JPH0460451B2 - - Google Patents

Description

Claim 1 In separating allergens from arnica flowers, dried arnica flowers are subjected to high-pressure extraction using supercritical CO ₂ at 31-90°C and 70-500 bar, and the eluted allergen substances and optionally A method for separating allergens from arnica flowers, which comprises separating the entrained extracted essential oil by reducing pressure and/or temperature.

2. Process according to claim 1, in which the separation is carried out at 50-70°C and 300 bar.

3. The method according to claim 1 or 2, wherein unground dried arnica flowers are used for the extraction.

4. The method according to any one of claims 1 to 3, for separating the allergenic sesquiterpene lactone from dried arnica flowers.

5. The method according to any one of claims 1 to 4, for producing an allergen-free Arnica flower medicinal species.

TECHNICAL FIELD The present invention relates to a method for separating allergens from arnica flowers by CO ₂ high pressure extraction.

Background technology Arnica montana L.
(Asteraceae) has been one of the most important medicinal plants in the medicinal treasures of central Europe since several centuries. Regarding the Arnica flower (Flores Arnicae), monographs are found in all German Pharmacopoeia. Many arnica preparations contain extracts of Arnica montana. The emphasis of the application of arnica preparations is external use. The action is characterized as antiseptic, anti-inflammatory and procoagulant. Arnica extracts are therefore used primarily in wound treatment ointments, but also in hair oils, lotions, herbal shampoos. When applied internally, respiratory and circulation promoting properties as well as absorption promoting properties have been observed.

The pharmacological action of arnica preparations is based on the specific substances it contains,
This is mainly due to essential oils, flavonoid glycosides and sesquiterpene lactones found in recent studies. A detailed article on the substances contained in Arnica flowers can be found in the journal “Falmatui in Unzerer”
Phamazie inunserer Zeit”, No. 10
Volume, pages 1-7 (1981).

However, improper application of arnica extracts, such as arnica tincture, poses a risk of allergic contact dermatitis. Many such cases are described in "Der Hautarzt", pages 10-17 (1980). Contact allergens include sesquiterpene lactones, helenalin, helenalin acetate, and helenalin-methacrylate.

By the way, by appropriate treatment of arnica flowers with liquid CO ₂ , the sesquiterpene lactones can be removed without trapping together with other contained substances important for pharmacological action, namely flavonoid glycosides and part of the essential oil. , it was found that it can be extracted quantitatively.

DISCLOSURE OF THE INVENTION The object of the present invention is therefore to isolate allergens from arnica flowers by applying 31%
High-pressure extraction with supercritical CO ₂ at ~90° C. and 70-500 bar and separation of the eluted allergenic substances and optionally entrained extracted essential oils by pressure and/or temperature reduction. The method for separating the allergen is characterized by:

For extraction, dried arnica flowers may be crushed, for example by crushing at high pressure using sliding rollers. However, this method has the disadvantage that essential oils are quantitatively extracted in addition to the allergen sesquiterpene lactone. It is therefore advantageous to perform the extraction on unpulverized flowers, in which case the sesquiterpene lactone content is likewise quantitatively extracted, but the main part of the essential oil is extracted along with other substances with pharmacological activity. It remains in the drug. This is usually an allergen-free arnica extract,
Used for extraction and manufacturing of tinctures, etc.

Extraction under pressure with carbon dioxide is known in principle and is described in numerous publications, such as Chemistry and Industry 12, pp. 385-405 (1982) and European Patent No. It is described in specifications No. 23680 and No. 58365.

The extraction is carried out at a pressure of 70-500 bar, preferably 300 bar and at a temperature of 31-90 °C, preferably 50-70 °C.
Performed using supercritical _CO2 at °C. Separation of the extract is carried out by reducing pressure and temperature. The extraction time of the unground drug species is approximately 3-6 hours.

The substances contained in the drug species before and after extraction are quantitatively measured by thin layer chromatography. The completeness of the extraction can be judged by corresponding examination of the _CO2 extract.

In principle, flavonoid glycoside testing is
Performed by DAB7 (Appendix 2, p. 116 et seq.): Dried arnica flowers (Arnica montana, Flos)
Then, extraction is performed using methanol (20 ml of methanol per 1 g of drug) for flavonoid testing.
It is then filtered and the filtrate is evaporated to 2 ml. This extract was mixed with chloroform/methanol (3:1).
Dissolve inside. Thin layer chromatography is performed by spotting 100 μl of a sample onto a silicic acid gel GF ₂₅₄ plate and developing it with ethyl acetate/formic acid (anhydrous)/acetic acid/H ₂ O (100:11:11:26). Detection is carried out by spraying a 1% solution of diphenylboryloxyethylamine in methanol.

Sesquiterpene lactone testing was performed by C. Willuhn and HD Schafer, Pharmazeutische Zeitung 123,
It can be carried out according to pp. 1803-1808 (1978): 40 g of benzol to 5 g of the drug, that is, the residue after extraction.
ml for 10 minutes, filtered and washed with 5 ml of benzene. benzol extract
It is concentrated by evaporation at 60-70°C, and the residue is extracted with 30 ml of 60% ethanol and then filtered. _CO2
- The extract (100 mg) is dissolved hot in 10 ml of benzol, filtered and shaken out four times with the same volume of 60% ethanol.

After removing the ethanol and replenishing the aqueous phase to 20 ml, shakeouts are carried out five times with 5 ml of chloroform each. The combined chloroform extracts are dried over CaCl ₂ and concentrated by evaporation. To remove the yellow entrainment, the residue was taken up in 0.5 ml of benzol and
Neutral aluminum oxide 90 [Merck,
Activation step 1] 20g of petroleum ether/benzole/chloroform/methanol (5:4:1:
2) Perform chromatography using the mixture.
Discard the first 10ml of eluent. Evaporate the next 20 ml to dryness and take up in 0.3 ml of benzene for DC analysis.

Samples (20-30 μl) are spotted on silicic acid gel F ₂₅₄ -thin plates (Merck & Co.) and developed with n-pentane/ether (8:17). Detection was performed using Zimmermann's reagent [m in ethanol.
- dinitrobenzole and 1.25N KOH (1:1)
2% solution] under ultraviolet light.

The essential oil content is measured using DAB8.

Examples Example 1 1000 g of unground dried arnica flowers containing 0.12 ml/100 g of essential oil were extracted for 6 hours at a temperature of 70° C. and a pressure of 300 bar in an extraction vessel of a high-pressure extraction device. The separation was carried out at 20°C and 60 bar.

A yellow-brown pasty oil with a hay-like aroma was obtained.

The yield of extract was 2.8% by weight. more water
30ml was separated and removed by decantation and squeezing. The following results were obtained from the analysis of the extract and the extracted drug species: a. Extract Essential oil: 0.06 ml/100 g dry raw material Water: 3.1 ml/100 g dry raw material Sesquiterpene lactone: Detectable by thin layer chromatography Flavonoid glycosides: Not detectableb Drug species to be extracted Sesquiterpene lactones: Only trace amounts can be detected Flavonoid glycosides: Isoquercitrin, astragalin, luteolin-7-glycoside, etc. can be detected by thin layer chromatography Essential oils: 0.05ml/100g Example 2 of extracted drug species 1000 g of dried arnica flowers were crushed using a sliding roller at a pressure of 60 tons, and then extracted at 70° C. and 300 bar for 4 hours. Separation was carried out at 22°C and 65 bar.

6.5% by weight of extract and 25ml of water were separated. Water was removed by decantation and squeezing.

The extract contained total sesquiterpene lactones and essential oil 2.2 ml/100 g (corresponding to 0.14 ml/100 g dry raw material). Flavonoid glycosides could not be detected.

Sesquiterpene lactones can no longer be detected in the extracted drug species, while the essential oil content is 0.02ml/100g.
It was hot. Flavonoid glycosides were completely obtained.