versions of composition for heat transmission. One of composition versions contains (i) from about 20 to about 90 wt % of R-1234yf; (ii) from about 10 to about 60 wt % of R-134a and (iii) from about 1 to about 20 wt % of R-32. Invention also relates to a number of versions of composition application.
EFFECT: composition has lower value of global warming potential and at the same time is characterised by productivity and energy efficiency.
56 cl, 7 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmaceutical industry, particularly to a method for recovering pinosylvin and methylpinosylvin from common pine (Pinus sylvestris) wood. The method for recovering pinosylvin and methylpinosylvin consists in extracting ground knots of common pine (Pinus sylvestris) wood, common pine (Pinus sylvestris) in isopropyl alcohol mixed with water, separating the extract and removing the solvent.
EFFECT: method enables a higher yield of pinosylvin and methylpinosylvin from common pine (Pinus sylvestris) wood.
3 cl, 5 ex, 2 dwg
SUBSTANCE: agent possessing the anti-inflammatory, antipyretic and antimicrobial action representing a dry extract of drug hedge hyssop leaves and blossom by grinding them, extracting in 96% alcohol on a water bath to a boil, and boiling, evaporating, diluting the evaporated residue by distilled water first, adding chloroform then, cooling to a room temperature and centrifuging, separating a water fraction and drying it in the certain environment.
EFFECT: agent possesses the pronounced anti-inflammatory, antipyretic and antimicrobial action.
5 dwg, 5 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the pharmaceutical industry, in particular to a medication, possessing a nephroprotective action. The medication with the nephroprotective action, contains an extract of grass with roots and rhizomes of slender-leaf iris (Iris tenuifolia), stabilising agents and preservatives, taken in a certain ratio, where the extract is obtained by extraction of the grass with roots and rhizomes of slender-leaf iris (Iris tenuifolia) with a 30-70% water solution of polyol.
EFFECT: medication possesses an effective nephroprotective action.
3 cl, 3 dwg, 7 tbl, 14 ex.
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to chemotherapy of cancer and represents a composition for hepatic cancer in individuals and containing a complex of formula [M(RCS3)2(RCS2)], wherein M represents 188Re having an activity of more than 3.7 GBq and a lipophilic organic phase emulsified with a water phase; as well as a method for preparing the above composition.
EFFECT: invention provides higher stability and efficacy of the composition for treating hepatic cancer in the individuals.
17 cl, 1 dwg, 9 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to the pharmaceutical industry, particularly to a composition for treating or preventing the human immunodeficiency virus (HIV) or hepatitis C. The herbal composition used for treating or preventing the patients infected by the HIV or hepatitis C viruses and containing a herbal ingredient with tannin agents and catechin, and a pharmaceutically acceptable carrier; the herbal ingredient is Agrimonia Eupatoria (GAFT) and/or gambier (Uncaria gambir). A method for preparing the herbal composition for treating or preventing the patients infected by the HIV or hepatitis C viruses.
EFFECT: composition is effective for treating or preventing the patients infected by the HIV or hepatitis C viruses.
12 cl, 6 dwg, 2 tbl
SUBSTANCE: group of inventions refers to medicine, namely to hepatology and neurology, and can be used for using rifaximin for preparing a drug preparation for maintaining hepatic encephalopathy (HE) remission in a subject. That is ensured by daily administration of rifaximin into the subject for approximately 12 months or more, or approximately 1095 days or longer, thereby maintaining the HE remission.
EFFECT: group of inventions provides the stable HE remission, as well as a low antibiotic resistance of the patient's bacterial flora.
16 cl, 34 dwg, 28 tbl, 6 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medicine and can be used as a hepatotrophic, lipotropic agent in gastroenterology, as well as for treating cardiac and cerebral atherosclerosis, in neurology in the lower extremity artery diseases (endarteritis). The agent contains tabletted powders of diisopropylammonium dichloroacetate, microcrystalline cellulose, lactose and excipients presented by Aerosil, Primogel and calcium stearyl fumarate.
EFFECT: agent possesses no toxicity, has the high adsorption properties, the apparent anti-inflammatory action, and provides metabolism and the liver regeneration.
3 tbl, 2 ex
SUBSTANCE: according to a known method of treating the liver disease accompanying type 2 diabetes mellitus involving the baseline therapy of diabetes mellitus and prescribed hepatoprotectors, the above hepatoprotector is presented by Mexicor in a daily therapeutically effective dose of not less than 16 weeks. The therapeutically effective dose of Mexicor makes 100 mg 4 times a day.
EFFECT: higher clinical effectiveness ensured by eliminating the liver disease more prominently, reducing the length of treatment, normalising the liver function test results over a short period of time, and avoiding any side effects.
2 cl, 2 tbl
SUBSTANCE: invention refers to medicine and aims at treating the non-alcoholic fatty liver disease. Increasing a transaminase level to three normal values inclusively requires applying essential phospholipids for 2-3 months twice a year that is followed by using statin 10-20 mg in a combination to ursodeoxycholic acid 15-20 mg/kg for 3-6 months. If the transaminase level exceeds three normal values, the treatment is isolated and includes ursodeoxycholic acid 15-20 mg/kg for 3-6 months.
EFFECT: method enables providing the higher clinical effectiveness of the non-alcoholic fatty liver disease.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medicine, namely to endocrinology, and can be used for treating non-alcoholic liver disease accompanying type 2 diabetes mellitus. The declared preparation Mexicor provides reducing manifestations of cytolysis and cholestasis, decreasing the steatosis index, enables improving metabolic lipid and glycaemic values and reducing insulin resistance. Mexicor is applied in a daily therapeutically effective dose of 100 mg 4 times a day for at least 16 weeks.
EFFECT: high pharmacological activity of Mexicor has been shown by achieving the pronounced and stable elimination of fatty liver disease that enables reducing the length of treatment with no side effects.
2 cl, 2 tbl
SUBSTANCE: invention refers to medicine, namely to surgery, and describes a method for compensating a disturbed intestinal bile inflow in patients with the external biliary drainage. A method for the deficient bile replacement with the external biliary drainage consists in conducting a background therapy, prescribing a therapeutic formulation containing the following ingredients: ursodeoxycholic acid 12-15 mg/kg/day (up to 20 mg/kg) in 2-3 doses, Eslidin (soya lecithin phospholipids 300 mg; methionine 100 mg, soya oil up to 550 mg) 1 capsule 3 times a day at mealtimes; Milaif 0.2 g 3 times a day.
EFFECT: invention enables improving the patient's health condition and relieving a pain syndrome by a safe and technically easy method.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to a novel derivative of N-acylanthranilic acid, represented by the following general formula 1, or to its pharmaceutically acceptable salt, in which R1, R2, R3, X1, X2, X3, X4 and A are determined in the invention formula.
EFFECT: invention relates to an inhibitor of collagen production, a medication for treating diseases, associated with the excessive production of collagen, containing N-acylanthranilic acid derivative Formula 1.
SUBSTANCE: hepatoprotective agent based on a lipid fraction from an alcohol extract of perforated thallome ulva - Ulva fenestrate P. et R., containing less than 70% membrane-active lipid components, including not less than 20% essential phospholipids with content of polyunsaturated fatty acids of the n-3 series of not less than 55%.
EFFECT: agent has effective hepatoprotective action, speeds up restoration of stages of metabolic reactions, thereby providing normalisation of biochemical properties of carbohydrate and lipid exchange.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to cosmetology and dermatology and represents a skin care composition applicable for local skin application, wherein the above composition contains salicylic acid or its salt in a combination with glycyrrhizic acid, or its salt or its derivative, cetylhydroxyproline palmitamide, lactic acid or its salt, bisabolol and niacinamide.
EFFECT: invention provides extending the range of effective skin care agents.
41 cl, 11 ex, 11 tbl