Method of obtaining vegetable preparation, which has diuretic and anticoagulation activity

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and deals with creation of medication of vegetable origin based on biologically active substances of Linaria vulgaris, which has diuretic and anticoagulation activity. Method of obtaining medication, which has diuretic and anticoagulation activity, consists in the following: extraction of milled grass of Linaria vulgaris with ethyl alcohol is carried out with successive drawing with 3 parts of extractant by method of remaceration with heating and connecting vacuum at the stage of discharge, under specified conditions.

EFFECT: method makes it possible to obtain medication, which has high diuretic and anticoagulation activity.

3 tbl, 1 ex

 

The invention relates to the field of medicine and pharmaceutical industry and for the creation of plant-based bioactive substances lnjanki ordinary Linaria vulgaris Mill (SEM. Scrophulariaceae), which has diuretic activity.

Diuretics are widely used in medical practice. They are frequently prescribed for diseases of the cardiovascular system: edema associated with chronic heart failure, in the treatment of hypertension. Very often these diseases are accompanied by the formation of vascular thrombosis than due to the use of anticoagulation in the treatment of. Currently, the proven efficacy of medicinal products from vegetable raw materials for the treatment of diseases of the cardiovascular system, and the herbal remedies do not have side effects and harmless with long-term use.

Known for a number of plant resources that have diuretic effects. Diuretic activity was detected in extracts from the underground organs of cinquefoil marsh [Patent RU 2318531 C1], the leaves of pear and herbs highlanders unnoticed and heterophyllous [Denisenko, O. Pharmacognostically study of plants diuretic action of a species of pear and knotweed in the conditions of South-in the current Ukraine, abstract. Diss. on saisc. degree Cand. Pharm. Sciences, 1991, Lviv], common bearberry, cowberry [Turov A.D., E. Sapozhnikova. Medicinal plants of the USSR and their use. M., 1982.]. Know the use of leaf horsetail as diuretics. The decoction is made from cut grass, briquettes and pellets from the herb horsetail [Mashkovsky PPM "Medicinal plants", Kharkov, Torching, 1997, Ed. 13, vol. 1, s]. Anticoagulant activity was detected in extracts from plants dandelion [Patent RU 2085205 C1], peony tree [Patent RU 93033687 A].

The closest to the complexity of the action and the method of obtaining a remedy, including dry extracts of grass knotweed, leaves of Indian, leaves of bearberry leaf and possessing diuretic and hypoazotemic activities [Patent RU 2008142861 AND]made for the prototype.

The main disadvantages of the prototype

1. The technology of obtaining funds multistage: each raw material is extracted separately.

2. The temperature of extraction may reach 80°C, which leads to additional costs.

3. The agent has no anticoagulant activity.

The aim of the invention is the obtaining of a product based on grass lnjanki ordinary and identifying its diuretic and anticoagulant action.

To achieve the decree is Noah purpose raw materials lnjanki ordinary, crushed to the size of particles passing through a sieve with holes of 2 mm diameter and disqualified from dust were extracted by the method of remuneratio division of the extractant into 3 parts with heat and connecting the vacuum at the stage of discharge. Raw materials consistently insisted with the first part of the extractant (5 parts), then the second and third (2.5 parts each), each time merging the hood. As extractant using 60%ethanol. Time infusion from each part of the extractant 40 minutes at a temperature of 42°C. the resulting extract was agglomerated in a rotary evaporator apparatus at a temperature of 60-70°C, then dried in a drying Cabinet at 60°C. the Yield is 24% by weight of vegetable raw materials. Further, the goal of the experiments that characterize the strength diuretic effect lnjanki ordinary.

The final product has the following qualitative and quantitative characteristics.

Analysis on the content of flavonoids:

Dry extract (0.1 g) dissolved in 10 ml of 60% ethanol

1. To 3 ml of the solution add 1 ml of 2% solution of aluminum chloride, appears yellow staining.

2. To 3 ml of the solution was added 10 mg of crystalline magnesium and 5 drops of concentrated chloroethanol acid, heated on a boiling water bath for 3 min, appears orange-red color.

If the natural enemy of the contents of flavonoids:

An analytical sample was air-dried raw material is crushed to a particle size passing through a sieve according to GOST 214-83 with holes with a diameter of 1 mm, About 0.5 g (accurately weighed) of the raw material is placed in a conical flask with a capacity of 200 ml with cut, add 100 ml of 60%ethanol. The flask is attached to the back of the fridge and heated in a boiling water bath for 60 minutes, computed from the boiling point of the extractant. The flask was cooled to room temperature, the extract was filtered through a cotton pad with flat bottom volumetric flask 100 ml capacity and through the same filter to bring the volume of solution of 60%ethanol to the mark. The obtained extract was filtered into a flask through a paper filter (solution A), the first 10 ml of filtrate drop; 2 ml And placed in a volumetric flask with a capacity of 50 ml, the volume was adjusted solution of 60%ethanol to the mark and mix (solution B). Measure the optical density of a solution in the spectrophotometer SF-2000 at a wavelength of 332 nm in a cell with the thickness of the absorbing layer 10 mm as the reference solution using 60%ethyl alcohol. The content of total flavonoids in the grass lnjanki ordinary in terms of acetylecholine calculated by the formula:

x(%)=A10050100%379the mV(100-w)

where a is the optical density of the investigated solution; m is the weighed raw materials, g; V - volume of solution taken for dilution; 100 volume of solution, ml; 50 - volume of solution, ml; W is the loss in weight on drying of raw materials, %; 379 - specific absorption acetylecholine at a wavelength of 332 nm.

The analysis of the content of iridoids:

Dry extract (0.1 g) dissolved in 10 ml of 60% ethanol.

1. To 5 ml of the resulting solution add 5 drops of reagent Trim-hill (mixture consisting of 5 ml of concentrated hydrochloric acid, 10 ml of 0.2% solution of copper sulphate and 100 ml glacial acetic acid), heated in a boiling water bath for 10 minutes, you receive the blue colouring.

2. To 5 ml of the resulting solution add 5 drops of reagent Stahl (1 g n-diethylaminobenzaldehyde, 5 g of phosphoric acid and 50 g of acetic acid, dilute with water to 100 ml), bring to the boil on a water bath, appears blue staining.

The quantitative content of iridoids:

An analytical sample of the raw material is crushed to a particle size passing through a sieve with holes of diameter 1 mm to About 2.0 g (accurately weighed) of crushed and screened from dust raw material is placed in a conical flask with rack VM is the cost of 100 ml, add 0.05 calcium carbonate and 50 ml of 20% ethyl alcohol. The flask is attached to the back of the fridge and heated in a boiling water bath for 40 minutes Hot extract was filtered through cotton wool in a volumetric flask with a capacity of 50 ml After cooling, the volume of the extract was adjusted to the mark with ethanol 20% and mixed. The resulting solution was transferred into a flat-bottomed flask with a capacity of 100 ml with 1.0 g of neutral aluminium oxide, mixed on a shaker for 20 min and filtered through a paper filter.

For photocolorimetric determination take 1 ml of the resulting solution into a test tube, add 0.5 ml of reagent Trim-hill (HCl conc.; 0,2% CuSO4; CH3COOH ice.) and 2.5 ml of 50%acetic acid. The obtained volume is heated on a water bath for 15 min, cooled and determine the optical density at a wavelength of 590 nm (yellow filter) in a cell with a layer thickness of 0.5 cm as the reference solution using a mixture of 1 ml of ethyl alcohol 20%, 0.2 ml of reagent Trim hill and 2.5 ml of 50% acetic acid. The content of iridoids determined by a calibration curve where the y-axis indicated the optical density of the solution (D), and the axis of abscisses - concentration aucubin (µg/ml).

The content amount of iridoids in terms of aucubin in percent of dry raw material is calculated by the formula:

X=S*50*100*100/m*(100-W)*1000*1000, where

With the obsession amount iridoids in terms of aucubin, found on schedule, µg/ml; m - weighed raw materials, g; W is the loss in weight on drying of raw materials, %.

The method of obtaining funds is illustrated by the following example:

0.1 kg of grass lnjanki ordinary (only aerial parts) are ground to a particle size <0,25≤2,0. The crushed raw material is loaded into the extractor, pour 500 ml of 60%ethanol and extracted for 40 minutes at a temperature of 42°C. the resulting extract is drained when connecting the vacuum. Partially exhausted raw again pour 250 ml of 60%ethanol and insist similar to the first stage of extraction. The entire infusion procedure is repeated 3 times. The total flow of extractant 1000 ml, which corresponds to the ratio of raw materials - extractant 1:10. The resulting extract is condensed in a rotary evaporator apparatus at a temperature of 60-70°C, then dried in a drying Cabinet at 60°C. Receive 0,024 kg of the finished product, which is 24% of dry extract by weight of the feedstock. The alcoholic extract is a dry mass of black and brown. The content of flavonoids in the extract is 23.7±0.20%, iridoids 0.28±0.001%, moisture 7.9±0.10%.

The proposed method for obtaining permits to obtain a product of constant composition, and prevents gelation of the obtained extraction and loss of biologically active substances. The method of obtaining can be implemented at the enterprises is yusko drugs.

Test

The experimental investigations of the toxicity and potency herbs lnjanki ordinary.

1. Study of acute toxicity.

Study of acute toxicity of the extracts were performed on white mice on Prozorovsky [Prozorovsky V.B. have been, Prozorovsky BTW, Demchenko V.M. rapid method for determination of the average effective dose and its errors. // Pharmacol. and toxicol. - 1978 - No. 4 - S-503.]. Dry extract lnjanki ordinary diluted in distilled water, was investigated after a single oral administration at doses of 1000 mg/kg 2000 mg/kg 5000 mg/kg

Studies on white mice with a wide range of doses, aimed at the detection of toxic effects of varying severity and even fatal outcomes. Lnjanka ordinary has no toxic effect on experimental animals even with the introduction of high concentrations. The results are presented in table 1.

Table 1
The results of determination of acute toxicity of dry extract lnjanki ordinary (Linaria vulgaris)
Extract (extractant)The number of animals in the experimentSTA is in dead animals Surviving animals, %LD50mg/kg
Herb horsetail (water)60100>5000
Grass lnjanki common (60% ethanol)60100>5000

As can be seen from the table, oral administration of extracts of the studied plants in these doses did not cause the death of animals. In five experimental groups of animals treated with the extracts, there was no visual changes compared to the control group of animals. The definition of LD50for mice is not possible. Further increase in the dose of the extracts is impractical due to lack of toxicity at the dose of 5000 mg/kg and the complexity of introducing more extract. Based on the obtained results we can conclude about the practical safety of extracts lnjanki ordinary and they belong to the 4th class of hazard (low hazard substances) according to GOST 12.1.007-76, (median lethal dose when injected into the stomach of more than 5000 mg/kg).

2. The study diuretic (diuretic) activity.

Diuretic actively the TB extract of the herb lnjanki ordinary evaluated in accordance with the "guidelines" (1989). Experiments carried out on white rats male Wistar weighing 190-200, an extract of the herb lnjanki ordinary without the use of water load was administered once orally at a dose of 50 mg/kg dissolved in 5 ml of distilled water. Animals of control group received equiano amount of distilled water on a similar scheme. As the comparison drug used horsetail, cooked GF XI followed by distillation of solvent (distilled water) until dry. The obtained extract was administered at a dose of 50 mg/kg in the diagram above. The collection of urine was performed in metabolic cages for 6 hours after administration of the extract. Measured amount of allocated urine. The results obtained are presented in table 2.

Table 2
The study of the diuretic activity of the dry extract of the herb lnjanki ordinary (Linaria vulgaris)
Extract (extractant)Urine volume
The first 3 hoursTotal 6 hours
Control mlExperience mlIzmenenijem urine, %Control mlExperience mlThe change in the volume of urine, %
Herb horsetail (water)1.22±0.442.0±0.046641.30±0.452.61±0.16100
Grass lnjanki common (60%ethanol)2.3±0.87863.14±0.38141

As follows from the data in table 2, a single injection of dry extract in the dose had a pronounced stimulatory effect on the excretion of water. This diuretic activity of the test extract was superior to the 6 hours that 41% of the comparison drug.

3. The study of anticoagulant activity.

Assessment of anticoagulant activity was performed in vitro using coagulometer "Minilab-701 in a concentration of 1 mg/ml For the study used the blood of dogs, stable and 3.8% solution of sodium citrate in the ratio of 9:1. As the inductor coagulation used 0.28% solution of calcium chloride. The standard anticoagulant activity served as heparin, which is felt in Kona is entrale 1 U/ml under the same conditions. The effect of the pharmacological tests were considered significant at P≤0,05 [Belenky, M. elements of a quantitative evaluation of the pharmacological effect, 2nd ed., HP: the Medical literature, 1963]. The data obtained are given in table 3.

Table 3
Anticoagulant activity of dry extract lnjanki ordinary
No.Extract, drug comparisonThe clotting timeChanging coagulation, %P
controlexperience
1Extract lnjanki ordinary21.9±0.9125.2±0.65-15.1<0.01
2heparin29.9±0.4836.6±1.82-22.4<0.01

As can be seen from table 3, the extract lnjanki ordinary has anticoagulant activity. Ondiscover slows the clotting time of blood by 15%.

It is established that the grass lnjanki vulgaris is a high diuretic and anticoagulant activity. The simultaneous combination of anticoagulant and diuretic properties allows you to use the drug for the prevention and treatment of diseases of the cardiovascular system, is often complicated by thrombosis and accompanied by increased circulating blood volume.

The essence of the invention is that the method for obtaining the drug from a plant possessing diuretic and anticoagulant action, not previously used in scientific medicine.

Literature

1. Belenky, M. elements of a quantitative evaluation of the pharmacological effect, 2nd ed., HP: the Medical literature, 1963.

2. Denisenko, O., Pharmacognostically study of plants diuretic action of a species of pear and knotweed in the conditions of South-Eastern Ukraine, abstract. Diss. on saisc. degree Cand. Pharm. Sciences, 1991, lions.

3. Mashkovsky PPM "Medicinal plants", Kharkov, Torching, 1997, Ed. 13, vol. 1, s.

4. Prozorovskiy V.B. have been, Prozorovsky BTW, Demchenko V.M. rapid method for determination of the average effective dose and its errors. // Pharmacol. and toxicol., 1978, No. 4, S-503.

5. The method of producing complex with fibrinolytic, thrombolytic and anticoagulant activity Patent RU NO. 93033687 AND from 20.11.1995.

6. The method of obtaining funds, possessing diuretic and anti-inflammatory activity Patent number 2318531 C1 from 10.03.2008.

7. A means of having anticoagulant effect of Patent RU 2085205 C1 from 27.07.1997.

8. Means possessing diuretic and hypoazotemic activity (options), and method thereof Patent number 2008142861 And from 10.05.2010.

9. Turov A.D., Sapozhnikova E.N., medicinal plants of the USSR and their use. M., 1982.

The method of obtaining funds, possessing diuretic and anticoagulant effect, consisting in the fact that the extraction of crushed grass lnjanki ordinary spend 60% ethyl alcohol in the ratio of raw material:solvent 1:10 in sequential infusion with 5, 2.5 and 2.5 parts of extractant method remuneratio division of the extractant into 3 parts with heat and connecting the vacuum at the stage of discharge, and the time of infusion, with each part of the extractant 40 minutes at a temperature of 42°C, and the resulting extract is condensed in a rotary evaporator apparatus at a temperature of 60-70°C, and then dried in a drying Cabinet at 60°C.



 

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