CH667180A5 - METHOD FOR PRODUCING THE NEW MANZANA CHAMOMILE. - Google Patents

Description

DESCRIPTION Preparations of the flower heads of chamomile (Chamomilla recutita (L.) Rauschert, synonymous with Matricaria chamomilla L.) are widely used for therapy because of their anti-inflammatory and spasmolytic effects and form an important part of the herbal medicinal product. The active ingredients (-) - a-bisabolol and chamazulen are of particular therapeutic importance. A good chamomile drug should therefore have as high a content of these two substances as possible.

Under the name «DEGUMILL» (this is the chamomile variety mentioned in the patent claim of German Patent 2 402 802; GDR variety protection right Degumill; Italian patent 1 035 096) an io chamomile variety is known which also has a high content of ( -) - has a-bisabolol and chamazulen. However, the chamazulen * and bisabolol content of this chamomile variety DEGUMILL is only stable if any cross-breeding or cross-pollination with other quark mill varieties whose bisabolol and chamazulen content is considerably lower, or with the wild camomile low in active ingredients, is prevented. This risk of cross-breeding through cross-pollination is almost always present, as wild chamomile occurs practically everywhere. 20 Various tetraploid chamomile types are also known (for example BODEGOLD, DDR; POHORE-LICKY, CSSR; ZLOTY LAN, Poland; BD-2, Hungary). These tetraploid chamomile varieties have a chamazule content that is not significantly lower than that of the new chamomile 25 variety Manzana. On the other hand, in these known tetraploid chamomile varieties, the important active ingredient (-) - a-bisabolol is only present in extremely small amounts and instead the other bisaboloids (bisa-bolol oxide A and B and bisabolon oxide) predominate, which together make up 30 more than half of the essential oil in the well-known tetraploid chamomile varieties.

A method has now been found for producing a new tetraploid chamomile variety with advantageous properties.

35 The invention relates to the objects or facts defined by the claims.

The new procedural tetraploid chamomile variety called Manzana is surprisingly distinguished from the well-known chamomile variety DEGUMILL 40 and the other known tetraploid chamomile varieties by a number of new and advantageous properties.

In contrast to DEGUMILL, it is no longer susceptible to cross-pollination with the naturally occurring chamomile populations (wild chamomile). In addition, it has a considerably higher content of the important active ingredient (-) - a-bisabolol than the DEGUMILL variety. Compared to the well-known tetraploid chamomile varieties, the Manzana variety according to the process quickly distinguishes itself by an incomparably higher content of the important active ingredient (-) - a-bisabolol and a very low content of the other bisaboloids.

In addition, the Manzana variety according to the method differs from the known tetraploid chamomile 55 varieties, surprisingly, by the following properties: improved germination capacity of the seeds; less accumulation of unusable herb mass (that means higher flower yield); lower water content of the flowers (that means better yield of dried flowers and shorter drying times); better suitability for mechanical harvesting because the flowers are largely localized in one plane and most flowers bloom at the same time (this also enables a uniform harvest date); better shelf life of the drug (less tendency to decay and horror 65 formation); particularly aromatic and typical camomile scent.

Furthermore, the Manzana chamomile variety according to the process is in contrast to the previously known tetraploi-

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the chamomile varieties are homogeneous in terms of content, the properties being resistant to chamazulen and (-) - a-bisabolol (i.e. homozygous).

In contrast, the single plant test of the well-known tetraploid chamomile varieties shows that the individual individuals can be assigned to different types of ingredients, i.e. each sample of a drug produced from it produces a qualitatively and quantitatively different, inhomogeneous result.

The object of the invention is to produce a new tetraploid chamomile variety with improved properties, in particular an increased content of (-) - a-bisabolol.

The new process-related chamomile variety is produced by tetraploidizing the chamomile variety DEGUMILL (gene mutation) and subsequent further selection and propagation steps (mother tree breeding for vegetatively reproducible cross-pollinators). The Manzana variety is composed of 34 strains or lines, which were processed separately in the maintenance breeding.

The tetraploidization can be carried out, for example, in a manner known per se by treating plant parts (seeds, roots, shoot tips, germs, armpit buds) or tissue of the DEGUMILL chamomile plant with chemicals, X-rays, gamma rays or UV rays. It can also be carried out by the decapitation callus method, by anther culture or by applying high or low temperatures to the chamomile plant DEGUMILL or plant parts or tissue of the chamomile plant DEGUMILL. For example, reference is made to Werner Gottschalk's book "The importance of polyploidy for the evolution of plants", Gustav Fischer Verlag, Stuttgart, 1976, in particular pages 13 to 22.

Chemical tetraploidization

Possible chemicals for tetraploidization are, for example: colchicine, acenaphthene, alkaloids such as atropine, veratrine, nicotine, sanguinarine, derivatives of benzene, diphenyl and phenanthrene, naphthalene and naphthalene derivatives, diphenylamine, tribromaniline, paradichlorobenzene-ol, methylnaphononethol, methylnaphononethol, methylnaphononethol, methylnaphononethol, methylnaphthononol, methylnaphthononol, methylnaphonolon, methylnaphoninol, methylnaphthonol, methylnaphoninol, methylnaphonol, methylnaphonol, methyl, naphthalenethynonol, methylnaphonol, methyl, naphthalenethynol, methylnaphonol, methyl, naphthalenethynol, methylnaphonol, methyl, naphthalenethynol, methylnaphonol, methyl, naphthalenethynon, methyl, -licylic acid and related substances, hexachlorohexane, per-vitin (hydrochloride), alkyl-alkali carbamates such as isopropyl sodium carbamate, phenyl urethane, salts of cacodylic acid (for example sodium salt), glycosides from Convallaria such as convonarin, convallatoxin and Convallamarin, He-teroauxin, Germisan (phenylmercuric catechol), organic mercury compounds such as ethyl mercury phosphate, ethyl mercury chloride, phenyl mercury hydroxide, phenyl mercury dinaphthyl methane disulfonate, chloroform, nitrous oxide (N2), and nitrous oxide (N2) . Oil cakes, compost and cow manure are also considered.

The treatment with the substances mentioned here takes place, for example, at temperatures between 0 and 35: C, preferably between 12 and 30 7C, in particular 15 and 25 C.

The application is carried out, for example, by treating seeds, shoot tips, roots (in particular root tips or roots of seedlings), ovaries, cut surfaces on leaves or stems, cell suspensions from meristem tissue, callus cultures or also by injection into the basal stem region or in the Area of armpit buds. The chemicals are generally used 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, whereby the

Acidification takes place, for example, using lower organic aliphatic acids such as acetic acid. If alcoholic solutions are used, they can also be slightly acidic. The concentrations of the chemicals in these solutions can be, for example, 0.01 to 0.5%, preferably 0.02 to 0.2%, in particular 0.05 to 0.1%. Gaseous substances are used as such, optionally under pressure (for example 1 to 10 bar). The treatment time is, for example, 1 to 36, preferably 2 to 12, in particular 4 to 6 hours.

The most effective concentrations and the duration of exposure should be tested in preliminary tests.

For example, treatment with colchicine at temperatures between 0 and 35 ° C., preferably 12 to 30 ° C., in particular 15 to 25 ° C., is particularly advantageous %, in particular 0.02 to 0.1%, preferably 0.05%, colchicine solution are made to swell or that germinated, 5 to 7-day-old well-developed seedlings of the chamomile variety DEGUMILL (with the cotyledons down) turn into a 0 0.1 to 0.2%, in particular 0.02 to 0.1%, preferably 0.05% colchicine solution are immersed in. In the latter method, the surrounding atmosphere should have almost 100% relative atmospheric humidity is, for example, 3 to 36 hours, in particular 4 to 10. In the case of seedlings, an exposure time of up to 10 hours is generally sufficient. If seeds are used, the exposure time may be up to Extend 36 hours.

After treatment with the chemicals, the swollen seeds, seedlings, or other parts of plants are rinsed several times with water. The swollen seeds are sown, for example. The plants with treated roots or other parts of plants or the treated seedlings are pricked out in plant boxes, for example. The plants are grown from the seeds or seedlings treated in this way (for example in a greenhouse: temperature between 18 to 25 ° C per day and 10 to 16 ° C at night) and the plants selected whose pollen is approximately 1 2 times larger than the pollen of the starting material or have a chromosome number of 36 somatic cells. If other parts of plants (above or below ground) are subjected to chemical treatment, only the shoots, roots or flowers / seeds resulting from the treated parts are subjected to a later examination for tetraploidization. If, for example, sprout treatment or armpit treatment has been carried out, only the new shoot arising from this armpit or from this sprout and the flowers or seeds formed on it are examined for chromosome numbers.

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

Tetraploidization by rays

The action takes place, for example, on seeds or root tips at temperatures between 0 and 35; C, preferably 10 and 30 C, in particular 15 and 25 "C. Irradiation sum: 5 to 50 Krad. Gamma * or X-rays are preferred.

Suitable UV rays are, for example, those with a wavelength of 400 to 30 nm, preferably of 350 nm.

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The plants or plant parts irradiated in this way are then further treated as after treatment with chemicals.

Use of high and low temperatures

As high temperatures, for example, temperatures between 33 and 50 C, preferably 42 to 45 C in question. For example, these temperatures are exposed to swollen seeds, seedlings, shoot tips and meristatic tissue. Duration of treatment: for example 1 to 48. preferably 12 to 24 hours.

Possible low temperatures are, for example: 0 to 5 C, preferably 0.5 to 4 C, in particular 2 C. These temperatures are exposed, for example: swollen seeds, seedlings, shoots and meristematic tissues. Duration of treatment: for example 1 to 100, preferably 20 to 40 days. The plants or parts of plants treated in this way are treated as well as after the treatment with chemicals.

The decapitating callus method (decapitating = decapitation or relaxation or cutting back)

The decapitation is carried out, for example, on young plants on the stem, preferably on the top vegetation cone, after the formation of 4 to 6 leaves or also on petioles or side shoots. The buds or shoots arising from the wound tissue (callus tissue) are cut off, brought to rooting, further cultivated in pots and the tetraploidized plants are selected in the same way as in the chemical treatment.

Anther culture (production of plants with a simple set of chromosomes from the anthers with subsequent spontaneous or artificial tetraploidization)

Closed tubular flowers are harvested from flowering plants, their anthers (anthers) are in the stage before the first pollen mitosis. The anthers are removed from the flower buds using a micromanipulator and transferred to Petri dishes, for example. are filled with the nutrient medium according to Nitsch and Nitsch (Table 1). The Petri dishes are then stored in a culture room for 16 hours a day at 28 C day and 20 C night temperature. After about four weeks, the anthers start to break open and plants grow out. These are haploid for diploid parents and dihaploid for te-traploid parents; After roots have formed, they are potted in garden soil, for example, and made to bloom in the greenhouse. These (di) haploid plants are sterile, but can be polyploidized by treating stem ends, roots or stems with chemicals such as colchicine, producing homozygous plants which can then be propagated via seeds. For further procedure see treatment with chemicals (for example colchicine treatment).

Table 1

Culture medium according to Nitsch and Nitsch (Science, 1969)

mg 1

KNO, 950

NH4NO, 720

MgS04 x 7 H ^ O 185

CaCl ^ 166

KH ^ P04 68

MnS04 x 4 HiO 25

H3BO3 10

ZnS04 x 7 HiO 10

Na-> Mo04 x 2 H.O 0.25

5 CuS04 x 5 H20 0.025 5 ml of a solution of 7.45 g of ethylenediaminetetraacetic acid disodium salt and 5.57 g of FeS04 x 7 H20 per 1000 ml of myo-inositol 100

10 glycine 2

Nicotinic acid 5

Pyridoxine HCl 0.5

Thiamine HCl 0.5

Folic acid 0.5

15 biotin 0.05

Sucrose 20 g

Ready-made medium (DIFCO-Bacto-Agar) 8 g

Indole acetic acid 0.1

20 pH of the medium adjusted to 5.5

From the tetraploid chamomile plants selected by pollen size measurement and / or chromosome counting, which can be obtained according to the possibilities described above, the plants are again selected which meet the following conditions a) approximately simultaneous flowering,

b) uniform, basic branching and a narrow flowering zone of approximately 5 cm,

30 c) large flower heads with an outside diameter of approximately 30 mm (25 to 35 mm),

d) Minimum content of chamazulen of 150 mg% and bisabolol of 300 mg% and a content of other bisaboloids (especially bisabolol oxides) below 50 mg% (based on the dried flowers, see example).

The plants selected in this way are then vegetatively propagated (cloned) by means of cuttings and selected over 3 to 5 generations, the selection always being made according to the criteria a) -d) given above. This means that the plants selected according to criteria a) - d) are cloned, seeds are obtained from the cloned plants, the plants obtained from them are selected according to criteria a) - d) and seeds are obtained again from the latter.

The seeding sequence - selection according to a) - d) - 45 seed extraction can be repeated 2-4 times. This is followed by another seeding sequence - selection according to a) - d) - cloning - seed extraction.

The last seed obtained is the propagation material of the Manzana chamomile variety. 50 The propagation of cuttings takes place in the following way: To propagate cuttings, the starting plants (clone mother plants) have to form short buds under short day conditions. This takes place in the winter half-year in the greenhouse without additional lighting 55 or in climatic chambers with a day length of 6 to 10, preferably 8 hours and temperatures of 10 to 15 C, preferably 12 C. For cloning or propagation, leaf, shoot and especially short shoot (side shoot) cuttings. They are rooted at 6012 to 18 "C, preferably 15" C and day lengths of 12 to 16, preferably 14 hours in a tense atmosphere (approx. 100% relative air humidity). For example, a peat-sand mixture in a ratio of 1: 1 can be used as the substrate; but pure quartz sand, rock wool 65 cuttings cubes, peat cuttings cubes and the like are also suitable.

The following soils are suitable for sowing, for example: garden soil; humic, medium-heavy loam soils; loamy or humus sandy soils.

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

The new, procedural chamomile variety Manzana belongs to the cultivated plant species of real chamomile with the botanical name Matricaria Chamomilla L. (synonym Chamomilla recutita (L.), Rauschert) and is defined by the active ingredient details of claim 1. The determination of the active substance content required in the method according to the invention is carried out on flowers which have a development stage such that 30 to 70%, in particular 40 to 60%, of all tubular flowers of a flower head are open (i.e. the flowers used for determining the active substance were picked at this time and then dried in a drying cabinet at 40 ° C. for 72 hours).

If the chamomile flowers are harvested at a point in time in which the flower development stage has progressed, i.e. where, for example, 100% or up to 100% of all the tubular flowers of a flower head are open (for example the stage of full bloom) and / or if that Drying the harvested flowers at a temperature higher than 40 C, the content of the active ingredients (-) - a-Bisabolol and Chamazulen is lower. So the harvest takes place in a vegetation stage where 30-100% of the tubular flowers of a flower head are open and is dried here at a temperature up to 60 ° C (preferably in the absence of sunlight, for example under such drying conditions, as is stated a few pages later) ), the chamomile drug thus obtained contains at least 120 mg% chamazulen and at least 200 mg% (-) - a-bisabolol, while the content of other bisaboloids remains below 50 mg%.

The term “other bisaboloids” in claim 1 means in particular:

(-) - a-Bisabolol oxide A and B; (-) - a-bisabolone oxide A; Other constituents of the essential oil of the process-like chamomile variety are en-in-dicycloether, Farnesen, Spathulenol and in various concentrations various volatile terpene hydrocarbons.

For example, the flowers dried in the drying cabinet at 40 ° C (harvested at the flower development stage, where 30 - 70% of the tubular flowers of a flower head are open), the Manzana variety according to the process contain between 150 - 200 mg% Chamazulen, between 300 - 450 mg% (- ) -cc-bisabolol and only a little, that is between 5 to 50 mg% of other bisaboloids based on the dry substance (i.e. 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 Manzana variety according to the method in a drying cabinet at 105 CC 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 basis of the dry mass of the flowers determined at 105 CC.

In terms of its phenotype, the Manzana variety according to the method is similar to the hitherto known tetraploid chamomile varieties (for example Bodegold, Zloty Lan, BK-2, Pohorelicky Vel-kokvety); It differs from these in particular in that in the process-based variety Manzana (-) - a-bisabolol, the main component of the essential oil of the flowers is that the chamazulen content is also higher and the content of the other bisaboloids (bi-

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sabololoxides A and B; Bisabolonoxid) is significantly lower. Other constituents of the Manzana essential oil are Farnese, Spathulenol and En-In-Dicyclo-ether.

The chamomile variety according to the process can be successfully grown on all soils with the exception of soils with a content of more than 20% of organic matter (humus substances and soil organisms); no special agrotechnical processes or cultural measures are required; Only a long day with a maximum day length of more than 13 hours is necessary for cultivation, which means that the temperate zones and subtropics are particularly suitable for cultivation.

The procedural chamomile variety Manzana has a medium late harvest date, a uniform height with a narrow flowering zone and large flower heads and is therefore particularly suitable for mechanical harvesting. In addition, plants of the Manzana variety that are sown at the same time generally bloom together at practically the same time, so that this also makes harvesting much easier and easier.

The Manzana chamomile variety according to the process produces a high yield. Possible uses include: Obtaining chamomile drug, chamomile oil, chamomile extracts and natural (-) - a-bisabolol.

A drug made from the chamomile variety Manzana according to the process has the maximum content of the active ingredients chamazulen and bisabolol if the chamomile flower heads are harvested in the vegetation stage, for example 30 to 70%, preferably 40 to 60%, that is to say generally 50% of the tubular flowers of a flower head are opened and drying takes place at an air temperature of at most 50 ° C, for example 35 to 50 ° C. Drying can take place either by artificial air supply or by drying in the shade, possibly also in the sun, but care should be taken to ensure that the heat supply does not go beyond what is necessary for complete drying. It is therefore advantageous to be convinced of the weight consistency achieved by means of checkweighing. Drying can be spontaneous or artificial (for example with artificial warm air). The active ingredient yield is greatest in the case of spontaneous drying in the absence of sunlight. The drying process should take place as soon as possible or immediately after the harvest. The drying should be carried out in thin layers, for example from 5 to 20 cm, preferably 10 cm thick.

Chamazulen is determined spectrophotometrically in a manner similar to that used for chamomile extracts. The (-) - a-bisabolol and the other ingredients of chamomile oil are determined using the gas chromatographic method that is customary for this purpose.

Appendix A contains a detailed description of the analytical methods of determination.

The active ingredient chamazulen is not in the chamomile flowers as such, but in the form of the sesquiterpene lactone matricin. Matricin has a pharmacological effect that corresponds to that of chamazulen. From this Matricin precursor, for example, chamazulen is formed immediately when heated (e.g. steam distillation, tea infusion). It is therefore customary to indicate not the content of matricin in chamomile, but the analytically detectable content of the chamazulen formed from it.

example

DEGUMILL chamomile seeds were placed on a filter soaked in 0.05% aqueous colchicine solution.

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paper and let swell at room temperature (20 ° C) for 6 hours. They were then removed from the filter paper, rinsed several times with water and sown in seed boxes (greenhouse): soil: peat-sand mixture 1: 1, temperature: 18 to 20 ° C, relative humidity: approx. 60%, day length with artificial lighting: 14 hours.

The germinating plants were observed until flowering, the success of polyploidization was determined by comparative measurement of the pollen and seed size and by counting the chromosomes of the plants (Fi progeny = first progeny grown from seeds of the colchicinated plants confirmed as tetraploid).

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

5 to 7 days old, well-developed DEGUMILL chamomile seedlings germinated on water-soaked filter paper were placed in a 0.05% colchicine solution at room temperature (20 ° C) for 4 to 6 hours with the cotyledons facing down. The sensitive germ roots were spared; In order to avoid damage to them dry, the surrounding atmosphere must have almost 100% relative humidity. After the treatment, the seedlings were rinsed several times with water and pricked into plant boxes. The further treatment was carried out as for the seeds.

The pollen size measurements are carried out with a Leitz binocular research microscope with a micrometer measuring eyepiece and a 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 grown in the greenhouse, 5 hours in 0.002 molar hydroxyquinoline solution and then placed in IN HCl for 15 minutes. For the examination, approximately 1 mm of the root tips are stained with 2% orcein acetic acid and examined microscopically in oil immersion. In the cells in mitosis, the 4-fold chromosome set of the somatic cells can be determined (4n = 36).

Those plants in which the pollen diameter is about 50% larger than that of the diploid starting material (about 30 instead of about 20 (im) and in which the chromosome set of the somatic cells was doubled to 36 (the corresponding number is 18 for the diploid starting material) Tetraploid: These were selected. Approximately 0.1 to 0.5% of the seeds or seedlings were tetraploidized using the above-mentioned method and developed flowerable, intact plants. The following steps now follow:

Step 1:

From the (as described above) selected tetraploid chamomile plants, those individuals were selected which a) bloom approximately at the same time,

b) have a uniform, basic branching and a narrow flowering zone of approx. 5 cm,

c) have large flower heads with an outside diameter of approx. 30 mm,

d) achieve or exceed a minimum content for chamazulen of 150 mg% and bisabolol of 300 mg% and their content of other bisaboloids (especially bisabolol oxides) is well below 50 mg%. (All values refer to flower heads that were dried at 40 ° C and harvested at the stage where only about 50% (40 to 60%) of the tubular flowers of a head are blooming).

These plants have been cloned. For this purpose, the plants (mother clone plants) were first raised to approximately 15 cm

Cut back shoot length, under 8 to 10 hours of day length and 12 to 14 C brought to new shoots (short side shoots). The short shoots were cut and placed in a peat-sand mixture. At around 100% relative humidity, 15 ° C air temperature and a day length of 14 hours, the rooting of the cuttings took 7 to 14 days.

Instead of the specified conventional cloning (cutting propagation), the in vitro propagation of tissue parts of the plant capable of division can also be used (so-called meristem propagation). Various parts of the plant are suitable for establishing a camomile culture, preferably the shoot tips or the armpit buds.

After rinsing the plants with H2O2, shoot tips of the leaf axillary buds are removed under aseptic conditions in the "laminar flow" (high-performance suspended matter filter with low-turbulence displacement flow) and transferred to test tubes that are mixed with a nutrient medium, for example according to Murashige and Skoog (Physiol. Plant 15.473 - 497.1962) are filled. The test tubes are placed in a climate room with a length of 12-18, preferably 16 hours (achieved by fluorescent fluorescent tubes), a light intensity of 500-10,000 lux, preferably 1000-3000 lux and a temperature of 15-30 ° C, preferably 22- 27 ° C.

As soon as the explants show good growth, they are transferred to the above-mentioned nutrient medium, but with a higher cytokine concentration (30 mg / 1 N6-isopentene-nyladenine) and little or no auxin (0-0.3 mg / 1 indole acetic acid) . As a result, the axes stretch and adventitious organs and more axillary buds are formed. These can be removed and tightened in the manner mentioned above.

The explants intended for the multiplication of plants (the 3rd passage = 3rd generation of propagation) are transferred after growth and formation of the leaves on the first-mentioned nutrient medium to a nutrient medium which contains 10 mg / 1 indole acetic acid or 3-indole butyric acid or 0, Contains 1 - 0.3 mg / 1 a-naphthylacetic acid. The plants take root and after about 4 weeks can be planted in pots filled with sterilized garden soil (steamed at 120 ° C for 12 hours) and further cultivated in the greenhouse (under conditions as is customary for conventional cuttings).

Murashige & Skoog culture medium:

mg / 1 mg / 1

NH4NO3 400 indole acetic acid 2.0

Ca (N03) 2 • 4H2O 144 furfuryladenine 0.1

KNO3 80 thiamine 0.1

KH2PO4 12.5 nicotinic acid 0.5

MgS04 • 7H20 72 pyridoxine 0.5

KCl 65 glycine 2.0

NaFe-EDTA 25 myo-inositol 100

H3B03 1.6 casein hydrolyses. 1000

MnS04 • 4H20 6.5 Saccarose 2%

ZnS04 • 7H2O 2.7 cleaned

KJ 0.75 agar powder 1%

Step 2:

The plants obtained after step 1 flowered together under isolated conditions in the greenhouse. The plants stood in 11 cm pots, filled with garden soil, at 18 to 24 ° C day and 12 to 14 ° C night temperature. The day length was at least 14 hours and was supplemented by additional exposure (200

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Watts / m2) reached. The water supply was carried out as needed.

From the totality of the selected individuals, those heads that had faded and were about to decay were continuously harvested over a period of 4 weeks for seed production. After drying the harvested flowers at room temperature, the seeds were obtained by blowing out the lighter flower components.

Step 3:

A sample was taken from the seed obtained after step 2 and approximately 2000 offspring were drawn from it (ecological conditions as in step 2) and selected from step 1 according to the same criteria a) to d). The individuals selected in this way were then operated according to step 2.

Step 4:

A part of the seed according to step 3 was sown at two different locations in autumn.

Location A)

450 m NN, 48.53 N / 11.5 ° E, 750 mm annual precipitation,

humid-temperate climate,

January —10 to 0 ° C,

July +10 to + 20 ^ C,

NN = north normal zero = sea level CN = northern latitude (degrees)

O = eastern longitude (degrees)

Location B)

200 m NN, 42e N / 1 ° E,

400 mm annual precipitation amount,

Mediterranean climate,

January 0 to +10 ° C,

July + 20 to + 30 ° C.

Sowing was carried out at both locations in late September / early October.

The field trial stocks obtained in this way were checked and assessed (rated) with regard to homogeneous growth, flower size and harvest date, and random samples of the flowers were also examined for the active substance content. From the field inventory, those individuals were selected that correspond to the parameters mentioned in step 1. From these seeds are obtained according to step 2, last sentence.

Step 5:

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

Step 6:

About 1500 individuals were grown from the seeds obtained after step 5 (ecological conditions as in step 2) and selected according to the same principle as indicated in step 1. 34 individuals were selected from the plants selected in this way and cloned according to step 1. 10 plants from each clone were planted in a random distribution at a distance of 40 x 30 cm outdoors (location A, see step 4) at an isolated location. The soil was loess loam, pH 7.0; planting took place at the beginning of June, the first harvest of the seeds in mid-July, then the plants were cut back, bloomed again and delivered a second seed harvest from mid to late August.

This material was harvested according to step 2.

The seed obtained after step 6 is the propagation material of the Manzana chamomile variety according to the method.

Flowers of plants from this propagation material (sowing September / October, harvest 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 are open, and which are then immediately in the drying cabinet 40 "C were dried for 72 hours, based on the weight of the dried flowers (dry substance), for example: 150 mg% chamazulen, 300 mg% (-) - a-bisabolole and at most 50 mg% other bisaboloids.

Further exemplary information on the plants of the chamomile variety obtained according to the example:

1. Growth uniform height (balance) with a narrow flowering zone, therefore particularly suitable for mechanical harvesting, large flower heads, medium-high yield;

Form: basically branched (3 to 5-fold);

Stems: upright, little branched:

2. Foliage

Leaf: pinnate, 2 to 3 fold;

Strength: medium;

Leaflet, color medium green;

Leaflet (middle of stem); Pinnation: medium to strongly pinnate;

3. Inflorescence

Flower heads (flower heads):

30 mm outer diameter,

15 mm inner diameter; Single basket weight (dry): approx. 45 mg; Flower sprout, length (mm): approx. 700, but depending on the place of cultivation (climate), sowing date, soil, fertilization, plant treatment, weather;

Beginning of flowering (day from January 1st):

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

Flowering: mid-June (see above);

Inflorescences without peduncle, essential oil content (% of dry matter): approx. 1.0%; Azulene content in essential oil: at least 15%;

Disintegration of the dried inflorescences, flower drug: low if harvested before opening the last tubular flowers; Pollen diameter: approx. 30 | xm;

Seed length: approx. 1.25 mm;

Chromosome number of somatic cells: 4 n: 36;

4. Fruit

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

5. Germination (KF)

75%;

6. Purity

94-95%;

7. Other features

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

Appendix A Extraction of essential oil The starting material is a drug from the chamomile variety Manzana. To produce the drug, only those flower heads are used in which 30 to 70%, in particular 40 to 60%, of the tubular flowers are open. The drying

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The drying takes place in a drying cabinet at 40 C for 72 hours.

The essential oil of chamomile flowers is obtained by steam distillation of the drug for two hours as described below:

2.0 g of non-comminuted drug is mixed with 250 ml of deionized water in a round-bottomed flask and subjected to a two-hour reflux distillation in a Clevenger apparatus (water vapor distillation reflux apparatus for the gravimetric determination of small amounts of essential oils). 1 ml of pentane per analysi is used as a template. The return speed is 40 + 4 drops / minute. After the distillation has ended, the essential oil dissolved in pentane is drained as free of water as possible into test tubes and any remaining essential oil adhering to the apparatus is rinsed with pentane. To remove any water residue, a spatula tip of dried Na ^ SC ^ is added to the solution and the solution is then sucked off through a glass filter of porosity D 3 or D 4 in crimped rim vials. After the pentane has evaporated at 40 ° C. in a water bath and dried in a desiccator, the amount of oil is determined gravimetrically.

The chamazulen and the bisabolol are then determined in the oil thus obtained (approx. 20 mg).

Spectrophotometric determination of chamazulen

Measurement solution:

Measuring device:

Wavelength:

Cuvette:

Specific absorbance of chamazulen (1 g 100 ml; 1 cm):

Compensation liquid:

Found Chamazulen content in mg 100 g:

All of the essential oil obtained from 2 g of the drug (about 20 mg) (obtained as described above) is dissolved in 25 ml of n-hexane or cyclo-hexane.

Filter photometer (for example Eppendorf).

578 nm

1 cm

20.8

n-hexane or cyclohexane

120 • E578

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

Measuring device:

Wavelength:

Cuvette:

Specific absorbance of chamazulen (1 g 100 ml: 1 cm):

Compensation liquid:

Found Chamazulen content in mg 100 g:

Spectrophotometer (for example PM Q II: or PM Q III «ZEISS»)

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1 cm

24.5

n-hexane or cyclohexane

The bisabolol is then determined in the measurement solution.

Gas chromatographic determination of the bisabolol and the remaining 5 bisaboloids

Gas chromatograph:

io ^

Detector:

Carrier gas:

Pillar:

i5 column filling:

20 temperature: detector: injection block: column:

Temperature-25 programming:

Sample solution:

Comparative solution:

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Injection quantity:

35 evaluation:

Bisabolol content in mg per 100 g drug:

Hewlett Packard model 5750, Erba Fractovap 2350 or similar device

Helium flame ionization detector

Ys inches; 200 cm; stole

3% nitrile silicone rubber "XE 60" on kieselguhr silanized "Chromosorb WAW HP" 125 to 150 \ im as carrier material

320 ° C 220: C 85-220 C

4 '/ minute

The measurement solution for the Chamazulen is used

About 15 mg of standard bisabolol are dissolved in 25 ml of cyclohexane

5 nl each of sample solution and reference solution

The evaluation is carried out by comparing the peak areas

10 x sample weight (comparison) area (sample)

[mg] x

Area (comparison)

The other bisaboloids can also be determined 45 by gas chromatography, for example under the following recording conditions:

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Equipment:

)

Pillars: Filling:

Carrier gas:

60 temperature program:

Injector / detector temperature:

65 detector:

Injection quantity:

102 ■ E,

Packard, model 7721, series 800 or Erba Fractovap series 2350

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

3% methylphenyl silicone rubber "OV 1" on kieselguhr silanized "Gaschrom Q" 125 to 150 µm as a carrier material

30 ml / minute Ni

80 to 180 C, 2.5 (3)? C / minute

200 C.

Flame ionization detector approx. 2 (il of the approx. 1:50 diluted essential oil

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The evaluation is partly without, partly with an internal standard. As an internal standard, methyl laurate or hexadecane are suitable for low-chamazulen or chamazulen-free oils. For oils with a chamazule

content above 5% is preferable as the internal standard, the content being determined photometrically (at 578 nm).

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Claims (6)

667 180 2nd PATENT CLAIMS 1. Process for the production of plants or propagation material of the tetraploid chamomile variety Manzana of the cultivated plant species Real chamomile (Chamomilla recutita (L.) Rauschert, synonymous with Matricaria chamomilla L., Astera-ceae), whose flowers dried at 40 ° C, based on the dry matter , at least 150 mg% chamazulen, at least 300 mg% (-) - a-bisabolol and less than 50 mg% of other bisaboloids, characterized in that the diploid chamomile variety «DEGUMILL» is tetra-ploidized and the selected tetraploid plants on the required active ingredient content to be subjected to further selection and propagation steps. 2. The method according to claim 1, characterized in that the tetraploidization by means of chemicals at temperatures between 0 and 35 ° C, by means of gamma rays, X-rays or UV rays at temperatures between 0 and 35 C, by means of high temperatures 33 - 50 C, by means of low temperatures of 0 - 5 ° C, by means of the decapitation callus method or by anther culture. 3. The method according to claim 1 or 2, characterized in that after the tetraploidization a) a selection of the tetraploid plants based on active ingredient content, namely with at least 150 mg% chamazulene, at least 300 mg% bisabolol and less than 50 mg% other bisaboloids, all related on the dry matter, at the same time flowering, even basic branching and a flower head size of 25 - 35 mm takes place, the plants selected in this way are cloned and seeds are obtained from the plants obtained by the cloning, b) offspring are obtained from the seed obtained in this way, then a selection according to a) is carried out and seed is again obtained from the selected plants, c) the measures in b) are repeated 2-4 times, d) offspring are obtained from the seed obtained in accordance with c), these are selected in accordance with a), the plants selected in this way are cloned and seed is obtained from the plants obtained by the cloning. 4. Chamomile or chamomile propagation material, obtained according to one of claims 1-3. 5. A method for producing a chamomile drug from flowers of chamomile according to claim 4, which at least Contains 150 mg% Chamazulen, at least 300 mg% (-) - a-Bisabo-lol and less than 50 mg% of other bisaboloids, characterized in that the flowers are harvested in the vegetation stage, where only 30 - 70% of the tubular flowers have a Flower heads are open and the flowers are dried at an air temperature of not higher than 50 ° C. 6. Chamomile drug containing at least 150 mg% chamazulen, at least 300 mg% (-) - a-bisabolol and less than 50 mg% of other bisaboloids, characterized in that it contains flowers of the chamomile according to claim 4.