WO2011072981A1 - Method for isolating stilbenoids from solvents - Google Patents

Abstract

The invention relates to a method for isolating stilbenoids from solvents and to the use of stilbenoids thus obtained.

Description

Process for the isolation of stilbenoids from solvents

Field of the invention

 The invention relates to a process for the isolation of stilbenoids from solvents and the use of the stilbenoids obtained therewith.

State of the art

 Stilbenoids are derivatives of stilbene. Among the stilbenoids is in particular the resveratrol (3, 4 ', 5-trihydroxystilbene), which also belongs to the class of polyphenols (more precisely the polyphenolic phytoalexins). In plants, stilbenoids, such as resveratrol, in particular

Fungal infections or strong UV exposure formed; Here they are the product of two different biosynthetic pathways, namely the shikimic acid pathway and fatty acid biosynthesis.

The stilbenoids are said to have antioxidant effects. Resveratrol, a known stilbenoid, which is also formed, for example, during wine fermentation and especially in red wine in high concentrations, acts as an antioxidant and also reduces the risk of coronary heart disease (see, for example, Becker John VW et al .: "Metabolie engineering of Saccharomyces cerevesiae for the synthesis of the wine-related antioxidant resveratrol ", FEMS Yeast Research, 4 (1), pages 79-85 (2003) and Jang et al." Cancer chemopreventive activity of resveratrol, a natural product derived from grapes ", Science, 275, pages 218-220 (1997)).

 Due to the beneficial effects of stilbenoids, these compounds are increasingly used in cosmetic or

used pharmaceutical compositions. In doing so, the Providing stilbenoids mostly by extraction

Plants; There are also in the prior art methods

in which stilbenoids are recombinant

Microorganisms are produced and in which the stilbenoid is usually isolated by extraction with an organic solvent from the culture broth and / or the cells.

Regardless of the manufacturing method used

Stilbenoids are these substances temporarily present as dissolved in an organic solvent.

 Conventional methods for isolating the stilbenoids from the non-water-miscible organic solvents optionally used in the above-mentioned extraction

For example, chromatography, evaporation, cooling,

Displacement crystallization or back-extraction with another organic solvent.

 Thus, WO 2009/016108 describes an isolation of

Stilbenoids by a sequential extraction from n-hexane with first ether, then acetone, then methanol and finally water.

 In WO0191763 stilbenoids are obtained from the solvent

Ethyl acetate crystallized with hexane. Thus, a classical displacement crystallization is performed around the

Isolate stilbenoids from the water immiscible solvent.

 A disadvantage of the current state of the art is the

additional use of other solvents in the

applied back extraction and the need for a high energy input for the evaporation of solvents or other purification steps.

The object of the invention was to provide a method with which without the above-described disadvantages of the prior art the stilbenoids can be isolated from an organic solvent.

Description of the invention

 Surprisingly, it was found that despite the

known poor water solubility of stilbenoids, in particular resveratrol, by an aqueous phase, the stilbenoid from a substantially water-immiscible solvent back-extract.

The present invention therefore provides a process for the isolation of at least one stilbenoid from a

Reaction mixture containing at least one with water in

Substantially immiscible solvent comprising the

Process steps A) bringing the reaction mixture into contact with an aqueous phase, B) separating the aqueous phase from the water-immiscible solvent and optionally C) isolating the stilbenoid from the aqueous phase.

 Another object of the invention is the use of stilbenoids obtained by this process for the production of everyday consumer products.

An advantage of the present invention is the reduction in energy input and the reduction of waste streams, particularly non-aqueous and thus often problematic solvent streams.

All percentages (%) are percentages by weight unless otherwise specified. In the context of the invention, "stilbenoids" are hydroxylated structural hydroxyl groups having at least one hydroxyl group

Derivatives of stilbene (1,2-ethenediyl bisbenzene) understood, where "stilbene" the E and Z isomer or cis and trans isomer

includes; Examples of stilbenoids are therefore resveratrol, piceatannol, pinosylvin and pterostilben.

The term "essentially not miscible with water"

describes according to the invention the properties of liquids not to be miscible with water in any ratio, but to form a multi-phase system when reaching a saturation concentration. In particular, this is understood to mean the property that the solvent can dissolve a maximum of 15% by weight, preferably not more than 10% by weight, particularly preferably not more than 2% by weight, of water, based on the weight of the

Solvent.

 For the purposes of the present invention, the term "reaction mixture" is to be understood as meaning a system at the time at which the stilbenoid to be isolated for the first time is essentially immiscible with water

Solvent is located. Such a reaction essentially comprises the substantially water-immiscible solvent resulting in stilbenoid to be isolated and reactions required from the formation of the stilbenoid

By-products. For clarification and not limiting are mentioned here: The first extraction phase with an im

Substantially water immiscible solvent from a fermentation broth, in particular that of in situ extraction or a first extraction phase with a substantially water immiscible solvent, wherein the stilbenoid is isolated from a plant part.

 Preferably, the inventive method consists of the

Process steps A), B) and optionally C). It is possible according to the invention to isolate all stilbenoids by the process according to the invention; 3, 5, 4 '-trihydroxystilbene, in particular the trans-form (resveratrol), is preferably isolated in this connection.

It is preferred in the invention that the solvent used in the process is an organic solvent.

As solvents, which are used in the process according to the invention, in principle all come with water in the

Substantially immiscible solvents in question. Such are, for example:

branched and unbranched, optionally double bonds containing hydrocarbons having carbon chains with a length of 6 to 16 carbon atoms such as nonane, decane, undecane, dodecane;

 Esters of branched and / or unbranched, optionally double bonds containing carbon chains with a

Total carbon number per molecule from 4 to 26

Carbon atoms such as ethyl acetate, octyl acetate, dibutyl adipate, especially fatty acid esters such as

for example, ethyl decanoate, ethyl laurate, ethyl oleate,

Ethyl myristate;

branched and unbranched, optionally double bonds containing carbon chains with a length of 5 to 16 carbon atoms such as 3-pentanone, 2-heptanone, 2-undecanone, 2-octanone, methyl isobutyl ketone;

branched and unbranched, optionally double bonds containing alcohols having carbon chains with a length of 4 to 18 carbon atoms such as 1-nonanol, 1-octanol, 1-butanol, oleyl alcohol ... etc; Ether of branched and / or unbranched, optionally double bonds containing carbon chains with a

Total carbon number per molecule from 4 to 12

Carbon atoms such as tert. methyl butyl ether,

Diethyl ether, diisopropyl ether;

branched and unbranched, optionally double bonds containing carbon chains having a length of 5 to 12 carbon atoms such as caprylic acid, capric acid, oleic acid, decanoic acid;

 Polyethers such as polypropylene glycol and polyethylene glycol;

vegetable oils such as olive oil, soybean oil, palm oil,

Rapeseed oil.

In the method according to the invention are preferred

Solvents used selected from the group comprising, preferably consisting of, methyl isobutyl ketone, 3-pentanone,

Ethyl butyrate, myristyl alcohol polypropoxylate (available as Tegosoft APM from Evonik Goldschmidt), diethyl adipate,

Octyl acetate, ethyl acetate, 2-heptanone, 1-octanol,

Tritolyl phosphate, triacetin, oleyl alcohol, nonanol, dodecane, dibutyl adipate, diethyl sebacate, polypropylene glycol 1000 to 4000, 2-octanone and 2-undecanone.

It is apparent that it may be advantageous to use mixtures of two or more of these solvents in the process according to the invention.

In process step A), the reaction mixture is brought into contact with an aqueous phase; In this case it is preferred that the reaction mixture directly with the aqueous phase in

Contact is brought; it is therefore preferable that no prior extraction with a non-aqueous solvent such as For example, be carried out ether, acetone or methanol.

The aqueous phase is preferably a phase which contains at least 50% by weight, preferably at least 80% by weight and particularly preferably at least 95% by weight, of water, based on the total weight of the aqueous phase.

 The aqueous phase is preferably a basic, aqueous phase.

 Thus, the aqueous phase preferably has a pH of greater than 7, preferably greater than 10, in particular greater than 12.

 The aqueous phase preferably contains compounds which ensure a stable pH behavior in the abovementioned range. Such buffer substances are selected from the group encompassing, preferably consisting of: phosphate buffer,

Ammonia buffer (NH ₃ aqueous solution), TRIS: tris (hydroxymethyl) aminomethane, HEPES: 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, HEPPS: 4- (2-hydroxyethyl) piperazine-1-propanesulfonic acid, carbonate buffer.

 The aqueous phase particularly preferably contains alkali metal and / or alkaline earth metal hydroxides in a concentration of 0.1 mol / L to 20 mol / L, preferably 0.5 mol / L to 15 mol / L, particularly preferably 0.9 mol / L to 10 mol / L based on the entire aqueous phase. In this context, the hydroxides are in particular selected from the group consisting of NaOH and KOH.

In process step B), the aqueous phase of the im

Substantially water immiscible solvent

separated, this come all known in the art

Separation process for two immiscible liquids such as For example, the use of separators, centrifuges,

Decanters and separators in question.

 In method step C) the stilbenoid may be preferred

Resveratrol be isolated from the aqueous phase for example, further use. For this purpose, methods such as displacement crystallization offer.

In process step C), the isolation of the stilbenoid is preferably carried out by evaporation of the surrounding surrounding stilbenoid

Solution.

 In a particularly preferred embodiment of the

Process step C), the pH of the aqueous phase is adjusted to less than 7, preferably less than 4, more preferably less than 2. This leads to a precipitation of the

Stilbenoids, preferably resveratrols, from the aqueous phase. To reduce the pH to the aforementioned values, the person skilled in the art knows many possible acids, in particular water-soluble organic, mineral and / or inorganic acids such as, for example, acetic acid, formic acid, sulfuric acid and hydrochloric acid. In particular, the use of hydrochloric acid, preferably in final concentrations of greater than 0.01 mol / L, preferably greater than 0.1 mol / L based on the aqueous phase, is advantageous here.

It may be advantageous if the lowering of the pH is accompanied by a lowering of the temperature of the

aqueous phase, to the precipitation of stilbenoid, preferred

Resveratrols, from the aqueous phase to promote. Therefore, process step C) comprises, in addition to the pH reduction

Step, in which the temperature of the aqueous phase is lowered, based on the temperature at the beginning of the process, wherein preferably the temperature is lowered to less than 20 ° C, preferably to less than 10 ° C, more preferably to less than 5 ° C. If the stilbenoid, preferably resveratrol, is present as a solid in the aqueous phase, it can be separated by conventional, simple means known to those skilled in the art for the separation of solid / liquid mixtures.

 Therefore, a preferred process according to the invention in process step C) comprises a step in which the stilbenoid, preferably resveratrol, is separated as a solid from the aqueous phase. According to preferred separations of the invention

Stilbenoids, preferably resveratrol are in this context

Centrifugation, filtration, sedimentation and squeezing.

 For example, in the filtration techniques to be used

Gravity filters, vacuum filters, filter centrifuges are used.

The stilbenoids isolated by the process according to the invention, in particular resveratrol, are advantageously to be incorporated into daily use products.

 Therefore, a further contribution to achieving the object stated at the outset is the use of at least one stilbenoid, in particular resveratrol, obtained by the process according to the invention for the production of cosmetics, foodstuffs,

Nutritional supplements, pharmaceuticals and animal feed.

In the examples given below, the present invention is described by way of example, without the invention, whose scope of application is apparent from the entire description and the claims, referred to in the examples

Embodiments should be limited. Examples:

Back extraction by means of aqueous phase from PPG1000

 20g / L of Resveratrol was dissolved in PPG1000. 10 ml of this

Solution was added 10 ml of 4M aqueous NaOH solution, 30

Shaken for a few seconds and separated again by centrifugation (1 min, 5000 x g). The NaOH phase was removed and treated with 4M HCl until a pH <6 was reached.

 When lowering the pH, the resveratrol precipitated out as a solid.

 The solid was separated by vacuum filtration, washed with water and dried.

Yield: 83%; Purity (HPLC): 83%

A defined amount of the resulting solid is dissolved in ethanol. 2 μΐ of the solvent sample was injected onto a Luna PFP (2) column (150 x 4.6 mm, 5 μm, Phenomenex) using the Agilent 1200 HPLC system equipped with a photodiode array detector and a binary gradient mixer. Resveratrol was treated with a mobile phase consisting of 65% methanol and 35% water with 0.5 ml trifluoroacetic acid per liter, with a

Dilution rate of 0.6 ml / min and a column temperature of 25 ° C eluted. Resveratrol was identified by the retention time and the UV / VIS spectrum of a reference substance (Sigma-Aldrich). To quantify the resveratrol in the

Samples became the peak areas with those of an external area

Standard curve of known concentration compared.

Back extraction by means of aqueous phase from diethyl adipate

20 g / L resveratrol were dissolved in diethyl adipate. 10 ml of this solution was mixed with 10 ml of 1M aqueous NaOH solution shaken, shaken for 30 seconds and separated by centrifugation (1 min, 5000 xg) the phases again. The NaOH phase was removed and treated with IM HCl until a pH <6 was reached. When lowering the pH, the resveratrol precipitated out as a solid.

 The solid was separated by vacuum filtration, washed with water and dried.

Yield: 88.9%; Purity (HPLC): 95%

Re-extraction by aqueous phase of octyl acetate

 There were 4.5 g / L Resveratrol dissolved in octyl acetate. 10 ml of this

Solution was added 10 ml of 1M aqueous NaOH solution, 30

Shaken for a few seconds and separated again by centrifugation (1 min, 5000 x g). The NaOH phase was removed and treated with IM HCl until a pH <6 was reached.

 When lowering the pH, the resveratrol precipitated out as a solid.

 The solid was separated by vacuum filtration, washed with water and dried.

 Yield: 83.5%; Purity (HPLC): 96.4%

Claims

Claims: 1. A method for isolating at least one stilbenoid  a reaction mixture comprising at least one water-immiscible solvent comprising the process steps  A) contacting the reaction mixture with a  aqueous phase,  B) separating the aqueous phase from that with water in the  Substantially immiscible solvents and  possibly  C) Isolation of stilbenoid from the aqueous phase. 2. The method according to claim 1, characterized in that the solvent is an organic solvent. 3. The method according to at least one of the preceding claims, characterized in that the solvent is selected from the group comprising methyl isobutyl ketone, 3-pentanone, ethyl butyrate, Myristylalkoholpolypropoxilat, diethyl adipate, octyl acetate, ethyl acetate, 2-heptanone, 1-octanol,  Tritolyl phosphate, triacetin, oleyl alcohol, nonanol, dodecane, dibutyl adipate, diethyl sebacate, polypropylene glycol 1000 to 4000, 2-octanone and 2-undecanone. 4. The method according to at least one of the preceding claims, characterized in that the aqueous phase contains at least 50 wt .-% water, based on the total weight of the aqueous phase. Method according to at least one of the preceding claims, characterized in that the aqueous phase preferably has a pH greater than 7. 6. The method according to at least one of the preceding claims, characterized in that the aqueous phase alkali and / or alkaline earth metal hydroxides in a concentration of 0.1 mol / L to 20 mol / L, based on the total aqueous phase. 7. The method according to at least one of the preceding claims, characterized in that in step B) the separation of the aqueous phase by the use of  Separators, centrifuges, decanters and separators takes place. 8. The method according to at least one of the preceding claims, characterized in that in step C) the isolation of stilbenoid by evaporation of the  Stilbenoid surrounding solution takes place. 9. The method according to at least one of the preceding claims, characterized in that method step C) comprises a step in which the pH of the aqueous phase is adjusted to less than 7. 10. The method according to claim 9, characterized in that in step C) the temperature of the aqueous phase is lowered, based on the temperature at the beginning of the process. Use of at least one stilbenoid obtained by a process according to at least one of claims 1 to 10 Manufacture of cosmetics, food,  Nutritional supplements, pharmaceuticals and animal feed.