RU2270202C1 - Method for preparing betulin and lupeol - Google Patents

Abstract

FIELD: natural substances, chemical technology.

SUBSTANCE: invention relates to the improved method of betulin and lupeol representing pentacyclic triterpenoids of series lupan-betylin from white-stem birch bark. Betulin and lupeol are separated by boiling with hexane of product obtained by extraction of birch bark with methyl-tert.-butyl ester and treated with alkali solution. After removing a solvent hexane-soluble lupeol is recrystallized one time from ethyl acetate. Hexane-insoluble residue represents practically pure betulin. Method provides preparing indicated compounds with high yields with purity above 95% and without using labor-consuming and expensive chromatography methods. Invention provides rational utilization of vegetable raw.

EFFECT: improved preparing method.

2 cl, 2 ex

Description

The invention relates to a method for isolating betulin (20,29-lupen-3β, 28-diol; 3β, 28-dihydroxy-lup-20,29-ene) and lupeol (20,29-lupen) from the outer layer of the cortex (birch bark) -3β-ol; 3β-hydroxy-loop-20.29-ena).

Betulin and lupeol, pentacyclic triterpenoids of the lupane series, have a wide spectrum of biological activity, and are also synthetic precursors of a large number of compounds with valuable pharmacological properties.

The results of the invention allow the rational use of plant materials and can be used in chemistry, medicine, pharmacy.

The content of betulin in birch bark varies from 10 to 40%, depending on the type of birch, locality and season of collection of bark; in the total birch bark extract, the proportion of betulin is approximately 70%. The main component of the extract accompanying betulin is lupeol, its content in the total extracts is about 3%.

Known methods for producing betulin from birch bark suggest its crystallization from the extract, the methods for which are divided into two groups. In the first group, birch bark extraction is carried out with various organic solvents - chloroform in the presence of silica gel [1], 90-95% ethyl alcohol in the presence of activated carbon [2], toluene [3], acetone [4]. The second group includes methods involving the use of alkali to separate the acid components of the extract: a) birch bark is treated with hydroalcoholic alkali (alcohol - MeOH, EtOH, iso-PrOH); b) the extract is washed with a solution of alkali of various concentrations [5-8].

Both groups of methods allow to obtain low-quality betulin. The preparation of a compound with a purity> 95%, which is necessary for carrying out synthetic work and biological tests, requires repeated (from one to five times) recrystallization, which significantly reduces its yield (from 11 to 5%) [1, 9].

The main disadvantages of these methods are the complexity of obtaining high-purity betulin, the lack of data on the number of recrystallization steps, for which it is possible to obtain highly purified betulin, the lack of information on betulin yield of the indicated qualification, and the lack of data on the method for monitoring the purity of obtained betulin.

Known methods for the isolation of lupeol from birch birch by extraction with benzene followed by chromatography of the obtained product [8] or by the method of supercritical fluid extraction with a CO ₂ solvent system at 40-50 ° C (P 3-5 atm). These methods also involve the use of column chromatography to obtain fractions enriched with lupeol from the total extract, and then recrystallization of the obtained concentrates. The yield of lupeol thus obtained is 1.0–1.5%, and the content of the basic substance is 90–95% [10, 11].

The disadvantage of these methods is the need to use special equipment, time-consuming and inefficient chromatographic methods of isolation.

Closest to the proposed invention is a method for producing betulin from birch bark, with chromatographic isolation of the associated lupeol. The method consists in the extraction of birch bark with a mixture of petroleum ether (70-100 ° C) - toluene in a ratio of 1: 1-7: 3 at the boiling point of the extractant, subsequent crystallization from a cooled extract of the product, which is a mixture of betulin and lupeol, and separation of this mixture by the method column chromatography [6].

The disadvantages of the method include the use of high-boiling and toxic solvents, the ineffectiveness of the methods for isolating the target compounds, the lack of data on methods for controlling the purity of betulin, and the lack of information about the quality of lupeol.

The problem to which the claimed invention is directed is to develop an effective method that eliminates the use of time-consuming and costly chromatographic methods of isolation and purification and allows you to get two compounds from birch bark at the same time - betulin and lupeol always accompanying it, with high yields in the form of individual substances suitable for synthetic modifications and the study of biological activity.

In the inventive method, lupeol and betulin with high selectivity are separated by boiling with hexane a product obtained by extraction of birch bark with methyl tert-butyl ether (MTBE) and treated with an alkali solution. In this case, lupeol, which has a lower polarity compared to betulin, passes into a solution of hexane, and the insoluble residue is almost pure betulin. By recrystallizing lupeol from ethyl acetate (EtOAc) once, a compound with a purity of more than 95% is obtained.

MTBE has a number of characteristics valuable for the extractant: first of all, it selectively dissolves natural compounds of medium polarity in the studied object, has a low boiling point (52 ° С), does not form peroxides (unlike other ether solvents widely used for extraction of natural compounds) ), does not mix well with water, has low toxicity, low cost, and is available.

The result of the invention is the extraction of plant material betulin and lupeol with a purity of more than 95%.

The invention consists in the following. Chopped birch bark (humidity <5%) is extracted with MTBE, or first with hexane (removing lupeol containing an admixture of betulin), and then MTBE extraction is continued. In both cases, the ether extract is further treated with a 4N NaOH solution to remove the sodium salts of the acid components of the extract, then it is washed with water and dried with a desiccant, the solution is filtered and the filtrate is evaporated. The obtained crystalline residue, which is a mixture of betulin and lupeol, is boiled with hexane, the insoluble part in hexane is filtered off. After distillation of the solvent from the filtrate, a mixture of lupeol and betulin is obtained, by recrystallization of which lupeol is isolated from EtOAc, with a mass fraction of the main substance> 95%. The hexane insoluble crystalline residue is betulin with a purity> 95%. In the obtained samples of betulin and lupeol according to thin layer chromatography (TLC), ¹ H and ¹³ C NMR spectra were not detected.

The invention is confirmed by the following examples.

Example 1

10 g of crushed birch bark are boiled for 2 hours in 100 ml of MTBE, the extract is filtered off, then 80 ml of MTBE is added and boiled for 40 minutes (the operation is repeated 4 times). The combined extracts are washed with 25 ml of 4N aqueous NaOH solution, then with water, dried with MgSO ₄ , the solution is filtered, the solvent is evaporated on a rotary evaporator, 1.9 g of a white crystalline product are obtained, which is boiled with 60 ml of hexane for 1 hour (the extraction procedure is repeated 4 times) the insoluble part in hexane is filtered off, dried in air, 1.6 g (16% by weight of dry birch bark) of betulin, individual according to TLC, ¹ H, ¹³ C NMR are obtained. The solvent is evaporated from the combined hexane extracts, 0.3 is obtained g single product recrystallization of which from EtOAc (15 ml) yields 0.28 g (2.8%) of luteol, individual according to TLC, ¹ H and ¹³ C NMR spectra.

Betulin: T. pl. 252-254 ° C (lit. 254-256 ° C, EtOH) [11]; [α] _D ²⁰ + 20 ° (c 0.016, pyridine), lit. [α] _D ²⁰ + 20 ° (pyridine). NMR ¹ H (δ, ppm): 0.76 s (3H, CH ₃ ); 0.83 s (3H, CH ₃ ); 0.97 (3H, CH ₃ ); 0.98 s (3H, CH ₃ ); 1.03 s (3H, CH ₃ ); 1.69 s (3H, 30-CH ₃ ); 3.20 dd (1H, 3-H, J 10.7 Hz, 5.0 Hz); 3.35 d (1H, 28-H _a J 5.4 Hz); 3.80 d (1H, 28-H _b J, 5.4 Hz); 4.59 s (1H, 29-H _a ); 4.69 s (1H, 29-H _a ). NMR ¹³ C (δ, ppm): 14.73; 15.33; 15.96; 06/16; 16.28; 06/19; 20.60; 25.16; 03/27; 27.35; 27.96; 29.15; 29.72; 33.94; 34.21; 37.14; 37.27; 38.67; 38.64; 40.69; 42.66; 47.77; 47.77; 48.73; 50.37; 55.26; 60.50 t (28-C); 78.96 (3-C); 109.66 (29-C); 150.45 (20-C).

Lupeol. T. pl. 213-215 ° C (EtOAc). Lit. 212-213 ° C [11]. [α] _D ²⁰ + 21 ° (c 9.3, CHCl ₃ ). ¹ H NMR Spectrum (δ, ppm): 0.76 s (3H, CH ₃ ); 0.79 s (3H, CH ₃ ); 0.83 s (3H, CH ₃ ); 0.95 s (3H, CH ₃ ); 0.97 s (3H, CH ₃ ); 1.03 s (3H, CH ₃ ); 1.69 s (3H, 30-CH ₃ ); 3.19 dd (1H, 3-H, J 10.7 Hz, 5.5 Hz); 4.57 s (1H, 29-H _a ); 4.69 s (1H, 29-H _b ). ¹³ C NMR spectrum (δ, ppm): 14.52; 15.39; 15.94; 09/16; 17.99; 18.29; 19.29; 20.91; 22.64; 11/25; 27.40; 27.96; 29.82; 31.57; 34.25; 35.55; 37.14; 38.03; 38.84; 39.97; 40.81; 42.80; 42.97; 47.97; 48.27; 50.40; 55.28; 78.98 (3-C); 109.29 (29-C); 150.97 (20-C).

Example 2

10 g of crushed birch bark are boiled in 60 ml of hexane for 1 hour, the solution is filtered off (the extraction procedure is repeated 5 times), the solvent is evaporated on a rotary evaporator, 0.18 g of luteol containing an admixture of betulin is obtained. Birch bark is poured with 60 ml of MTBE and boiled for 40 minutes (repeat the procedure 4 times). The combined extracts were washed with 25 ml of 4N NaOH, dried with MgSO ₄ , the solvent was evaporated, and 1.75 g (21%) of the product was obtained, which was boiled with hexane (35 ml × 2). The insoluble part in hexane is filtered off, dried in air, and 1.6 g (16%) of betulin are obtained as an individual compound. After evaporation of the filtrate, 0.15 g of lupeol containing an admixture of betulin is obtained. The combined fractions of lupeol (0.33 g) were recrystallized from EtOAc (see Example 1) to obtain 0.28 g (2.8%) of lupeol, the sample of which is identical to that described above.

Literature

1. Pat. US 5750578, May 12, 1998.

2. Pat. RU 2172178 C1, 08.20.2001.

3. Pat RU 2192879, 11.20.2002.

4. Izv. Universities of the North Caucasus. region. Tech. Sciences. 1993, N 1-2, p.24-26.

5. Pat. US20030153776, 08/14/2003.

6. Pat. RF 2184120, 06.27.2002.

7. Pat. RF 2138508, September 27, 1999.

8. Housing and communal services, 1967, T.40, s.920-921.

9. Pat. US 5804575, 10/10/1998.

10. Pat. US 6392070; 05/21/2002.

11. Pat. US 6586021, 06/01/2002.

12. Phitochemistry, 1980, N 19, 2707.

Claims (2)

1. A method of producing betulin and lupeol from birch bark, including extraction with an organic solvent, crystallization of a product representing a mixture of betulin and lupeol and subsequent separation of the mixture, characterized in that methyl tert-butyl ether is used as the organic solvent, and the separation of betulin and lupeol is carried out boiling the mixture in hexane, separating insoluble pure betulin and recrystallizing luteol isolated from the filtrate from ethyl acetate. 2. The method of producing betulin and lupeol according to claim 1, characterized in that the extraction of birch bark is preliminarily carried out with hexane.