RU2601917C1 - Method of producing medicinal agent for correction of thyroid functions - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, and method of preparing drug of herbal raw material for correction of thyroid function. Method of producing medicinal agent for correction of thyroid functions by extraction of roots and rhizomes of potentilla white, Baikal skullcap roots and roots and rhizomes of rosewort at ratio of 50 wt%, 25 wt%, 25 wt%, respectively, which are milled and extracted by 50-60 % ethanol under certain conditions.

EFFECT: agent prepared as described above is characterized by high content of biologically active substances, effectively corrects thyroid dysfunction.

1 cl, 7 tbl, 3 ex

Description

It is known that an increase in thyroid diseases is associated with geochemical, social, environmental and other factors [4]. The thyroid gland is not only one of the largest endocrine organs in terms of size and weight, but also one of the most significant from the standpoint of the role for the body of secreted hormones - iodothyronines. The level of iodine consumption by the population most affects the frequency of thyroid pathology. Around the world, an additional means of prevention is iodization of edible table salt, as well as the administration of iodine tablets to the most vulnerable segments of the population (children, pregnant and lactating women). In Russia, more than 30% of the territory where almost 80% of the population has a massive lesion of the thyroid gland, mainly endemic goiter. According to world statistics, at least 3% of the population suffer from various diseases of the thyroid gland (increased or decreased glandular function, nodular formations). It is known that the insufficient content of thyroid hormones in the body leads to a disorder of the structural and functional relationships of the nervous, cardiovascular, immune and other systems [5]. Thyroid hormones are essential for tissue growth, in the process of laying and functioning of the brain, the formation of intelligence, regulation and maintenance of metabolism. Pharmacological correction of the hypothyroid state of the body is based on the replacement of iodine deficiency and thyroid hormones. Despite the vast arsenal of modern drugs with thyrotropic action used to treat thyroid diseases, the need for effective and safe drugs remains. Given the clinical manifestations, with hypothyroidism it is advisable to use an integrated approach to treatment, the purpose of which is not only the normalization of thyroid hormonal status, but also the improvement of the state of the neurovegetative, cardiovascular, immune systems, correction of cognitive impairment and emotional-affective disorders. In the complex treatment of patients with the specified profile of disorders, effective sedative and metabolic therapy is prescribed. For this purpose, herbal sedatives, anabolic drugs, tranquilizers, mainly benzodiazepine series, antidepressants are used. The drugs used do not always have sufficient clinical efficacy and can cause side effects with prolonged use. In this regard, significant interest is justified in the search for new drugs that can prevent and correct hypothyroidism.

Medicinal plants are the most promising source of biologically active substances of thyrotropic action. One of the plants used in folk medicine for the treatment and prevention of thyroid diseases is white cinquefoil (Potentilla alba L., family Rosaceae - Rosaceae). It has been established that extracts from Potentilla alba L. can be used in the correction of hypofunction and hyperthyroidism [3, 6]. Along with this, taking into account the dependence of mental processes and clinical manifestations of hypothyroidism on the level of thyroid hormones in the blood, the development of secondary immunodeficiency, it seems appropriate to correct pathological manifestations of hypothyroidism with agents that have immunomodulatory (Rodiola) and nootropic (Scutellaria,) effects. The object of the study was the complex herbal remedy “Tireoton-1” (conditional name), consisting of dry white cinquefoil extract, Baikal dry scutellaria extract and dry Rhodiola rosea extract in the ratio of 50%, 25%, 25%, respectively, and its effect on the course of experimental hypothyroidism . In Tireoton-1, the main active ingredient is white cinquefoil, and Baikal skullcap and Rhodiola rosea are intensifying and modulating the effect of the former.

As a prototype, kelp was used in tablets (Evalar, Russia) at a dose of 3 mg / kg, which compensates the daily norm of iodine by 133% (according to the annotation to the drug).

The technical result of the invention is the expansion of the range of herbal medicines that have a reliable and secure raw material base and corrective disorders of the thyroid gland, affecting the main links in the pathogenesis of the disease due to the multicomponent nature of the drug. The inventive drug includes dried shredded extract from the following plants: rhizomes and roots of a white cinquefoil, rhizomes and roots of Rhodiola rosea, roots of Scutellaria baicalensis. To achieve this goal, the plant material is the rhizomes and roots of the white cinquefoil, meeting the requirements of the project GOST R 51074-2003, rhizomes and roots of Rhodiola rosea, meeting the requirements of GF XI issue. 2, Art. 75, and the roots of Scutellaria baicalensis, corresponding to the requirements of FS 42-453-91, taken in a ratio of 50, 25, 25%, respectively, are ground in a mill for grinding dry samples MPP-2 to a particle size of not more than 2 mm and extracted 50-60% ethyl alcohol in the ratio of raw material-extractant equal to 1: (14-16) (taking into account the coefficient of water absorption), at a temperature of 55-65 ° C and constant stirring. The process is repeated three times. The extraction time at the 1st and 2nd phase contacts is 60 minutes, at the 3rd phase contact - 30 minutes. The extracts are filtered through sulphide cloth, combined, evaporated to 1/3 of the original volume and purified by separation. The purified extract is doubled to 1/5 of the original volume, dried in a vacuum dryer and ground in a propeller-type mill. The yield of the finished product is 27.0 ± 0.5% by weight of the plant material. The final product is a fine brown powder with a pleasant odor and a bitter astringent taste, with a moisture content of not more than 5%. The product we obtained is characterized as a means of correcting thyroid dysfunction.

For a qualitative assessment of the obtained phytoextract, we used: chromatography in a thin layer of sorbent. Chromatographic analysis was performed on Sorbfil PTSX-AF-A-UV plates using various solvent systems: chloroform - ethanol - water (26: 16: 3), n-butanol - glacial acetic acid - water (4: 1: 2) .

Phytosterol compounds were detected after processing the chromatographic plates with a 20% sulfuric acid solution and heating at 100 ° C for 15 minutes in the form of pink spots with an Rf value of about 0.7 at the level of a standard sample of β-sitosterol. Identified - β-sitosterol and oleanolic acid.

Phenolic compounds were detected after processing the chromatographic plates with a solution of aluminum chloride in the form of yellow-green spots. Identified routine; quercetin; cinaroside.

Phenols derivatives were detected in a solvent system (chloroform-methanol-water (26: 14: 3)) after the chromatogram was processed with 10% sodium carbonate solution and heated in an oven at 100 ° C for two minutes, then the plate was sprayed with diazotized sulfacyl and heated at 110 ° C for 2 minutes. On the chromatogram 1, a reddish spot with Rf 0.42, corresponding to salidroside [1].

Quantitation.

Simple phenols (salidroside). To 0.5 g of dry Tireoton-1 extract was added 10 ml of water and heated in a water bath under reflux for 15 minutes. The cooled filtrate was placed in a 50 ml flask, 6 ml of a 10% solution of lead acetate, 2 ml of a saturated solution of sodium sulfate were added, stirred, brought up to the mark (A) with water. 5 ml of solution A was transferred to a flask with a volume of 25 ml, 2.5 ml of a 2% sodium carbonate solution, 2.5 ml of diazotized sulfanyl were added, the volume of the solution was adjusted to the mark with water, mixed and the optical density of the solution was measured at a wavelength of 486 nm, in a cuvette with a layer thickness 10 mm, water was used as a comparison solution. The content of salidroside (X%) was calculated by the formula:

, где

where

D is the optical density of the test solution;

k ^v is a dilution factor of 250;

M - sample of the extract;

253 - specific absorption rate of salidroside;

W is the weight loss upon drying.

Steroids (β-sitosterol). To 0.5 g of dry Tireoton-1 extract was added 10 ml of chloroform and heated in a water bath under reflux for 15 minutes. The chloroform extract, evaporated to a dry residue, was placed in a 25 ml flask and dissolved in concentrated sulfuric acid. The resulting solution was mixed and the absorbance was measured at a wavelength of 310 nm, in a cuvette with a layer thickness of 10 mm, concentrated sulfuric acid was used as a comparison solution. The content of sterol compounds (X%), in terms of β-sitosterol, was calculated by the formula:

, где

where

D ₀ is the optical density of the test solution;

k ^v - dilution factor of the extract, equal to 25;

D ₁ is the optical density on β-sitosterol;

k ^vst - coefficient of dilution of β-sitosterol equal to 25;

M - sample of the extract;

W is the mass loss upon drying of the extract.

It was determined that the quantitative content of sterol compounds in terms of β-sitosterol was about 3.4%.

Method for the quantitative determination of the total content of flavonoids.

0.5 g of dry extract “Tireoton-1” was placed in a 100 ml conical flat-bottomed flask with a ground stopper, 60 ml of 60% ethyl alcohol was added, the contents of the flask were heated in a boiling water bath for 1 h. After cooling, the extract was filtered into a volumetric flask with a capacity of 200 ml through a paper filter, which was washed with 10 ml of 60% ethyl alcohol. The extraction was repeated under the same conditions again and the volume of the combined filtrate was adjusted to the mark with 60% ethyl alcohol (solution A). 2 ml of solution A was transferred to a 25 ml volumetric flask and adjusted to the mark with 60% ethyl alcohol (solution B). The optical density of solution B was measured on a spectrophotometer at a wavelength of 281 nm in a cell with a layer thickness of 10 mm. As a comparison solution, 60% ethyl alcohol was used. At the same time, the optical density of the solution of a standard baikalin sample was determined at a wavelength of 281 nm. The total content of flavonoids (X,%) in terms of Baikalin and absolutely dry raw materials was calculated by the formula

, где

where

D ₀ is the optical density of the test solution;

k ^v - dilution factor of the extract, equal to 2500;

E is the specific absorption coefficient of baikalin, equal to 610;

M - sample of the extract, g;

W is the mass loss upon drying of the extract.

It was determined that the quantitative content of flavonoids in terms of Baikalin in the dry extract “Tireoton-1” was 4.15%.

A quantitative assessment was carried out on the yield of flavonoids, in terms of baicalin, phenols, in terms of salidroside, and extractive substances. Metrological characteristics are presented in table 1.

A known method of obtaining a dry extract "Tireoton" [1], however, we have proposed a more economical resource-saving option that allows to increase the yield of biologically active substances (table. 2)

Example 1

500 g of rhizomes and roots of white cinquefoil meeting the requirements of GOST R 51074-2003, 250 g of rhizomes and roots of pink rhodiola, meeting the requirements of GF XI issue. 2, Art. 75, and 250 g of the roots of Scutellaria baicalensis meeting the requirements of FS 42-453-91, taken in a ratio of 50, 25, 25%, respectively, are crushed in a mill for grinding dry samples MRP-2 to a particle size of not more than 2 mm (sieve No. 20 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 14 liters of 50% ethyl alcohol in a ratio of raw materials-extractant equal to 1:14, taking into account the coefficient of water absorption of the raw materials. It is extracted at a temperature of 60 ° C with constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. Two more extractions are carried out for 60 and 30 minutes, each time feeding 50% alcohol to the extractor in an amount equal to the volume of the drained extraction (1st discharge - 10.85 L, 2nd discharge - 11.05 L, 3rd discharge - 9.87 L ) The combined extracts are evaporated to 1/3 of the original volume and purified by separation. The purified extract is doubled to 1/5 of the original volume, dried in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Get 270.3 g of the finished product, which is 27.3% of the weight of the feedstock. The dry extract is a fine powder of brown color with a pleasant smell and a bitter astringent taste, crumbles, the moisture content is 4.5%.

The acute toxicity of Tireoton-1 was determined using the Pershin method [6] on white Wistar rats weighing 180 ± 11 g. Animals were under constant observation for 14 days. As a result of the experiments, it was found that the DL50 "Tireoton-1" - 450 mg / kg Animals died mainly on 1-2 days. At the same time, in the first hours after the administration of the test substance, signs of intoxication were observed in animals in the form of decreased motor activity, tachycardia, and increased breathing. The death of animals occurred from respiratory arrest. Macroscopic examination of the internal organs revealed vascular congestion with stasis, small-pointed hemorrhages under the epicardium, bloating of the intestines, plethora of the mesenteric vessels and liver. During a pathomorphological study of the internal organs, sharp plethora of the examined organs was noted, more pronounced in the lungs and liver.

When Tireoton-1 per os was introduced to white rats at the maximum possible doses of 4500 mg / kg, no animals died within 14 days. In the first 2-5 hours after administration to the animals, “Tireoton-1” noted the phenomena of inactivity, rapid urination, refusal to feed.

Thus, the data obtained allow us to attribute “Tireoton-1” to the group of low toxic substances according to the current classification.

Given the DL50 Tireoton-1, which is 450 mg / kg, the results of previous experiments used doses of 10 and 50 mg / kg to study its effect on the course of experimental hypothyroidism in rats. As a comparison drug, kelp was used in tablets (Evalar, Russia) at a dose of 3 mg / kg, which compensates the daily iodine rate by 133% (according to the annotation to the drug).

To study the thyrotropic effect of “Tireoton-1”, a model of experimental hypothyroidism was reproduced [7]. As a result of the administration of mercazolil orally to white Wistar rats at a dose of 10 mg / kg for 28 days, there was a significant decrease in T ₃ by 15%, T ₄ by 43%, an increase in TSH by 6.0 times compared with data from animals of the intact group. The administration of Tireoton-1 to animals orally at a dose of 50 mg / kg for 21 days is accompanied by an increase in the level of T ₄ in the blood of rats by 2.2 times, T ₃ by 47%, TSH decreases by 50% compared with the control group . The deiodation index was 7.02, which almost corresponds to the deiodation index of the intact group of animals (7.12). Tireoton at a dose of 10 mg / kg has a less pronounced pharmacotherapeutic efficacy compared to that at a dose of 50 mg / kg. The level of T ₄ increases by 55.1%, T ₃ - by 15%, but the level of TSH remained high, decreasing by only 5% compared with the indices of the animals of the control group. The body mass of rats decreased slightly compared with the control. The data obtained are given in table. 3.

Thus, as a result of the experiment, it was found that "Tireoton-1" increases the functional activity of the thyroid gland, normalizing the level of thyroid hormones. The course administration of Tireoton-1 at a dose of 50 mg / kg leads to an increase in the deiodination index and practically corresponds to that of the intact group of rats. "Tireoton-1" has a similar effect in comparison with kelp.

Example 2

500 g of rhizomes and roots of white cinquefoil meeting the requirements of GOST R 51074-2003, 250 g of rhizomes and roots of pink rhodiola, meeting the requirements of GF XI issue. 2, Art. 75, and 250 g of the roots of Scutellaria baicalensis, corresponding to the requirements of FS 42-453-91, taken in a ratio of 50, 25, 25%, respectively, are crushed in a mill for grinding dry samples MRP-2 to a particle size of no more than 2 mm (sieve No. 20 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 14 liters of 60% ethyl alcohol in a ratio of raw materials-extractant equal to 1:15, taking into account the coefficient of water absorption of the raw materials. It is extracted at a temperature of 60 ° C with constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. Two more extractions are carried out for 60 and 30 minutes, each time supplying 60% alcohol to the extractor in an amount equal to the volume of the drained extraction (1st discharge - 10.95 L, 2nd discharge - 11.35 L, 3rd discharge - 9.95 L) . The combined extracts are evaporated to 1/3 of the original volume and purified by separation. The purified extract is doubled to 1/5 of the original volume, dried in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Obtain 27.1 g of the finished product, which is 27.1% of the weight of the feedstock. The dry extract is a fine brown powder with a pleasant odor and a bitter astringent taste; crumbles, the moisture content is 4.12%.

Next, the possibility of the influence of Tireoton-1 on the state of the central nervous system of rats during experimental hypothyroidism is considered. The study was carried out using the methods: “open field”, “elevated cross-shaped labyrinth” (PCL), passive avoidance reaction.

A three-week administration of Tireoton at a dose of 50 mg / kg to white rats leads to an increase in almost all indicators. General motor activity (horizontal and vertical) increases by 4.5 times, a mink reflex appeared, indicating the appearance of research activity. A 31% decrease in grooming acts with previous research activity indicates the “comfortable” nature of grooming. The level of bowel movement is also reduced in the experimental group. Approximate research activity of rats of the experimental group exceeds those in the comparison group.

The “elevated cruciform labyrinth” reveals the degree of anxiety of the animal. In this section, we compare the performance of the PKL test of the experimental groups who received Tireoton, Tireoton-1 at a dose of 50 mg / kg and laminaria at a dose of 3 mg / kg against experimental hypothyroidism.

As can be seen from table 5, the course introduction of “Tireoton-1” at a dose of 50 mg / kg for 21 days reduces the time spent in the closed arms of the labyrinth by 20% compared with the control. The time spent in the open arms and on the central platform of the labyrinth is significantly increased by 4.0 times in comparison with the indices of the control group of animals. It should also be noted that with the introduction of “Tireoton-1”, the time spent in the open arms of the labyrinth increases, the number of exits into the open arm of the labyrinth as compared to the animals in the control group. The highest research activity of rats was noted, expressed in the number of hanging and peeping under the maze. Thus, it is possible to confirm the presence of a more pronounced anxiolytic activity of Tireoton-1, in comparison with the extract of white cinquefoil, which is due to extracts of Scutellaria baicalensis and Rhodiola rosea, which enhance and modulate the effect of the former.

In the passive avoidance reaction test it was reliably noted that “Tireoton-1” contributes to the preservation of a memorable trace in the experiment. So, it was found that after 24 hours the latent period increased by 70%, on the 3rd day - 2.7 times, on the 7th day - 2 times compared with the control group (table. 6). These indicators were similar to those of the comparison drug.

Thus, the introduction of “Tireoton-1” against the background of experimental hypothyroidism contributes to an increase in orientation-research activity, stimulation of anxiolytic activity and the development and preservation of a memorable trace.

Example 3

500 g of rhizomes and roots of white cinquefoil meeting the requirements of GOST R 51074-2003, 250 g of rhizomes and roots of pink rhodiola, meeting the requirements of GF XI issue. 2, Art. 75, and 250 g of the roots of Scutellaria baicalensis, corresponding to the requirements of FS 42-453-91, taken in a ratio of 50, 25, 25%, respectively, are crushed in a mill for grinding dry samples MPP-2 to a particle size of no more than 2 mm (sieve No. 20 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 16 liters of 65% ethyl alcohol in a ratio of raw materials-extractant equal to 1:16, taking into account the coefficient of water absorption of the raw materials. It is extracted at a temperature of 60 ° C with constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. Two more extractions are carried out for 60 and 30 minutes, each time feeding 65% alcohol into the extractor in an amount equal to the volume of the drained extract (1st discharge - 11.85 L, 2nd discharge - 11.65 L, 3rd discharge - 10.00 L ) The combined extracts are evaporated to 1/3 of the original volume and purified by separation. The purified extract is doubled to 1/5 of the original volume, dried in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Get 26.6. g of the finished product, which is 26.6% of the weight of the feedstock. The dry extract is a fine brown powder with a pleasant smell and a bitter astringent taste, crumbles, the moisture content is 4.55%.

The study of the functioning of the oxidative-antioxidant systems of the rat organism was carried out under experimental hypothyroidism and under the conditions of correction of the drugs Tireoton and Tireoton-1. The concentration of MDA, the level of catalase and reduced glutathione in blood serum and SOD in blood erythrocytes were determined.

With the course administration of “Tireoton-1” for 21 days with experimental hypothyroidism, there is a tendency to a decrease in the content of MDA in blood serum by 21% with a simultaneous increase in the activity of catalase in blood serum by 1.4 times and SOD - by 2.1 times compared to with these indicators in the control group. The level of reduced glutathione increases by 7% compared with the control (table. 7). Such data allow us to conclude that an increase in the antioxidant system of the body and a decrease in free radical processes in the correction of experimental hypothyroidism "Thyroton-1".

Thus, the course administration of Tireoton-1 at a dose of 50 mg / kg increases the functional activity of the thyroid gland during experimental hypothyroidism, increasing the level of thyroid hormones in the blood serum of rats and decreasing the TSH level by half compared to the control group. Also, “Tireoton” contributes to the improvement of the behavioral responses of animals against the background of a hypothyroid state: an increase in orientation-research activity, a decrease in animal anxiety, preservation of a memorable trace in the experiment, and stimulation of the antioxidant system of the body during experimental hypothyroidism.

Thus, it was experimentally established that “Tireoton-1” has a complex effect on the body during experimental hypothyroidism, affecting the main links in the pathogenesis of the disease.

Used Books

1. Arkhipova E.V. The effect of the extract of Potentilla alba L. and the complex agent “Tireoton” on the course of experimental hypothyroidism. Abstract of dissertation for the degree of candidate of medical sciences / Institute of General and Experimental Biology of the Siberian Branch of the Russian Academy of Sciences. - Ulan-Ude, 2012 .-- 20 p.

2. The State Pharmacopoeia of the USSR: issue. 2. General methods of analysis. Medicinal plant material. 11th ed. - M., 1987 .-- 400 p.

3. Zakharia A.V. The study of white cinquefoil as a promising tool for the treatment of thyroid diseases: Abstract. diss ... cand. biol. sciences. - Lviv, 1997 .-- 24 p.

4. Melik-Huseynov VV Atlas of plants. Plants in folk medicine of Russia and neighboring states. Pyatigorsk: SNOW, 2011.607 s.

5. Prikhodko EI Treatment of patients with thyrotoxicosis with pestra white grass used in diseases of the thyroid gland // Medical business. No. 6. 1976, p. 66-71.

6. Semenova E.F. The chemical composition of white cinquefoil and its use for therapeutic purposes / E.F. Semenova, E.V. Presnyakova // Chemistry and Computational Simulation. Butlerov messages. - 2001. - No. 5. - S. 32-34.

7. Requirements for the preclinical study of the general toxic effects of new pharmacological substances. - M., 1984. - 49 p.

8. Chugunova L.G., Ryabkov A.N., Savilov K.V. A method for modeling hypothyroidism // Patent 2165648 Russian Federation. IPC G09B 23/28, A61K 31/4164. - Ryazan State Medical University. - No. 97120428/14. - 2001.

Claims (1)

A method of obtaining a medicinal product for the correction of thyroid dysfunction by extracting plant material, characterized in that the roots and rhizomes of the white cinquefoil, the roots of Scutellaria baicalensis and the roots and rhizomes of Rhodiola rosea are used in the ratio of 50 wt.%, 25 wt.% , 25 wt.%, Respectively, which are crushed to a particle size of not more than 2 mm and extracted with 50-60% ethanol in the ratio of plant material - extractant equal to 1: (14-16), the first and second time for 60 minutes, the third - 30 minutes at a temperature of 60-65 ° C.