RU2484839C1 - Method for preparing golden rhododendron extract with reduced steroid (cardiac) glycosides - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to preparing an anti-inflammatory, antibacterial, cytostatic, stimulating and tonic effect of golden rhododendron with reduced steroid (cardiac) glycosides. A method for preparing a golden rhododendron extract with reduced steroid (cardiac) glycosides by pre-extraction treatment of dry leaves of golden rhododendron in aqueous sodium hydroxide (pH≈13), addition of ethanol into the reaction mixture, keeping, filtration and reduction to neural pH (pH≈7), under certain conditions.

EFFECT: method allows reducing steroid (cardiac) glycosides in aqueous-alcoholic extract in 3,0 times.

1 dwg, 1 tbl

Description

The invention relates to the field of biotechnology industry and medicine, can be used to create biologically active drugs and drugs anti-inflammatory, antibacterial, cytostatic, stimulating and tonic effects.

Golden Rhododendron (Rhodendron aureum Georgi) fam. Heather {Ericaceae) are widely used in folk medicine in Russia, Europe, Southeast Asia. The plant gained particular popularity in Eastern Siberia, the population of which uses it in the treatment of a variety of diseases, including malignant tumors (popular names: Kashkara, Kashkarnik golden, black mane) [Usov et al., 1995].

Golden rhododendron leaves contain sitosterols, triterpenoids, flavonoids, tannins, organic acids, sesquiterpenic alcohols, glycosides (andromedotoxin, rhododendrin, arbutin) [Belousov et al., 1992, Belousov et al., 2000], as well as cytostatic compounds, for example colchifoline [Filippova, 2003]

In relation to strepto- and staphylococci and pathogenic intestinal flora, rhododendron golden preparations have a strong bactericidal effect [Chulanova, 1959, RF Patent No. 2182829]. Water decoctions and leaf extracts turned out to be effective for the treatment of gum disease, stomatitis, with halitosis and found application in gynecology [Lopatin, 1950].

It has been established that preparations of golden rhododendron have a pronounced cardiac effect and, when prescribed in an authorized therapeutic dose, patients with cardiovascular insufficiency normalize heart function, reduce arterial and venous pressure (have a hypotonic effect), and increase blood flow velocity. At the same time, drugs increase urination, reduce edema [Velyatiev et al., 1987].

However, due to the high content of cardiac glycosides that can cause heart rhythm disturbances, arrhythmias in case of overdose, this plant is not included in the Russian Pharmacopoeia. Therefore, the urgent task in the development of certified biologics compositions from golden rhododendron tissues is the development of a deglycosylation method while maintaining the content and bioactivity of aglycones.

The literature contains information on three possible ways of hydrolysis of cardiac glycosides: acidic, alkaline, enzymatic [Bochkov et al., 1978, Grinkevich, Safronich, 1983]. In acid or alkaline hydrolysis, deep cleavage immediately occurs to aglycon and sugar components [Grinkevich, Safronich, 1983], and in case of enzymatic hydrolysis, the degree of hydrolysis depends on the reaction time and to stop it at a certain stage, an additional operation is required - enzyme inactivation [RF patent No. 2249042 ].

An analogue of the proposed method of deglycosylation is acid hydrolysis, based on the action of strong mineral acids [Lobanova, Turetskova, 2011, Tolkacheva et al, 2011] (scheme). However, the disadvantage of this method, for example, when using sulfuric acid, is the formation of sulfates, as a rule, having low solubility, which creates additional difficulties in cleaning the final product.

Descriptions of the methods of alkaline hydrolysis of cardiac glycosides in the literature available to us have not been found.

The closest analogue (prototype) is a method based on the use of enzymatic hydrolysis of steroid glycosides Ruscus aculeatus (ruscosaponins) using crude hydrolases from Aspergillus niger; The method allows to obtain desglucodedesramnoruzcin with a high degree of efficiency [RF patent No. 2249042]. According to this method, crude hydrolase preparations are commercial materials that are used in the winemaking and food industries, for example, Gist Brocades under the brand name Cytolase PCL5.

The disadvantages of this method of isolation and obtaining derivatives of cardiac glycosides is the need to purchase enzyme preparations and expensive equipment, including for conducting and monitoring the reaction, as well as the need to inactivate hydrolases after hydrolysis.

The essence of the invention lies in the fact that before receiving the extract from the dry leaves of golden rhododendron, they are subjected to grinding with subsequent alkaline hydrolysis in order to deeply destroy glycosides, including steroid (cardiac).

The optimal conditions for the pre-extraction of powder of dry leaves of golden rhododendron are soaking in 0.1 M (pH≈13; table) aqueous solution of sodium hydroxide in a ratio of 1:10 by weight for 4 hours at a temperature of 35-40 ° С.

The extraction is carried out by adding ethyl alcohol to the reaction mixture, bringing its concentration to 50%, after which this mixture is infused for 14 days at a temperature of 20-25 ° C. The resulting extract is filtered to remove suspended particles and the pH is adjusted to neutral (pH≈7). Determination of the content of cardiac glycosides and flavonoids is carried out by spectrophotometric method [Rogozhin, 1999].

As a result of alkaline pre-extraction treatment of the golden powder of rhododendron leaves, the content of steroid (cardiac) glycosides in the extract decreases by a factor of 3.0, while the concentration of flavonoids increases by a factor of 3.0 compared to the extract obtained without preliminary treatment (table).

The technical effect of the invention is the simplicity of technological operations, the absence of expensive equipment and reagents. Pre-extraction of raw materials of golden rhododendron allows you to get extracts with a low content of cardiac glycosides, which do not have a pronounced adverse side effect on cardiac activity.

The effect of the invention is achieved by the fact that the method of pre-extraction treatment of golden rhododendron tissues leads to a 3.0-fold decrease in the content of steroid (cardiac) glycosides while a 3.0-fold increase in the concentration of flavonoids (aglycones) compared to the control extract obtained without preliminary processing of raw materials .

The diagram shows the reaction mechanism of acid hydrolysis of glycosidic bonds.

Table. The content of cardiac glycosides and flavonoids in golden rhododendron extracts obtained under various conditions of pre-extraction processing of coarse raw materials Pre-extraction conditions, pH The content of cardiac glycosides in the extract, mg / ml The content of flavonoids in the extract, mg / ml 1.0 0.65 ± 0.03 1.20 ± 0.04 2.0 0.50 ± 0.03 1.18 ± 0.03 3.0 0.29 ± 0.02 1.11 ± 0.03 7.0 0.32 ± 0.02 1.29 ± 0.04 11.0 0.42 ± 0.02 1.15 ± 0.03 12.0 0.39 ± 0.02 1.59 ± 0.12 13.0 0.11 ± 0.01 3.88 ± 0.01

Claims (1)

A method of obtaining a golden rhododendron extract with a reduced content of steroidal (cardiac) glycosides, characterized in that the pre-extraction treatment of dry leaves of golden rhododendron is carried out for 4 hours with a 0.1 M aqueous solution of sodium hydroxide (pH≈13) in a ratio of 1:10 by weight, after which Ethyl alcohol is added to the reaction mixture to a concentration of 50% and insisted for 14 days at a temperature of 20-25 ° C, the extract is filtered to remove suspended particles and the pH is adjusted to neutral (pH≈7), resulting in the content of steroid (cardiac) glycosides in the water-alcohol extract decreases by 3.0 times.

Images