DE3728506A1 - Process for obtaining alkaloids - Google Patents

Abstract

The present invention describes an effective process for obtaining tropane alkaloids, such as atropine and scopolamine, by culturing fibrous roots of plants of the family Solanaceae. The process consists of the following working steps: Transformation of the cells of the plants of the family Solanaceae by an Ri plasmid occurring in Agrobacterium rhizogenes, culturing of the fibrous roots and extraction of the alkaloids which the fibrous roots discharge into the liquid or solid medium.

Description

The invention relates to a method for producing alkaloid according to the preamble of claim 1. In particular The present invention relates to an effective method for alkaloids, i.e. H. biogenic and medical substances, in particular tropane alkaloids that are caused by Plants of the Solanaceae family are synthesized by cultivating hair roots of these plants Family Solanaceae to win.

As an industrial process for obtaining secondary herbal products with the help of herbal cell and Tissue cultures is a method (A) for cultivation undifferentiated tissue, such as B. cells and calluses, and a method (B) for cultivating plant organs such as B. roots and shoots known. The method However, (A) has the following difficulties (I) - (IV) on:

• I. Characteristics of cell lines that are highly productive, and which have been obtained by selection easily lost if cultivated further.
• II. The products obtained by applying method (A) are sometimes slightly different of the targeted products through cultivation of the original plant.
• III. The usable products obtained in this way have safety problems on because there is a need for growth regulators, who have security problems, during the cultivation process.
• IV. Since the desired products are generally in the Cell are piled up, it is for extracting this Products necessary to destroy the cells. The separation of the targeted products from the rest Components of the cell increase the cost of their extraction.

On the other hand, the method includes (B) Method (B-1) using shoots and roots which have been proliferated in vitro and a Method (B-2) in which Agrobacterium rhizogenes in Plants are inoculated and the roots formed then be cultivated. The method (B) is based on the following: As soon as A. rhizogenes in stem, leaves and roots of the plants etc. are inoculated so-called hair roots in the infected areas. It was known that the roots grow in cases where a Part of a gene, a very large plasmid (Ri plasmid), that occurs in the rhizogenes artery, in a gene of the plant is incorporated, the growth of the roots very quickly progresses and that the amount of secondary products, produced in this way, larger than ordinary ones Roots is. So that's the (B-2) method  a method using the knowledge mentioned above, and comprises the following steps: A. rhizogenes is inoculated in plants that have useful substances in their Produce roots, collect the roots produced under clipping, cultivated in various devices, such as B. a tank and crushed to the useful substances to preserve the grown roots. This method has attracted attention as one Method of extracting natural products, and them is for practical use on some plants considered feasible.

However, although the method (B-1) does not have the above problems (I) and (II), and the method (B-2) not the Problems (I) through (III) above have both methods Disadvantage. First, it is difficult to achieve the one you are targeting To separate substances from the cell components, and the Total costs become high because the targeted substances, accumulated in the cells by cell disruption must be removed, and secondly, this procedure only be carried out in the batch process, because it is difficult to grow plant organs continuously to remove.

It is therefore an object of the present invention Develop processes through which alkaloids are used Hair roots are produced easily and continuously can be extracted.

This problem is solved by the characteristic Features of claim 1.

A preferred embodiment of the invention Method represents a method that cells that transformed by the Ri plasmid occurring in A. rhizogenes are used, the hair roots in Cell culture is cultivated and the alkaloids in the culture medium  eliminated, won by extraction.

The present invention is based on the discovery that in plants of the Solanaceae family general view does not apply to infection a plant with A. rhizogenes at the infected sites Hair roots are formed, and that these hair roots not only grow quickly, but that they are also alkaloids produce that in the cells of the hair roots accumulated and not removed from the cells of the hair roots will; d. H. the present invention is based on the discovery that in plants of the family Solanaceae the hair roots formed in this way discharge the alkaloids produced into the medium.

Advantageous further developments of the invention result from the subclaims.

A major advantage of the present invention is that the alkaloid extraction takes place with high productivity.

The method of the present invention is on plants of the Solanaceae family applicable and this limitation of Planting is very important in the context of the present invention. Because, contrary to popular belief, can Hair roots of plants of the family Solanaceae alkaloids discharge into the medium, the alkaloid extraction a has high productivity. Although all plants in the family Solanaceae can be used for the present invention , it is preferable to use plants that of the genera and Atropa spec., Datura spec., Hyoscyamus spec., And Scopolia spec. come.

Examples of where A. rhizogenes is used to educate of hair roots in the plants listed above amplify, include A. rhizogenes ATCC 25818, A. rhizogenes  ATCC 15834, A. rhizogenes ATCC 43057 and the like. There can also be bacteria used in which the Ri plasmid or T-DNA, which forms part of the plasmid and in Escherichia coli or other bacteria is integrated.

According to the present invention, plants with A. rhizogenes treated so that part (T-DNA) of the Ri plasmids integrated into the genome of the plant cell becomes (transformation).

Examples of methods for obtaining hair roots, with cells from stems, roots, leaves, etc. of Plants of the Solanaceae family by integrating the T-DNA of the Ri plasmid are transformed, the following procedures 1) to 5):

• 1) Direct inoculation procedure in plants,
• 2) leaf blade transformation regeneration process, that uses leaf pieces (R. B. Horsch et al., SCIENCE 227, 1227 (1985)),
• 3) coculture method, the protoplast of the plants used (Z. M. Wei et al., Plant Cell Rep., 5: 93-96 (1986)),
• 4) spheroplast method, the A. rhizogenes and protoplasts used by plants (R. Hain et al., Plant Cell Rep., 3, 60 (1984)),
• 5) Method for direct injection of the Ri plasmid from A. rhizogenes or the T-DNA part of the plasmid in the Plant cell by microinjection or the like.

In the case where the Ri plasmid is in the genome of the  Plant cell integrated by one of the methods 1) to 4) it is necessary to add A. rhizogenes after the Eliminate integration. The elimination of A. rhizogenes can be done by one of the following methods will:

• - high temperature treatment (40 ° C),
• - treatment with antibiotics,
• - Further cultivation of the root tip and the Hair roots with short growth cycles.

Preferably, the hair roots are said to be caused by the above Procedures can be obtained under the following conditions be cultivated:

• - Media: 1. liquid medium; 2. solid medium, solidified with agar etc.,
• - Cultivation: 1. culture at rest; 2. Shake culture (Rotary, linear shaker); 3. rotation culture; 4. Cultivation on a large scale Scale in a tank,
• - Components of the media: it can be a fixed or Liquid medium are used, which is the usual Macro and micro elements, sugar and vitamins used.

Examples of media used:

• 1. Murashige & Skoog Medium,
• 2. Gamborg B5 Medium,
• 3. Nitsch-Nitsch medium,
• 4. White Medium.

As described above, conventional media or Modifications thereof for the cultivation of plant organs, Calluses, cells or the like can be used.  

In the present invention, however, it is preferable to use a liquid medium containing phosphoric acid in a concentration of 1.5 to 6.5 mmol / l (mM), in particular 1.7 to 4.5 mmol / l (mM), Fe ²⁺ in a concentration of 1.0 to 90 µmol / l (µM), in particular 5 to 85 µmol / l (µM), Cu ²⁺ in a concentration of 0.15 to 1 µmol / l (µM), in particular 0.2 to 0.9 µmol / l (µM), Co ²⁺ in a concentration of 0.3 to 1.5 µmol / l (µM), in particular 0.35 to 1.2 µmol / l (µM), gibberellin in a concentration from 2.9 × 10 ^-3 to 28.8 µmol / l (µM), in particular 0.03 to 20 µmol / l (µM), salicylic acid in a concentration of 0.07 to 3700 µmol / l (µM), in particular 0.7 to 1500 µmol / l (µM), malic acid in a concentration of 0.07 to 3800 µmol / l (µM), in particular 0.7 to 1500 µmol / l (µM), succinic acid in a concentration of 0.08 to 4300 µmol / l (µM), in particular 0.8 to 1700 µmol / l (µM), indole butyric acid in a concentration of 0.05 to 500 µmol / l (µM), in particular 0.05 to 250 µmol / l (µM) and / or abscisic acid in a concentration of 0.04 to 380 µmol / l (µM), in particular 0.04 to 190 µmol / l (µM).

In this liquid medium, phosphoric acid, sodium dihydrogen phosphate, Disodium hydrogen phosphate, trisodium phosphate or the like as a source of phosphate, iron (II) sulfate, Iron (III) sulfate or the like as a source of iron ions, Copper sulfate or the like as a copper ion source, and cobalt chloride or the like as a source of cobalt ions be used. As a source of the salicylic acid ion, Malic acid ion, succinic acid ion, indole butyric acid ion and abscisic acid ion can be equally Compounds are used that have the corresponding ion dissociate in water. Examples of such connections are the acids themselves and their alkali salts.

In particular, it is preferable to use the above liquid medium with the addition of special amounts of the above  To form ions to a conventional medium which inorganic components and a carbon source as contains essential components, growth regulators, Vitamins and / or amino acids, and if necessary, contains other components.

In addition to phosphorus, iron, copper and cobalt, inorganic Components such as nitrogen, potassium, calcium, magnesium, Sulfur, manganese, zinc, boron, molybdenum, chlorine, Sodium, iodine and the like are added to the medium, Examples of such components are potassium nitrate, sodium nitrate, Calcium nitrate, potassium chloride, calcium chloride, Magnesium sulfate, sodium sulfate, magnesium sulfate, Zinc sulfate, boric acid, sodium molybdate, molybdenum trioxide and potassium iodide.

Examples of carbon sources are hydrocarbons like sugar and derivatives thereof, organic acids like Fatty acids and primary alcohols such as ethyl alcohol.

Examples of growth regulators are auxins such as indole acetic acid (IAA), naphthylacetic acid (NAA), p-chlorophenoxyisbutyric acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and cytokinins such as kinetin, zeatin and dihydrozeatin.

Examples of vitamins are biotin, thiamine (vitamin B1), Pyridoxine (vitamin B₆), pantothenic acid, ascorbic acid (Vitamin C), inositol and nicotinic acid.

Examples of amino acids are glycine, alanine, glutamic acid and cystine.

The concentrations of components other than those mentioned above specific components added to the liquid medium can vary over a wide range. Usually the liquid medium contains inorganic ones  Components in a concentration of approximately 0.1 µmol / l (µM) to 100 mmol / l (mM), carbon sources in a concentration of approximately 1 to 120 g / l, growth regulators in a concentration of 0.01 to 10 µmol / l (µM), vitamins in a concentration of approximately 0.1 to 100 mg / l and amino acids in one concentration from about 0.1 to 100 mg / l.

According to the present invention, this can be done to the medium added fresh weight on hair roots over a wide Range vary. Generally, it is preferable, roughly 10 mg to 1 g (fresh weight) of hair roots to 50 ml of the To add medium.

Should the hair roots according to the present invention be cultivated, light is not absolutely necessary, and it is actually preferable to produce them of alkaloids such as e.g. B. scopolamine on one cultivate darkened place. It is also preferable the temperature of the culture between about 10 ° C and 35 ° C, especially between 23 ° C and 28 ° C because the growth rate of the hair roots below the temperature of 10 ° C and above the temperature decreases from 35 ° C.

According to the present invention, the medium is covered by a Process of continuous replacement of a part the medium through fresh medium during cultivation the hair roots, or collected by a procedure, that the whole medium after the sufficient growth who exchanges hair roots. In any case, the target Then alkaloids by various methods isolated from the medium.

The present invention allows easy production on an industrial scale of tropane alkaloids like atropine, Scopolamine (hyoscin) and 1-hyoscyamine from Solanaceae.  Therefore, the present invention has the following Advantages 1) to 5) on:

• 1) Properties of high-producing cell lines that obtained through selection go to further cultivation not easily lost
• 2) It can be the same substance continuously the original plant is also produced, obtained,
• 3) It is not necessary to have growth regulators Have security issues to use
• 4) It is not necessary to target substances of the other substances that the cell contains to separate if the hair roots are not destroyed are,
• 5) Alkaloids can be obtained continuously because the medium continuously from the device that is used to cultivate the hair roots can be.

The present invention is based on the following examples to be explained further.

Example 1

The Ri plasmid contained in A. rhizogen was in Stengel, Leaves, roots or other parts of the aseptic Plant inoculated by meristem cultures and aseptic Seedlings were obtained.

Two to five weeks later, hair roots appeared on the infected areas collected by clipping and  on Murashige & Skoog (MS for short) solid medium, the 1st contains g / l carbericillin or 0.5 g / l claforan, and after a week or two, go for a fresh one MS solid medium, the same as described above Contains components that have been implemented.

The above process was repeated two or three times, to remove bacteria and a small part of the so sterilized hair roots became aseptic on one MS liquid medium implemented in an Erlenmeyer flask and in a rotary shaker at 100 rpm at 25 ° C below Permanent dark conditions incubated.

The medium was in periods of one week to 4 months Cultivation time collected to the alkaloids extract. The solid material was removed from the medium first removed by filtration and the medium thus obtained with Chloroform at pH 2, adjusted with 1 N sulfuric acid was extracted and thus the aqueous phase obtained. The aqueous phase was then adjusted to pH 10 with ammonia, to perform the chloroform extraction. The chloroform phase thus obtained was obtained using Dehydrated sodium sulfate anhydride, and then until Evaporated to dryness to obtain the alkaloid fraction.

The amount of alkaloids was determined using high performance liquid chromatography (HPLC) determined. It was TSK-Gel ODS-120T (Toyosoda Co.) for column packing, a mixture of methanol and 10 mmol / l (mM) sodium 1-heptanesulfonate at pH 4 with acetic acid (48:52) as eluent and a wavelength of 215 nm for Detection used. The results obtained are in Table 1.  

Table 1

Example 2

To obtain seedlings, seeds of Datura innoxia Mill. With a sterilizing agent such as sodium hypochlorite solution sterilized and then on a MS solid medium brought. The Ri plasmid isolated from A. rhizogenes (strain No. ATCC 15834) was obtained in the thus obtained Stems or leaves or other parts of the aseptic Plant inoculated.

Two to five weeks later, hair roots appeared on the infected areas, were collected by clipping, on a solid MS medium containing 1 g / l carbenicillin, implemented, and again after a week or two fresh MS solid medium implemented using the same components contained. The process was done once or twice repeated to obtain sterile hair roots.

On the other hand, 50 ml of the liquid medium, into a 100 ml Erlenmeyer flask as indicated in Table 2 given and at a temperature of Sterilized at 120 ° C for 15 minutes. The hair roots were converted into the liquid medium and in a rotary shaker at 100 rpm and at 25 ° C under permanent dark conditions incubated for 30 days.

After this cultivation, the hair roots were cut through Filtration collected, weighed and freeze-dried subject. The dried hair roots were too weighed and ground to powder. The powder was then with a mixture of chloroform, methanol and ammonia (15: 5: 1) extracted. Then the mixture was filtered to get the extract. The extract obtained was then an extraction with chloroform at pH 2 (adjusted with 1 N sulfuric acid) to the aqueous Obtain phase, again with chloroform at pH 10 (adjusted with ammonia) was extracted. The so  Chloroform phase obtained was with sodium sulfate anhydride dehydrated and evaporated to dryness to the alkaloid fraction to obtain.

The liquid medium was weighed and then the pH to pH 2 set to do the same extraction process subject.

The amount of scopolamine in the alkaloid fraction so obtained was included with N, O-bis (trymethylsilyl) acetoamide converted to a silyl derivative and determined by gas chromatography. As a pillar (Temperature 235 ° C) was an OV-17 (2 m × 3 mm), as Carrier gas nitrogen (flow rate 50 ml / min) and as Detector uses an FID.

The total scopolamine content was determined by adding the Levels of scopolamine from hair roots and liquid medium were obtained.

The concentration of phosphoric acid in the liquid medium and the results obtained are shown in Table 3.  

Table 2

Table 3

From Table 3 it can be seen that compared to the conventional MS medium (No. 1) by using the Liquid media (No. 2 to 4) a high content of scopolamine can be won.

Example 3

Cultivation and extraction were carried out in the same manner as shown in Example 2, except that the phosphoric acid concentration was adjusted to 1.25 mmol / l (mM) and the concentration of Fe ^{2+ was} varied. The results are shown in Table 4.

Table 4

Example 4

Cultivation and extraction were carried out in the same manner as shown in Example 2, except that the phosphoric acid concentration was adjusted to 1.25 mmol / l (mM) and the concentration of Cu ^{2+ was} varied. The results are shown in Table 5.

Table 5

Example 5

Cultivation and extraction were carried out in the same manner as shown in Example 2, except that the phosphoric acid concentration was adjusted to 1.25 mmol / l (mM) and the concentration of Co ^{2+ was} varied. The results are shown in Table 6.

Table 6

Example 6

Cultivation and extraction were the same as in example 2 shown, except that the phosphoric acid concentration set to 1.25 mmol / l (mM) and Gibberellin was further added to the liquid medium. The results are shown in Table 7.

Table 7

Example 7

For the extraction of atropine, the culture and Extraction carried out exactly as shown in example 2, except that the concentration of phosphoric acid  1.25 mmol / l (mM) and the components were added to the liquid medium. In this case the content of atropine by the same method as scopolamine certainly. The results are shown in Table 8.

Table 8

Claims (6)

1. A process for the production of alkaloids, characterized by the following process steps: cells of plants of the Solanaceae family are transformed by the Ri plasmid occurring in the Agrobacterium rhizogenes; one cultivates the hair roots and extracts the alkaloids that have given the hair roots into a medium. 2. The method according to claim 1, characterized in that that the plant Atropa spec., Datura spec., Hyoscyamus spec. and / or Scopolia spec. is. 3. The method according to claim 1 and / or 2, characterized in that Agrobacterium rhizogenes after Removal of transformation removed. 4. The method according to any one of claims 1 to 3, characterized characterized in that the medium is a solid or is liquid medium.   5. The method according to any one of claims 1 to 4, characterized characterized in that the medium is a common medium is. 6. The method according to any one of claims 1 to 5, characterized in that the medium is a liquid medium and as an ion phosphoric acid in a concentration of 1.5 to 6.5 mmol / l (mM), Fe ²⁺ in a concentration of 1.0 to 90 µmol / l (µM), Cu ²⁺ in a concentration of 0.15 to 1 µmol / l (µM), Co ²⁺ in a concentration of 0.3 to 1.5 mmol / l (mM ), Gibberellin in a concentration of 2.9 × 10 ^-3 to 28.8 µmol / l (µM), salicylic acid in a concentration of 0.07 to 3700 µmol / l (µM), malic acid in a concentration of 0.07 up to 3800 µmol / l (µM), succinic acid in a concentration of 0.08 to 4300 µmol / l (µM), indole butyric acid in a concentration of 0.05 to 500 µmol / l (µM) and / or abscisic acid in a concentration of Contains 0.04 to 380 µmol / l (µM).