DE1695266A1 - New method of treating certain plant species to extract organic nitrogen compounds - Google Patents

Description

INSTITUT NATIONAL DE RECHERCHE CHIMIQUE APPLIQUEE

Paris, France

New method of treating certain plant species to extract organic nitrogen compounds.

The invention relates to a novel method for treating poppy seeds in order to produce the alkaloids contained therein to extract.

It is known that the yields of opium alkaloids differ greatly in the various poppy species, with papaverum Album or Papaverum somniferum generally give optimal yields, while other species such as Papaverum nigrum or 'Pavot oeillette only supply small amounts of alkaloid, whose extraction is not economical.

It has already been tried Pavot oeillette in improved species to breed, thus better Alkaloidausbeute would be achieved and the cultivation in the temperate climates would be possible this research are lengthy mid difficult

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and the production of Opiura alkaloids remains essentially so far limited to zones with favorable climatic conditions in which Papaverum album delivers maximum yields.

It has now been found - and that is the essential content of the invention - that the yields can be obtained in an advantageous manner of the various types of poppies on aicaloids can be improved by using the crop harvested under normal conditions before all processing steps that are necessary to obtain the alkaloids, fermentation by microorganisms subject; this fermentation process significantly increases the extractable alkaloids content of the poppy species.

The processed poppy varieties can have different degrees of ripeness.

A benefit of this approach is that one material can be used under economical processing conditions that comes from poppy species that can be grown under wide-ranging climatic conditions, in particular under the usual cultivation conditions of the temperate zone.

Another major advantage is that with certain types of poppy, for example with Papaverum nigru *;: ^ the poor

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of alkaloids are, the harvest is practically inapplicable until a fermentation has been carried out, the must be carried out with industrial effort, whereby an effective control of the alkaloid production can be carried out leaves.

Another characteristic of the invention is that for the fermentation of the plant matter (primarily the poppy seed capsules, % which are particularly rich in alkaloids, but also the stems and roots), microorganisms are used that are isolated from naturally occurring mold contamination. Of the cloned fungi, fungi of the species Alternaria come into consideration, for example Alternaria tenuissima and Alternaria Chartoru. > However, this is not intended to limit the scope of the invention to these fungi.

Another feature of the invention relates to the storage u. A breeding of the above strains of microorganisms. It is known that the strains of such microorganisms, in particular of the various Alternaria species, tend to multiply in a vegetative manner through culture and "transplanting" at the expense of spore reproduction. It has been shown for the practical application, which forms the subject of this invention, that the strains of microorganisms characteristic of the invention are kept in a satisfactory manner.

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Renewed and propagated by placing them in a natural environment based on poppy seed powder, or in a Artificial milfeu poor in nutrients, so that the microorganisms remain under greatly reduced living conditions. The freshly isolated strains can also be freeze-dried be kept.

^ The usual methods were used to ferment the poppy seeds. For this purpose, after the plant substance was immersed in water, that is to say in the submerged state, it was incubated and an aerobic fermentation was carried out, which is why previously sterilized air had to be admitted. The vegetable matter distributed in the water was naturally inoculated with microorganisms. For the fermentation a temperature between 5 ° C and 4o C was followed, on vorteilhaftesten was to maintain a constant temperature within the limits between 25 ⁰ C and 3o ° C au. The incubation period can vary between a day and a month

"ken, the cheapest are 6-10 days. At the end of the incubation period the liquid portion is separated from the solid portion by filtration or centrifugation, this latter portion being separated consists of treated vegetable matter and fungal mycelium; liquid and solid parts are then subjected to extraction treatments in order to separate off all alkaloids.

A special feature of the fermentation process is in fact-

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thing to see that it works just as well on a previously sterilized plant material as in a similar, non-sterile one Milieu can be made, provided that from the beginning an inoculation (with mycelium and with spores) with selected Microorganisms is made in such large quantities that a vigorous, rapid and dominant reproduction occurs.

The extraction of total alkaloids in the various parliamentary groups μ functions after the fermentation proceeds according to methods known per se in front of him, such as by a modified method according to STAS-OTTO Annalen der Chemie, Vol loo, 1856, p 44; this includes three successive extidctions with organic solvents, for example with chloroform in the case of fractions made alkaline beforehand, and subsequent extraction with chloroform after acidification,

In the examples given below to illustrate the invention, the overall results achieved in the extractions in an alkaline medium and the extractions in an acidic medium are given in order to take into account that it has happened that in the extracts of the fermented material after acidification gawöimeasn small amounts of . Found non-alkaloids that were detected by chromatography. Such substances can. on. Fungal mycelia return ₉ which at the same time contains the extract ic-n. subjects »sin4 | sb these substances

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include tenuazonic acid (l'acide tenuazonique), one in acidic Extractable amino acid environment.

example 1

400 g of dried and powdered capsules of a portion of the harvest Papaverum nigrum were dissolved in 5 liters of dist. Suspended water; the suspension was sterilized for 20 minutes at 120 ° C. and then inoculated with a special type of Alternaria. Eight days was long bebrütst at 25 to 27 ⁰ C under aerobic conditions. The material obtained by fermentation was treated to extract all alkaloids compared to 400 g capsules of the same harvest quantity which had not been fermented. The results of the extractions, expressed in extract%, based on the initial amount, are given in the following table:

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Extraction pH at the material Material not middle extraction fermented fermented (% extracted) (% extracted) chloroform alkaline o, 24o o, o28 benzene alkaline o _f lol o, ol3 Isobutyl alkaline o, 289 o, o5o alcohol Partial total o, 63o o, o91 chloroform angry o, 212 o, o88 Total o, 842 0.179

In this case, the alkaline extraction yield is 6.9 times and the total yield is 4.7 times the yield from the non-fermented material.

Example 2

400 g dried and powdered capsules of a harvest quantity

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Papaverum album were fermented as in Example 1, and the same with respect to the material obtained after fermentation of the extraction result (%, based on the initial amount of capsules) compared with non-fermented material of the same harvest. The results can be found in the following table:

Extraction pH at the material Material not middle extraction fermented fermented (% extracted) (% extracted) chloroform alkaline o _t 295 o, 138 benzene alkaline o, 13o o, o42 Isobutyl alkaline o, 262 o, o6o alcohol Partial total 0.687 o, 24o chloroform angry 0.238 0.125 Total o, 925 o, 365

In this example, the alkaline extraction yield is 2.8 times and the total yield is

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Increase 2.5 times that of unfermented material.

Attempts have also been made to determine the importance of the individual factors for the advantageous course of the fermentation process and thus clarify the alkaloid yield. These factors include the fermentation conditions and the state of grinding the capsules, the mechanical mixing of the cooling substrate and the type of strains of microbes used. Through the extraction treatments that fermented poppies are subjected to were, among other things, the ratio of isoquinoline to phenantrene components in the total alkaloid yield specify.

It was confirmed that the fermentation of the vegetable Substances can be controlled advantageously through previous sterilization. Without thereby restricting the invention want, the plant material was exposed to a temperature of 120 ° C for three hours for sterilization; these The sterilization method should not use other sterilization methods in use, such as radiation applications exclude. The previous sterilization offers the advantage that the development of different, the increase of the Alkaloid content of the fermented substance unfavorably influencing Bacteria are prevented, as well as the appearance of useless fungi «

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It has also been shown that the aerobic fermentation of the
Poppy seeds in the submerged state, ie when the vegetable material is fermented dispersed in water, as described above, a certain amount of nutrient solution for the fermentation-causing microorganisms is advantageously added to the fermentation substrate, for example - without wishing to restrict the invention to this - the / in the following examples 3 and 4 specified nutrient solutions.

In addition, it was shown that the fermentation process on the submerged poppy seed components ran better if mechanical means were used to mix them during the fermentation period. Such agitation or mixing in the fermentation substrate resulted in an increase in the alkaloid yield. The mixing can take place mechanically, but can also take place under the action of sterile air blown into the substrate or in any other way ψ . In this way, the heat generated by the fermentation process is also advantageously better dissipated.

It was also found that the state of crushing of the plant material played an important role in increasing the alkaloid content in the fermented medium, and that this The more finely divided the vegetable matter was, the greater the increase. Incidentally, the better crushing of the

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Starting material in the eventual alkaloids extraction a livelier course of the extraction.

It was also found that the fermentation process under conditions other than those described, for Example of fermentation of poppy seeds in a submerged state, resulting in a significant increase in the extractable content arene alkaloids in poppy seeds.

It has been shown that the fermentation was also successful with ground and possibly sterilized poppy seeds, which then simply varies in extent (between 2o and loo weight percent) with water or, better, with a nutrient solution for the microorganisms and then moistened was inoculated with microorganisms «In this case the poppy plants are only coarsely ground so that the proportionally Large pieces of capsule let sterilized air pass between them, which was previously passed through Water has been moistened. During such aerobic fermentation of the poppy seed material in a humid environment, quantities are required of about 1 kg of vegetable matter in fermentation, amounts of sterilized and humidified air - without this information should bring about a limitation of the invention - between 0.5 and 50 l / min., preferably between 6 and 8 l / min, required. Temperature conditions and fermentation

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ons time are the same as for submerged poppies. It is to be regarded as an advantage that the volume of the fermentation medium and thus the size of the equipment required compared the fermentation in water is reduced.

Studies have also been carried out on the isolation of fungi of the genus Alternaria from the plant material. During three years cultures and transplantations of about ten species, among them the species P1 (according to I, RCHA), were carried out; P1 was used in the poppy seed fermentation experiments reported in Examples 3 and 4 described below. This strain was handed over to the collection of the Laboratoire de Cryptogamie du Muse * um d ^f Histoire Naturelle, Paris, and registered there under the number 1927.

By the way, can be inventively poppy plants various w which use maturity, but plants are preferred in the peak of maturity.

The examples described below are not intended to be limiting bring about the invention. In particular, other fungi that cause fermentation can be used.

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ORIGINAL

Example 5

The test was carried out with three samples a, b, c of 1 kg each from one and the same amount of capsules of Papaverum album, which were crushed to a size of 0.5 to 2 cm. The natural water content of the plant material was included about Io%

The comparative sample a was subjected to the extraction operations without being fermented beforehand.

Sample b, like sample c, was fermented for 6 days, starting from its natural state, without prior sterilization or inoculation.

Sample c was sterilized at 120 ° C. for 3 hours. After the sterilization and the humidification to be described below sample c was inoculated with an Alternaria (strain P1, according to I.R.C.H.A, as stated above) and fermented for 6 days.

During the fermentation of samples b and c, no mechanical mixing took place because the relatively large and previously humidified capsule fragments allowed the passage of sterilized and, as described below, humidified air.

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ORIGINAL INSPECTED

tete, which by the mass in an amount of (at the treated Amount of vegetable substance) about 0.5 to 5 l / min., In the present case 6 to 8 l / min, flowed.

The capsules were moistened by bringing about a moisture ratio of 75% with the aid of a nutrient medium consisting of ammonium nitrate ( 0.3 g), potassium nitrate tk (0.3 g), dipotassium phosphate (1 g), an addition of 20 cm an oligo-element solution and normal water (made up to 1 liter). The nutrient medium was adjusted to pH = 6.

The oligo-element solution was prepared by dissolving the following substances in normal water: 0.05 g potassium olybdate, o, o5 g sodium borate, o, o5 g cobal nitrate, o, o5 cadmium sulfate, o, o5 g copper sulphate, o, o5 zinc sulphate, o, o5 manganese sulphate · Then a drop of ferric chloride was added and made up to 1 liter topped up with normal water. A stream of carbon dioxide was passed through the solution and then filtered.

The injected sterilized air was humidified by it penetrated a layer of water under the capsule mass.

The temperature of the mass subjected to fermentation was kept ^{at about 26 ° C. by a control system.} Normally

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there will be a temperature increase of a few degrees Celsius during the observed on the first day of fermentation.

The three samples became the one described below after fermentation Subject to extraction process which differs from that described in Examples 1 and 2.

The three samples were washed twice in pure water, ^ a third time in a SaIζsäurelösung with 2.23 weight percent.

The filtrates from the three washes were pooled for each sample by adding ammonia to about pH = 9 alkaline mixed and extracted by means of a solvent, d as from chloroform and isobutyl alcohol in a volume ratio 3: 1. Evaporation of the solvent gave a dry residue which corresponded to the total alkaloids and which was redissolved in chloroform. This solution of the alkaloid bases in chloroform was then treated with hydrochloric acid (2.23 Weight percent) extracted. As a result, all of the alkaloids change into an acidic, aqueous phase.

This aqueous acidic phase is washed with chloroform, whereby the alkaloid bases of the isoquinoline group are in the organic phase let extract (narcotine and papaverine). This

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ORIGINAL fNSPECTED

Chloroform extract is washed with water over sodium sulfate dried and evaporated to dryness. The dry residue obtained essentially contains the isoquinoline group.

The aqueous, acidic extract obtained by chloroform is made alkaline with ammonia to pH = 9. The basic one The aqueous solution is then washed with a mixture of chlorine form and isobutyl alcohol (volume ratio 3: 1), whereby the phenantren group of the alkaloid bases (morphine and codeine) can be extracted in the organic phase, After washing with water, this organic phase is dried over sodium sulfate and evaporated to dryness. Of the The dry residue obtained essentially contains the phenantrene group.

The weights of the dry extracts obtained in this way are used to calculate the yield.

By thin layer chromatography it can be shown that the Extracts of the acidic aqueous solution correspond very precisely to the narcotine-papaverine group, while the extracts of the alkaline Solution essentially corresponds to the group morphine-codeine.

The results from Example 3 are in the table below

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summarized:

sample

Example 3 Alkaloids (in g per kg capsules)

Extract Extract Extract from acidic from alka- _aam + rem milieu 8 ^{es and} milieu

Multiples of the total alkaloid yield

a comparative _fiC _

nrohA ° » ^{8S 3} »

sample

b natural state

o, 76

c vaccinated with ₅ . Alternaria ^ t ¹ * P1 (1)

2.62

4.47

3.38

o, 75 opposite a

5, oo lo, 4o (2.33 "a ⁽²⁾ (3, ο8" b

(1) Multiple of the yield compared to a = 6.35

The fermentation at the fersuch b, starting from the natural one Condition, has led to a strong multiplication of bacteria. The decrease in the yield compared to the reference experiment a reveals the weakening effect of the bacterial population. Therefore, it is desirable to use certain strains of mushrooms, such as those of the Alternaria group.

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In experiment c, the plant material was sterilized, then inoculated with Alternaria P1 and then fermented. In this static environment (mechanical mixing did not take place) the fungi only developed in certain surface areas of the mass. Despite this restricted The overall yield in the extraction has increased by a factor of 2.33 compared to comparative experiment a elevated.

Example 4

The test was carried out with two samples of 1 kg papaverum album from the same stock from which the samples for example 3 were taken.

The two samples of capsules were finely ground and pulveri- * Siert to a grain size between about l / mm and 5o to obtain about 1 mm; these sizes do not represent any restriction.

Comparative sample a was extracted without prior fermentation.

The previously sterilized sample b was fermented in

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was subjected to a non-immersed state, namely, 1 kg of capsules with Io weight percent moisture for 15 to 2o 1 of the nutrient solution specified in connection with Example 3 used.

The fermentation medium was inoculated with the same fungus as indicated above and the fermentation was carried out with simultaneous mechanical stirring; for mixing, for example, used a motor with two propellers, arranged at the bottom or in the medium-sized range M of the fermentation plant; the speed was between 15 and 20 rpm; These figures do not represent any limitation of the invention. The conditions for temperature, aeration and fermentation time were the same as in Example 3.

The extraction process is the same as that mentioned in Example 3, the liquid medium after fermentation, equivalent to the first of the two washings with ^ water, in which the extraction process was switched on.

The results from Example 4 can be found in the table below:

Tabel:

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Example 4 Multiples of the alkaloids (in g per kg of capsules) yield Total alkaloid

sample

Extract Extract total from sour from alkali extract rem lischen
Milieu milieu

a comparative sample 1.42

b vaccinated with Alternaria P1

lo, 51

4.65

lo, 63

6, o7

21.14 3.48 versus a

(1) Multiple of the yield compared to a = 7.4

If the results from Example 4 are compared with those of Example 2, the beneficial effects of the following, Recognize interacting factors: extensive comminution of the plant material, mixing of the Fermentation environment, use of a nutrient solution, type of mushrooms used.

Claims

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Claims (1)

■ 189 * 266 Patent claims; 1. Process for treating different types of poppy seeds, including those that can be grown in the temperate climate zone, to increase their content of extractable alkaloids, characterized in that the under normal conditions harvested poppy seeds are subjected to fermentation by microorganisms before each treatment to extract the alkaloids with the result that the poppy seed's extractable alkaloids content is considerably increased. 2. The method according to claim 1, characterized in that fungi are used as microorganisms, which are made from poppy seed capsules naturally contaminated by fungi are won * 3, method according to claim 2, characterized in that as Microorganisms fungi of the species Alternaria are used. 4, method according to part 3, characterized in that the 109812/1619 _{BAD 0R1G | NAL} 169F266 PiIz Alternaria tenuissima is used. 5. The method according to claim 3, characterized in that the fungus Alternaria chartorum is used. 6. The method according to claim 2, characterized in that the mushrooms isolated from the poppy seed capsules are preserved and propagated by transplanting them into a natural environment based on poppy seed powder, 7. The method according to claim 2, characterized in that the isolated from the poppy capsules mushrooms preserved and are propagated by transplanting into a strongly ψ depleted nutrients artificial milieu. 8. The method according to claim 2, characterized in that the mushrooms freshly isolated from the poppy seed capsules by freeze-drying to be preserved " Process according to Claim 1, characterized in that the poppy seeds are subjected to aerobic fermentation in the submerged state, and in that for this purpose 10 9 8 12/1619 BATH ORIGINAL the existing from poppy plant material is dispersed in water so that the plant material is inoculated men with Mikroorganis that an aerobic fermentation is carried out while introducing sterile air at a temperature between 5 ⁰ C and 4o ° C in the immersed state, and that the fermentation takes between a day and a month, after which the fermented product is subjected to a process for the extraction of the alkaloids. Iu, method according to AnT ₁ "I 9, characterized in that the incubation is carried out for 6-1 * ge at temperatures between 25 and 30 ° C. 11. The method according to claim 9, characterized in that * the dispersed vegetable material is sterilized with microorganisms prior to inoculation. i.A. The method according to claim 9, characterized in that the Vegetable material is not sterilized, but is inoculated in abundance with microorganisms. 13. The method according to claim 9, characterized in that in the 109812/1619 "" .24-1695268 fermentable milieu a nutrient solution for the Fermentation causing microorganisms is given. 14. The method according to claim 9, characterized in that the fermentation substrate in any suitable manner is mixed through. 15. The method according to claim 9, characterized in that the Vegetable material is finely ground and pulverized prior to initiating fermentation. 16. The method according to claim 1, characterized in that the poppy seeds are subjected to aerobic fermentation in the moist state and that for this purpose the poppy seeds are ground and then moistened with water in a weight ratio of 2o to 10o% , or that instead of water one for the microorganisms Suitable nutrient solution is used that the poppy seed is inoculated with microorganisms that it is then subjected to fermentation in the so simply moistened state, using sterilized air previously humidified by passing through water and for a period of between a day and a month a temperature between 5 and 40 ° C is maintained 10 9 812/1619 OFUGINAU INSPECTED -25- 169P266 is, and that then the fermentation product is subjected to an extraction treatment to obtain the alkaloids. 17. The method according to claim 16, characterized in that the ground poppy seeds are sterilized before moistening 18. The method according to claim 16, characterized in that the poppy seeds are only coarsely ground. 19. The method according to claim 3, characterized in that the fungus Alternaria Pl (according to I.R, C.H.A.) Is used will. 20. The method according to claim 1, characterized in that the poppy seeds used are in varying degrees of ripeness. 109812/1619