CS260347B1 - Method of lichenan insulation - Google Patents

Abstract

The solution relates to the method of isolation and purification lichenan from Icelandic moss (Cetraria islandica). A new effect is achieved so that in the first stage Cetraria islandica extracts stationary at twenty times an aqueous solution containing 0.4 % to 0.6 wt. sodium hydroxide and 0.025 % to 0.05 wt. sodium sulfide for a period of time 12 hours at 20-25 ° C, then after filtering off the insoluble portion thereof extract again for 1 hour at RT 45-50 ° C with a new extraction solution about half the concentration of ingredients used the base and in the second step, the lichenan is extracted from the washed insoluble fraction a multiple of an aqueous solution of 0.1 to 0.2% wt. acetic acid and / or citric acid] and / or tartaric acid for 30 hours minutes at 95 ° C to 100 ° C the third stage from the cooled filtrate precipitates lichenan with an equal volume of ethanol o 96% by weight. solution can find application most commonly in biotechnology in the preparation of chromolytic substrates for the determination of lichenase enzyme activities and affinants of general type and human medicine in the preparation of preparations with antitumor activity.

Description

The invention relates to a method for the isolation and purification of lichenan from Icelandic moss (Cetraria islandica).

Lichenan belongs to rare plant polysaccharides containing β (1 → 4) α, β (1-3) bonds in a 2.5: 1 ratio [I. Tvaroska, K. Ogawa, Y. Deslandes, R.H. Marchessault: Can. J. Chem. 61, 1,608 (1983)]. It occurs mainly in Icelandic moss and cereal bran, from which it is usually isolated by extraction with hot water. According to the method of extraction, a lichenate of varying yield and very different purity is obtained. fs. Peat, W.J. Whelan and J.G. Roberts: J. Chem. Soc. 3,916 (1957)], (Morris, J. Biol. Chem. 142, 881 (1942)), (A.S. Perlin and S. Suzuki: Can. J. Chem. 40, 50 (1957)).

The disadvantages of hitherto known methods of isolation and purification of lichenan are the relatively high labor and unsatisfactory purity of the final preparation. Purity of lichenan is of particular importance, especially in recent years, when lichenan has been shown to exhibit antitumor activity in addition to the known pharmaceutical effects. [Takeda T, Funutsa, M., Shibata, S., Fukuoka, F .: Chem. Pharm. Bull. 20, 2,445 (1972)]; [Nishikawa, J., Ohki, K., Takahoshi, K., Kurono, G., and Fukuoka, F .: Chem. Pharm. Bull. 22, 2692 (1974)]; [Konopa J., Privett O. S., Jenkin Η. M., Goldin A., Curr. Chemother. Why. Int. Congr. Chemother 2, 1095 (1977)].

The aforementioned drawbacks of the known methods are eliminated by a novel process which provides lichenan by high purity sentences using relatively simple isolation methods.

The principle of the invention is that in the first step (Cetraria islandica) is extracted stationary in twenty times the aqueous solution containing 0.4 to 0.6% by weight. % sodium hydroxide and 0.025 to 0.05 wt. sodium sulphide for 12 to 20 hours at 20 to 25 ° C, after filtering and washing the insoluble matter, this is again extracted for 1 hour at 45 to 50 ° C with a new extraction solution with half the concentration of the used constituents and in a second step the lichenate is extracted from the washed insoluble portion by a factor of 5 times an aqueous solution of 0.1 to 0.2 wt. acetic acid or citric acid or tartaric acid for 30 minutes at a temperature of 95-100 ° C, whereupon in the third stage a lichenate is precipitated from the cooled filtrate with an equal volume of ethanol at a concentration of 96% by weight.

We have found that the most effective method of eliminating undesirable accompanying substances, in particular tannins, is extraction with aqueous sodium hydroxide solution and sodium sulphide at the indicated concentration. At higher concentrations of sodium hydroxide, lichenate is already partially extracted, which would reduce the yield of the main product.

The addition of sodium sulfide has proven to be an effective means of creating a reducing environment, eliminating the oxidative degradation of lichenan during the alkaline extraction stages, ensuring efficient extraction of unwanted concomitants in the feedstock.

Furthermore, we have also found that the alkaline extraction solution used swells the cell walls, whereby the diffusion processes of the lichenate extraction into the aqueous solution are substantially reduced. The actual effective extraction of lichenane at the boiling point of the solution is therefore completed as early as 30 minutes, as opposed to the known processes where the extraction of hot lichen into hot water is carried out for up to 24 hours.

Another important finding of the proposed process is that if the extraction of lichenate from the purified raw material is carried out in an acidic environment, the obtained lichenate has a relatively high whiteness (above 80 ° / o MgO). The hitherto known processes provided products of cream to brown, which had to be purified in subsequent stages.

It is also not negligible that it is expedient to pour the solution of the lichenate into the ethanol and not vice versa when precipitating the lichenate. This gives a lichenate precipitate which is very good filterable and after clearing for one hour the clear supernatant can be separated from the product without filtration.

In particular, the advantages of the proposed method are that the process is time-consuming in terms of technology and time, and the resulting product is characterized by high chemical purity and whiteness.

Example 1

100 g of Icelandic moss (Cetraria islandica) is quenched with 2 liters of an aqueous solution containing 0.4 wt. % sodium hydroxide and 0.025 wt. sodium sulphide and allowed to be stationary extracted with occasional stirring at 20 ° C for 20 hours. The extraction solution was then separated by filtration and the insoluble material was collected on the filter. Wash with water until the filtrate is clear. Thereafter, the insoluble matter was again quenched in the reactor with 2 liters of a new extraction solution with half the concentration of sodium hydroxide and sodium sulfide as in the first step and extracted from, and stirring at 50 ° C + 2 ° C for one hour. The extraction solution is then removed by filtration and the insoluble moss is washed with water until the filtrate is clear and alkaline. After washing, the insoluble matter is quenched with 1.5 liters of boiling distilled water acidified with 2 ml of acetic acid and heated to boiling for 30 minutes, after which time the reaction mixture is filtered on a glass filter S _t and the insoluble matter is washed with 0.5 liters of hot water. A total of 2 liters of the filtrate was obtained, which was cooled to 20 ° C and poured into 2 liters of 96 pb ethanol with stirring. wt. The precipitated lichenate is filtered off, washed with ethanol, finally with acetone and air-dried. A white powdered lichenate of high chemical purity was obtained, yield 22 g, which, after total hydrolysis, gave a single chromatographic spot of glucose. The whiteness of the product was 82% MgO.

Example 2

The eye procedure of Example 1, except that a 0.6 wt.% Solution was used in the first extraction step. % sodium hydroxide and 0.05 wt. sodium sulfide for 16 hours. In the second main extraction step, 4 g of tartaric acid was used instead of acetic acids. 20 g of lichenate of similar chemical purity to Example 1 were obtained with a brightness of 83% MgO.

EXAMPLE 1 a d 3

Procedure as in Example 2 except that water acidified with 4 g citric acid was used in the main extraction step. The yield of lichenate was 21 g and the whiteness of 82% MgO with similar chemical purity as in Example 2.

The invention can find application particularly in biotechnology in the preparation of chromolytic substrates for the determination of lichenase enzyme activity and affinity of the general type and in human medicine in the preparation of preparations with antitumor activity.

Claims (3)

Process for the isolation of lichen from Icelandic moss Cetraria islandica characterized in that in the first step, Cetraria islandica is extracted stationary in a twenty-fold aqueous solution containing 0.4 to 0.6% by weight. % sodium hydroxide and 0.025 to 0.05 wt. sodium sulphide for 12 to 20 hours at 20 to 25 ° C, after which the insoluble matter is filtered off again for 1 hour at 45 to 50 ° C with a new extraction solution of ynAlez at half the concentration of the base components used and The lichenate is extracted from the washed insoluble fraction by five times the aqueous solution of 0.1 to 0.2 washes. wt. of acetic acid or citric acid or tartaric acid for 30 minutes at a temperature of 95-100 [deg.] C., in which, in the third step, a lichenate is precipitated from the cooled filtrate with an equal volume of ethanol at a concentration of 96% by weight.