RU2696867C1 - Method for preparing medicinal substances from dracocephalum moldavica l. - Google Patents

Abstract

FIELD: technological processes.

SUBSTANCE: invention relates to a method for preparing an extract of Dracocephalum moldavica L. herb containing flavonoids having neurotropic activity, consisting in extraction of ground raw material with ethyl alcohol, filtration, evaporation, wherein ground herb of Dracocephalum moldavica L., in budding and flowering phase, is extracted three times with 50 % ethyl alcohol, with ratio of raw material : extractant equal to 1:10, at room temperature, while feeding to each subsequent extraction a fresh extractant equal to the fused extraction, obtained extracts are successively evaporated as supplied to volume up to 1/20, water vat concentrates are combined and treated three times with chloroform in ratio of chloroform : aqueous phase equal to 1:2, organic phase is separated, and the aqueous still residue is re-precipitated with ethanol of 96 % in ratio of 1:10, the settled precipitate is settled, the evaporated supernatant from the previous step is dissolved at 65 °C in distilled water with addition of cationite Ku-2-8-chs(H⁺) to pH equal to 3, filtered, the obtained solution is extracted with n-butanol three times at ratio of 1:2, the aqueous layer is separated, the butanol extract is washed with distilled water twice, and then the butanol extract washed with water is distilled in the form of an azeotrope with water to obtain an extract.

EFFECT: obtaining an extract from a grass of the Moldovan's hippopotamus, which is harvested during a flowering or budding phase, to create an agent possessing neurotropic activity and affecting the cardiovascular parameters.

1 cl, 3 tbl, 1 ex

Description

The invention relates to the chemical and pharmaceutical industry, in particular, to a method for producing medicinal substances of Moldavian snakehead (Dracocephalum moldavica L.) to create a drug with neurotropic activity and affecting the performance of the cardiovascular system.

Moldavian snakehead (Dracocephalum moldavica L.) of the family Lamiaceae (Lamiaceae), introduced on the territory of the branches of the FSBIU VILAR of the Russian Federation. It is cultivated as an essential oil plant in Ukraine, Bulgaria and Romania. In addition, recently cultivated in the south of Russia. The components that make up the Moldovan snakehead are biologically active substances and determine the therapeutic effect. The antibacterial activity of essential oil and alcoholic extracts from the grass of Moldavian snakehead, as well as the presence of expectorant, diuretic and sedative effects in alcoholic extracts was established. It is used in perfumery, cosmetology and cooking [1]. In traditional medicine, a plant is used in the form of teas, infusions, as well as dietary supplements in combination with other components [2].

Since ancient times, plants of the snakehead family have been used in Chinese folk Uyghur medicine to treat heart diseases (heart palpitations, coronary heart disease), arterial hypertension, tracheitis, atherosclerosis, neuralgia, migraine, headache and toothache.

Studies of the chemical composition of extracts from the aerial parts of the Moldavian snakehead are widely covered in world literature [3-6]. The grass of the plant contains flavonoids, essential oil, phenolic acids, tannins and other substances. Of particular importance in the literature is rosmarinic acid (caffeic acid ester and 3,4-dihydroxyphenyl lactic acid), which has a wide range of therapeutic and prophylactic effects: antioxidant, hepatoprotective, cardioprotective, nephroprotective, immunomodulating, antiallergic and anti-tumor. The acid inhibits the activity of acetylcholinesterase, glutathione reductase, and reduces the genotoxic and cytotoxic effects of ionizing radiation [7].

As a prototype, the method of obtaining the total extract of flavonoid type flavonoids from the Moldavian snakehead with a yield of 50% of the content in the raw material, which consists in processing the crushed raw material with ethyl alcohol, diluting the extract with water, passing through a column of microporous resin or polyamide, washing with water and aqueous alcohol, was selected , selection of the fraction of target compounds, filtration, evaporation and further for the production of treatment for atherosclerosis, heart diseases, tonsillitis, as well as their complex armatsevticheskih forms (tablets, granules, powders, tinctures, capsules, etc.), which is adopted by us as the prototype [CN 101 537 039 A]. The disadvantages of the prototype the duration of the process and the complexity of the selection.

The objective of the invention is to develop from the grass of the Moldavian snakehead, prepared in the flowering and budding phase of a method for producing a dry purified extract containing phenolic compounds (flavonoid type flavonoids and phenylpropanoids) in terms of rosmarinic acid providing neurotropic activity, as well as funds from the Moldavian snakehead with Moldavian content compounds in terms of (+) - catechin, which has a reliable anti-inflammatory effect, inhibiting exudative and alternative components of the pathogenesis of acute inflammation.

The technical result of the invention of the present invention is to obtain from the grass of the Moldavian serpentine, prepared in the flowering or budding phase of the product under the working name Rosmatin (hereinafter Rosmatin), which provides neurotropic activity with phenolic compounds in terms of rosmarinic acid, - The proposed method allows to obtain a high yield of the target product .

The technical essence of the proposed solution lies in the use of a method for extracting an extract from the grass of the Moldovan dragonhead (Dracocephalum moldavica L.) containing flavonoids with neurotropic activity, consisting in the extraction of crushed raw materials with ethyl alcohol, filtration, and evaporation. At the same time, the ground grass of the Moldovan snakehead, in the budding and flowering phase, is extracted three times with 50% ethanol, with a ratio of raw materials: extractant equal to 1:10, at room temperature, with a fresh extractant equal to the drained extract supplied for each subsequent extraction, the extracts obtained are evaporated sequentially as they reach a volume of up to 1/20, aqueous bottoms are combined and treated three times with chloroform in the ratio of chloroform: aqueous phase equal to 1: 2, the organic phase is separated, and the aqueous cubic the residue was reprecipitated with ethyl alcohol 96% in a ratio of 1:10, the precipitated precipitate was sedimented, the evaporated supernatant from the previous stage was dissolved at 65 ° C in distilled water with the addition of Ku-2-8-hs cation exchanger (Н ⁺ ) to a pH of 3, filtered, the resulting solution was extracted with n-butanol three times in a 1: 2 ratio, the aqueous layer was separated, the butanol extract was washed with distilled water twice, and then the butanol extract washed with water was distilled off as an azeotrope with water to obtain an extract.

The proposed technical solution is illustrated by the following examples 1 and 2 of obtaining substances and preclinical experiments 1 and 2.

EXAMPLE 1. The method of obtaining Rosmatin

Feedstock: Moldavian snakehead grass, harvested during the flowering phase or budding phase. The crushed raw material to a particle size passing through a sieve with a hole diameter of 0.5-5 mm, with a moisture content of 8.57%, in an amount of 200 g is loaded into a flat-bottomed flask with a capacity of 10 l, extracted three times with 50 ± 5% aqueous ethyl alcohol in the ratio ( raw materials - extractant, 1:10) at room temperature and with constant stirring for 3 hours. For each subsequent extraction serves fresh extractant equal to the drained extraction. Water-alcohol extracts are filtered on a Buchner funnel. Obtain from the first extraction of 1.530 liters of water-alcohol extraction, from the second extraction of 1.480 liters and from the third extraction, 1.490 liters of water-alcohol extracts are obtained. The resulting extracts were evaporated to 1/20 of the original volume. Evaporation of water-alcohol extracts is carried out sequentially upon receipt. Aqueous vat concentrates are combined, an aqueous vat residue is obtained in an amount of 240 ml with a density of 1.113 g / cm and is treated 3 times with chloroform in the ratio of chloroform: aqueous phase (1: 2). The organic phase is separated, and the residual organic solvent from the aqueous phase is removed in vacuo. An aqueous bottoms residue is reprecipitated from ethyl alcohol 96% in a ratio of 1:10 for further purification.

To do this, 2.4 l of ethyl alcohol 96% is poured into a 5.0-liter flat-bottomed flask, with vigorous stirring, 240 ml of aqueous bottoms from the previous step are added in portions (ratio of aqueous bottoms: ethyl alcohol of 96% (1: 10). The precipitated sandy-beige precipitate was sedimented for 30 minutes, then the supernatant water-alcohol solution was filtered by decantation through a No. 3 Schott filter. The precipitate was compacted and remained at the bottom of the flask. 250 ml of ethyl alcohol 96% were added while stirring and washed and filtered twice. through Schott filter No. 3. The alcohol from the washing of the precipitate is added to the main filtrate.The supernatant water-alcohol solution has a transparent brown color, the weight of the evaporated supernatant is 29.31 g.

The washed precipitate weighing 63.64 g is dried in air for two days or in a vacuum oven at 40 ° C for four hours.

The one-off supernatant water-alcohol solution from the previous stage in the amount of 29.31 g is dissolved in 160 ml of distilled water (in a water bath at a temperature of 65 ° C. Ku-2-8-hs (H ⁺ ) cation exchanger is added to the aqueous solution until acidic a solution of pH equal to 3, filtered through a filter POR 40 or POR 100.

The resulting acidic mother liquor was extracted with n-butanol, saturated with water, 3 times in a 1: 2 ratio. The aqueous-organic phase is easily stratified. The butanol phase is washed twice with distilled water in a ratio of 1: 1. Further, butanol is distilled off in the form of azeotropes with water in vacuum at 60-70 ° C, and 50 ml of water is consumed per 100 ml of extract. Obtain 4.6 g of the substance "Rosmatin", the yield of the finished product is 2.51%, which is the yield of the substance 33.15% from the beginning of the process with the content of polyphenols in terms of rosmarinic acid 60.9%.

The use of KU-2 cation exchanger followed by processing of the acidic mother liquor with butyl alcohol allows carbohydrates to be removed, which reduce the shelf life of the substance of Rosmatin.

The resulting substance of Rosmatin is a dry purified extract containing, as the dominant component, rosmarinic as well as caffeic acid, flavonoid-type flavonoids: glycoside tilianin, cinaroside, luteolin, apigenin. Rosmatin is used to prepare a dosage form (tablets, sprays, tinctures, capsules).

The resulting substance of Rosmatin is a dry purified extract containing the dominant component of rosmarinic, as well as caffeic acid, flavonoid type flavonoids, tilianin glycoside, cinaroside, luteolin, apigenin.

EXPERIENCE 1. The study of neurotropic activity and the effect on the indicators of the cardiovascular system of Rosmatin

A dry purified extract of Rosmatin from the Moldavian snakehead was presented for the study.

The effects of the course administration of Rosmatin in doses of 10 and 100 mg / kg on the performance of the cardiovascular system (heart rate, systolic and diastolic blood pressure) and the nervous system (in tests to study spontaneous motor activity and the effect on chloral hydrate sleep parameters) of mice were studied .

Research methods

The experiments were carried out on white nonlinear mice weighing 18-22 g in an amount of 32 individuals. The mice were kept under vivarium conditions with the FGBNU VILAR standard diet. Laboratory animals were divided into four groups of 8 animals: the first - control animals, the second, third and fourth groups - experimental. The second and third experimental groups of mice received the test substance Rosmatin, the fourth group received a drug comparing valerian extract (tablets containing 20 mg of valerian thick extract) at a dose of 100 mg / kg. All substances were dissolved in 1% starch suspension and administered intragastrically for 4-5 days, control animals received an equivalent volume of 1% starch paste.

On the 4th day of the experiment, 30 minutes after the intragastric administration of substances and 1% starch suspension, we studied the dynamics of the spontaneous locomotor activity of mice according to the standard method using an actometer from Ugo Basile (Italy) with automatic registration. During the experiment, mice were placed four individuals in each of the two chambers of the device for five minutes to study spontaneous motor activity in dynamics for two hours: after 30, 60, 90 and 120 minutes.

On the same day, heart rate, systolic and diastolic blood pressure were measured in animals using the Systola and Phlogiston hardware and software systems (manufacturer of Neurobotics LLC, Moscow). Before measuring blood pressure, mice were placed in restrictor containers for 30 minutes, heated to 37 ° C on a Phlogiston heating platform. The Sistola non-invasive blood pressure measurement system for laboratory animals using the built-in electric air pump automatically pressurizes the air in the tail cuff until the pulsation of the blood flow stops, and then, slowly lowering the pressure in the cuff, it automatically measures systolic and calculates diastolic pressure based on infrared readings pulse sensor, worn on the tail of the animal after the cuff. The Phlogiston system is designed to maintain the set temperature when heating small laboratory animals. Heating mice is necessary for the correct measurement of pressure, since heating provides stable, with sufficient volume, blood circulation in the tail.

On the 5th day of the experiment, we studied the effect of Rosmatin on sleep parameters caused by a "cortical hypnotic analyzer" chloral hydrate. A solution of chloral hydrate was prepared immediately before use on a physiological solution of sodium chloride 0.9%. 350 mg / kg chloral hydrate was injected intraperitoneally to the control and experimental groups of mice 30 minutes after intragastric administration of 1% starch suspension and the studied sample of Rosmatin and the drug for comparison of valerian extract at a dose of 100 mg / kg, respectively. The time of falling asleep was noted by the lateral position of the animals, the time of awakening was fixed by the animals adopting the usual posture with open eyes and grooming. The observation period for the duration of sleep was 4 hours.

The results of a study of the effect of course administration of Rosmatin on the spontaneous motor activity of mice in dynamics are presented in table 1.

1. As can be seen from table 1, the test sample of Rosmatin at a dose of 10 mg / kg significantly enhances the spontaneous locomotor activity of mice - 1.72 times compared with the control group - 2 hours after the start of testing.

The action of the comparison drug - tablets of valerian extract at a dose of 100 mg / kg was characterized by a significant (at the 60th minute) decrease in spontaneous motor activity of mice by 44% compared with control animals.

Table 2 shows the effects of course administration of a sample of Rosmatin in doses of 10 and 100 mg / kg on the indicators of the cardiovascular system of mice: systolic, diastolic and pulse blood pressure (BP) and heart rate (HR).

As can be seen from table 2, after a course of administration of Rosmatin at doses of 10 and 100 mg / kg, a significant increase in systolic (by 13% and 11%, respectively) and pulse (by 36% and 31%, respectively) blood pressure in experimental mice was observed compared with the control group, and also recorded a significant positive chronotropic effect - increased heart rate by 24% and 20%, respectively, compared with control animals. The comparison drug did not cause any significant changes in blood pressure and heart rate in mice compared to control animals.

Table 3 shows data on the effect of 5-day course administration of Rosmatin in doses of 10 and 100 mg / kg on sleep parameters caused by the "sleeping pill analyzer" chloral hydrate.

As can be seen from table 3, a 5-day course administration of the test sample of Rosmatin led to an increase in the duration of sleep caused by the “cortical hypnotic analyzer” chloral hydrate, that is, it stimulated the cortical inhibition and hypnotic effect of chloral hydrate, and at a dose of 100 mg / kg, the enhancement of hypnotic effect wore reliable nature (duration of sleep increased by 36% compared with control animals). The course administration of the comparison drug, valerian extract at a dose of 100 mg / kg in our experiment was accompanied by a less significant (13% compared with the control group) increase in the duration of chloral hydrate sleep.

The studied sample of Rosmatin at a dose of 100 mg / kg has a significant phase effect on sleep parameters caused by the “cortical hypnotic analyzer” chloral hydrate: after the phase of the activating effect on the central nervous system, characterized by an increase in the time of falling asleep (sleep latency) by 1.4 times compared to with the control group, then potentiation of cortical inhibition caused by chloral hydrate was observed, and an increase in the total duration of sleep by 1.36 times compared with control animals.

Thus, in this experiment, the phase nature of the neuropsychotropic activity of the studied sample of Rosmatin at a dose of 100 mg / kg was revealed - after the phase of the activating effect on the central nervous system, potentiation of cortical inhibition caused by chloral hydrate was observed.

Thus, the test sample of Rosmatin has an activating, tonic effect on the cardiovascular and nervous systems.

Bibliography

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Claims (1)

A method of obtaining an extract from the grass of the Moldavian dragonhead (Dracocephalum moldavica L.) containing flavonoids having neurotropic activity, which consists in extraction of the crushed raw material with ethyl alcohol, filtration, evaporation, characterized in that the crushed grass of the Moldavian snakehead, in the phase of budding and flowering, - 50% ethyl alcohol, with a ratio of raw materials: extractant equal to 1:10, at room temperature, when a fresh extractant equal to the drained extraction is fed to each subsequent extraction, the irradiated extracts are evaporated sequentially as they reach a volume of up to 1/20, the aqueous bottoms are combined and treated three times with chloroform in the ratio of chloroform: the aqueous phase is 1: 2, the organic phase is separated, and the aqueous bottoms are reprecipitated with 96% ethyl alcohol in a ratio of 1 10, the precipitate is settled supernatant The evaporated solution from the previous step was dissolved at 65 ° C in distilled water with the addition of KU-2-8-ES (H ⁺⁾ to pH of 3, filtered and the resulting solution was extracted with n-butanol three rows in the ratio 1: 2, the aqueous layer was separated, butanol extract was washed with distilled water twice, and then washed with water, butanol extract was distilled off as azeotropes with water, to obtain an extract.