RU2615356C1 - Method for lutenurinum preparation manufacture from yellow water-lily (nuphar lutea (l) smith) - Google Patents

Abstract

FIELD: pharmacy.

SUBSTANCE: method for manufacture of Lutenurinum preparation with antimicrobial activity from yellow water-lily (Nuphar lutea L.) rhizomes, consisting of raw materials extraction, alkaloids extraction and hydrochlorides preparation by alkaloid solution saturation with hydrogen chlorides, at that, the extraction of crushed raw material is performed three times by 1.0-5.0% phosphoric acid aqueous solution, the extracts are concentrated and combined upon reception, neutralized with calcium hydroxide, and then the dried calcium phosphate precipitate containing alkaloid bases is extracted 3 times by ethanol, the extracts are combined, the obtained reaction mixture of total alkaloids is chromatographically separated on a column with silica gel using chloroform as eluent, alkaloid hydrodichloride is obtained from the resulting nuflein-enriched fraction.

EFFECT: method allows to increase efficiency of technology and content of the desired alkaloid with antimicrobial activity.

1 dwg, 6 ex

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" (i.e. 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, which has a protistocidal and spermatocidal effect, is active against trichomonads, pathogenic fungi, has a bacteriostatic effect against gram-positive microorganisms, is used to treat urogenital diseases, in particular for the treatment of trichomonas colpitis and urethritis, as well as contraceptive (Ya.A. Aleshkina, S.A. Vichkanova, M.A. Rubinchik, T.N. Ilyinskaya. The drug Lutenurin. Auth. certificate. 145858, 05/04/1967, bull. No. 10, 07/28/1967; Lutenurin , L utenurinum, FS 42-948-75; Lutenurin, Lutenurin, Lutenurine, Prospect TsBNTI MP, 89 pp .; Lutenurin, in the book: Complete modern directory of drugs, 2nd ed., S.A. Krzhizhanovsky and MB Vititnova , Publishing House "Rinol Classic", M., 2002, S. 950). The drug has a wide spectrum of antimicrobial activity against gram-positive bacteria (staphylococci, streptococci), spore-forming and acid-resistant bacteria and pathogenic fungi such as Candida albicans, Microsporum lanosum, Trichophyton gypeum, as well as anti-trichomonas and spermatocardial activity: , Borus Press, 2009, 256 pp.).

Used in medical practice, the drug Lutenurin, which is the sum of the alkaloids of thiobinufaridine, C ₃₀ H ₄₂ N ₂ O ₂ S, and nuflein, C ₃₀ H ₄₂ N ₂ O ₄ S (6,6'-dihydroxythiobinufaridine) in the form of hydrochlorides. The active principle of Lutenurin’s drug is nuklein alkaloid (1) (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 Edited by A.I. Bankovsky, Kolos, Moscow, 1969, p. 322-333).

The technology for producing Lutenurin was first developed at the All-Russian Research Institute of Medicinal and Aromatic Plants (VILAR). The raw material for the preparation of the preparation was used rhizomes of yellow capsule, harvested in the territory of the Russian Federation, containing 0.4% of the total alkaloids (nuflein, thiobinufaridin, nufarin, nufamine, neotiobinufaridin, isomeric nufaridins and deoxynufaydins, thionuflutin, and others (Atlas of medicinal plants in Russia, Moscow, 2006, pp. 155-156) The study of the structure of egg capsule alkaloids was also conducted at Mc Master (Ontario - Canada) and Warsaw (Poland) (La Londe and Wrobel) (RT La Londe, CF Wong, KC Das, J. Am. Chem. Soc. 95, 6342 (1973). JT Wrobel, B. Bobeszko, TI Martin, DB Maclean et al. Can. J. Chem., 51, 2810 (1 973). Ch.F. Wong, RT LaLonde, Phytochemistry, 9 (8), 1851-1854 (1970); 9 (11), 2417 (1970). A. Iwanow, K. Woltasiewicz, JT Wrobel, Phytochemistry, 25 (9), 2227-2231 (1986). RT. LaLonde, CF Wong, Phytochemistry, 11, 3305-3306 (1972).) The drug and its preparation were patented in several countries (Pat. France No. 1417969, Pat. England No. 968042, US Pat. No. 3,147,246, Pat. Japan No. 447778). The structure of the active component of the drug nouflein was confirmed by NMR spectroscopy (M.E. Perelson, T.N. Ilyinskaya, O.N. Tolkachev, Chemistry of Natural Compounds, 1975, No. 6, 768-770). The capsules, which are close in structure to alkaloids, have similar physicochemical characteristics.

Initially developed production technology of Lutenurin (Ya.A. Aleshkina, S.A. Vichkanova, M.A. Rubinchik, T.N. Ilyinskaya. Auth. Certificate. No. 148858, 11.09.09.1959), published in the monograph by V.P. Zakharova, N.I. Libizova and H.A. Aslanova “Medicinal substances from plants and methods for their production”, Tashkent, Ed. "FAN", UzSSR, 1980, pp. 109-112, includes the stages of preparation of raw materials, extraction, salt and purification of the finished product. To do this, the dried and crushed rhizomes are made alkaline with a 10% ammonia solution with stirring, and then extracted with dichloroethane, successively five times, each time filtering the extract through the cloth. The filtrates are treated with sulfuric acid, the acid phase is separated, neutralized with ammonia, the precipitated precipitate is filtered, washed with water and dried in air. The resulting powdery amount of alkaloids is dissolved in sulfuric ether, the solution is separated from the precipitate by decantation. The combined extracts are dried, filtered, evaporated to 1/10 of the initial volume, a five-fold volume of petroleum ether is added, the precipitate is separated by decantation, the supernatant is again dried with potash, filtered, and dry hydrogen chloride is passed into the resulting solution until acidic. Lutenurin precipitate is filtered off, washed with petroleum ether, wiped through a nylon mesh and dried on baking sheets for 2-3 days until the solvent is completely removed, and then sieved through a nylon sieve. The finished product yield, according to the regulations, is 65%.

The disadvantages of this technology are: the duration of the process, the use of toxic and flammable solvents.

In a previously published patent, the technology of obtaining the drug Lutenurin (Sheychenko O.P., Sheychenko V.I., Tolkachev O.N., Fateeva T.V., Shipulina L.D., Bykov V.A., Sokolskaya T.A. ., patent of the Russian Federation 2292218, 01/27/2007), which consists in the following: the dried and crushed rhizomes of the yellow capsule are extracted three times with an aqueous-alcoholic solution of phosphoric acid, the combined extracts are evaporated to an aqueous bottoms, the latter is treated with butanol, the aqueous-acid phase is separated, neutralized sequentially with a solution of ammonium hydrate then - the calcium carbonate is filtered, washed with water, alkaloid base is extracted with alcohol. The target fraction was evaporated and the hydrochlorides were obtained by saturating the alkaloids solution with hydrogen chloride. The yield of the finished product, calculated on absolutely dry raw materials - 0.35% or calculated on the content of nouflein in the raw materials - 52%. The specified technology for the preparation of Lutenurin is selected by us as a prototype.

The disadvantages of the known method of the prototype are the pricing and emission of carbon dioxide in the process of neutralizing phosphoric acid, requiring the use of additional technological methods, as well as the use of ethyl alcohol at the head stage.

The technical task of the proposed method is to increase the efficiency of the technology, simplify the process operations and increase the content of the target alkaloid 6,6'-dihydroxythiobinufaridine (nuflein), which has antimicrobial activity.

The solution to the technical problem is achieved by using the method of obtaining the drug Lutenurin, which has antimicrobial activity, from the rhizomes of the yellow capsule (Nuphar lutea L.), which consists in the extraction of raw materials, the extraction of alkaloids and the production of hydrochlorides by saturating the alkaloids solution with hydrogen chloride. In this case, the extraction of the crushed raw materials is carried out with a 1.0-5.0% aqueous solution of phosphoric acid at room temperature in the ratio of raw materials: extractant equal to 1:20, the obtained extract is concentrated and neutralized with calcium oxide hydrate, then the dried calcium phosphate precipitate containing alkaloids base extracted with aliphatic alcohol or chloroform, the resulting reaction mass of the sum of alkaloids is chromatographically separated on a silica gel column using chloroform as an eluent from the obtained fraction enriched in th nufleinom receive gidrodihlorid alkaloids.

The technical result obtained: emissions of volatile substances of carbon dioxide and ammonia and foaming are eliminated, the production technology of Lutenurin is increased due to the elimination of the use of aliphatic alcohols at the head stage of extraction, simplification of technological operations, and an increase in the content of the amount of quinolizidine alkaloids in terms of nuflein to 81% .

The proposed new technology includes the following stages:

1. As a raw material, rhizomes of yellow egg capsules containing nuflein are used.

2. The extraction of the dried and crushed rhizomes of the yellow capsule is carried out with an aqueous solution of phosphoric acid 1-5%.

3. The aqueous acid extract containing alkaloids is concentrated in vacuo.

4. The resulting concentrate is treated with calcium hydroxide.

5. The precipitated precipitate of calcium phosphates in a mixture with alkaloids in the form of bases is separated, washed with water and dried, preferably at room temperature or in a heater at a temperature of 60-80 ° C.

6. Process the dried precipitate of mineral salts in a mixture with alkaloids with an aliphatic alcohol, for example ethanol, or chloroform at 45 ° C or at room temperature, and filter.

7. Evaporate the alcohol filtrate in vacuo.

8. Separate the impurities of mineral salts.

9. Chromatographically separate the resulting amount of alkaloids on a column of silica gel, chloroform is used as eluent. The fraction with the predominance of nouflein and thiobinafaridine enriched with nouflein is evaporated and then hydrochlorides are obtained as in the prototype.

10. The step-by-step control over the process is carried out by TLC, and the quantitative content of the sum of quinolysidine alkaloids in intermediates and the finished product is determined spectrophotometrically and the content of nouflein in the finished product by ¹ H NMR spectroscopy.

A description of the method for producing Lutenurin is given in the following examples.

Example 1. The crushed raw material of a yellow capsule in an amount of 100 g was extracted with 3% phosphoric acid (phosphoric acid) at room temperature three times for 3 hours in a ratio of 1:20, with stirring, each time adding a new portion of the extractant equal to the drained mother liquor. The third extract is used to extract fresh raw materials of the next boot. The combined extracts (2.41 L) are concentrated to 1.12 L, neutralized by adding calcium oxide hydrate in an amount of 100 g, with stirring for 60 minutes, the precipitate Ca ₃ (PO ₄ ) _{2 is} separated, washed with distilled water 3 times 30 times ml and dried at room temperature. Dried calcium phosphate, containing alkaloids bases, is extracted with ethyl alcohol 3 times in 500 ml with a working stirrer. The combined extracts were evaporated in vacuo. Obtain 0.84 g of the amount of alkaloids from 100 g of raw materials excluding moisture. Chromatography of alkaloids is carried out on a column of silica gel L 40/100 for column chromatography (Chemapol) in a ratio of 1:10 (substance sorbent). Elution of alkaloids is carried out with chloroform.

The first two eluate-free eluate fractions are separated, and the noufle-containing fractions 4-15 are combined and evaporated to give 0.45 g of bases containing nuflein. The selection of fractions is carried out under control on TLC.

Alkaloids hydrochlorides are obtained by passing dry gaseous hydrogen chloride (from a generator for its production from sulfuric acid and ammonium chloride) into a solution of a mixture of sulfur and petroleum ethers (1: 5) of alkaloids dried over potash. A precipitate of hydrochlorides with a total content of quinolizidine alkaloids in terms of 78-80% nuflein (spectrophotometric method of analysis) is precipitated. The yield of the finished product is 0.45%.

Example 2. Extraction with an aqueous solution of phosphoric acid in the head stage is carried out as in Example 1. Then it is filtered, the acid solution of 2.100 L is evaporated in vacuo to a volume of 0.60 L and extracted with butanol 3 times 0.70 L each; 0.60 l; and 0.50 L, the aqueous phase is separated off, butanol residues are removed in vacuo, the residue is diluted with water and evaporated again to remove butanol residues. The acid bottoms are neutralized with calcium oxide hydrate with stirring. The precipitated precipitate of calcium phosphates and alkaloids is separated, washed with water and dried at room temperature. The resulting product was extracted with chloroform, dried with potash, the extracts were combined, filtered and evaporated, then the nouflein-containing fraction was separated on a chromatographic column. Hydrochlorides of the total alkaloids are obtained, as in Example 1, with a yield of 0.42-0.45% with the content of quinolysidine alkaloids in them, calculated as nouflein bisanhydrodichloride, not less than 60%.

Example 3. Carried out analogously to example 1, using 5% phosphoric acid in the head stage of extraction. The dried precipitate of calcium phosphates in a mixture with alkaloids is extracted with chloroform, then hydrochlorides of the total alkaloids are obtained in a known manner with a yield of 0.42% and the content of quinolizidine alkaloids in them in terms of nouflein - 77%.

Example 4. The method is carried out as in example 2, characterized in that the precipitated precipitate of calcium phosphates together with alkaloids is treated with ethanol. The alcohol extracts were evaporated, and the resulting gummy extractive substances were separated, as above, on a silica gel column. The hydrochloride is obtained, as in example 1. The yield of alkaloids hydrochlorides is 0.40-0.42% with the content of furanoquinolisidine alkaloids in them, in terms of nouflein, bisanhydrodichloride not less than 60% (78-81%).

Example 5. The method is carried out analogously to example 1, except for the use of 1% phosphoric acid in the head stage of extraction. At the stage of extraction of the dried precipitate of calcium phosphates in a mixture with alkaloids, isopropyl alcohol is used. Hydrochlorides of the total alkaloids are obtained in a yield of 0.4% with a content of 78% of the quinolysidine alkaloids in them.

Example 6. The method is carried out as in example 2. It is characterized in that the combined ethanol extracts of alkaloids are evaporated after processing the solid neutralization products, the residue is dissolved in a 5% hydrochloric acid solution, the combined acid solutions are alkalinized to pH 9 with a 25% solution ammonium hydroxide, and then evaporated in vacuo. The dry residue is treated with chloroform. The chloroform extract was washed with water, dried with sodium sulfate and evaporated in vacuo.

The residue is chromatographed on a silica gel column, collecting chloroform fractions containing nuflein, from which alkaloids are obtained, as in Example 1. The finished product is 0.42-0.45% containing furanoquinolisidine alkaloids in terms of nouflein bisanhydrodichloride 79 %

The following is the antimicrobial activity of the resulting product. In the laboratory of microbiological research FGBNU VILAR was studied the antimicrobial activity of the drug obtained by the present method (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 one of the indicative pathogenic test microorganisms.

Wednesday

When determining the bacteriostatic activity of the samples of the preparation, 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 drug samples, a culture medium 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 double dilutions, a series of decreasing concentrations of the drug samples were prepared. The last test tube with a clean medium (without adding the drug) 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 three times.

Accounting for results.

The antibacterial effect of the studied samples of the drug was determined by the minimum concentration inhibiting the growth of bacteria (MIC), at which the growth of microorganisms was not visually observed. The results of the study are presented in the table.

Research results

Table 1 shows the high bacteriostatic activity of all the studied samples of the drug at a concentration of 1 μg / ml in relation to the studied strain of Staphylococcus aureus 209-P. It should be noted that the activity of the drug obtained by the present method (in the form of salt) does not differ in activity from the prototype.

FINDINGS

1. The proposed method is simple to implement, increases the efficiency of the production technology of the drug Lutenurin.

2. Studied the antibacterial activity of the samples of the drug lutenurin obtained by the present method in comparison with the prototype. A high antibacterial activity of all studied samples of the drug at a concentration of 1 μg / ml against Staphylococcus aureus 209-P was established.

3. Based on the results obtained, it can be concluded that it is advisable to obtain the drug Lutenurin according to the claimed method.

DETERMINATION OF AUTHENTICITY AND QUANTITATIVE DETERMINATION OF ANTIMICROBIAL DRUG FROM YELLOW YELLOW

The drug Lutenurin, obtained from the rhizomes of the yellow capsule, is a mixture of substances belonging to the class of quinolysidine alkaloids. According to ¹ H NMR data (Fig. 1), the substance Lutenurin is dominated by alkaloid nuflein, which has biological activity.

In the representative region of the spectrum, the most intense signals belong to the alkaloid nouflein. In the ¹ H NMR spectrum of a solution of nouflein in a benzene-trifluoroacetic acid solvent mixture (Fig. 2), the chemical shifts of the signals have the following meanings: 6.31 and 6.44 ppm. (β protons of furan rings), 7.41 and 7.57 ppm. (and protons of furan rings) and 7.73 and 8.03 ppm. (protons of the ammonium fragment). A weak dependence of the position of the signals on the ratio of solvent components (benzene - trifluoroacetic acid) was noted.

Claims (1)

The method of obtaining the drug Lutenurin, having antimicrobial activity, from the rhizomes of the yellow capsule (Nuphar lutea L.), which consists in the extraction of raw materials, extraction of alkaloids and obtaining hydrochlorides by saturating the alkaloids with hydrogen chloride, characterized in that the extraction of 100 g of crushed raw materials is carried out 1.0- 5.0% aqueous solution of phosphoric acid three times for 3 hours at room temperature in the ratio of raw materials: extractant equal to 1:20, each time adding a new portion of extractant equal to the drained mother liquor, extract as it is received, it is concentrated and combined, neutralized with calcium oxide hydrate, then the dried calcium phosphate precipitate containing the alkaloids base is extracted with ethyl alcohol 3 times 500 ml of extractant, combining the extracts, the resulting reaction mass of the total alkaloids is chromatographically separated on a silica gel column using as an eluent of chloroform, from the obtained fraction enriched with nouflein, alkaloids hydrodichloride is obtained.

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