DE102006004829A1 - Obtaining Glucosinolates from Capparales Exudates - Google Patents

Abstract

The present invention relates to a method for obtaining at least one glucosinolate from a root exudate of at least one plant of the order Capparales, comprising the steps: (a) growing the plant in an ungrounded system; (b) administering a nutrient solution with an increased sulfur content; (c) stimulating glucosinolate formation by administering at least one elicitor; (d) Obtaining the glucosinolates from root exudates of the plant. In addition, the invention relates to the glucosinolates obtained by the process and to their use for food supplements, as a starting or raw material for functional food or as a pharmaceutical.

Description

Environment of invention

The The present invention relates to the technical environment of phytochemicals, especially pharmaceutically valuable compounds produced in plants, which can be used as a medicine or nutritional supplement. Especially The present invention relates to a process for obtaining at least a glucosinolate from a root exudate of plants of the order Capparales and the glucosinolates thus obtained and their use for nutritional supplements or as a pharmaceutical.

background the invention

plants are a valuable source for secondary Plant substances or secondary Metabolites, many of which are considered nutritional supplements in so-called "fuctional food "as well as starting or raw material compounds for pharmaceutically active compounds (Pharmaceuticals) and health-promoting compounds used can be. (Mulabagal and Tsay, 2004; Borisjuk et al., 1999). The extraction from secondary Metabolites are conventional from vegetable residual or waste material that is not for consumption suitable is. Examples of this are broccoli or cauliflower stalks, outer cabbage leaves and turnip greens, which are mechanically minced and being open-minded to the desired phytonutrients to extract. In this plant material are often only very small Concentrations of secondary Plant substances, so that big Quantities of starting material must be used. That's why procedures have been designed to be secondary Extract metabolites from individual plant organs. For example can secondary Metabolites are obtained from exudates of plants.

by virtue of their health-promoting Effects, in particular their anti-carcinogenic properties are Glucosinolates and their hydrolyzed products pharmacologically and nutritionally interesting (Talalay and Fahrey, 2001, Zhang, 2001). glucosinolates are a group of plant compounds that are in order the capparales, to which the agriculturally important vegetables belonging to the family Brassicaceae, being found. Glucosinolates consist of a β-D-thioglucose reduced group, a sulfonated oxime residue and a variable Side chain derived from amino acids is derived (Kneer et al., 1999). Based on the chemical Structure of their side chains, the glucosinolates in different Classes such as aliphatic glucosinolates, aromatic glucosinolates and indole glucosinolates (Bennett et al., 1994; Vallejo et al., 2003).

The WO 98/27806 A1 describes a Brassica plant which is an artificial one generated, opposite the wild-type plant contains reduced glucosinolate content. Height Levels of glucosinolates are due to their toxic by-products as undesirable because they negatively affect the enzyme myrosinase. myrosinase is the enzyme released by the digestion of the Brassica plant and the degradation of glucosinolates to glucose, thiocyanates, isothiocyanates and nitriles catalyzes.

The WO 94/19929 A2 also relates to Brassica plants with reduced Glucosinolate content. There are Brassica seeds with a reduced Glucosinolate content of a maximum of 3.4 mmol per gram of seed produced. These seeds can Particularly suitable as animal feed and are there advantageous because less degradation products of glucosinolates the nutritional value of Reduce animal feed. The reduction of glucosinolates takes place through targeted intervention in their biosynthetic pathway.

The WO 2004/089065 A1 describes a Brassica plant with elevated glucosinolate Salary sought because of the anticancerogenic properties of glucosinolates becomes. Disclosed is a method of making Brassica plants with elevated Glucosinolate content in edible plant parts, such as Broccoli, cauliflower and cabbage. For the production of glucosinolates all aboveground plant parts harvested, destroyed and mechanically and enzymatically open minded. A disadvantage is the low glucosinolate content in aboveground plant parts of Brassica plants.

The FR 2 819 376 A1 Brassica describes plants with an increased content of glucosinolates. The plants are created by targeted crossing and breeding. For the isolation of the glucosinolates the plants are harvested, destroyed and opened up. A disadvantage of this method is the destruction of the starting plant, so that always new plant material must be provided.

WO 99/52345 A1 likewise describes a method for producing a Brassica plant with an increased content of anticancerogenic glucosinolate derivatives. The glucosinolates are freeze-dried gained material. A particular disadvantage is that the glucosinolate content in aboveground plant parts of Brassica plants is relatively low.

Consequently There is a need for an effective method for obtaining Glucosinolaten from plants of the order Capparales, in which the Starting plant in the recovery of glucosinolates are not destroyed got to. The solution This technical problem is solved by providing the method according to the invention for obtaining at least one glucosinolate from a root exudate solved at least one plant of the order Capparales, the in the claims is specified and which is described in more detail below.

Summary of the invention

• (a) Anzucht der Pflanze in einem erdlosen System;
• (b) Verabreichen einer Nährlösung mit erhöhtem Schwefelgehalt;
• (c) Stimulieren der Glucosinolat Bildung durch Verabreichen mindestens eines Elicitors;
• (d) Gewinnen der Glucosinolate aus Wurzelexsudaten der Pflanze.

The present invention relates to a method for obtaining at least one glucosinolate from a root exudate of at least one plant of the order Capparales, comprising the steps:

• (a) growing the plant in a soilless system;
• (b) administering a nutrient solution with increased sulfur content;
• (c) stimulating glucosinolate production by administering at least one elicitor;
• (d) recovering the glucosinolates from root exudates of the plant.

Besides the invention relates to the method according to the invention glucosinolates and their use as nutritional supplements, as Starting or raw material for Dietary supplements (functional food) or as a pharmaceutical.

Summary the figures

1 shows a graph of glucosinolates obtained from root exudates from beet plants (Brassica rapa) after 30 days of growth in the hydroponic system. 1H - Hoagland solution, 2H - double concentrated Hoagland solution, 2H2S - double concentrated Hoagland solution plus double sulfur, 2H2S-SA ₀ - double concentrated Hoagland solution plus double sulfur plus salicylic acid applied (applied) at the beginning, 2H2S-MJ ₀ - Twofold concentrated Hoagland solution plus twofold sulfur plus methyl jasmonate, applied initially, 2H2S-SA ₂₅ - double concatenated Hoagland solution plus twofold sulfur plus salicylic acid applied (applied) on the 25th day of the experiment.

2 shows the aliphatic, aromatic and indole glucosinolates which were exuded in 30 days from the beet plants (Brassica rapa) grown in the hydroponic system. Dark bars: aliphatic glucosinolates, light gray bars: aromatic glucosinolates; medium gray bars: indol glucosinolates; Meanings of the abbreviations as in 1 ,

3 shows the kinetics of glucosinolate exudation on beet plants (Brassica rapa, var. rapifera, subvar pigmeae teltowiensis) grown in the hydroponic system. Left side, 3a : without elicitors, right side, 3b : with elicitors; C = control (= 2H2S - double concentrated Hoagland solution plus double sulfur, without elicitor treatment); Meaning of the abbreviations as in 1 ,

4 shows the dynamics of the total glucosinolate content in exudates of the beet plant (Brassica rapa, var. rapifera, subvar pigmeae teltowiensis). SA: salicylic acid, MJ: methyl jasmonate.

detailed Description of the invention

• (a) Anzucht der Pflanze in einem erdlosen System;
• (b) Verabreichen einer Nährlösung mit erhöhtem Schwefelgehalt;
• (c) Stimulieren der Glucosinolat Bildung durch Verabreichen mindestens eines Elicitons;
• (d) Gewinnen der Glucosinolate aus Wurzelexsudaten der Pflanze.

The present invention relates to a method for obtaining at least one glucosinolate from a root exudate of at least one plant of the order Capparales, comprising the steps:

• (a) growing the plant in a soilless system;
• (b) administering a nutrient solution with increased sulfur content;
• (c) stimulating glucosinolate production by administering at least one elicitone;
• (d) recovering the glucosinolates from root exudates of the plant.

The present invention is based on the scientific finding that, when cultivated (= cultivating) a plant of the order Capparales in a soil-less (= soil-free) system to which a special nutrient solution with increased sulfur content is administered by stimulation of glucosinolate formation Glucosinolates from root exudates can be obtained in a simple manner and in high concentration via elicitors. In addition, the plant does not need to be destroyed to recover the glucosinolates, so it is a non-destructive process.

Without to be bound or limited by any theory, the inventors take the present invention that the increased sulfur content of the nutrient solution together with the administered elicitors combinatorial the plant to a increased Glucosinolate stimulates formation.

The inventive method has the particular advantage that the plant is used to obtain glucosinolates not harvested and destroyed must become. Rather, you can the glucosinolates continuously from one and the same plant the root exudates are recovered. This is the procedure cost-effective, because no new vegetable raw material - as in the procedures of State of the art - for extraction the glucosinolates must be provided. Next to it is the glucosinolate Expression by using the nutrient solution according to the invention with increased sulfur content together with the elicitors just raised in the roots of the plants. Thereby the extraction of glucosinolates from root exudates becomes particularly interesting, so on elaborate Digestion and isolation of glucosinolates - as in the prior art described - off the aboveground plant parts can be dispensed with.

So As used herein, the term "increased sulfur content" means a sulfur content, which is higher as in a simple Hoagland solution. In particular, a sulfur content of between about 300 mg / l to about 1,000 mg / l an "increased sulfur content".

So As used herein, the term "exudate from roots" or "root exudate" means the predominantly liquid waste products the living plant root. The exudates are from the plant root mainly continuous Lich delivered so that they caught and their ingredients can be concentrated and analyzed.

Of the Term "elicitor" or "elicitors" refers to triggering, specific Substances. "Elicitoren" derives from Lat .: Elegere = read, select from. Elicitors are substances of plant origin that are a defense system or induce a defense response. They induce hypersensitive Responses, lignin formation and protective proteins can. in the Within the scope of the present invention, elicitors in particular salicylic acid (SA) and methyl jasmonate (MJ).

In a preferred embodiment of the invention, the glucosinolates are aliphatic glucosinolates, aromatic glucosinolates and / or indole glucosinolates. All glucosinolates have a β-D thioglucose reduced Group, a sulfonated oxime moiety and a variable side chain, those of amino acids is derived on. The aliphatic glucosinolates are in particular from the amino acids Methionine, alanine, valine, leucine and isoleucine derived. The aromatic Glucosinolates are derived in particular from tyrosine and phenylalanine as the basic amino acid from. The indole glucosinolates can in particular be derived from tryptophan.

In a further embodiment In the invention, the aliphatic glucosinolates are selected from Group consisting of progoitrin, gluconapine, gluconapoleiferin, glucobrassicanapin; sinigrin; Glucoalysin and glucoraphanin; the Indol glucosinolates are selected from the group consisting of 4-hydroxyglucobrassicin, 4-methoxy-glucobrassicin, Neoglucobrassicin and glucobrassicin; and the aromatic glucosinolate is selected from the group consisting of gluconasturtiin and glucotropaeolin.

indole glucosinolates, in particular glucobrassicin and its derivatives are preferred because They can be used particularly well as anti-cancer drugs. From the aromatic glucosinolates gluconasturtiin and glucotropaeolin It is assumed that they are strong anticancerogenic substances, which is why they are also preferred. The aliphatic glucosinolate 4-methylsulfinylbutyl isothiocyanate (as a derivative of glucoalysin) induces the detoxification of enzymes, like quinone reductase and glutathione transferase, both in the suppression of Cancer cells act.

In a further embodiment According to the invention, the roots are primary and / or secondary roots the plant. The primary Root has a rhizodermis, a primary cortex, an endoderm, a pericycle with lateral roots for secondary growth in thickness as well as a periderm, radial vascular bundles as well as a bark around the Central cylinder on. secondary Roots are roots that are the sprout and not the primary root arise from. Both types of roots are particular to the invention Suitable because they have an increased content of beneficial Have glucosinolates.

Next the function as an organ for the nutrient uptake The plant root is also capable of a variety of compounds excrete into the root environment. In one-year plant species will 30 to 60% of the photosynthetically fixed carbon in the roots and the considerable proportion of up to 70% of this carbon can over the root into the rhizosphere be delivered. In addition to the extraction of plant organs, substances also be obtained from exudates of plants. Especially advantageous here are root exudates. For the commercial use can some secondary Metabolites by continuous rhizone secretion from the roots obtained by hydroponic (see below).

In a further embodiment In the present invention, the glucosinolates are derived from the roots of live plants, preferably obtained by rhizone secretion. The Use of roots of living plants has the special Advantage that the starting material can be used continuously. Unlike many prior art methods in which green Plant material is used, mechanically and / or enzymatically unlocked and consumed by glucosinolate production, can the plants are continuously used in the present invention. If one and the same plant over several weeks or months for the extraction of glucosinolates is used, this is special environmentally friendly and economical. Preferably, the method The rhizone secretion used in the desired phytochemicals (= Metabolites) is released into the surrounding medium, which in particular Advantages in the subsequent Purification and recovery of the segregated substance possesses.

In a further embodiment is the plant used in the process according to the invention a plant of the order Capparales. The plant may also be selected from the group consisting of Capparaceae, Brassicaceae, Resedaceae and Tropaeolaceae.

Examples of members of the family Brassicaceae (= cruciferae) are Brassica (cabbage), Sinapis (mustard), Raphanus (radish), Lepidium (cress), Cardaria (arrow cress), Capsella (shepherd's purse), Camelina (camelina), Arabis (goose cress), Cardaminopsis (foam cress), Nasturdium (watercress), Rorippa (Marsh cress), Erysimum (pagan), Arabidopsis (narrow wall) and sisymbrium (Rauke).

When The turnip, Brassica rapa, has proved particularly advantageous. Especially good is the variety Brassica rapa L. var. Teltow.

The inventive method is carried out in a unglooded (= floating) system. It has proven to be particularly advantageous, the floating system as an aerobic or hydroponic system.

When Earth-free systems are particularly suitable for water-based systems, so that the glucosinolates are recovered from the root exudates can. In the aeroponic system, the roots are constantly using humidified air nutrients provided. The nutrient solution will as wet dust at short intervals, usually intervals of a few Minutes, sprayed on. The solution is taken from a storage tank and pumped by a extremely accurate timers in very short cycles of a few seconds reach to a few minutes, administered to the plants.

In an advantageous embodiment is the aeroponic system with at least one spray device Mistake. In this system, the roots in a film or Fog kept from nutrient solution, the Nearly 100% relative humidity ensures drying out to prevent the plant roots. Particularly advantageous, a sprayer of type Dan Fogger 7800, Dan Sprinklers, Kibbutz Dan, Israel become. In particular, the nutrient solution with be pumped a two-stage centrifugal pump. First, will the liquid from the storage tank over pumped a filter on the plant bed, from where they are in the Tank flows back.

It has proven to be particularly advantageous, the nutrient solution constant over the sprayer spray. This procedure is not typical for an aeroponic system, that with a spraying device is equipped for usually with intervals in minute intervals work. However, it has been advantageous due to the root structures proved not to lower the water content by an interval. The pressure of the pump is advantageously 2 bar, which is about half compared to values from the state of Technology is degraded. This low pump pressure has turned out to be advantageous for The roots proved mechanical due to higher pump pressure damaged can be.

In an alternative embodiment the aeroponic system is equipped with at least one defensor. Advantageously, the rotating Defen sensor forms a very fine Moisture mist. The water particles are of the order of magnitude from approx. 5-10 μm. Especially It is advantageous if the pump is switched on every 15 minutes and then join for 30 minutes rest.

The Plants are advantageously hydroponic or aeroponic System cultivated. Borisjuk (1999) suggests plants for exudates in hydroponic systems. The inventors of the present However, the invention has made the observation that an aeroponic System excellently suitable because the amount of water in the system minimized can be. In this way it is possible to compare one from the beginning obtain high exudate concentration, so that the recovery of the Glucosinolates from the exudates over less expensive Cleaning and concentration stages can be done. So that's it aeroponic system particularly economical, because the operating costs are lower.

In an alternative embodiment becomes a hydroponic system with a floating platform, preferably used in the form of a polystyrene plate, the plants anchored directly in the floating platform on the surface the nutrient solution floats. The hydroponic system can be ventilated by means of a compressor, the bubbles through the nutrient solution in a flow rate of about 100 ml / min. realized on each of the approximately ten ventilation tubes.

In a particularly preferred embodiment The invention is the nutrient solution one approx. 1.5 times to approx. 3 times concentrated Hoagland solution, whereby an approximately 2-fold concentrated Hoagland solution is particularly preferred.

In particular, it has been found that the sulfur content of the Hoagland solution must be significantly increased in order to obtain optimal yields of glucosinolates. In particular, the sulfur (S) is added as sulfate (SO ₄ ) directly to the nutrient solution. In particular, the salts MgSO ₄ , ZnSO ₄ and / or CuSO ₄ are suitable. It is also possible to use K ₂ SO ₄ , Na ₂ SO ₄ and / or FeSO ₄ .

• – ca. 610 mg bis ca. 1.230 mg Ca(NO₃)₂;
• – ca. 456,7 mg bis ca. 909,3 mg KNO₃;
• – ca. 23,1 mg bis ca. 46,2 mg Eisenchelat (FeChe);
• – ca. 204 mg bis ca. 408 mg KH₂PO₄;
• – ca. 339,3 mg bis ca. 1.000 mg MgSO₄ × 7 H₂O;
• – ca. 2,1 mg bis ca. 4,3 mg H₃BO₃;
• – ca. 0,7 mg bis ca. 1,3 mg MnCl₂;
• – ca. 0,09 mg bis ca. 0,2 mg ZnSO₄;
• – ca. 0,04 mg bis ca. 0,08 mg CuSO₄; sowie
• – ca. 0,12 mg bis ca. 0,24 mg H₂MoO₄.

In a particularly preferred embodiment, the nutrient solution has the following constituents, based on 1 l:

• About 610 mg to about 1230 mg Ca (NO ₃ ) ₂ ;
• About 456.7 mg to about 909.3 mg KNO ₃ ;
• About 23.1 mg to about 46.2 mg iron chelate (FeChe);
• About 204 mg to about 408 mg KH ₂ PO ₄ ;
• About 339.3 mg to about 1000 mg MgSO ₄ .7H ₂ O;
• About 2.1 mg to about 4.3 mg H ₃ BO ₃ ;
• - about 0.7 mg to about 1.3 mg MnCl ₂ ;
• About 0.09 mg to about 0.2 mg ZnSO ₄ ;
• About 0.04 mg to about 0.08 mg CuSO ₄ ; such as
• About 0.12 mg to about 0.24 mg H ₂ MoO ₄ .

In this embodiment, the amount of MgSO ₄ .7 H ₂ O can be varied in the wide range from about 339.3 mg to about 1000 mg.

• – ca. 820 mg Ca(NO₃)₂;
• – ca. 606,2 mg KNO₃;
• – ca. 30,8 mg FeChe;
• – ca. 272 mg KH₂PO₄;
• – ca. 492,4 mg MgSO₄ × 7 H₂O;
• – ca. 2,9 mg H₃BO₃;
• – ca. 0,8 mg MnCl₂;
• – ca. 0,1 mg ZnSO₄;
• – ca. 0,05 mg CuSO₄; sowie
• – ca. 0,16 mg H₂MoO₄.

In a particularly preferred embodiment, the nutrient solution has the following constituents, based on 1 l:

• - about 820 mg Ca (NO ₃ ) ₂ ;
• - about 606.2 mg KNO ₃ ;
• - about 30.8 mg FeChe;
• - about 272 mg KH ₂ PO ₄ ;
• About 492.4 mg of MgSO ₄ .7H ₂ O;
• - about 2.9 mg H ₃ BO ₃ ;
• - about 0.8 mg MnCl ₂ ;
• - about 0.1 mg ZnSO ₄ ;
• - About 0.05 mg of CuSO ₄ ; such as
• - About 0.16 mg H ₂ MoO ₄ .

This is a 2-fold concentrated Hoagland solution. In a further preferred embodiment, the Hoagland solution 1.5 times to about 3 times concentrated can be supplemented with additional sulfur, advantageously in the form of MgSO ₄ .7H ₂ O. Particularly preferably, the nutrient solution contains about 984.6 mg of MgSO ₄ × 7 H ₂ O, based on 1 liter of liquid.

In a further preferred embodiment Certain advantageous elicitors are used. advantageous Elicitors are glucosinolate-influencing elicitors. The glucosinolate Profiles can by physiological treatment (= application) with / by elicitors to be influenced. Elicitors mimic the effects of stressors how to activate and activate the infestation of pathogens and pests thereby the biochemical defense system of the plant, what too quantitative and qualitative changes in the profile of secondary metabolites the plant leads. Elicitors also influence gene expression and therefore can Used as molecular tools to study the glucosinolate profiles to modify the plant.

Salicylic acid (SA) and methyl jasmonate (MJ) serve as signaling molecules in pathogen attack or mechanical injury to the plant. The application of either salicylic acid and / or methyl jasmonate may increase the glucosinolate content in the plants change a lot, in particular, greatly enrich. These elicitors solve signal cascades made various defense responses such as cell wall reinforcement, induction of PR proteins or the synthesis of secondary metabolites, such as glucosinolates Influence on the expression of genes in the glucosinalate Induce synthesis. Be under PR proteins Pathogenesis related proteins (= pathogenesis related proteins) understood that are undetectable in healthy plants and accumulate only after certain stress conditions. Furthermore can salicylic acid and Methyl jasmonate effective the content of indole, aliphatic and aromatic Increase glucosinolates in species of the order Capparales. The Use of the elicitors salicylic acid and methyl jasmonate is especially for Plants of the species Brassica rapa advantageous, wherein the combined Application especially proven Has.

In a further preferred embodiment the elicitor (s) are administered together with the nutrient solution. In this way contains the nutrient solution both a desired one Concentration of sulfur as well as the elicitors or the elicitor, so that uses only a single nutrient solution which, in addition, can only be applied in a single step becomes. A complicated switching on and off of different nutrient solutions or Interval switching can be avoided in this way.

In an advantageous development comprises the inventive method to obtain glucosinolates from root exudates the additional Step of purifying and concentrating the glucosinolates. Because the glucosinolates from the plant continuously over the Root exudates are released into the surrounding nutrient solution medium, the purification and recovery of glucosinolates is very easy. For example, an HPLC can be used.

Farther The present invention relates to the glucosinolates obtained by the inventive method to be obtained. The glucosinolates are especially useful as dietary supplements, as raw materials for Functional food or as pharmaceuticals suitable.

detailed Description of the Preferred Embodiments

A. Detailed description the figures

1 shows the overall yield of glucosinolates in exudates of beet plants (Brassica rapa). The yield increased from 2.3 mg / plant to 7.8 mg / plant. The abbreviations used are as follows: 1H - Hoagland solution, 2H - double concentrated Hoagland solution 2H2S - double concentrated Hoagland solution plus two fold increased sulfur 2H2S-SA ₀ - two times concentrated Hoagland solution plus two fold increased sulfur plus salicylic acid applied at the beginning, 2H2S-MJ ₀ - Twofold Concentrated Hoagland Solution plus Twofold Sulfur plus methyl jasmonate initially applied, 2H2S-SA ₂₅ - Twofold Concentrated Hoagland Solution plus Twofold Sulfur plus Salicylic Acid applied on the 25th day of the experiment.

The increase in the concentration of nutrient solution caused an increase in glucosinolate content in exudates to 3.8 mg / plant, which represents a 1.8-fold increase. The use of additional sulfur resulted in an increase in glucosinolate content in exudates to 4.6 mg / plant. When the elicitors salicylic acid (SA) and methyl jasmonate (MJ) were co-administered with increased levels of sulfur, the maximum levels of glucosinolate increased to 7.8 mg / plant and 7.3 mg / plant, respectively. Administration of the elicitors at a later stage of plant growth did not cause such significant changes as compared to administration at the beginning of the experiment: 6.6 mg / plant glucosinolates were detected in exudates in which salicylic acid was added on the 25th day of the experiment ( 2H2S-SA ₂₅ ).

2 shows the distribution of aliphatic, aromatic and indole glucosinolates in plants grown for 30 days in the hydroponic system. The increase in the amount of nutrient increased the amount of aliphatic and aromatic glucosinolates while the amount of indol glucosinolates remained approximately the same. Above all, additional sulfur increased the yield of aliphatic glucosinolates. Addition of the elicitors salicylic acid and methyl jasmonate, added at the beginning of plant growth, reduced the level of aliphatic glucosinolates in exudates. The administration of salicylic acid on the 25th day of plant growth did not substantially alter the content of aliphatic glucosinolates.

Gluconasturtiin was the only aromatic glucosinolate identified in beet plants (Brassia rapa). Its content in the exudates was increased 2-fold in a double concentrated Hoagland solution compared to a single concentrated Hoagland solution, and the concentration of the aromatic glucosinolate was reduced with additional administration of sulfur. The addition of salicylic acid and methyl jasmonate at the beginning of the culture (SA ₀ and MJ ₀ ) increases the gluconasturtiin content by 50 to 70% compared to 2H2S. This was also the case with administration of salicylic acid on the 25th day of the experiment.

The level of indol glucosinolates in exudates virtually did not change as the concentration of the Hoagland solution increased, but it increased about 1.5-fold after the administration of additional sulfur. Both elicitors, salicylic acid and methyl jasmonate, given at the beginning of the culture, caused a surprisingly high increase in indole glucosinolates in exudates (3.3-fold more than 2H2S for SA ₀ and 2.8-fold more for MJ ₀ ). Even with the administration of salicylic acid on the 25th day of culture, the content of indol glucosinolates still increased to 1.8-fold compared to 2H2S. The markedly disproportionate increase in indol glucosinolates on administration of salicylic acid or methyl jasmonate is surprising and was not expected by the inventors. Thus, the method according to the invention is particularly suitable for the recovery of high indol glucosinolates, which can be used particularly advantageously in cancer therapy, as a dietary supplement or as a starting or raw material for Funcitonal Food.

3 shows the kinetics of rhizosphere secretion, in particular the kinetics of glucosinolates in exudates of plants grown in the hydroponic system. Exudation kinetics studies showed that the level of exuded glucosinolates in the first 10 days of the experiment was not significantly affected by the nutrient concentration ( 3 - left side). During the next 10 days, the exudate glucosinolate content increased in those plants treated with initial Hoagland solution (1H) as well as those treated with the modified, double concentrated Hoagland solution (2H). Between the 20th and 30th day of the experiment, the glucosinolate content increased for 1H and 2H2S, but it decreased slightly for 2H. The increase in glucosinolate content in exudates following administration of both elicitors was already observed at the beginning of the experiment ( 3 - right side). In the system in which MJ was used, the highest level of exuded glucosinolates was measured immediately after the start of treatment (3.1-fold increased over the untreated variant), while the content rapidly decreased towards the end of the experiment. During the first 10 days after the salicylic acid treatment, the plants exuded 2.4-fold more glucosinolates than the control plants ("C" = 2H2S - double concentrated Hoagland solution plus double sulfur) without Elicitor treatment. The content of glucosinolates exuded in the period between the 10th and the 15th as well as between the 15th and the 20th day of the experiment was 2.1 and 1.9 times more than the control in the same Period. The intensity of exudation decreased for salicylic acid treated plants as well as for methyl jasmonate treated plants toward the end of growth. Thus, the herbal response to elicitor administration is a one-time event that occurs during the first days after treatment and then gradually decreases. Therefore, it is particularly preferred within the scope of the present invention to carry out the elicitors treatment with salicylic acid and / or methyl jasmonate immediately at the beginning of the experiment, it is best to administer the elicitor (s) together with the nutrient solution at the beginning of day zero of the experiment.

4 shows the results of one too 3b parallel experiment, with the difference that the plants were grown in the aeroponic system. In particular, treatment with elicitors has been shown to result in enhancement of glucosinolate rhizone secretion as compared to the control. Examination of exudation kinetics of secondary roots showed that the increase in glucosinolate content in exudates after administration of both elicitors was already observed immediately after the start of the experiment. When treated with methyl jasmonate, the highest level of exuded glucosinolates was measured immediately after treatment, while the glucosinolate content decreased at the end of the experiment (day 30). The glucosinolate content exuded from the plants during the first ten days after treatment was 2.1 mg / plant, which was 3.1 times greater than the values of the control. Between the 10th and the 15th day as well as between the 15th and 20th day, the glucosinolate Ge halt to 1.5 mg / plant and was thus only 2.0 or 1.8 times the value of the control. For the next five days, it was almost at the value of control. During the last five days of plant growth in this treatment, the glucosinolate content reached 1.0 mg / plant, which was 0.2 mg / plant below the control value. During the first ten days after salicylic acid administration, the plants exuded 1.6 mg / plant of glucosinolate, which was 2.4-fold greater than control values. The content of glucosinolates exuded during the period between the 10th and 15th as well as the 15th and 20th day of the experiment was 1.6 mg / plant. These values are 2.2 and 1.9 times the value of the control over the same time period. Between the 20th and 25th day and between the 25th and the 30th day of the experiment, the plants exuded 1.4 mg glucosinolate / plant after salicylic acid treatment, so that the value is 1.2-fold and 1.3, respectively -fold above the control. At the beginning of the experiment, the administration of methyl jasmonate caused a greater increase in glucosinolate content in exudates compared to salicylic acid, which the inventors believe may explain that salicylic acid not only inhibits glucosinolate synthesis but also its degradation by affecting the activity of the enzyme myosinase strengthened.

B. Detailed description the materials used and the results

1. Cultivation the plants

The beet seeds In Grodan rockwool were plant cubes (5 cm wide × 5 cm deep × 5 cm high), the in standard greenhouse Plastic bowls (52 cm wide × 35 cm deep × 7 cm high) were put on. The seeds were half-concentrated Hoagland solution above above irrigated plant systems attached moisture systems. she were pre-germinated until the roots of the seedlings about 2 to 4 cm the bottom of the dice had emerged. This was after about 15 to 20 days with 2 leaves and a root weight of about 3 g of the case. The cubes were subsequently in 10x11 cm intervals in the holes of Polystyrene plates (1 m × 1 m tall, 4.5 cm thick), which are then in the aeroponic or hydroponic Systems were used.

2. Technical Characteristics of the aeroponic and hydroponic systems

in the Within the scope of the present invention, both aeroponic and also used hydroponic systems. These ungloved (= ungrounded) Water-based systems have the advantage that the nutrients dissolved directly in water and the plants are the nutrients can record at any time. Due to the ubiquitous Availability the desired amount of nutrients the plant can supply the individually required amount at any time directly from the plant Take root. Furthermore The floating systems are beneficial because of the pH and the nutritional value the water is easily measured and the desired Values can be maintained. The water-based systems increase plant growth and growth Yield per area across from the earth-containing sys- tems (= earth systems) clearly. Furthermore is the risk of pest infestation as well as the occurrence of diseases on the plants significantly reduced. Furthermore deleted the casting the plants according to a pouring plan. In the present experiments, in addition to the hydroponic Systems also used aeroponic systems with the goal of quantity of the water used in the system maximally minimize what the Exudate extraction makes it easier. In this way, the aeroponic System can be operated very economically. Within the scope of the invention Three different systems were installed, namely an aeroponic system with a spraying device, an aeroponic system with a mist sprayer or a defensor and a hydroponic system. The spraying device of the aeroponic Systems was designed in the specific example as spray nozzles.

• 1H – Hoagland Lösung;
• 1,5H – 1.5-fache Konzentration der Hoagland Lösung;
• 2H – doppelte Konzentration der Hoagland Lösung;
• 2S – doppelte Schwefel-Konzentration;
• SA₀ – Salicylsäure, appliziert am 1. Tag;
• MJ – Methyljasmonat, appliziert am 1. Tag;
• SA₁₅ – Salicylsäure, appliziert am 15. Tag;
• SA₂₀ – Salicylsäure, appliziert am 20. Tag;
• SA₂₅ – Salicylsäure, appliziert am 25. Tag.

The experiments carried out in the aeroponic or hydroponic system are shown schematically in Table 1. Table 1 Schematic representation of the experiments

• 1H - Hoagland solution;
• 1.5H - 1.5-fold concentration of the Hoagland solution;
• 2H - double concentration of Hoagland solution;
• 2S - double sulfur concentration;
• SA ₀ - salicylic acid, applied on the 1st day;
• MJ - methyl jasmonate, applied on the 1st day;
• SA ₁₅ - Salicylic acid, applied on the 15th day;
• SA ₂₀ - salicylic acid, applied on the 20th day;
• SA ₂₅ - salicylic acid, applied on the 25th day.

2.1 Aeroponic system

In In aeroponic systems, the plants are moisturized with nutrients Air supplied. The roots are in the growth chamber, where they are at short intervals, for usually Intervals of a few minutes were sprayed with the moisture mist of the nutrient solution. The solution was taken directly from their storage tank via a pump and sprayed on the plant roots. The pump was timely accurate controlled so that the spray cycles exactly from a few seconds to a few minutes were enough.

2.2.1 Aeroponic system with spray nozzles

in the aeroponic system equipped with spray nozzles the roots are kept in a mist of the nutrient solution. The fog of the Nutrient solution contains one relative humidity of nearly 100% and effectively prevents dehydration of plant roots. In the context of the invention, the aeroponic system with spray nozzles of the Type Dan Fogger 7800, Dan Sprinklers, Kibbutz Dan Israel. The nutrient solution was with the two-stage centrifugal pump CH-60 (Model A-CVBV, Grundfos, Erkrath, Israel) from a storage tank over a Bitron 15 filter (type G15T8, Oase, Berlin, Germany) too pumped to the breeding bed. From there, the excess nutrient solution could be returned to the storage tank. In the context of the present invention, the aeroponic system with spray nozzles continuously used, i. the nutrient solution became constant and continuous without intervals on the plant roots sprayed. This use of the aeroponic system is uncommon, but the Inventors of the present invention have found that the beet root very sensitive to responds to an occurring lack of water. The pressure of the pump was 2 bar, wherein in the prior art usually 4 bar are used. However, the inventors of the present invention have found that the pressure used in the prior art to a mechanical damage the root of the beet leads.

2.1.2 Aeroponic system with Defensor

The in the context of the present invention used aeroponic system with Defensor contained the Defensor type 505, WMH Walter Meier Holding, Pfäffikon, Germany. The rotating or rotating atomizer formed finest fog from nutrient solution. The Size of the water particles in the sprayed nutrient mist is about 5 to 10 microns. A timer switched the pump (Type 505, Axair AG, Pfäffikon, Germany) for 15 Minutes at a 30-minute Interval. The Defensor (Type 505) itself contains a rotating gyroscope (Centrifuge), a room where dry air is located rotating plate as well as a moisture ring that moisturizes Air as finely atomized Moisture mist (aerosol) emits to the outside.

2.2 Hydroponic system

in the hydroponic system with floating platform and a ventilation system the plants were located directly in the floating platform, the on the surface the nutrient solution floated. The hydroponic system was powered by a compressor (model 40A-10011, Hiblo, Germany). The compressor blew air through the nutrient solution at a flow rate from about 100 ml / min. A total of 10 ventilation tubes were provided, all operated at the same time.

3. greenhouse conditions for the plant growth

3.1 light

The plants were grown with an exposure of 12 hours corresponding to the daytime from 8 o'clock in the morning to 8 o'clock in the evening and hv 400 μmol / m ² s. Quantum sensors (Model Li-190 SZ, LI-COR Lincoln, Nebraska, USA) were used to measure the photosynthetic or active radiation. When the natural radiation outside the greenhouse exceeded 450 μmol / m ² s, the plants were shaded by louvers placed above them. As an artificial light source SRG 140 (Philips, Germany) lamps were used which promote plant growth with light that has a natural spectrum. The lamps were mounted 1.5 m above the growth or cultivation systems and covered about 2 m ² of surface area per lamp.

3.2 temperature

The Air temperature in the greenhouse was 16 ° C while of the day and 12 ° C while the night. In the root zone exceeded the temperature the air temperature around 1 to 2 ° C. The greenhouse was ventilated, though the temperatures exceeded the above values.

3.3 Humidity

The Humidity in the greenhouse reached 60%. It was created by the fog system HNS (Type 7, Osberma, Engelskirchen, Germany) with HNS nozzles (type 07.1, Osberma, Engelskirchen, Germany). The temperature and humidity in the Glasshouse were with air vents, Type 77001 (Henrich Zeiseniss Water Technology, Herrsching, Germany) determined and recorded. The temperature in the root zones within The aeroponic systems were measured with the Psychrometer PT 100 (Typ WTE 10, Geraberg GmbH, Geraberg, Germany). The temperature, the exposure and the humidity conditions in the growth chamber were equipped with RAM CC 600 equipment (type DT 500, Ltd Rowville, Victoria, Australia). The measurements were performed once a minute, and the average mean was saved once an hour. The whole above Conditions became constant during of the vegetative period.

4. Production the exudate samples

Four replicate samples each of 1 L of sample solution containing plant root exudates were taken from each system every 10 days for the first experiment and every 5 days beginning on day 10 for the second and third experiments. During the sampling, the roots were additionally washed with the solution of the growth or cultivation system using the compressor (Type 3590, Agrotop GmbH, Obertraubling, Germany). 20 μM / l AgNO _{3 was} added to the sample solution in order to prevent the destruction of the glucosinolates by the enzyme myrosinase, which is also released into the growth medium. AgNO _{3 has been shown to} stop the activity of myrosinase. After Sampling, the nutrient solution was renewed in each system. The samples were first filtered using paper filters (N 604, Schleicher and Schuell, Dassel, Germany). The exudate samples were processed by scaleup ultrafiltration and reverse osmosis in the tangential flow filtration system (ProScale System with Helicon-RO-4 spiral wrap modules containing a Nanomax-95 membrane, obtained from Millipore GmbH, Eschborn, Germany). The samples were then transferred to 600 ml glass bottles (Christ, Osterode am Harz, Germany), frozen at -28 ° C and freeze-dried.

links having a molecular weight of 250 to 2000 Da were selected and concentrated and monovalent salts were removed from the solution. The process carried out in the context of the invention for the production the exudate samples needed less steps than the conventional one Process for the production of above-ground plant material. The classical manufacturing processes include the steps of cleaning, Freezing and freeze-drying, grinding and extracting the needed Links. In the context of the invention was an additional Process step in the process for preparing the exudate samples for the HPLC introduced. The additional step includes prefiltration and the concentration of the liquid. Nevertheless, the conventional Steps of grinding and extracting the plant material avoided, so that the inventive method over the Prior art has advantages.

5. Glucosinolate Extraction and determination by HPLC

For the determination the glucosinolate by HPLC became the Krumbein HPLC method et al. (2005). Freeze-dried specimen that consisted of dried substances of 1 l of the solution with exudates, was heated and at 75 ° C for 1 min. incubated with 4 ml of a methanol / water mixture (v / v = 7: 3, t = 70 ° C) extracted and after adding of 1 ml of 0.4 M barium acetate at 400 rpm for 10 min. centrifuged. Of the Residue was extracted twice more with 30 ml of methanol / water mixture (v / v = 7: 3, T = 70 ° C). The extracted material was combined and made up to 10 ml. From that 5 ml of the extract was applied to a 250 μl DEA-Sephadex A-25 ion exchanger (with acetic acid activated, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) applied and rinsed with 10 ml of double distilled water. When next 250 μl a purified solution of arylsulfatase (Boehringer-Mannheim GmbH, Mannheim, Germany) and for 12 h on the exchanger before desulfo compounds are doubly distilled with 5 ml Water were eluted. The desulfoglucosinolate analysis was performed by HPLC (Merck HPLC pump L-7100, DAD detector L-7455, automatic Sample Processing AS-7200 and HPLC Manager Software D-7000) Using a Spherisorb ODS2 column (5 μm, 250 × 4 mm). It became a gradient of 0-20 % Acetonitrile in water for 2 to 34 min., Followed by 20% acetonitrile in water until the 40th Minute and then 100% acetonitrile for 10 min. elected until the 50th minute. The provision was included a flow rate of 1.3 ml / min and one wavelengths performed by 229 nm. Sinigrin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and glucotropaeolin (Appli. Chem GmbH, Darmstadt, Germany) were used as standards.

The Individual glucosinolates were compared by comparing their retention times (Retention times) with individual glucosinolates in the rapeseed oilseeds (BCR-190C and BCR-367R) used as standard reference materials were identified. The glucosinolate content was determined using Sinigrin as internal standard and the response factor (response factor) calculated each compound compared to Sinigrin. The determination the glucosinolates were each done twice, and the concentrations The glucosinolates were expressed as mg exuded glucosinolates per plant expressed. The data show the dynamics of glucosinolate content in both Plants as well as in exudates during of the experiment as well as in exudates lasting during the 30 days Experiments were collected.

The in turnips (Brassia rapa) occurring glucosinolates are shown in Table 2.

Table 2: Glucosinolates in beet

Cex:

Gehalt an einzelnen Desulfoglucosinolaten in Exsudaten pro mg Pflanze

Vol:

Volumen der Lösung im System in l

Npl:

Menge der Pflanzen im System pro Pflanze

C:

Gehalt an einzelnen Deslufoglucosinolaten in der Lösung aus dem Gesamtsystem in mg, wobei

S:

Quadrat der Peakfläche von Desulfoglucosinolaten

Sis:

Quadrat der Peakfläche des internen Standards

RF:

Response Faktor von Desulfoglucosinolat

RFis:

Response Faktor des internen Standards

Nis:

Menge des internen Standards in mg/l

Vol':

Probenvolumen in l.

The respective glucosinolate content was calculated over the respective peak areas using sinigrin as the internal standard and the response factor of each compound relative to sinigrin. The content of glucosinolates in exudates of a plant was calculated by the following formula:

Cex:

Content of individual desulfoglucosinolates in exudates per mg of plant

vol:

Volume of solution in the system in l

nPL:

Quantity of plants in the system per plant

C:

Content of individual Deslufoglucosinolaten in the solution from the total system in mg, where

S:

Square the peak area of desulfoglucosinolates

Sis:

Square of the peak area of the internal standard

RF:

Response factor of desulfoglucosinolate

RFI:

Response factor of the internal standard

Nis:

Amount of internal standard in mg / l

Vol ':

Sample volume in l.

The fresh weight, the dried material, the total weight and the individual glucosinolate contents in plants and exudates were calculated using descriptive statistics and analysis of variance. The mean comparison was performed according to the Tukey's Honest Significant Difference Test (HSD, significance level α = 0.05). All statistical analysis was done with Statistica ^TM for Windows ^TM (Version 6.1, Statstoft Inc.).

7. Plant growth with different nutrient supply

• 1H: Ausgangs Hoagland Lösung (1-fach)
• 2H: 2-fach konzentrierte Hoagland Lösung
• 2H2S:2-fach konzentrierte Hoagland Lösung plus zweifach gesteigerte Schwefelkonzentration

The inventors have first selected a nutrient solution which ensures sufficient growth of the plants of Brassica rapa. This solution was modified according to the invention to increase the glucosinolate content in plants as well as in exudates. The Hoagland solution was chosen as the starting nutrient solution for the growth of beet (Brassica rapa). Table 3 shows the ingredients of the Hoagland solution and its modifications. Table 3: Hoagland solution and its modifications

• 1H: output Hoagland solution (1-fold)
• 2H: 2-fold concentrated Hoagland solution
• 2H2S: 2-fold concentrated Hoagland solution plus twice the sulfur concentration

The Salts can as two individual solutions (A and B) are prepared and stored. Immediately before use become the two solutions united.

Of the nutrient content the output Hoagland solution has been raised, because the inventors of turnips (Brassica rapa), which were grown in Hoagland solution, one yellowing the leaves (Chlorosis). Because of this fact, the nutrient concentration in the Hoagland solution elevated, Care was taken to ensure that a balance between the individual components remained. The beet plants needed high Amounts of sulfur in a balanced ratio to nitrogen. This heightened Sulfur requirement of plants of the family Brassicaceae was surprising and was observed and analyzed within the scope of this invention. The inventors therefore have a 2-fold concentrated Hoagland solution, the contains a 2-fold increased sulfur concentration and used.

8. Use of elicitors

When Elicitors were salicylic acid (SA) and methyl jasmonate (MJ). Salicylic acid was in a concentration of 1.1 mg / L (= 110.0 mg / L), while methyl jasmonate in one Concentration of 100 μg / l has been used. Both substances were obtained from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany. The elicitors became too the nutrient solution too Beginning of plant growth in aeroponic systems, although shown in previous studies (Van Dam et al., 2003) was that treating the leaves with elicitors the glucosinolate Salary in the shoot, but not increased in the roots.

9. Results

9.1 Total glucosinolates

The total glucosinolate content as well as the content of individual aliphatic, aromatic and indole glucosinolates was quantified in the leaves, primary roots, secondary roots and exudates of plants of the order Capparales, in particular beet plants (Brassica rapa). Progoitrin, gluconapine, gluconapoleiferin and glucobrasscianapine were determined as representatives of the aliphatic glucosinolates. Gluconasturtiin was determined as a representative of the aromatic glucosinolates. Prominent representatives of indol glucosinolates were 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin and glucobrassicin. The total content of glucosinolates in plants and their exudates after 30 days was 22.1 mg / plant for the control treatment, and was increased by about one-third for treatment with the elicited salicylic acid (Sa) and methyl jasmonate (MJ), respectively the values were 30.1 and 30.7 mg / plant, respectively. The glucosinolate content found in exudates increased from 4.6 mg / plant (control) to 7.8 and 7.3 mg / plant for salicylic acid and methyl jasmonate treatments, respectively at. The inventors believe that the increase in glucosinolates in the exudates is due to the release of plant defense products triggered by the addition of the elicitor.

• Aliphatische Glucosinolate: Gluconapin, Progoitrin, Glucobrassicanapin, Gluconapoleiferin;
• Aromatische Glucosinolate: Gluconasturtiin
• Indolglucosinolate: Glucobrassicin, 4-Hydroxy-Glucobrassicin, 4-Methoxy-Glucobrassicin, Neoglucobrassicin

Treatment with salicylic acid and methyl jasmonate increased the glucosinolate content in secondary roots to 10.2 and 7.8 mg / plant, respectively, an increase of 2.1 and 1.8-fold over the control treatment (4.8 mg / Plant) corresponded. Methyl jasmonate was able to increase the glucosinolate content in leaves up to 6.1 mg / plant (1.7-fold), whereas the salicylic acid treatment resulted in a lower level (3.6 mg / plant) than in the control. The inventors believe that these results can be explained by increased expression of genes involved in glucosinolate synthesis as well as by the mobilization of the shoot glucosinolates by phloem transport into the roots. The changes in the metabolic profiles of the aliphatic, aromatic and indole glucosinolates caused by salicylic acid and methyl jasmonate treatments were more significant in the exudates than in the plant tissues. The inventors believe that this is due to elicitor-induced rhizosphere secretion, which is due to de novo synthesis of the secondary metabolites and non-elicitor-induced loss from the root tissues. The results are summarized in Table 4. Table 4 Glucosinolates in plants and exudates (mg / plant)

• Aliphatic glucosinolates: gluconapine, progoitrin, glucobrassicanapine, gluconapoleiferin;
• Aromatic glucosinolates: gluconasturtiin
• Indol Glucosinolates: Glucobrassicin, 4-Hydroxyglucobrassicin, 4-Methoxyglucobrassicin, Neoglucobrassicin

The differences were determined for each treatment, in particular for the control of the SA and MJ treatment, as well as for each plant organ and for the exudates. The values followed by the same letter (a, b, c) are not significantly different.
nnwb: not detectable

9.2 Aliphatic glucosinolates

The aliphatic glucosinolates were the major class of glucosinolates in beet plants (Brassica rapa) and exudates of the control at 12.2 mg / plant, while the levels of the aromatic and indol glucosinolates were 3.0 and 6.6 mg / plant, respectively (Table 4). , The present results show that Brassica rapa has a glucosinolate profile comprising butenyl and pentenyl glucosinolates and their hydroxylated homologues. Progoitrin is the most commonly identified in the turnip aliphatic Glucosi nolat. Its concentration reached 4.6 mg / plant in plants and exudates for control treatment (Table 5). The administration of salicylic acid and methyl jasmonate lowered the progoitrin content in plants and exudates to 2.5 or 2.6 mg / plant. The progoitrin content found in exudates after a methyl jasmonate treatment was equal to the control treatment, 0.6 mg / plant, while it dropped to 0.4 mg / plant after salicylic acid treatment. In the plants the progoitrin content fell mainly at the expense of the primary roots, which meant that its content in the control was 2.5 mg / plant, compared to 1.2 mg / plant for the salicylic acid treatment and 1.4 mg / Plant for the methyl jasmonate treatment.

the same changes were also for Gluconapine observed where the total content in plants and in exudates 1.7 mg / plant in the control compared to 1.1 mg / plant for the salicylic acid treatment and 1.3 mg / plant for the methyl jasmonate treatment reached (Table 5).

Of the Content of gluconapoleiferin and glucobrassicanapine in both plants and exudates were 2.0 and 1.2 mg / plant for the control, respectively. These values were by a salicylic acid Treatment not affected. However, a methyl jasmonate increased Treatment of the content of gluconapoleiferin to 1.3 mg / plant, while the Methyl jasmonate treatment the glucobrassicanapine content to 2.4 increased mg / plant in the plants and the exudates. The biggest increase was in the leaves observed where gluconapoleiferin has a value of 0.9 mg / plant and Glucobrassicanapine has a content of 1.1 mg / plant, respectively the methyl jasmonate administration compared to 0.6 or 0.1 mg / plant for the Reached control. The results of analyzes of aliphatic Glucosinolates are shown in Table 5.

Table 5: Aliphatic glucosinolates in plants and exudates (mg / plant)

The Differences were made for each treatment determines, in particular the control, salicylic acid (SA) and Methyl jasmonate (MJ), each for every plant organ as well as the exudates. The values, respectively followed by the same letter (a, b, c) are not significant different.

• Butenylglucosinolate: Gluconapin und Progoitrin
• Pentenylglucosinolate: Glucobrassicanapin und Gluconapoleiferin
• Hydroxylalkenylglucosinolate: Progoitrin und Gluconapoleiferin
• Alkenylglucosinolate: Glucobrassicanapin und Gluconapin

The Brassica rapa Gls-elong genes regulate the production of elongase, resulting in chain extension of synthesized glucosinolates. This could explain the ratio of butenyl to pentenyl glucosinolates in plants and exudates of Brassica rapa of 2: 1 in the control treatments (6.2: 3.1 mg / plant) (see Table 6). However, the ratio of butenyl to pentenyl glucosinolates in plants and exudates changed dramatically under the influence of salicylic acid and methyl jasmonate. The ratio was 1.1: 1 (3.7: 3.3 mg / plant) for salicylic acid and 0.8: 1 (3.9: 4.7 mg / plant) for methyl jasmonate. The inventors believe that the elicitors affect expression of the Gls-elong genes after administration of the elicitors. The alkenyl glucosinolates gluconapine and glucobrassicanapine undergo hydroxylation and thus conversion to the hydroxyalkenyl glucosinolates progoitrin and gluconapoleiferin. The ratio of hydroxyalkenyl glucosinolates to alkenyl glucosinolates was 2.3: 1 for the control treatments (6.6: 2.9 mg / plant). However, the ratio changed to 1.8: 1 (4.5: 2.5 mg / plant) in plants and exudates after salicylic acid treatment and to 1.4: 1 (4.9: 3.6 mg / plant) in plants and exudates after a methyl jasmonate treatment. The inventors assume that the Elicitoren the Her effecting under-regulation of Gls-oh genes that reduced the levels of alkenylglucosinolates for salicylic acid and methyl jasmonate treatments and increased the hydroxyalkenyl glucosinolates after methyl jasmonate treatment. The results are summarized in Table 6. Table 6: Relationship between the subclasses of aliphatic glucosinolates in plants and exudates (mg / plant)

• Butenyl glucosinolates: gluconapine and progoitrin
• Pentenylglucosinolates: glucobrassicanapine and gluconapoleiferin
• Hydroxoalkenyl glucosinolates: progoitrin and gluconapoleiferin
• Alkenyl glucosinolates: glucobrassicanapine and gluconapine

9.3 Aromatic glucosinolates

gluconasturtiin was the only aromatic glucosinolate that was detectable in Brassica rapa is. His salary reached levels in controls and exudates of 3.0 mg / plant and rose to 7.8 (2.6-fold) after salicylic acid treatment and even to 9.1 mg / plant (3.0-fold) after a methyl jasmonate Treatment (Table 4). This increase is especially in primary roots the plants considerable. There the content of the aromatic rose Glucosinolate gluconasturtiin of values of 0.6 mg / plant 3.4 mg / plant after salicylic acid treatment and at 3.8 mg / plant after a methyl jasmonate treatment. In the exudates In the control treatment, the gluconasturtiin content was 1.1 mg / plant and rose to 1.8 and 1.9 mg / plant after administration of salicylic acid or methyl jasmonate. This means a 60 or 70% increase in the Compared to the control.

Interestingly, were aromatic glucosinolates in the leaves only after administration the elicitors detectable (SA: 0.4 mg / plant and MJ: 0.6 mg / plant). This observation leaves The inventors suspect that the elicitors may be the cause of glucosinolates in organs in which they were not present before. The Inventors further suggest that the increase in aromatic glucosinolates in all plant parts after the administration of the elicitors over the Induction of CYP79A2 explained which converts phenylalanine into aromatic aldoxime and even in leaves and Roots is expressed.

9.4 Indol Glucosinolates

In In this experiment, the total content of indole glucosinolate in the plants 5.1 mg / plant and 1.5 mg / plant in the exudates for the Control treatment (Table 4). Both elicitors caused a rise of indole glucosinolate content in plants (SA: 9.3 mg / plant (2.2-fold Increase) and MJ: 7.7 mg / plant (1.8-fold increase)). The effect was most evident in the secondary roots (SA: 6.3 mg / plant (2.7-fold increase) and MJ: 3.9 mg / plant (1.7-fold increase)) and in the exudates (SA: 4.9 mg / plant (3.3-fold increase) and MJ: 4.2 mg / plant (2.8-fold Increase)) (Table 4).

The indole glucosinolates found in Brassica rapa are 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin. These are derivatives of glucobrassicin after undergoing hydroxylation and methoxylation. In the experiments conducted in the present invention, salicylic acid and methyl jasmonate administration also induced a specific increase in indol glucosinolate content (Table 7). However, it is important to note that the elicitors affected the various indole glucosinolates to different extents. Salicylic acid increased the levels of glucobrassicin, 4-methoxy-glucobrassicin and neoglucobrassicin in plants much more than methyl jasmonate. In the plants and the exudates for the control, 1.3 mg / plant glucobrassicin and 3.3 mg / plant neoglucobrassicin were observed. Surprisingly, after the elicitor treatments were observed much higher amounts, namely 3.4 mg / plant glucobrassicin and 2.2 mg / plant 4-methoxy-glucobrassicin after salicylic acid treatment and 2.6 mg / plant glucobrassicin and 1.3 mg / plant 4-methoxy-glucobrassicin after methyl jasmonate treatment. The increase in glucobrassicin content occurred mainly in the secondary roots. The values were 0.3 mg / plant for the control, 2.2 mg / plant for the salicylic acid treatment and 0.9 mg / plant for the methyl jasmonate treatment. In the exudates, the glucobrassicin content was increased from 0.4 mg / plant (control) to 0.7 mg / plant (SA treatment) and 0.9 mg / plant (MJ treatment).

In Brassica rapa was neoglucobrassicin the most prevalent indol glucosinolate. Its content reached in the plants 2.6 mg / plant while it in the exudates in an amount of 0.7 mg / plant. It was also most influenced by the two elicitors used. In plants treated with salicylic acid his salary rose to 3.4 mg / plant, while his salary was lower than methyl jasmonate Treatment even to 3.6 mg / plant, each compared to the control rise. This compares to an increase of 31% and 40%, respectively for control. In the exudates were treated for salicylic acid treatment also big climbs observed. The absolute value was 3.4 mg / plant (4.9-fold Rise). After methyl jasmonate treatment, the value was 2.4 mg / plant, which was a 3.4-fold increase. The highest increase Neoglucobrassicin after the administration of elicitors was in the secondary Roots observed where the content was 2.3 mg / plant after salicylic acid treatment and 2.6 mg / plant by methyl jasmonate compared to 1.7 mg / plant for the Control reached. There was no methoxy-glucobrassicin in the leaves and the primary Roots of untreated plants demonstrated, but the administration led by elicitors to an accumulation of 0.1 and 0.3 mg / plant respectively in the Scroll and the primary Roots after a salicylic acid Treatment and 0.1 or 0.1 mg / plant in the leaves and the primary roots after a methyl jasmonate treatment. The inventors assume that this finding is due to the induction of glucosinolate synthesis in the Scroll or by transporting these glucosinolates from the roots in the phloem was effected. The results are summarized in Table 7.

Table 7: Indol glucosinolates in plants and exudates (mg / plant)

The differences were determined for each treatment, in particular the control, salicylic acid (SA) and methyl jasmonate (MJ), for each plant organ as well as the exudates. The values, each followed by the same letter (a, b, c), are not significantly different.
nnwb = undetectable

9.5 Application of elicitors

The use of both elicitors greatly increased the levels of aromatic and indole glucosinolates (Table 8). The roots of untreated plants exuded 0.1 mg / plant gluconasturtiin during the first 10 days after treatment, while the value after salicylic acid treatment was 0.3 mg / plant and after the methyl jasmonate treatment reached 0.5 mg / plant. The increase in indolglucosinolate content to 1.2 mg / plant after salicylic acid treatment and to 1.3 mg / plant after methyl jasmonate treatment was particularly pronounced, while the control was 0.2 mg / plant. However, the content of the exuded aliphatic glucosinolates fell after the treatments with the elicitors compared to the control. For salicylic acid treatment, it reached 0.1 mg / plant, and for methyl jasmonate treatment, it reached 0.3 mg / plant, while for control, it was 0.5 mg / plant. Therefore, the inventors conclude that the elicitor administration affects the individual glucosinolates differently (= differentiated) in exudates as well as in plant tissues. This provides the ability to target the profiles of glucosinolates by elicitor administration to increase the yield of glucosinolates with the desired health benefits. The results are shown in Table 8.

Table 8: Glucosinolate content in exudates on the 10th day of the experiment (mg / plant)

The Differences were made for each experiment determines, in particular the control, salicylic acid (SA) and Methyl jasmonate (MJ), each for every plant organ as well as the exudates. The values, respectively followed by the same letter (a, b, c) are not significant different.

10. Summary

investigations which were carried out in the context of the present invention, showed that the use of salicylic acid and methyl jasmonate one strong influence on the glucosinolate content in the plant organs as well as in the roots of the test plants (Brassica rapa). In particular, the levels of the aromatic and the in dolglucosinolates were which both have anticarcinogenic properties, greatly increased. Both Elicitors reduced the content of aliphatic glucosinolates, especially in the primary Roots and in the exudates. The inventors of the present invention to explain the different effects of the elicitors on the individual glucosinolates by their biological function within the defense reaction, caused by interaction between the plant and the herbivore or the plant and the microorganism takes place. A salicylic acid and / or Methyl jasmonate treatment stimulated the rhizone secretion of glucosinolates. The plant's response to the administration of the elicitors occurred mainly during the the first days after the treatment, followed by a gradual decrease joined. Thus, the present invention, in particular the inventive method suitable, desired Produce targeted glucosinolates in high concentration. These Glucosinolates can subsequently because of their beneficial health-promoting, anti-carcinogenic and other properties as phytochemicals (= metabolites), Pharmaceuticals, dietary supplements or raw materials for Functional food can be used.

References

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Claims (20)

Method for obtaining at least one Glucosinolate from a root exudate of at least one plant of the Order Capparales, comprising the steps: (a) cultivation of Plant in a soilless system; (b) administering a nutrient solution with increased sulfur; (c) stimulating glucosinolate formation by Administering at least one elicitor; (d) recovering the glucosinolates from root exudates of the plant. The method of claim 1, wherein the glucosinolates aliphatic glucosinolates, aromatic glucosinolates and / or indol glucosinolates are. The method of claim 2, wherein the aliphatic Glucosinolates selected are from the group consisting of progoitrin, gluconapine, gluconapoleiferin, Glucobrassicanapine, sinigrin, glucoalysin and glucoraphanin; the Indole glucosinolates selected are from the group consisting of 4-hydroxy-glucobrassicin, 4-methoxy-glucobrassicin, Neoglucobrassicin and glucobrassicin; and the aromatic glucosinolates selected are from the group consisting of gluconasturtiin and glucotropaeolin is. Method according to one of the preceding claims, wherein the roots primary and / or secondary roots are. Method according to one of the preceding claims, wherein the glucosinolates from the roots of live plants, preferably be obtained by rhizosphere secretion. Method according to one of the preceding claims, wherein the plant of the order Capparales is selected from the group consisting from Capparaceae, Brassicaceae, Resedaceae and Tropaeolaceae. Method according to one of the preceding claims, wherein the floating system is an aeroponic or hydroponic system is. The method of claim 8, wherein the aeroponic System with at least one spray device and / or equipped with a defensor. The method of claim 8, wherein the hydroponic System is a floating hydroponic system. Method according to one of the preceding claims, wherein the nutrient solution one about 1.5 times to about 3 times concentrated Hoagland solution, preferably is an approximately 2-fold concentrated Hoagland solution. Method according to one of the preceding claims, wherein the sulfur (S) as sulfate (SO ₄ ), in particular as MgSO ₄ , ZnSO ₄ and / or CuSO ₄ . Method according to one of the preceding claims, wherein the nutrient solution comprises the following constituents, based on 1 l: - about 610 mg to about 1230 mg Ca (NO ₃ ) ₂ ; About 456.7 mg to about 909.3 mg KNO ₃ ; About 23.1 mg to about 46.2 mg iron chelate (FeChe); About 204 mg to about 408 mg KH ₂ PO ₄ ; About 339.3 mg to about 1000 mg MgSO ₄ .7H ₂ O; About 2.1 mg to about 4.3 mg H ₃ BO ₃ ; - about 0.7 mg to about 1.3 mg MnCl ₂ ; About 0.09 mg to about 0.2 mg ZnSO ₄ ; About 0.04 mg to about 0.08 mg CuSO ₄ ; and - about 0.12 mg to about 0.24 mg H ₂ MoO ₄ . The method of claim 12, wherein the nutrient solution comprises the following components, based on 1 l: - about 820 mg Ca (NO ₃ ) ₂ ; - about 606.2 mg KNO ₃ ; - about 30.8 mg FeChe; - about 272 mg KH ₂ PO ₄ ; About 492.4 mg of MgSO ₄ .7H ₂ O; - about 2.9 mg H ₃ BO ₃ ; - about 0.8 mg MnCl ₂ ; - about 0.1 mg ZnSO ₄ ; - About 0.05 mg of CuSO ₄ ; and - about 0.16 mg H ₂ MoO ₄ . The method of any one of claims 11 to 13, wherein the nutrient solution comprises about 984.6 mg MgSO ₄ .7H ₂ O. Method according to one of the preceding claims, wherein the elicitor selected is from the group consisting of methyl jasmonate, salicylic acid, jasmonic acid, chitosan and KCN. Method according to one of the preceding claims, wherein the elicitors, in particular methyl jasmonate and salicylic acid, together be administered. Method according to one of the preceding claims, wherein the elicitor (s) are administered together with the nutrient solution. Method according to one of the preceding claims, wherein the procedure additionally the step comprises: (e) purifying and concentrating the glucosinolates. Glucosinolates obtained by a method one of the preceding claims. Use of the glucosinolates according to claim 19 nutritional supplements or as a pharmaceutical.