DE2358434A1 - METHOD FOR PURIFYING VEGETABLE FLAVONE EXTRACTS - Google Patents

Description

MÖ4LER-SÖFL £ DIPL.-PHYS.EfFi, ^ ÄJtflTZ ÖIPL-dWßfcl, ^ 0EUPEL j = iNSTERWÄILl!> DIPL.-fNG,

235M34

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77130 Etrepiliy
France

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is "bekajini ;, certain Fflanzerti e.g. the Friichthülleii Öitrüsffaölilieiij the leaves of Hamamelis Virginiana and the 4S -unripe berries of various ßhamnus species (e.g. Amygdalinus _t Φ öieoides, saxatilis and infectöria), subject to certain conditions in order to to extract from it the flavone pigments, which are in abundance *

^. For the determination of these extracts, after the reconciliation * d'ensten methods known from the literature use the "plant source

Df.Müller-Borfr Dr. Manilz Dr. Deufel Dipl.-Ing. Finslerwald Dipl.-Ing. Grärnkow

33 Bfäqnsäiweig, Am BOrgeipärk ä 8 Munich 22, Robert-Koch-Strasse 1 7 Stuttgart-Bad Cannstatt, Marktstrasse 3

telöforr (0531) 7388? Tölefon (0811) 293645, TelexS-220S0 mbpat Telephone (0711) 567261

"Bähk: Central Cash Office Bayer, Völfebanken, Munich, Account No. 9822 Posfettei *: Müft» enK4S5 -

materials are crushed and then extra- liiert j to which alcohols or ketones are optionally added where "work is carried out at a suitable temperature.

The extracts obtained in this way are very impure and contain a certain number in addition to the flavone pigments of substances originating from the plant tissue as well as degradation products of such substances, which in the course of the extraction operation are formed.

The flavone pigments can be made from these complex extracts be separated by conventional methods of natural product chemistry, z. B. by distribution between solvents, d. H. that is, partition chromatography, by chromatography on different carriers or by precipitation with selective reagents.

However, it was found that certain impurities the Piavon pigments in the course of these 'known cleaning operations accompany and are difficult to separate from these, so that the flavonoids obtained, although purified are unsuitable for certain uses in human medicine. The cleaned products usually show a pyrogenic and allergenic effect what they are from Use as injectable preparations and from transcutaneous applications.

The contaminants responsible for these undesirable effects are, on the one hand, vegetable proteins and in particular the nucleobase proteins or histones, which are either directly bound to the flavonoids or via certain divalent cations, e.g. B. Ga ⁺ *, Ou ⁺ * or oligo elements, which form mixed chelates with the proteins and the flavonoids, and on the other hand to products with comparative

h 0 9 8 2 3/11 1 3

wise high molecular weight, which are related to leucocyantannins, those in the oxidative condensation of flavonoids since is formed, which are easily adsorbed by both flavonoids and proteins, and their formation during the first stages of treatment of the Plant material is practically unavoidable.

These impurities, even if they are only in Traces are present, unfold their disadvantageous, biological effect, must therefore be completely removed. One treatment the crude extract before each fractionation operation therefore proves to be inevitable.

The invention provides a method for purifying Herbal raw extracts created, which are easy to use Feasibility and results in apyrogenic flavone extracts that can be used as they are or as a starting material for separating the flavone pigments can serve. The extracts obtained can be used directly in human medicine and also for administration forms suitable for parenteral or transcutaneous application,

The method of the invention for the purification of crude aqueous or aqueous-alcoholic flavone extracts is characterized in that the extract solution is selectively adsorbed one after the other on a complex-forming ion exchange resin, an acidic clay and activated alumina, and after After adsorption, the purified solution is either chemically treated to separate the flavonoids it contains .or dries for the production of pharmaceutical preparations transferable powders.

The three stages of adsorption enable and have to Aims, sequentially the metals plus chelating agents,

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the soluble proteins and finally the condensed ones To remove tannoids, i.e. compounds similar to tannins. The pure flavonoids can be extracted from the purified solution by chemical means according to known chemical methods Process to be separated. The obtained by drying Powders are used in human therapy. .

According to a preferred embodiment, it is the ion exchange resin is a resin with active phosphonic acid groups, and in the case of acidic clay around montmorillonite.

According to a further advantageous embodiment of the invention, the solution is cooled to 0 to 2 ⁰ G decanted into an isothermal cooling vessel after the adsorption of resin and aluminum oxide.

According to a further advantageous embodiment of the invention is adsorption on clay and alumina under a stream of nitrogen in a closed isothermal Cooling vessel carried out.

According to a further advantageous embodiment of the invention the final treatment of the cleaned solution is done within 2 hours after cleaning carried out.

The cleaning method according to the invention comprises three successive ones Adsorption stages to the chelating metals, the soluble proteins and the condensed tannin-like To remove tannoids from the raw aqueous or aqueous-alcoholic flavone extracts by classic extraction processes were obtained.

AO 9823/1113

The first: purification stage involves fixing the oligomer elements on a suitable ion exchange resin, e.g. on a resin known under the trade name Buolite ES 63 ^f , which is a special phosphorus group-containing nickel, Cobalt, copper and sinking resin is involved, starting with a 10 to 15 parts aqueous extract solution which is practically neutral, whereupon the resulting solution is acidified with sulfuric acid to a pH value of 2 and the acidic solution is cooled to OG and is kept at this temperature for about 48 hours. During storage, a complex precipitate forms, which mainly contains mixed sulfates of basic proteins and calcium as well as moderately condensed polyuronides filtered »whereupon its pH value is adjusted to 7 with the help of sodium or potassium hydroxide solution · '

The second purification step is the basic j P ro-r proteins at a Ädsoa ^ ptiionston to fixiereii, which may be, for ^ ', B * involve montmorillonite * ice are 2? S montmorillonite added to the _above-f ia , better dispersed and. wary has been swollen and the (iemiseh is stirred for at least 2 Sfemden: Then the ΐοη is removed by centrifugation and the treatment is repeated a second time with t f ^a 3 ^ a =. These operations are preferably carried out in a vessel closed with a lid while introducing an inert gas test, e.g. nitrogen ... .'-: .-

The third purification stage consists in removing the tannoids on neutral activated aluminum oxide such as: ÜlypSj as he is for ^; chromatographic separation method is used to fix; where 50/00 aluminum oxide has a particle size of 2%. 'Are used to 50 microns in which substantially by V / eg neutral extract. The mixture of aqueous extract solution and aluminum oxide is stirred for 1 hour in a closed

a vessel with the introduction of nitrogen as in the second cleaning stage. The alumina is then through The filtration is removed and the treatment is repeated with 2.5% alumina.

The extract obtained is made up with the help of sulfuric acid adjusted to a pH of 2 and cooled to 0 G and left at this temperature for 48 hours. A precipitate forms, which is separated off by filtration. The pH is then adjusted to 4.00 with potassium hydroxide solution.

The purified solution obtained can be used immediately to remove the flavonoids by any known means to separate chemical processes from it, as well as to be used directly for therapeutic Ehdanwendanwendent purposes. in the in the latter case it is essential to dry the solution without waiting, e.g. B. by atomization, d. H. Spray drying, or preferably by lyophilization. In any case it should be noted that the purified solution should not be longer than May be kept for a few hours, as it is subject to rapid changes that take advantage of the treatment can destroy.

The dried product obtained is suitable for all desired pharmaceutical forms, e.g. B. for tablets, gel capsules and granules, including for injectable formulations, if the physiological solvent and the lyophilized powder are mixed together just before the injection will. Bas dried product can also be converted into oils and ointments for medical and cosmetic purposes Use provided that the water content of such formulations is reduced to a strict minimum. That powder obtained by direct drying of the solution acts. not allergic and is not or only slightly pyrogenic. The flavonoids separated from the purified solution are

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allergic and apyrogenic. .

The following examples illustrate the invention in more detail,

example 1

It was made from an aqueous solution with $ 12 dry matter assumed that by exhaustive treatment of orange fruit skins had been obtained with water.

'Such solutions are z. B. simultaneously with the ethereal Oils in for superficial scraping and stripping Obtained and made by machines specific to fruits thus normal by-products of the fruit processing industries. "-" "" '

1OOO 1 of the solution with 10 fo dry extract, which was carefully filtered on kieselguhr and whose pH value must be between 6.5 and 7.5 ", was at a rate of

200 liters per hour passed through a column of 100 1,

the '-'

the resin known with / under the name "Duolite ES 63",

which had previously ^{been saturated with Na +} ions was filled * The solution impregnating the resin was replaced by an appropriate volume of water. In this way, 990 liters of a solution were obtained which were completely free of heavy metals.

The resulting solution was reduced to about 0 G using a
Plate heat exchanger cooled and then acidified to a pH of 2, which was precisely measured, which required about 6 liters of concentrated sulfuric acid.

The resulting solution was placed in a cooling chamber or a
isothermal cooling vessel placed in it for 48 hours

409823/1 113

was left standing. At the bottom of the jar sat down. a. Precipitation, which corresponded to about 50 kg wet weight. Of the The precipitate formed was separated off by decanting and washed with 100 l of water, followed by decanting again was separated.

The decanted liquid was filtered through kieselguhr and placed in a vessel with a removable lid, on the bottom of which a stirrer was provided pH of 7 neutralized, which required about 7 kg of sodium hydroxide lozenges. . .

There was then a stream of nitrogen with the aid of a porous Introduced distribution device and 100 1 of a dispersion containing 20 kg of montmorillonite were added with stirring added. The mixture was stirred for 2 hours, after which the mixture was allowed to stand still for 2 hours. Another Agitation allowed further suspension and separation of the clay in a spinner centrifuge.

The clear liquid obtained was returned to the specified Given a vessel into which, after saturation with nitrogen, 50 1 of a dispersion containing 10 kg of montmorillonite are introduced became.

It was stirred for 2 hours, followed by standing for 2 hours. Then it was suspended again by stirring, whereupon the clay was separated in a centrifugal separator.

The clay material obtained by centrifuging was treated with 100 l Washed water, whereupon it was separated again with a centrifuge. The washing water resulting from washing the clay was combined with the main liquid.

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While stirring and. During decanting, the introduction of nitrogen was interrupted and the "movable. Lid" was closed with With the help of its pneumatic seal, it is placed in a gastight manner.

The liquids were combined in a vessel of the specified type and, with maximum stirring and the introduction of nitrogen, 5 kg of activated aluminum oxide for chromatographic purposes were then added, which was ground and sieved to a particle size of 20 Ms 5 μm and previously dispersed in 25 1 of water was. _:

The stirring was stopped for 1 hour and then stopped, after which it was filtered and the filtrate dissolved in; was collected in a vessel of the specified type. 2.5 kg of aluminum oxide were then added and the mixture was again stirred for 1 hour. Stirring was then stopped and the mixture was filtered. At the outlet of the filter, the solution obtained was cooled in a plate heat exchanger mx £ 2 ^ o, whereupon it was collected in a column vessel. The solution was adjusted to a pH of 2.00 (+0.05) which requires about 4 "1 - concentrated sulfuric acid. The solution was kept at this temperature for 48 hours with a gentle introduction of nitrogen. A dense precipitate of about 30 kg wet weight formed, which was separated off by filtration The filtrate solution obtained was collected in a vessel equipped with a permanent nitrogen introduction device.

The still cold solution was neutralized to a pH value of 5.00 (I OjO5J, which required about 5 kg of sodium hydroxide in lozenge form. The solution, which was still cold and saturated with nitrogen, was immediately concentrated under a vacuum of ΙΟ to 20 Torr at a 4Ö ⁰ G temperature not exceeding that was accurately measured, until the content of dry substance amounted to 25 ° Ύ '. the resulting solution must have a

409823/1113

have a pH value of 4.00 (+ 0.05), the pH value at Adjusted as required with the help of sulfuric acid or caustic soda became.

The solution was immediately frozen and lyophilized or spray dried instead. A similar one was used for spraying Volume of methanol added to reduce the thermal effects on the product. It also turned out to be expedient to use a spray dryer with a cooling device for the powder and a continuous Packing device was equipped.

When lyophilization was carried out, the vacuum of the lyophilization chamber became after drying the product by nitrogen, canceled to saturate the product with nitrogen. Became sprayed, the resulting chilled product was placed under a vacuum bell jar and at 5 bis for 1/2 hour Maintained 10 torr, after which the vacuum was released with nitrogen to remove any trace of entrapped oxygen remove. In both cases, the powders obtained were conditioned under a nitrogen atmosphere and dried with a drying agent protected. . ";

The powder obtained in this way could be used directly for therapeutic purposes Purposes are used and corresponded to that product, which is referred to in the Propharmacopoe as "Gitroflavonoid". It did not cause any allergic reactions and proved to be apyrogenic if the raw extract had not yet undergone fermentation.

The solution, concentrated to 25 % in vacuo, could also be used as starting material in a flavonoid extraction circuit by liquid-liquid exchange or selective precipitation under such conditions that it could be kept temporarily under constant introduction of nitrogen

409823/1113

took place and lasted no longer than 2 hours.

Example 2

1000 kg immature berries of Rhamnus amygdalinus were to 2 mm crushed in a grinder made of stainless steel and at the exit of the grinding apparatus in 500 1 water of 95 ⁰ G collected on a particle size of 1 ". The mixture of water and crushed berries was at 95 ° G held by direct introduction of filtered and de-oiled steam.

The mass obtained was then placed in a percolator with a capacity of 2000 l and the percolation was carried out under pressure with 2000 l of water at 110 ^° C. The juice leaving the percolator was collected and immediately cooled to 20 C in a plate heat exchanger, whereupon it was concentrated under vacuum at 10 to 20 torr at a temperature of not more than 40 G in such a way that the volume obtained was 1000 liters. The resulting concentrate, which contained about 10 fo solutes and had a pH of 5.5 to 6.0, was centrifuged and filtered.

The 1000 liters of rhamnus concentrate obtained then became the same further treated like the 1000.1 flavone extract from citrus fruits according to Example 1, whereby point by point was carefully observed in all process stages.

The powder obtained, which was rich in oampherol derivatives, could be used directly for therapeutic purposes its properties, especially diuretic properties, it could also be used as a starting material for isolation of oampherol and its derivatives according to the usual known Procedure.

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The final solution with 25 ° dry substance could also be used to isolate the pure products, taking into account the conditions described in Example 1.

409823/1113

Claims (1)

1 $ "■;■"■■; .- Patents s ρ back e Process for the purification of plant-based flavone extracts, characterized in that "one the extract solution successively selectively adsorbed on a complexing ion exchange resin an acid clay and activated alumina, and after adsorption the purified solution either chemically treated to separate the contained therein Plavonoids or dries to obtain in pharmaceutical preparations convertible powders, 2t method .nach claim 1, characterized in that one as an exchange resin, a resin with active phosphonic acid groups used, 3, method according to claims 1 and 2, characterized in that that montmorillonite is used as clay> . 4. Process according to Claims 1 to 3 _1, characterized in that, after adsorption on resin and aluminum oxide, the solution obtained is reduced to 0 to 2 thermal cooling vessel decanted, solution obtained cools to 0 to 2 ^Q G and in one 5, process according to claims 1 to 4, characterized in that the adsorption on clay and activated aluminum ^ oxide under a stream of inert gas in a closed . its cooling vessel. : ■ _ "· .. 6. The method according to claims 4 and 5, characterized in that that after adsorption on activated alumina holding the solution in the cooling vessel for decanting while introducing inert gas. 4 09823/11 1'i 2358 ^ 34 7. The method according to claims 1 to 6, characterized in that that nitrogen is used as the inert gas. 8. The method according to claims 1 to 7 »characterized in that the final treatment of the purified solution within two hours after cleaning is finished performs. 9. The method according to claim 8, characterized in that the purified solution is concentrated and the concentrate to · a powder dries. 10. The method according to claim 9, characterized in that one. the drying is effected by spraying or lyophilization. 11. The method according to claims 9 or 10, characterized in that τ that the powder obtained is cooled and continuously dehydrated. 12. A pharmaceutical agent containing according to the method according to claims 1 to 11 purified extract. 4 0 9 8 2 3/1113