KR870001573B1 - Process for separation of tripenoid from centelly asiatica - Google Patents

Abstract

The isolation of tripenoid from centelly asiatica comprises (a) extracting leaves from centelly asiatica with MeOH or an aq. MeOH soln. at 40-80≰C and removing MeOH, (b) diluting the ext. with distld. H2O and extracting with EtoAc, (c) extracting the aq. layer with n-butanol and removing the undesired matter by adjusting the moisture content to 4-7%, and drying, (d) crystallizing the solids with H2O to yield asiaticoside, and (e) adjusting the moisture content of the EtoAc layer to 1-3.3%, to give gennin component.

Description

Separation of Trifenoid Components from Centella asiatica

The present invention relates to a method for separating a trifenoid component having a wound healing promoting effect from Centella asiatica.

Centella is a perennial herb belonging to the mountain family family, and grows in southern coastal islands such as Jeju Island and Jindo, and foreign mountainous regions include Madagascar, Sunda Island, India, and South China.

Bontems first separated one of the trifenoids from Epul in 1942 and named it Asiatigoside, and Fulonsky said in 1949 that the structure of this substance had the following structural formula (I) (Nature 163): , 258 (1949).

Since then, various trifenoids have been isolated from the plant, of which medicinal significance is asaticoside, asiatic acid and madeassic acid. Asiantic acid is the glycoside, the Asian Ticoside, is genine, and madecaic acid is the stereoisomer of Asiantic acid. In 1957, Thiers et al. Discovered that extracts of Centella asiatica-based extracts promote wound healing [M.H. Thiers: Lyon Medical 17 (1957).

In addition, although many studies have been conducted on the centella, no literature has been found on how to economically separate the wound healing accelerators contained in the plant. The separation methods known to date are structural identification or laboratory separations and cannot be transferred directly to production.

The present inventors earnestly studied in this regard, and invented a separation method capable of transferring medicinal ingredients, namely Asianticoside, Asiantic acid and medemic acid, contained in Centella as economical and directly to production.

Among the medicinal ingredients, asiaticoside is a glycoside, and asiatic acid and madeic acid are genine, the difference in solubility in solvents is expected to be very large depending on the general properties between glycoside and genine. The solubility was investigated. As a result, glycoside asiaticoside was dissolved in protic organic solvents such as water, and genine contained organic acid and hydroxyl group, solubilized in alcohols and nonpolar solvents, but not in water. In addition, the two kinds of organic acids have organic acids, and showed a sharp change in properties by forming salts with metal ions. In particular, alkali metal salts of these genes, ie sodium and potassium salts, are water soluble. As such, it is more convenient to separate and manipulate these two groups of substances using the contrast solubility between asiaticosides and the two genins.

Hereinafter, the separation method of these materials will be described in detail. The dried or fresh Centella asiatica is finely pulverized, then placed in a reactor, and about 3 to 5 times methanol or an aqueous solution thereof is added to the Centellar, followed by heating at 40 to 85 ° C. for 2 to 5 hours with stirring. After centrifugation of the extract and plant residues, the plant residues are repeated two more times in the same operation. After combining the extracts, methanol was distilled off under reduced pressure, and 2 to 3 times the amount of bottled water and ethyl acetate were added to the remaining material, which was put in a percolator and refluxed for 24 to 72 hours. As the aqueous layer obtained at this time, Asianticoside, and ethyl acetate contained genein, were separated and treated separately.

Separation of Asian Ticosides from Aqueous Layers

The aqueous layer obtained above is placed in an extractor and stirred under reduced pressure to remove dissolved ethyl acetate. An equivalent amount of n-butanol is added thereto and stirred at 20 to 40 ° C. for 2 to 5 hours. After standing for 1 hour, the butanol layer and the aqueous layer were separated and the aqueous layer was operated twice in the same manner as above. Dehydrated magnesium sulfate is added to the combined n-butanol extracts to adjust the water content to 3-7%. When the water content is less than 3%, a large amount of tartic material and a considerable amount of asiaticosides precipitate, which adversely affects the yield. On the other hand, when the water content is more than 7%, a large amount of tar material is n- Dissolved in butanol layer causes enormous disruption to the next step. As such, adjusting the water content in the n-butanol extract to the above range is a very important factor in the separation method of the present invention.

In addition, although it was used also for the mixed solvent of ethyl acetate and alcohols, and the mixed solvent of butanol, water, or alcohols, it was more preferable than n-butanol in the amount of elution, purity, etc. of Asianticoside. The n-butanol extract obtained above is dried and then distilled water of about 5 to 30 times the weight of the dried product is heated and dissolved for 30 minutes at 50 ° C. At this time, the insoluble content is removed by filtration at 50 ℃, the filtrate is allowed to stand for 24 to 72 hours at 5 ℃. At this time, the resulting crystalline material may be dried and used directly for medicinal purposes. A portion of this dried product is taken and crystallized three times in 60% methanol to obtain pure asiaticoside having a melting point of 235 to 238 ° C.

In the mother liquor obtained above, the content of asiaticoside is greatly reduced, while the tartar is relatively high. The mother liquor is dried under reduced pressure, and then added to n-butanol of about 1/2 to 1/5 of the amount of the mother liquor, and mixed with distilled water so that the water content is 3 to 7%.

The n-butanol solution is warmed at 50 ° C for 30 minutes and then left at room temperature for 12 hours. The resulting tar phase material is removed and the n-butanol solution is dried. This dried material is determined as 5 to 30 times the amount of distilled water.

The yield of asiaticoside from the stock solution and mother liquor ranges from 0.35 to 0.95% depending on the collection time, place of collection, dry condition and quality of the herbaceous plants. This yield demonstrates that the extraction efficiency of the present invention is very high in view of the highest Asian Ticoside content of the Centella as 1.20%.

Isolation of a Genine Mixture from Ethyl Acetate

When the combined ethyl acetate solution is allowed to stand at room temperature for 12 hours, a small amount of water layer is formed at the bottom, and an insoluble substance forms toward the ethyl acetate layer at the interface between water and the organic layer.

The material was obtained by filtration and then suspended in water and stirred with warming at 40 ° C for 1 hour. Finish stirring and filter at 40 ° C. This operation removes the water-soluble substance from the water-insoluble substance. The water insoluble substance is dried and then dissolved in 5 to 50 times anhydrous ethyl acetate by weight of the dry matter. Distilled water is added to the solution to adjust the water content to 1 to 3.3%. After stirring, the mixture was left at room temperature for 12 hours, and the resulting material was filtered and dried. 0.5-1.3% of white matter is obtained according to the collection time, place of collection and dry condition of the centella. This white substance is mainly composed of Asiantic acid and madeic acid, and its composition ratio is approximately 1: 1.

As described above, according to the method of the present invention, a mixture of asiatic acid and madecaic acid can be easily obtained by using a solubility difference according to the water content of the ethyl acetate solution. On the basis of the above-mentioned solubility difference, separation was attempted using various solvents such as ether, chloroform, benzene, cyclohexane, and n-hexanebutanol, but all were inferior to ethyl acetate.

The present invention is specifically illustrated by the following examples.

Example 1

100 kg of dried Centella leaf is added to the cutter to make an average size of 0.5 × 0.5 cm ² , put into the extractor and add 400% of 80% methanol and reflux for 3 hours under stirring. After extraction, centrifuge to separate the extract and plant residue. The plant residues are put back into the extractor and added to 300 l of 80% methanol and extracted under the same conditions as above. The combined extracts are placed in a vacuum distillation apparatus and the methanol is evaporated off under reduced pressure. Methanol is removed and the residue is added with water to make a total volume of 300ℓ, put into a percolator and extracted with 300 liters of ethyl acetate for 24 hours. After extraction, the water layer and ethyl acetate layer were carefully separated and treated separately.

Water treatment

The aqueous layer obtained above is put back into the extractor to remove the dissolved ethyl acetate under reduced pressure. 300 liters of n-butanol was added to the aqueous layer from which ethyl acetate was removed, shaken at 20 ° C for 1 hour, and allowed to stand. The aqueous layer and the n-butanol solution were separated. The aqueous layer is extracted twice with the same amount of n-butanol. To the combined n-butanol extract was added 28 kg of dry anhydrous magnesium sulfate, stirred for 30 minutes, and left at 20 ° C. for 12 hours. The n-butanol solution is decanted and separated and dried. 50 liters of distilled water was added to the dried product (about 2.2 kg), and the mixture was shaken at 50 ° C for 1 hour and left for 1 hour. The aqueous solution is filtered from the precipitated material and the filtrate is left at 5 ° C for 24 hours. The resulting crystals are filtered off and dried. Weight of dry crystals: 732g The crystals are filtered and the remaining mother liquor is dried again. Then, 300 l of n-butanol containing 4% water is added thereto, and the mixture is shaken vigorously and left to stand. At this time, n-butanol solution is poured from the tar-like material and dried. This dried material is dissolved in 10 liters of distilled water and left at 5 ° C. for 72 hours. The resulting crystals are filtered off and dried.

Weight of Dry Crystal: 219kg

Treatment of ethyl acetate layer

An insoluble solid is formed at the bottom of the ethyl acetate layer, which is obtained by centrifugation. This solid is suspended in 10 L of distilled water and warmed at 50 DEG C for 30 minutes while stirring. After cooling to 20-25 [deg.] C. it is filtered and the solid is dried. This dried material is dissolved in 50 L of anhydrous ethyl acetate. Ethyl acetate solution is added while stirring at 1.5 degreeC distilled water, maintaining 40 degreeC. After standing for 12 hours, the produced white solid is filtered and dried.

Weight of solids: 1.24 kg

Example 2

To the n-butanol solution obtained in the same manner as in "treatment of the aqueous layer" of Example 1, 10 kg of anhydrous magnesium sulfate was added, stirred, and left to stand for 12 hours. The n-butanol solution was taken out separately, dried and crystallized once in the same manner as in Example 1. 901g of dark brown crystalline material was produced. Recrystallization twice to purify this material yielded 527 g of white crystals.

Example 3

40 kg of anhydrous magnesium sulfate was added to the n-butanol solution obtained in the same manner as in "treatment of the aqueous layer" of Example 1, and stirred for 12 hours. After standing, dried with n-butanol solution and crystallized according to the method of Example 1, 177 g of white crystals were obtained.

Claims (3)

After extracting the leaves of Methanol at 40-80 ° C. with methanol or methanol aqueous solution, methanol was removed, the extract was diluted with distilled water, extracted with ethyl acetate, divided into ethyl acetate and water layers, and the aqueous layer was extracted with n-butanol and n Adjust the water content of the butanol extract to settle and remove unnecessary substances, dry the n-butanol solution, crystallize the dried product with a solvent such as water to obtain asiaticoside, and the ethyl acetate layer adjusts its water content. Separation and recovery method of the trifenoid characterized in that. The method according to claim 1, wherein the water content of n-butanol in the aqueous layer is adjusted to 4 to 7%. The method according to claim 1, wherein the moisture content of the ethyl acetate layer is adjusted to 1 to 3.3%.