RU2292218C1 - Method for lutenurine production - Google Patents

Abstract

FIELD: chemical and pharmaceutical industry.

SUBSTANCE: candock rhizome with roots are extracted with acidic 20-60 % aqueous alcohol solution, preferably ethanol or isopropanol aqueous solution acidified with 5 % phosphoric acid. Method further includes extract filtering; organic solvent separation in vacuum; neutralization with calcium carbonate; solid phase separation; treatment thereof with acetone; filtering; stripping; purification of sum alkaloid fraction via formation of respective sulfates and bases; base extraction with toluene and alkaloid hydrochloride production by solution saturation with hydrogen chloride.

EFFECT: method of increased product yield.

3 ex, 1 tbl

Description

The invention relates to the pharmaceutical industry, in particular to methods for producing yellow capsule alkaloids (Nuphar lutea (L.) Smith - (S.K. Cherepanov. Code of amendments to the "Flora of the USSR" (t.I-XXX) , L., Nauka, 1973) or (Nuphar luteum - Index kewensis, II, 1960) of the water lily family (Nymphaceae). The drug Lutenurin, obtained from the rhizomes of this plant, has a protistocidal and spermatocidal effect, active against Trichomonas, pathogenic fungi, bacteriostatic effect against gram-positive microorganisms using for the treatment of urogenital diseases, in particular for the treatment of trichomonas colpitis and urethrides, as well as a contraceptive (Y. A. Aleshkina, S. A. Vichkanova, M. A. Rubinchik, T. N. Ilyinskaya. The drug is lutenurin. Avt.Svid 145858, 04/04/1967, Bull. No. 10, 07/28/1967; Lutenurin, Lutenurinum, FS 42-948-75; Lutenurin, Lutenurin, Lutenurine, Prospect CBNTI MP, 89 pp .; Lutenurin, in the book: A complete modern directory of drugs, 2nd ed., S.A. Krzhizhanovsky and M.B.Vititnova, Izd. House "Rinol Classic", M., 2002, p.950). The drug lutenurin is a mixture of hydrochlorides, mainly dimeric sulfur-containing quinolysidine alkaloids: thiobinufaridine, neothiobinufaridine, nuflein. The active principle of the drug is the alkaloid nuflein (dihydroxy-thiobinofaridine) (T.N. Ilyinskaya, A.D. Kuzovkov, T.G. Monakhova. Chemical study of yellow capsule alkaloids, in the book: "Medicinal Plants. Chemistry", Volume 15, VILAR, under the editorship of A.I. Bankovsky, Kolos, Moscow, 1969, p. 222-333).

A known method for producing lutenurin, which consists in the following: dried and crushed rhizomes of yellow capsules containing 0.4% of the total alkaloids, is treated with ammonium hydroxide, extracted with dichloroethane, then extracted with sulfuric acid, the aqueous phase is made alkaline, with ammonium hydroxide, the alkaloids are extracted with ether, after alkaloids are extracted with ether drying the extract with potash it is concentrated, impurities are precipitated with petroleum ether, and then hydrochlorides are obtained by saturating the alkaloids solution with hydrogen chloride, the target product is separated by washing petroleum ether, dried and sieved through a sieve (T.N. Ilyinskaya, A.D. Kuzovkov, S.A. Vichkanova, M.A. Rubinchik, L.P. Tolstykh, Ya.A. Aleshkina. Method for producing lutenurin, Autosd. 216920, 04/26/1968, Bull. No. 15; V.P. Zakharov, N.I. Libizov, H.A. Aslanov, Lutenurin, in the book: "Medicinal substances from plants and methods for their production", Tashkent, FAN Uz SSR, 1980, pp. 10-12). Yield 65%. This method is selected as a prototype.

The disadvantages of the method of producing lutenurin is the use of a toxic solvent (dichloroethane), as well as the large loss of the target alkaloids in the cleaning process.

In order to simplify the technology of preparation and increase its yield, a new method is proposed, which consists in the following: the dried and ground rhizomes of the yellow capsule are extracted with an aqueous-alcoholic solution (preferably aqueous ethanol or isopropanol) acidified with phosphoric acid, filtered, the organic solvent is removed in vacuo then the aqueous acidic bottoms are treated with butanol, stirred, the organic phase is separated, washed with water, the combined acid extracts neutralize the guide ammonium atom, treated with a mixture of calcium carbonate with water, filtered, the precipitate was washed with acetone and the filtrate was evaporated in vacuo, the resulting technical amount of bases was purified by dissolving in dilute sulfuric acid, alkalizing with ammonium hydroxide, extraction with toluene, washing with an organic layer with water, drying with sodium sulfate and by evaporation. The resulting amount of alkaloids (0.3-0.4%) is converted into hydrochlorides as described in the prototype method. The yield of the target product 0.35%.

The method is simple to implement, increases the yield of the target product, eliminates toxic halogen-containing solvents at the head stage of extraction, reduces the overall duration of the process compared to the prototype, eliminates the formation of emulsions during liquid extraction and simplifies subsequent cleaning of the target product.

The production method is given in the attached examples 1-3.

Example 1. 200 g of dried (with a moisture content of 10.7%) and crushed rhizomes of yellow capsule is extracted with stirring (3 hours at 40-45 ° C) with an aqueous-alcoholic solution of phosphoric acid obtained by mixing 630 ml of 96% alcohol (60%) and 445 ml of 5% phosphoric acid (40%), with a ratio of raw materials - extractant (1: 7), filtered in vacuo. The operation is repeated 3 times, adding the extractant in an amount equal to the volume of the drained extract, the organic solvent is removed in vacuo, the aqueous acidic bottoms with a volume of 320-330 ml are combined, then the resulting suspension is mixed with butanol (1: 3 of the total solution volume), insist 1 h, butanol is separated, the operation is repeated 2 times (in this case, the precipitated fine precipitate goes into solution), the aqueous phase is filtered through cotton wool, then it is again shaken with butanol, the combined organic phase is separated and washed with water 4 times. The combined acid extracts with a volume of 1600 ml are neutralized with ammonium hydrate to pH 8, a mixture of 100 g of calcium carbonate and 20 ml of water is added, stirred for 5-10 minutes, filtered through a Buchner funnel, the aqueous filtrate is not used further, and the precipitate is thoroughly separated and washed with acetone ( 3 times 100 ml) and the filtrate is evaporated to dryness in vacuo. The residue was dissolved in dilute sulfuric acid (about 20 ml), made basic with ammonium hydroxide (pH 8), extracted with toluene, the organic layer was washed with water, dried with sodium sulfate and evaporated. The resulting amount of alkaloids bases (0.3-0.4%) are converted into hydrochlorides as described in the prototype method. The yield of the target hydrochloride of 0.35%.

The product meets the requirements of FS 1975 for lutenurin in the content of the active component (nuflein dihydrochloride) in it (at least 52%), the content of nuflein dihydrochloride in the preparation (according to the NMR spectrum in comparison with the known sample of the serial preparation), as well as antimicrobial activity of the sample in relation to Staphyllococcus aureus, studied by serial dilution, which was equal in activity to the known drug (0.98 μg / ml).

Example 2. The process is carried out as in example 1, characterized in that as the extractant in the head stage using 20% aqueous ethyl alcohol, acidified with 5% phosphoric acid. The base yield of 0.3-0.4%. The yield of hydrochloride is 0.35%.

Example 3. The process is carried out as in example 1, characterized in that 60% aqueous isopropyl alcohol, acidified with 5% phosphoric acid, is used instead of aqueous ethyl alcohol as an extractant in the head stage. The base yield of 0.4-0.6%. The yield of hydrochloride is 0.5%.

The study of the antimicrobial activity of lutenurin obtained by the present method in comparison with the prototype

In the laboratory of pharmacology and chemotherapy VILAR was studied the antimicrobial activity of lutenurin obtained by the present method (alkaloids in the form of a base and in the form of salt) in comparison with the prototype.

The study of bacteriostatic activity in in vitro experiments

In the study of bacteriostatic activity in in vitro experiments, the method of double serial dilutions of the drug in liquid nutrient media was used.

Microorganisms

Gram-positive cocci - Staphylococcus aureus 209-P was used as a pathogenic test microorganism.

Wednesday

When determining the bacteriostatic activity of lutenurin samples, meat-peptone broth (MPB) was used.

Research methodology

Bacteriostatic activity in experiments in vitro was studied by the method of serial dilutions of the drug in a liquid nutrient medium (BCH). In the study of lutenurin samples, a culture medium in an amount of 4 ml was added to the first test tube containing 4 mg of the drug to obtain an initial concentration of 1000 μg / ml and then, by diluting twice, a series of decreasing concentrations of lutenurin samples were prepared. The last test tube with a clean medium (without the addition of lutenurin) served as a control.

After that, all test tubes (experimental and control) were seeded with a test culture of the microorganism.

A suspension of the test culture was prepared in an isotonic sodium chloride solution according to the bacterial standard turbidity of 10 PIECES (10 ⁹ microbial bodies / ml), then 0.2 ml of a suspension containing 10 ⁴ microbial bodies / ml was added to each tube. Crops were incubated in a thermostat at a temperature of 37 ° C for 24 hours.

The experiments were performed in triplicate.

Profitability Analysis

The antibacterial effect of the studied lutenurin samples was determined by the minimum bacterial growth inhibitory concentration (MIC), at which the growth of microorganisms was not visually observed.

The results of the study are presented in the table.

Research results

The studies established (see table) the high bacteriostatic activity of all the studied samples of lutenurin at a concentration of 1 μg / ml in relation to the studied strain of Staphilococcus aureus 209-P. It should be noted that the activity of lutenurin obtained by the present method (alkaloids in the form of a base and alkaloids in the form of a salt) does not differ in activity from the prototype.

Table
Bacteriostatic activity of lutenurin samples Lutenurin Samples MIC activity (μg / ml) Sum of alkaloids (alkaloids in the form of a base) 1,0 Sum of alkaloids (alkaloids in the form of salt) 1,0 Lutenurin (prototype) 1,0

FINDINGS

As a result of the studies established the following.

1. Studied the antibacterial activity of samples of lotenurin obtained by the present method (alkaloids in the form of bases and alkaloids in the form of salt) in comparison with the prototype.

2. A high antibacterial activity of all studied was established.

samples of lutenurin at a concentration of 1 μg / ml against Staphylococcus aureus 209-P.

3. The activity of lutenurin obtained by the present method (alkaloids in the form of a base and alkaloids in the form of a salt) does not differ in activity from the prototype.

4. Based on the results obtained, it can be concluded that it is advisable to obtain lutenurin according to the claimed method.

BIBLIOGRAPHY

1. Vichkanova S.A. Inhibitors of microorganisms among natural substances of plant origin. // Diss. doc sciences. M., 1981.

2. Vichkanova S.A., Makarova L.V., Rubinchik M.A., Adgina V.V. // To the question of the study of the antimicrobial properties of essential oils. "Medicinal plants", t.14. "Pharmacology and chemotherapy", 1972.

3. Milovanova S. N., Stepanishcheva Z. G. // Methods of experimental chemotherapy. M., 1971.

4. Pershin G.N. // Methods of experimental chemotherapy. M., 1971.

Claims (1)

The method of producing lutenurin from the rhizomes of the yellow capsule (Nuphar Lutea (L.), which consists in extracting raw materials, extracting alkaloids and obtaining hydrochlorides by saturating the alkaloids with hydrogen chloride, characterized in that the raw materials are extracted at the head stage with 20-60% aqueous aliphatic alcohol , preferably ethyl or isopropyl, acidified with 5% phosphoric acid, filtered, the organic solvent is removed, the aqueous bottoms after distillation of the alcohol are treated with butanol, which is then separated, aqueous After neutralization to pH 8, the acid phase is treated with calcium carbonate to remove phosphate ions, the precipitate is filtered, washed with acetone and the filtrate is evaporated to dryness, the residue is dissolved in diluted sulfuric acid, alkalinized to pH 8, the alkaloids are extracted with toluene, the extractant is evaporated and hydrochlorides are obtained.