RU2337698C1 - Antidiabetic agent with hypolipidemic activity for treatment and prevention of insular diabetes ii type - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: invention can be used as new agent for correction of carbohydrate metabolic disorders to prevent and treat insular diabetes II type. Agent represents ecdysteroids-containing substance extracted from aerial saw-wort Serratula coronata L. (Asteraceae) representing mixed 20-hydroxyecdysone in amount not less than 75% and 25S-inocosterone in amount not less than 10%.

EFFECT: sugar-reducing and hypolipidemic activity of agent.

3 ex, 3 tbl, 12 dwg

Description

The invention relates to pharmacology, in particular to drugs and food additives of antidiabetic action, and can be used as a new tool for the correction of disorders of carbohydrate metabolism for the prevention and treatment of type II diabetes mellitus.

Diabetes mellitus is a consequence of relative or absolute insulin deficiency. In accordance with the WHO classification, two forms of the disease are distinguished: insulin-dependent (type I diabetes mellitus) and non-insulin-dependent (type II diabetes mellitus). Type I diabetes mellitus is characterized by absolute insufficiency of the formation of insulin by b-cells of the islets of Langerhans of the pancreas and requires hormone replacement therapy with insulin preparations. Patients with type II diabetes mellitus need to be prescribed oral antidiabetic drugs, which include sulfonylureas and biguanides. The sugar-lowering effect of sulfonylurea derivatives is due to increased insulin secretion by b-cells of the pancreas, increased action of insulin circulating in the bloodstream, increased glucose utilization by skeletal muscle and liver, inhibition of glucose formation from fat in the liver and lipolysis of adipose tissue. Treatment with sulfonylurea derivatives is indicated for type II diabetes mellitus in adults. These drugs are contraindicated in insulin-dependent diabetes, blood diseases, accompanied by a decrease in the number of leukocytes and platelets in the blood, and in a number of other cases. The least pronounced side effect of all drugs in this group is possessed by maninil, predian, and glurenorm. The second group of oral antidiabetic agents are biguanides. These drugs are effective in the presence of endogenous or exogenous origin in the body, since they do not affect its synthesis and release from the b-cells of the pancreatic islets. Biguanides potentiate the action of insulin by enhancing skeletal muscle glucose utilization, inhibiting the formation of glucose from fats, reducing the absorption of glucose in the small intestine, enhancing lipolysis and reducing the rate of fat synthesis. Contraindications to the use of biguanides are type I diabetes, depletion, damage to the liver, kidneys, coma, diseases accompanied by hypoxia (Makovetsky A.G., Velikhov V.K. Diabetes mellitus. M., 1987. 102 S.).

The objective of the invention is to expand the arsenal of means of lipid-lowering and anti-ischemic action, obtaining a low-cost drug from available plant materials with high efficiency.

The technical result of the present invention is the creation of funds from renewable cheap plant materials for the treatment of non-insulin-dependent diabetes (type II diabetes), which has not only a hypoglycemic effect, but also has lipid-lowering activity, since the development of diabetes is usually accompanied by a pronounced shift in the state of lipid metabolism.

The technical result is achieved by the fact that, as an agent with antidiabetic and lipid-lowering activity, in the treatment and prevention of type II diabetes mellitus, an ecdysteroid-containing substance isolated from the aerial part of the plants of the crown-bearing Serratula coronata L. (Asteraceae), which is a mixture of 20-hydroxyecdisone, is used in an amount of not less than 75% and 25S-inocosterone in an amount of not less than 10%.

A known mixture of ecdysteroids isolated by the method described in the patent of the Russian Federation No. 2153346, containing 20-hydroxyecdysone at least 75% and 25S-inocosterone at least 10% with the structural formulas indicated below, which are active substances that exhibit physiological activity.

The proposed tool, which we called "Serpisten", is a white crystalline powder, odorless, sparingly soluble in water, highly soluble in methanol and 70% ethanol.

A mixture of phytoecdysteroids is obtained in a known manner (according to patent 2153346) as follows.

Harvesting of raw materials, preferably leaves, is carried out in the phase of vegetation, mass budding or the beginning of flowering. It was experimentally established that in these phases of development the content of active ecdysteroids is maximum. A study of the distribution of ecdysteroids in plants of the crown-sickle was carried out in middle-aged generative individuals. Crowned Serpukha far surpasses safflower rape by the content of ecdysteroids. The use of the aerial parts of plants, unlike rhizomes of rapontica, greatly facilitates the harvesting and processing of ecdysteroid-containing plant materials.

The crushed plant material (the aerial part of the plant of the genus Serratula of the Asteraceae family) is repeatedly extracted with water at a temperature not exceeding 100 ° C for no more than 1 hour with constant stirring and the ratio of the raw material extractant is 1:10, respectively. The thick aqueous extract was evaporated under reduced pressure, treated with a mixture of ethyl acetate-methanol taken in a ratio of 4: 1, or chloroform-methanol in a ratio of 6: 1. The procedure is repeated until complete extraction of ecdysteroids from the thick extract. The organic extracts are evaporated to dryness, then the ecdysteroids are purified by chromatography on alumina in a chloroform-methanol or ethyl acetate-methanol system of increasing polarity and recrystallization in an ethyl acetate-methanol or ethyl acetate-ethanol system in a 9: 1 ratio.

An example of the method

6 kg of crushed leaves of a plant of the genus Serratula of the Asteraceae family are poured into 60 l of water and extracted for 1 hour at a temperature of 50 ° C with constant stirring, then the extract is drained, and the plant material is subjected to re-extraction. The combined extract is evaporated in a vacuum rotary film evaporator under reduced pressure to 2 L. 2 l of the thick extract is treated three times with 8 l of a mixture of ethyl acetate-methanol in a ratio of 4: 1 with constant stirring and filtered. The filtrate was evaporated to dryness, the residues were combined and transferred to a column of aluminum oxide and eluted with a mixture of chloroform-methanol of increasing polarity. The eluate is collected in flasks of 1 l and determine the content of ecdysteroids in them by thin layer chromatography (TLC). The desired fractions containing ecdysteroids are evaporated to dryness and recrystallized in ethyl acetate-methanol (9: 1).

Thus, the method allows to obtain the substance Serpisten, which is a mixture of phytoecdysteroids containing at least 75% 20-hydroxyecdysone and at least 10% inocosterone (white crystalline powder, odorless, sparingly soluble in water (1.9 g / l), good soluble in 70% ethanol (61 g / l) and methanol (75 (g / l)). Yield 42 g or 0.7% by weight of raw material, which is 70% of theoretically calculated yield.

A known method for the isolation of 20-hydroxyecdysone and 25S-inocosterone (RF patent 2155599, which allows to obtain individual compounds from a mixture of phytoecdysteroids and mix them in an effective amount.

The invention is confirmed by photographs.

Figure 1. The normal structure of the islet of the Langerhans pancreas of an intact rat. 20th day. Hematoxylin-eosin stain. SW 40 × 7.

Figure 2. The normal structure of the islet of the Langerhans pancreas of an intact rat. 20th day. Hematoxylin-eosin stain. SW 40 × 7.

Figure 3. The normal structure of the islet of the Langerhans pancreas of an intact rat. 20th day. Coloring aldehyde fuchsin. SW 20 × 7.

Figure 4. The normal structure of the islet of the Langerhans pancreas of an intact rat. 20th day. Coloring aldehyde fuchsin. SW 20 × 7.

Figure 5. Alloxan diabetes in rats. 20th day. Violation of the structure of the islets of Langerhans. Hematoxylin-eosin stain. SW 40 × 7.

6. Alloxan diabetes in rats. 20th day. Violation of the structure of the islets of Langerhans. Lysis of cells and a decrease in the number of nuclei. Hematoxylin-eosin stain. SW 40 × 7.

7. Alloxan diabetes in rats. 10th day. The destruction of the islet of Langerhans. Lysis of cells and a decrease in the number of nuclei. Hematoxylin-eosin stain. SW 40 × 7.

Fig. 8. Alloxan diabetes in rats. 20th day. The destruction of the islet of Langerhans. Lysis of cells, replacement with stromal elements. Coloring aldehyde fuchsin. SW 20 × 7.

Fig.9. Restoration of the islet of Langerhans of the pancreas of the experimental rat after 20 days of treatment with metformin. An increase in the number of nuclei and cells. Hematoxylin-eosin stain. SW 40 × 7.

Figure 10. Restoration of the islet of Langerhans of the pancreas of the experimental rat after 20 days of treatment with metformin. An increase in the number of nuclei and cells. Coloring aldehyde fuchsin. SW 20 × 7.

11. Restoration of the islet of the Langerhans pancreas of an experimental rat after 20 days of treatment with Serpisten. An increase in the number of nuclei and cells. Hematoxylin-eosin stain. SW 40 × 7.

Fig. 12. Restoration of the islet of the Langerhans pancreas of an experimental rat after 20 days of treatment with Serpisten. A noticeable increase in the size of the island itself, the number of nuclei and synthesizing insulin cells. Coloring aldehyde fuchsin. SW 20 × 7.

Verification of the antidiabetic and hypolipidemic activity of the substance Serpisten was carried out at ZAO Adaptogen (St. Petersburg). The experiments were performed on non-linear white rats with the intragastric route of administration on the model of alloxan diabetes. Animals were kept under standard vivarium conditions under natural light. Water and food (standardized briquetted feed) were provided to animals without restriction. The work was carried out in accordance with the "Methodological recommendations for the experimental study of new oral hypoglycemic pharmaceuticals" [1986].

Alloxan diabetes was caused by a single subcutaneous injection of an aqueous solution of alloxane hydrate (ICN Biomedicals, USA) at a dose of 150 mg / kg to rats weighing 180-200 starving for 24 hours. The following groups of animals were studied: intact; untreated rats with diabetes; animals with diabetes who received metmorphine (200 mg / kg) daily for 23 days, taken as a prototype, manufactured by Polfa-Kutno A / O, Poland., reg. No. 008422 from 08/22/1997; animals with diabetes receiving Serpisten (5 mg / kg) daily for 23 days. The drugs were administered intragastrically 1 time per day in starch gel.

The introduction of drugs began on the third day after the administration of alloxan, when the blood glucose level increased approximately 3 times compared with the control [Gatsura VV, Saratikov A.S., 1977; Nikolaychuk L.V., 1988]. Each group contained 20 animals of both sexes. During the experiment, the general condition of the animals, water consumption, body weight were recorded.

In the blood serum, the level of glucose, total lipids, triglycerides, and cholesterol was determined by conventional methods [Medical ..., 2002].

The concentration of hemoglobin and glycosylated hemoglobin was determined in red blood cells. The hemoglobin content was evaluated by the hemoglobin cyanide method proposed by M. Kushakovsky. [1968] using a standard set of reagents (NPF "Abris +"). Glycosylated hemoglobin was determined spectrophotometrically [Medical ..., 2002].

In addition, a study was made of the histological structure of the pancreas of experimental animals when staining sections with hematoxylin-eosin and aldehyde fuchsin.

The use of a new agent leads to an increase in the survival of animals in conditions of alloxan diabetes and reduces the manifestation of this pathology, such as polydipsia. In experimental type II diabetes mellitus, Serpisten reduces the level of hyperglycemia and the concentration of glycosylated hemoglobin in the blood of experimental animals, thereby exerting a preventive effect on the development of the main complications of diabetes mellitus. On the background of treatment with Serpisten, the level of total lipids, cholesterol and β-lipoproteins returned to the level of intact animals, which indicates the hypolipidemic effect of the studied substance.

Example 1. The effect of the drug Serpisten on the physical parameters of rats with experimental diabetes.

The positive effect of the study drug and the comparison drug (metformin - a group of biguanides) on the survival of animals was shown. A significant change in the weight of animals was recorded only after 20 days of the experiment and only in the group of untreated animals. At the same time, water consumption, an indicator that is considered one of the “big” symptoms of diabetes, significantly changed in all groups of animals. The most pronounced polydipsia was observed in the group of untreated animals on the 20th day of the experiment. The use of the drug Serpisten led to a significant decrease in water consumption (Table 1).

Table 1
Results of a physical study in rats with experimental diabetes Groups of animals The percentage of surviving animals Body weight g Daily water intake, ml After 10 days Intact animals (n = 20) one hundred 180 ± 5 15 ± 1 Untreated Diabetic Rats (n = 14) 70 185 ± 10 45 ± 2 ^* Animals with diabetes treated with metformin (200 mg / kg) (n = 20) one hundred 185 ± 5 25 ± 3 ^* _** Animals with diabetes treated with Serpisten (5 mg / kg) (n = 20) one hundred 180 ± 5 20 ± 3 _** In 20 days Intact animals (n = 20) one hundred 195 ± 10 17 ± 2 Untreated Diabetic Rats (n = 10) fifty 165 ± 10 ^* 55 ± 2 ^* Animals with diabetes treated with metformin (200 mg / kg) (n = 16) 80 180 ± 5 20 ± 3 _** Animals with diabetes treated with Serpisten (5 mg / kg) (n = 20) 75 182 ± 10 24 ± 6 _** Designation: ( ^* ) - hereinafter, the differences are statistically significant compared with intact animals, at p <0.05; ( _** ) - hereinafter, the differences are statistically significant compared with untreated animals, at p <0.05.

Example 2. The effect of the drug Serpisten on glucose and the degree of glycosylation of hemoglobin in rats with experimental diabetes.

As can be seen from the data presented in table 2, in experimental untreated animals, pronounced hyperglycemia is observed. During treatment with the studied drug Serpisten, a significant decrease in glucose levels was observed, almost to the level of intact animals. When using the comparison drug, the positive changes in glucose levels were similar, but somewhat less pronounced.

Like many proteins, hemoglobin in the process of functioning in the human body upon contact with glucose is capable of non-enzymatically glycosylation, i.e. add glucose. The effect of glycosylation depends only on the magnitude and duration of hyperglycemia. In experimental animals, the level of glycosylated hemoglobin naturally increases by the 10th day of the experiment and even more pronounced increases by the 20th day of the experiment. Under the influence of the drug Serpisten, by the 10th day of treatment, the level of glycosylated hemoglobin significantly decreases. By day 20, under the influence of the study drug and metformin, the concentration of HbA _{1c was} also 2 times lower than the level of untreated animals, but the level of intact animals was not reached.

At the same time, the level of unchanged hemoglobin decreased, significantly decreasing in the group of untreated animals; with the use of drugs, the level of hemoglobin increased slightly, but the level of intact animals was not reached (Table 2).

table 2
Glucose level and hemoglobin glycosylation level in rats with experimental diabetes Groups of animals Glucose, mmol / L HbA _1c , μmol fructose / g Hb Hemoglobin, g / l After 10 days Intact animals (n = 10 ⁺ ) 4.5 ± 0.4 4.5 ± 0.5 - Untreated Diabetic Rats (n = 10) 16.2 ± 1.5 ^* 8.4 ± 0.8 ^* - Animals with diabetes treated with metformin (200 mg / kg) (n = 10) 6.6 ± 0.8 _** 5.1 ± 0.6 _** - Animals with diabetes treated with Serpisten (5 mg / kg) (n = 10) 5.6 ± 0.2 _** 6.2 ± 0.3 ^* _** - In 20 days Intact animals (n = 10) 4.2 ± 0.5 4.5 ± 0.5 159 ± 2 Untreated Diabetic Rats (n = 10) 18.6 ± 1.2 ^* 12.5 ± 1.3 ^* 109 ± 9 ^* Animals with diabetes treated with metformin (200 mg / kg) (n = 10) 6.4 ± 0.6 ^* _** 6.2 ± 0.6 ^* _** 124 ± 15 ^* Animals with diabetes treated with Serpisten (5 mg / kg) (n = 10) 5.4 ± 0.3 _** 6.5 ± 0.5 ^* _** 112 ± 13 ^* Designation: ( ⁺ ) - Hereinafter: for the study, blood was taken from 10 animals randomly selected, with the exception of a group of untreated animals

Example 3. The effect of the drug Serpisten on lipid metabolism in rats with experimental diabetes.

With the development of diabetes, marked changes in the state of lipid metabolism are observed. There was an increase in total lipids and cholesterol in the blood serum of animals without treatment by day 10, which increased slightly by day 20. Against the background of treatment with Serpisten and the comparison drug, both indicators returned to normal and by day 20 decreased almost to the level of intact animals. Similar changes were observed in the level of β-lipoproteins, which also characterized the positive dynamics of the process during treatment with Serpisten.

Several other trends were established with respect to triglycerides, the level of which did not significantly change in any group of animals by the 10th day of the experiment. By the 20th day of the experiment in untreated rats, the level of triglycerides also did not change, and when Metformin and Serpisten were used, they significantly decreased, which indicates the lipolytic effect of the drugs.

Table 3
Lipid metabolism in rats with experimental diabetes Groups of animals Total lipids, g / l Cholesterol, mmol / L Triglycerides, mmol / L β-lipoproteins, g / l After 10 days Intact animals (n = 10) 10.2 ± 0.3 1.4 ± 0.3 2.5 ± 0.2 2.0 ± 0.3 Untreated Diabetic Rats (n = 10) 17.4 ± 1.2 ^* 7.2 ± 0.2 ^* 2.8 ± 0.4 4.5 ± 0.1 ^* Animals with diabetes treated with metformin (200 mg / kg) (n = 10) 10.6 ± 0.6 _** 2.6 ± 0.3 ^* _** 2.6 ± 0.1 2.2 ± 0.3 _** Animals with diabetes treated with Serpisten (5 mg / kg) (n = 10) 10.2 ± 0.5 _** 2.7 ± 0.4 ^* _** 2.5 ± 0.2 2.0 ± 0.2 _** In 20 days Intact animals (n = 10) 10.1 ± 0.3 1.2 ± 0.2 2.5 ± 0.2 2.0 ± 0.3 Untreated Diabetic Rats (n = 10) 18.5 ± 1.3 ^* 7.6 ± 0.3 ^* 2.9 ± 0.4 4.5 ± 0.2 ^* Animals with diabetes treated with metformin (200 mg / kg) (n = 10) 11.6 ± 1.2 _** 1.7 ± 0.3 _** 1.6 ± 0.1 ^* _** 2.2 ± 0.3 _** Animals with diabetes treated with Serpisten (5 mg / kg) (n = 10) 10.6 ± 1.0 _** 1.5 ± 0.2 _** 1.5 ± 0.2 ^* _** 2.1 ± 0.3 _**

Example 4. The data of histological examination of the pancreas.

The structure of the islets of Langerhans pancreas of intact rats is shown in Fig.1-4.

The results of histological studies of the pancreas of rats with experimental diabetes indicate violations of the structure of the islets of Langerhans (Fig.5-8).

The results of histological studies of the pancreas in the treatment of rats with experimental diabetes Serpisten and a comparison drug metformin clearly demonstrate the recovery of islets of Langerhans and indicate a pronounced antidiabetic effect of the drug Serpisten (Fig.9-12).

Thus, the substance Serpisten, which is a mixture of the ecdysteroids 20-hydroxyecdysone and 25S-inocosterone from the aerial part of the plants of the crown-sickle, has a pronounced antidiabetic effect:

- Sickleworm has a positive effect on the condition of animals, increasing the survival of animals in conditions of alloxan diabetes and reducing the manifestation of this pathology, such as polydipsia;

- in experimental diabetes mellitus, Serpisten reduces the level of glucose and the concentration of glycosylated hemoglobin in the blood of experimental animals, thereby exerting a preventive effect in relation to the development of the main complications of diabetes;

- with the development of diabetes, marked changes are observed in the state of lipid metabolism, against the background of treatment with Serpisten, the level of total lipids, cholesterol and β-lipoproteins normalizes almost to the level of intact animals, which indicates the lipid-lowering effect of the investigated substance and may be an additional criterion for the effectiveness of therapy.

Claims (1)

An antidiabetic agent with hypolipidemic activity for the treatment and prevention of type II diabetes mellitus, containing an ecdysteroid-containing substance isolated from the aerial part of the plants of the crown-bearing Serratula coronata L. (Asteraceae), which is a mixture of 20-hydroxyecdysone in an amount of at least 75% and 25S inocoster amount of at least 10%.

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