DD223064B5 - Process for the preparation of antiphlogistic agents - Google Patents

Description

The invention relates to a method for obtaining anti-inflammatory agents from Matricaria Chamomilla. Medicines and cosmetic preparations of preparations of flower heads of chamomile (Chamomilla recutita [L.] Rauschert, synonymous with Matricaria chamomilla L) find one because of their anti-inflammatory and spasmolytic effect

wide use. Particular importance is attached to the active ingredients (-) - a-bisabolol and chamazulen. An agent made from chamomile should therefore have the highest possible content of these two substances.

Known is the camomile DEGUMILL (DE-PS 2 402 802, IT-PS 1 035 096), the high content of (-) - a-Bisabolol and Has chamazulene. Chladek u. a. describe in the Pharmacy Volume 13 (1958), pages 712-718 the bloom fascination in the real chamomile

(Matricaria chamomilla L) and the possibilities of economic exploitation of such forms.

Phytochemical investigations of a newly cultivated camomile species were carried out by Poethke et al. a. in the pharmaceutical Central Hall, Volume 108, (1969), pages 733 and 813-823. For preserving the active ingredients in dried Chamomile flowers, in particular the important anti-inflammatory agents Azulene and (-) - a-bisabolol, is the Grusanfälligkeit such dried flowers, that is their zerbröselung, of the utmost importance. When dried Chamomile flowers, which crumble easily, that is to say form a strong growth, are followed by a much faster decrease of the Oil content and thus the active ingredients as the non-crumbled flower baskets. However, it is known that all currently known tetraploid or large-flowered camomiles, including those mentioned in the above References mentioned, in the dried state ι have very unstable flower heads. This means that here is one

Strong Grusbildung or Zerbröselung takes place. The percentage of crumbs can be up to 50%. By this decay of the flower baskets, the quality of the dried flowers is greatly reduced, the active ingredient content drops to half.

In addition, the dried flowers (drugs) lose their appearance. Another disadvantage of the previously mentioned or described camomiles is that they are all around Bisaboloxid-Kamillen acts, d. H. they contain either no or only very small amounts (-) - a-bisabolol. The aim of the invention is to provide an improved process for the preparation of new agents with

antiphlogistic effect of a new tetraploid chamomile variety.

The invention has for its object to produce a new agent with anti-inflammatory effect, which is

characterized in particular by an increased content of (-) - a-bisabolol and chamazulene, the content of chamazulen being at least 120 mg%, (-) - a-bisabolol at least 200 mg% and the content of other bisaboloids being less than 50 mg%

The invention relates to a method for obtaining anti-inflammatory agents, consisting of dried flowers of Matricaria chamomilla, said flowers at least 120 mg% Chamazulen, at least 200 mg% (-) - Ot-bisabolol and, less than 50 mg% of contain remaining bisaboloids, if the corresponding flowers are harvested at the vegetative stage, where 30 to 100% of the tubular flowers of a flower head are opened and the drying takes place at a temperature of at most 60 ⁹ C. The process according to the invention is characterized in that the diploid camomile Degumill Tetraploidiert in a conventional manner, selects the tetraploidized product in a conventional manner and thus obtained or grown plants subjected to further selection and propagation steps, always those plants are selected out, which

a) bloom at about the same time

b) have a uniform, basal branching and a narrow flowering zone of about 5 cm

c) have large flower heads with an outer diameter of about 30 mm (25 to 35 mm) and

(d) have a minimum content of the flowers dried at 40 ° C (in relation to the dried leaves) of at least 150 mg% chamazulen, at least 300 mg% (-) - ot-bisabolol and a content of other bisaboloids lower than 50 mg%, with Flowers are dried here at 40 ^° C. and harvested in the vegetation stage, where 40-60% of the tubular flowers of a flower head have flowered,

and the plants thus obtained are harvested at the stage where 30-100% of the flower heads of a flower head are opened and dried at a temperature of at most 60 ^° C.

Completely surprising and in contrast to all previously known tetraploid large - flowered chamomile flowers are the

By contrast, dried camomile flowers obtained by the process according to the invention are solid, and practically no or only very little crumbing takes place here. Since the agent produced according to the invention is an antiphlogistic agent, the very low degree of crumbing is a very decisive factor for the stability and storage of this agent and a completely surprising one over the previously known dried flowers

Progress. The tetraploidization takes place by means of chemicals at temperatures between 0 and 35 ⁹ C, by means of gamma rays, X-rays or UV rays at temperatures between 0 and 35 ⁹ C, by means of high temperatures of 33 to 50 ⁹ C or

using low temperatures of 0 to 5 ° C.

For the preparation of the composition according to the invention a camomile with the name DEGUMILL is used (it is

in this case in the claim of DE-PS 24 02 802 mentioned chamomile variety; GDR variety protection law Degumill;

IT-PS 1 035 096). This chamomile Degumill has a high content of (-) - O-bisabolol and chamazulene at the same time. The chamazulene and bisabolol content of this chamomile DEGUMILL is, however, stable only if any Incorporation or cross-pollination with other camomile varieties, their bisabolol and chamazulengehalt

is considerably lower, or is prevented with the universally available drug-poor wild chamomile. These

Cross-pollination by cross-pollination is almost always present, as wild chamomile occurs practically everywhere. The chamomile DEGUMILL is diploid. By tetraploidization according to the methods specified in the main claim

However, it is surprisingly achieved that the resulting tetraploid chamomile is no longer susceptible to the

Cross-pollination is, that is, the chamazulengehalt and the (-) - a-Bisabololgehalt becomes stable. By more Selection and amplification steps then succeed, in particular, in the content of (-) - a-bisabolol and also in chamazulene

continue to increase, while the content of the remaining bisaboloids decreases and is below 50 mg%.

The new chamomile so obtained from the chamomile DEGUMILL is surprisingly also distinguished from the

other known tetraploid camomile varieties by a number of new and advantageous properties, namely an incomparably higher content of the important active ingredient (-) - ot-bisabolol and a very low content of other bisabolites, as well as improved seed germination; less seizure of unusable

Herb mass (ie higher flowering yield); lower water content of the flowers (ie better yield

dried bleeds and shorter drying times); better suitability for the mechanical harvest because the flowers are largely localized in one plane and most flowers bloom at the same time (this also makes a uniform harvest date possible); better shelf life of the drug (less tendency to disintegration and bulking) and particularly aromatic and typical Kamillegeruch.

The tetraploidization can, for example, in a conventional manner by treatment of plant parts (seeds, Roots, sprouts, germs, axillary buds) or tissue of the camomile plant DEGUMILL with chemicals, X-rays, gamma rays or UV rays take place. You can continue by applying high or even

low temperatures on the camomile plant DEGUMILL or plant parts or tissue of the camomile plant

DEGUMILL done. For example, the book by Werner Gottschalk, The Importance of Polyploidy for

the evolution of plants ", Gustav Fischer Verlag, Stuttgart, 1976, in particular pages 13 to 22 referenced.

The tetraploidization by chemicals. Examples of suitable chemicals for tetraploidization include: colchicine, acenaphthene, alkaloids such as atropine, Veratrine, nicotine, sanguinarine, derivatives of benzene, diphenyls and phenanthrene, naphthalene and naphthalene derivatives, Diphenylamine, tribromoaniline, paradichlorobenzene, methylnaphthoquinone, methylnaphthohydroquinone, salicylic acid and

related substances, hexachlorohexane, pervitin (hydrochloride), alkyl-alkali carbamates such as isopropyl-sodium-carbamate,

Phenylurethane, salts of cacodylic acid (for example sodium salt), glycosides of Convallaria such as convallarin, convallatoxin

and convallamarin, heteroauxin, germisan (phenylmercuric catechin), organic mercury compounds such as ethyl

OuecksiierPhoiphat, Ethyl uuecksifcerChlorid, Phenylhydrosilbef Hydrotid, Phe ^ OeberiMhylinethandisiilfonat, chloroform, nitrous oxide (N ₂ O) and Mixtures of these substances. Furthermore, oil cake, compost and Kuhdung into consideration. The treatment with the substances mentioned here, for example, at temperatures between 0 and 35 ⁹ C, preferably

between 12 and 30 ^° C., in particular 15 and 25 ^° C.

The application takes place, for example, by treating seeds, shoot tips, roots (in particular root tips

or roots of seedlings), ovary, cut surfaces of leaves or stems, of cell suspensions

Meristem tissue, callus cultures or by injection into the basal stalk region or in the area of Axillary buds. The chemicals are generally in the form of solutions in water, weakly alcoholic

(Alcohol content usually below 5%) or weakly acidic solutions. The pH of the weakly acidic

Solutions is, for example, 5.5-6.5, wherein the acidification, for example by means of lower organic aliphatic Acids such as acetic acid, takes place. If you use alcoholic solutions, they may also be slightly acidic. The For example, concentrations of deV chemicals in these solutions may be 0.01 to 0.5%, preferably 0.02 to 0.2%,

especially 0.05 to 0.1%. Gaseous substances are used as such, optionally under pressure (for example 1 to 10 bar). The duration of treatment is, for example, 1 to 36, preferably 2 to 12, in particular 4 to 6 hours.

The most effective concentrations as well as the duration of exposure should be suitably tested in preliminary tests. For example, the treatment with colchicine at temperatures between 0 and 35 ⁰ C, preferably 12 to

30 ⁰ C, especially 15 to 25 ⁰ C. This can be done, for example, that seeds of the camomile DEGUMILL in a 0.01 to 0.2%, in particular 0.02 to 0.1%, preferably 0.05% Colchicine solution to swelling or sprouted, 5 to 7 day old good developed DEGUMILL camomile seedlings (with the

Cotyledons down) in a 0.01 to 0.2%, in particular 0.02 to 0.1%, preferably 0.05% colchicine solution

be dipped. In the latter method, the surrounding atmosphere should have nearly 100% relative humidity.

The duration of the colchicine treatment is for example 3 to 36 hours, in particular 4 to 10 hours. In the Use of seedlings is generally sufficient for up to 10 hours. When using

from semen, the duration of exposure may be up to 36 hours if necessary.

After treatment with the chemicals, the swollen seeds, seedlings or other plant parts,

rinsed several times with water. The swollen seeds are seeded, for example. The plants with treated roots or other plant parts or the treated seedlings are for example piked in plant boxes. From the so-treated seeds or seedlings, the plants are reared (for example, in the greenhouse:

Temperature between 18 to 25 ^° C. per day and 10 to 16 ^° C. at night) and the plants selected whose pollen is about 1 '/, times

are larger than the pollen of the starting material or have a somatic cell number of 36 chromosomes. If other parts of the plant (subterranean or subterranean parts) are subjected to the treatment with chemicals, only the shoots, roots or flowers / seeds resulting from the treated parts will become later

Examination subjected to tetraploidization. For example, if a sprout treatment or a Armpit treatment was made, then only the one from this armpit or from this shoot

emerging new shoot and the flowers or seeds formed on it for chromosome number examined.

The pollen size measurements and the chromosome counting can be carried out, for example, as in Example 1

is specified.

Treatment with chemicals can also be carried out by the following method: From flowering plants closed tubular flowers are harvested, whose anthers (anthers) in the stage before the

first pollen mitosis. The anthers are removed by micromanipulator from the flower buds and in

Transfer petri dishes, for example, with the nutrient medium after Nitsch and Nitsch (Table 1) are filled. Subsequently

The Petri dishes are stored in a 16-hour day culture room at 28 "C daytime and 20 ° C night temperature, and after 4 weeks the anthers begin to break up and plantlets emerge, which are diploid parent haploid, diaphloid parent tetraploid; For example, roots are planted in garden soil and flowered in the greenhouse, these (haploid) plants are sterile, but can be sprouted by sprouts.

Root or stalk treatment with chemicals such as colchicine polyploidized, homozygous Plants emerge, which can then be distributed via seeds. The next procedure is the same as for the treatment

with chemicals (for example colchicine treatment).

The tetraploidization by radiation. The action takes place, for example, on seeds or root tips at temperatures between 0 and 35 ° C., preferably

10 and 30 ° C, especially 15 and 25 "C. Irradiation sum: 5 to 50 Krad. Gamma- or

X-rays in question. Examples of UV rays are those having a wavelength of 400 to 30 nm, preferably 350 nm. The thus irradiated plants or plant parts are then treated as well as after treatment

with chemicals.

Application of high and low temperatures. As high temperatures, for example, temperatures between 33 and 50 ^e C, preferably 42 to 45 ⁰ C in question. These temperatures are for example exposed to: swollen seeds, seedlings, shoot tips and meristematic Tissue. Duration of treatment: for example 1 to 48, preferably 12 to 24 hours. Suitable low temperatures are for example: 0 to 5 "C, preferably 0.5 to 4" C, especially 2 ⁰ C. This Temperatures are exposed, for example: swollen seeds, seedlings, shoots and meristematic tissue. duration

the treatment: for example, 1 to 100, preferably 20 to 40 days. The so treated plants respectively

Plant parts are treated as well as after treatment with chemicals.

Culture medium after Nitsch and Nitsch (Science, 1969)

mg / 1

KNO ₃ 9To NH ₄ NO ₃ 720 MgSO ₄ XTH ₄ O 185 CaCl ₂ 166 KH ₂ PO ₄ 68 MnSO ₄ χ 4 H ₂ O 25 H ₃ BO ₃ 10 ZnSO ₄ χ 7H ₂ O 10 Na ₂ MoO ₄ × 2 H ₂ O 0.25 CuSO ₄ XSH ₂ O 0,025 5 ml of a solution of 7.45 g Ethylenediaminetetraacetic acid disodium salt and 5.57 g FeSO ₄ χ 7 H ₂ O to 1000 ml myo-inositol 100 glycine 2 nicotinic acid 5 Pyridoxine HCl 0.5 Thiamine HCI 0.5 folic acid 0.5 biotin 0.05 sucrose 20 g Ready-to-use medium (DIFCO-Bacto-Agar) 8g indole acetic acid 0.1

pH of the medium adjusted to 5.5

Of the tetraploid chickweed plants selected by pollen size measurement and / or chromosome counting, which can be obtained according to the possibilities described above, the plants are then selected again which fulfill the following conditions:

a) approximately simultaneous flowering,

b) a uniform, basal branching and a narrow flowering zone of about 5 cm,

c) large flower heads having an outer diameter of about 30 mm (25 to 35 mm),

d) minimum content of chamazulen of 150 mg% and bisabolol of 300 mg% and a content of other bisabolites (in particular bisabolol oxides) below 50 mg% (based on the dried flowers, see example).

The selected plants are now vegetatively propagated by cuttings (verklont) and read out over 3 to 5 generations, the selection always according to the above criteria a) -d). That is, according to the criteria a) -d) selected plants are verklont, from the verklonten plants seed is obtained, the plants obtained from the selected according to the criteria a) -d) and recovered seed from the latter.

The sequence sowing - selection according to a) -d) - seed extraction can be repeated 2-4 times.

This is followed once again by a sequence of seeding selection according to a) -d) cloning-seed extraction.

The last seed obtained is then the propagation of the method according to the chamomile variety Manzana.

The propagation of cuttings takes place in the following way: For cuttings propagation, the starting plants (clone mother plants) must form flower bud-free short shoots under short-day conditions. This takes place in the winter season in the greenhouse without additional lighting or in climatic chambers at a day length of 6 to 10, preferably 8 hours and temperatures of 10 to 15 "C, preferably 12 ° C. For cloning or propagation are leaf, shoot · and in particular short drive They are rooted at 12 to 18 ^° C., preferably 15 ° C. and day lengths of 12 to 16, preferably 14 hours in a taut atmosphere (about 100% relative humidity). As a substrate, for example, a peat-sand mixture in a ratio of 1: 1 can be used; But there are also pure quartz sand, rock wool cube cuttings, peat cutting cubes and the like.

For example, the following soils may be considered for sowing: Garden soil; humus, moderately heavy loamy soil; loamy or humus sandy soil.

It can be sown in the greenhouse or in the field. The temperatures for germination and growth of the plants are for example between 12 to 24 ° C, in particular 18 to 20 ° C. If sowing outdoors, it is preferred to sow in autumn (September / October) or in spring (March / April). These dates apply to all possible cultivation areas (for example, northern hemisphere, temperate to subtropical climatic regions).

The chamomile variety thus obtained belongs to the cultivated plant species of the true chamomile with the botanical name Matricaria Chamomilla L. (synonym Chamomilla recutita [L.], Rauschert). The material of the flowers of this chamomile, dried at a temperature not exceeding 60 ^° C and harvested at the stage of vegetation where 30-100% of the flower heads of a flower head are opened, contains at least 120 mg% of chamazulene, at least 200 mg% (-) -Ot-bisabolol and less than 50 mg% of remaining bisabolites. A particularly effective agent is obtained when such flowers are dried, which are harvested in the flower development stage, where only 30-70%, in particular 40-60% (that is generally 50%) of all flower heads of a flower head are open and under Exclusion of sunlight at an air temperature of at most 50 ° C, for example, at 35-50 ° C, especially dried 40 X.

The drying can be done either by artificial air or by drying in the shade, possibly also in the sun, but care should be taken that the heat does not exceed what is required for complete drying Maßediche. So it is advantageous that one convinces by respective control considerations of the achieved weight constancy. The drying can be done spontaneously or artificially (for example with artificial warm air). The active compound yield is as soon as possible or be held at its greatest in spontaneous drying in the absence of sunlight, preferably at temperatures between 40-60 ⁹ C, more preferably 40-50 "C. The drying process should immediately after harvesting. Drying should be in thin layers, for example from 5 to 20 cm, preferably 10 cm in thickness.

For example, included in a drying cabinet at 40 ⁸ C dried flowers (harvested when 40-60% of the florets of a flower head are open) at least 150 mg%, for example between 150 to 200 mg% chamazulene, at least 300 mg%, for example between 300 to 450 mg% (-) - α-β-isabolol and only slightly, that is between 5 to 50 mg% other bisabolites based on the dry matter (ie based on the absolute dry weight of the flowers). This absolute dry weight is determined by drying a separate sample of chamomile flowers of the chamomile according to the invention (variety Manzana) in a drying oven at 105 ° C. to constant weight (72 to 96 hours) The active substance content of the flowers (drug) dried at, for example, 35 to 50 ° C. is then calculated on the determined at 105 ⁰ C dry mass of flowers.

The term "other bisabololides" in claim 1 is understood in particular:

(-) - α-Bisabololoxide A and B; (-) - α-Bisabolonoxide A. Further constituents of the essential oil of the camomile variety according to the invention are enyne-dicycloether, farnesene, spathulenol and, in low concentrations, various volatile terpene hydrocarbons.

In their phenotype, the new tetraploid chamomile obtained according to the specified process steps from the variety Degumill is similar to the previously known tetraploid chamomile varieties (for example Bodegold, Zloty Lan, BK-2, Pohorelicky Velkokvety); it differs from these in particular in that now (-) - Oi-bisabolol is the main component of the essential oil of the flowers, that also the Chamazulengehalt higher and the content of the other bisabololides (bisabololoxides A and B, bisabolone) is considerably lower ,

The new chamomile obtained from Degumill can be grown successfully on all soils with the exception of soils containing more than 20% organic matter (humic substances and soil organisms); no special agro-technical or cultural measures are required; only a long day with more than 13 hours maximum day length is necessary for the cultivation, that means for the cultivation especially the temperate zones and the subtropics come into question.

The new chamomile from Degumill has a late harvesting date, a uniform stature height with a narrow flowering zone and large flower heads and is therefore particularly suitable for mechanical harvesting. In addition, at the same time, sown plants of the new chamomile obtained from Degumill generally bloom together at virtually the same time, thereby also greatly simplifying and facilitating the harvest.

The new chamomile obtained from the Degumill variety also provides a high yield and is therefore particularly suitable for the preparation of Bisabolol · and Chamazulenreichen agents with anti-inflammatory effect, which are used in medicine, cosmetics and in the form of teas. For example, camomile extracts or camomile extracts can be obtained from the dried material obtained according to the invention by extraction with ethanol or isopropanol or aqueous alcohol mixtures or by extraction with supercritical gases.

The determination of chamazulene is carried out spectrophotometrically similar to the usual way in chamomile extracts. The determination of the (-) - a-bisabolol and the other ingredients of camomile oil is carried out according to the customary gas chromatographic method.

A detailed description of the analytical determination methods is given in Appendix A.

The active ingredient chamazulene is present in the chamomile flowers not as such, but in the form of the sesquiterpene lactone matricin. The matricin has an effect similar to that of chamazulene. From this pre-stage matricin arises, for example, when heated (for example, steam distillation, tea infusion) immediately the Chamazulen. It is therefore customary in chamomile not to indicate the content of matricin but the analytically detectable content of the resulting chamazulene.

embodiment

The invention is explained in more detail below with reference to some examples.

Obtaining the agent with anti-inflammatory effect. (Submitted on 08.02.1985) A chamomile, which is obtained as described below, is sown in the usual way field. Once the plants have developed and the flower heads are in the vegetation stage, where about 50% of the tubular flowers are open, these flower heads are harvested with a chamomile picking machine, the comb spacing of the picking machine are set so that only the flower heads, their tubular flowers to 50% are open, stripped.

The crop is transported as quickly as possible to a shady place and spread there until further processing in a thin layer. Then the flower heads are freed from the stems in a sieve and up to the

Constant weight dried. The weight loss is about 80%. The drying takes place in the shade in a well-ventilated place on trays in a layer thickness of about 10 cm.

After about 2-3 days, the drying is completed. To remove stem parts and flower grass, the dried material is sieved and then pressed into bales.

analysis

Essential oil: 950 mg%

Chamazulene: 1 "50 mg%

(-) - α-Bisabolol: 300 mg%

If the drying takes place, for example, in a stationary strip drying plant by means of artificially heated air at a temperature between SO and 60 ° C., the drying is complete after about 4 hours. To remove stem parts and flower grass, the dried material is sieved and then pressed into bales.

The analytical values of such agent are, for example:

Essential oil: 850 mg%

Chamazulene: 80 mg%

(-) - a-bisabolol:

The starting tetraploid chamomile used is obtained as follows:

example 1

Seeds of chamomile variety DEGUMILL were applied to a 0.05% aqueous colchicine soaked filter paper and let at room temperature (20 ⁰ C) to swell for 6 hours. Then they were removed from the filter paper, rinsed several times with water and seeded in seed boxes (greenhouse): soil: peat-sand mixture 1: 1, temperature: 18 to 20 ° C, relative humidity about 60%, daylength of artificial exposure: 14 hours.

The germinating plants were observed until flowering, the polyploidization success was determined by comparative measurement of pollen and seed size and by chromosome count of the plants, progeny = first seed-derived progeny of the tetraploid-confirmed colchicinated plants.

The tetraploidization can also be carried out in the following way:

Germinated DEGUMILL chamomile seedlings germinated on water-saturated filter paper at room temperature (20 Ό for 4 to 6 hours with the cotyledons germinated downwards in a 0.05% colchicine solution, sprouting the sensitive sprouting roots; In order to prevent dry damage to them, the ambient atmosphere must have a relative humidity of close to 100% After treatment, the seedlings were rinsed several times with water and pimped in plant boxes, followed by the same treatment as for the seeds.

Pollen size measurements are made using a Leitz Binocular Research Microscope Micrometer and Micrometer Slide.

Chromosome counting takes place at root tips: 1 to 2 cm long fresh root ends are collected from the young plants or cuttings plants grown in the greenhouse, placed in 0.002 molar hydroxyquinoline solution for 5 hours and then in 1N HCl for 15 minutes. For examination, about 1 mm of the root tips with 2%

Orcein acetic acid stained and examined microscopically in oil immersion. In the cells in mitosis, the 4-fold set of somatic cells can be determined (4n = 36).

Those plants in which the pollen diameters are approximately 50% larger than those of the starting diploid material (approximately 30 instead of approximately 20 pm) and where the somatic cell chromosome set was doubled to 36 (where the diploid starting material is the corresponding number 18) are tetraploid , About 0.1 to 0.5% of the seeds or seedlings were tetraploidized in the above method and developed flowering, intact plants. The following steps now follow:

Step 1:

From the selected tetraploid chickweed plants (as described above) those individuals were selected,

a) bloom at about the same time

b) have a uniform, basal branching and a narrow flowering zone of about 5 cm,

c) have large flower heads with an outer diameter of about 30 mm,

(d) reach or exceed a minimum level for chamazulen of 150 mg% and bisabolol of 300 mg% and for which the content of other bisabolites (in particular bisabolol oxides) is well below 50 mg%. (All values refer to flower heads dried at 40 ° C. and harvested at the stage where only about 50% (40 to 60%) of the tubular flowers of a head bloomed.)

These plants were verklont. For this purpose, the plants (clone mother plants) were first cut back to about 15 cm rung length, brought under 8 to 10 hours of day length and 12 to 14 ° C to the new shoot (short side shoots). The short shoots were cut and put in a peat-sand mixture. At about 100% relative humidity, 15 "C air temperature and a day length of 14 hours, the rooting of the cuttings took 7 to 14 days.According to the specified conventional cloning (cuttings propagation), the in vitro propagation of dividable tissue parts of the plant can be used ( To establish a camomile culture, various parts of the plant are suitable, preferably the shoot tips or the axillary buds.After rinsing the plants with H ₂ O ₂ under aseptic conditions in the "laminar flow" (high-performance suspended solids filter with low-turbulence displacement flow) shoot tips of the Blattachselknospen and transferred into test tubes, which are filled with a nutrient medium, for example, according to Murashige and Skoog (Physiol Plant 15, 473-497, 1962). The test tubes are placed in a conditioning room with 12 -18, preferably 16 hours of daylight length (achieved by fluorescence fluorescent tubes), a light intensity of 500-10,000 lux, preferably 1,000-3,000 lux and a temperature of 15-30 "C, preferably 22-27 ⁰ C.

As soon as the explants show good growth, they are transferred to an above-mentioned nutrient medium but with a higher cytokinin concentration (30 mg / l N -Isopentenyladenine) and little or no auxin (0-0.3 mg / l indoleacetic acid). As a result, the axes stretch and adventitious organs and axillary buds are formed. These can be removed and tightened according to the aforementioned manner.

The explants intended for plant propagation in larger numbers (the 3rd passage = 3rd generation of propagation) are transferred after growth and formation of the leaves on the first nutrient medium to a nutrient medium containing 10 mg / l indoleacetic acid or 3-indol butyric acid or 0.1- 0.3 mg / 1 ct-naphthylacetic acid. In the process, the plantlets rooted and after about 4 weeks can be planted in sterilized garden soil (steamed at 120 ^° C. for 12 hours) and further cultivated in the greenhouse (under conditions as usual for conventional propagation of cuttings).

Nutrient medium according to Murashige & Skoog:

mg / l

mg / l

NH ₄ NO ₃ 400 indole acetic acid 2.0 Ca (NO ₃ ), 4H ₂ O 144 furfuryladenine 0.1 KNO ₃ 80 thiamine 0.1 KH ₂ PO ₄ 12.5 nicotinic acid 0.5 MgSO ₄ -7H ₂ O 72 pyridoxine 0.5 KCI 65 glycine 2.0 NaFe EDTA 25 myo-inositol 100 H ₃ BO ₃ 1.6 Casein Hydrolys. 1000 MnSO ₄ H ₂ O 4 6.5 sucrose 2% ZnSO ₄ 7H ₂ O 2.7 purified agar powder 1% KJ 0.75

Step 2:

The plants obtained after step 1 bloomed together under isolated conditions in the greenhouse. The plants

stood here in 11 cm pots, filled with garden soil, at 18 to 24 "C daytime and 12 to 14 ^C C night temperature

Day length was at least 14 hours and was achieved in the winter months by additional exposure (200 watts / m ² ). The Water was supplied as needed. Of the totality of the selected individuals, those heads were continuously admitted over a period of 4 weeks Seed crops harvested that were withered and about to disintegrate. The seed was after drying the

harvested flowers at room temperature by blowing out the lighter flower components won.

Step 3:

From the seed obtained after step 2, a random sample was taken and from this approximately 2,000 offspring were raised

(ecological conditions as in step 2) and selected according to the same criteria a) to d) of step 1. The selected individuals were then treated according to step 2.

Step 4:

From the seed according to step 3, one part was seeded at two different locations in the autumn.

Location A)

450 m above sea level, 48.5 »N / 11.5 ° O,

750 mm annual precipitation total,

humid-temperate climate,

January -10 to 0 ⁰ C,

July +10 to + 20 "C,

NN = north of sea level = sea level

⁰ N - northern latitude (ngrad)

⁹ O = eastern longitude (ngrad)

Location B)

200 m above sea level, 42 ° N / 1 ° O,

400 mm annual precipitation sum,

Mediterranean climate,

January 0 to + 10 ⁰ C,

July +20 to +30 ° C.

The sowing took place at both locations at the end of September / beginning of October. The field trial stocks thus obtained were checked with regard to homogeneous growth, flower size and harvest date

and assessed (scored), in addition, samples of the flowers were examined for drug contents. From the field population, those individuals corresponding to the parameters mentioned in step 1 were selected again. Of these will

Seed according to step 2, last sentence won.

Step 5:

With the seed according to step 4, steps 3 and 4 were repeated in the stated order and manner.

Steps:

From the seed obtained after step 5 approximately 1500 individuals were raised (ecological conditions as in step

2) and according to the same principle, as indicated at step 1, selected. Of the so selected plants were

Selected individuals and verklont according to step 1. Each 10 clones of each clone were spaced at random

40 χ 30 cm in the field (site A, see step 4) planted in an isolated place. The soil was loess clay, pH 7.0; the

Planting took place in early June, the first harvest of the seeds in mid-July, after which the plants were cut back, flowered

again and delivered mid-to late August, a second seed crop.

The seed extraction of this material was carried out according to step The seed obtained after step 6 is the propagation material. Flowers of plants from this propagation material (seeding September / October, harvest in early June of the following Year), which are picked at the time when about 50% (40 to 60% of the tubular flowers of a head open

are, and then immediately dried in an oven at 40 ⁸ C 72 hours, based on the

Weight of the dried flowers (dry substance), for example: 150 mg% chamazulen, 300 mg% (-) - OC-bisabolol and

at most 50 mg% remaining bisabolites.

Further exemplary information on the camomile obtained according to the example:

I.Wuchs

uniform growth height (balance) with narrow flowering zone, therefore particularly suitable for mechanical harvest, large flower heads, medium yield;

Form: basic branched (3 to 5fold); Stem: upright, not very branched;

2. Foliage

Leaf: pinnate, 2 to 3 times; Strength: medium; Leaflet, color middle green; Pinnate leaf (stem center); Foliage: medium to strong pinnate;

3. Inflorescence

Flower heads (flower heads): about 30 mm outer diameter, about 15 mm inner diameter; Single basket weight (dry): about 45 mg;

Flower bud, length (mm): about 700, but depending on location (climate), sowing date, soil, fertilization, Plant treatment, weathering; Beginning of flowering (day from 1 January):

circa 160th day (Sowing September, location Freising Federal Republic of Germany, otherwise depending on the factors mentioned above).

Flower: mid-June (see above); Inflorescences without stem, content of essential oil (% of dry matter): about 1.0%; Content of azulene in the essential oil:

at least 15%;

Decay of dried inflorescences, flower drug: low, if harvested before opening the last tubular flowers; Pollen diameter: about 30 pm; Seed length: about 1.25 mm; Somatic cell chromosome number: 4 η: 36;

4. Fruit

Thousand grain mass (TKM) of seeds: 0.06 to 0.13 g;

5. Germinability (KF) 75.75%;

6. purity 94-95%;

7. Other features

Drying ratio fresh: dry (flower) = 5.5 to 6: 1, characteristic aromatic smell of the drug, fine aromatic, typical taste of the tea infusion.

Annex A

Extraction of the essential oil Starting material is a drug from the chamomile variety Manzana. For the preparation of the drug only such Flower heads used in which 30 to 70%, in particular 40 to 60% of the tubular flowers are open. The drying

takes place in a drying oven at 40 ° C for 72 hours.

The essential oil of the chamomile flowers is described as follows by two hours of steam distillation of Drug won:

In a liter round bottomed flask, add 2.0 g of undecomposed drug to 250 ml of deionized water and leave for 2 hours

Return distillation in a Clevenger apparatus (water vapor distillation / reflux apparatus for gravimetric Determination of small amounts of essential oils). The template used is 1 ml of pentane per assay. The Return speed is 40 ± 4 drops / minute. After completion of the distillation that is dissolved in pentane

Essential oil drained as anhydrous in test tubes and possibly remaining adhering in the apparatus

essential oil rinsed with pentane. To remove any residual water, the solution becomes a spatula tip

dried Na ₂ SO _{4 was} added and the solution was then filtered through a glass suction filter of porosity D 3 or D 4 in Rollrandfläschchen. After evaporation of the pentane at 40 ⁰ C in a water bath and drying in a desiccator, the amount of oil is determined gravimetrically. In the oil thus obtained (about 20 mg), the chamazulen and the bisabolol are then determined.

Spectrophotometric determination of chamazulene

test sample:

instrument:

Wavelength:

cuvette:

Specific extinction of chamazulene (1 g / 100 ml, 1 cm):

Compensation liquid:

Found content of chamazulen in mg / 100 g:

The entire essential oil (about 20 mg) obtained from 2 g of drug (obtained as described above) is dissolved in 25 ml of η-hexane or cyclohexane. Filter photometer (for example Eppendorf). 578 nm 1 cm

20.8

η-hexane or cyclohexane

120 E

S78

If no filter photometer is available for the measurement, the determination can also be carried out with a spectrophotometer:

Spectrophotometer (for example PM Q II / or PM Q III .ZEISS ")

605 nm

1 cm

instrument:

Wavelength:

cuvette:

Specific extinction of chamazulene (1 g / 100 ml, 1 cm):

Compensation liquid:

Found content of chamazulen in mg / 100 g:

In the measurement solution, the bisabolol is then determined.

Gas chromatographic determination of bisabolol * and the remaining bisaboloid «

24.5

η-hexane or cyclohexane

102 · E ₁

'60S

Gas chromatograph:

Detector:

Carrier gas:

Pillar:

Column packing:

Temperature:

Detector:

Injection block:

Pillar:

Temperature programming:

Sample solution:

Reference solution:

Injection amount:

Evaluation:

Content of bisabolol in mg per 100 g drug:

Hewlett Packard Model 5750, Erba Fractovap 2350 or similar device

Flame Ionization Detector

helium

V ₈ inches; 200 cm; stole

3% nitrile silicone rubber "XE 60" silanized on kieselguhr

"Chromosorb WAW HP"

125 to 150 pm as support material

320 ⁹ C

220 "C

85-220 ⁰ C

4 ° / minute

It is used the measuring solution for the Chamazulen

Approximately 15 mg of standard bisabolol are dissolved in cyclohexane to 25 ml

Of sample solution and reference solution, 5 pl

The evaluation is done by peak area comparison

10 χ weight (comparison) [mg] χ area (sample)

Area (comparison)

The determination of the remaining bisaboloids can also be carried out by gas chromatography, for example under the following conditions:

Packard, Model 7721, Series 800 and Erba Fractovap Series 2350

Glass columns, 3 m / 2 mm in diameter; 2 m / 2 mm in diameter

3% methylphenylsilicone rubber "OV 1" silanized on kieselguhr

"Gas chromium Q" 125 to 150 pm as support material

30 ml / minute N ₂

80 to 180 ^° C, 2.5 (3) "C / minute

200 "C

Flame Ionization Detector

approx. 2 μΙ of the approx. 1:50 diluted essential oil

The evaluation is partly without, partly with internal standard. As internal standard lauric acid methyl ester or hexadecane are suitable for chamazulenarme or chamazulenfreie oils. For oils with a chamazulene content above 5 % , this is preferable as an internal standard, with the content being determined photometrically (at 578 nm).

Equipment: Columns: Filling:

Carrier gas:

Temperature program:

Injector / detector temperature:

Detector:

Einspritemenge:

Claims (2)

A method of obtaining antiphlogistic agents, consisting of dried flowers of Matricaria chamomilla, said flowers containing at least 120 mg% chamazulen, at least 200 mg% (-) - a-bisabolol and less than 50 mg% of other bisaboloids, if the corresponding flowers are harvested at the vegetation stage, where 30-100% of the tubular flowers of a flower head are opened and the drying takes place at a temperature of at most 60 ⁰ C, characterized in that the diploid chickweed Degumill tetraploidized in a conventional manner, the Tetraploidized product selected in a conventional manner and subjects so obtained or reared plants further selection and propagation steps, always those plants are selected out, which a) bloom at about the same time b) have a uniform, basal branching and a narrow flowering zone of about 5 cm c) have large flower heads with an outer diameter of about 30 mm (25 to 35 mm) and (d) have a minimum content of the dried flowers at 40 ^° C (in relation to the dried leaves) of at least 150 mg% chamazulen, at least 300 mg% (-) - ct-bisabolol and a content of other bisaboloids lower than 50 mg%, with Flowers are dried here at 40 ^° C. and harvested in the vegetation stage, where 40-60% of the tubular flowers of a flower head have flowered, and the plants thus obtained, harvested at the stage where 30-100% of the flower heads of a flower head are opened and dried at a temperature of at most 60 ⁰ C. 2. The method according to claim 1, characterized in that the tetraploidization by means of chemicals at temperatures between 0 and 35 ⁰ C, by gamma rays, X-rays or UV rays at temperatures between 0 and 35 ⁹ C, by means of high temperatures of 33 to 50 ⁰ C. or by means of low temperatures of 0 to 5 ⁰ C.