FI80982B - FOERFARANDE FOER FRAMSTAELLNING AV EN NY KAMOMILLSORT (BENAEMNING MANZANA) OCH DESS ANVAENDNING. - Google Patents

Description

1 80982

Method of production and use of a new variety of chamomile (name Manzana)

The invention relates to a method for the use of chamomilla recutita (L.) Rauschert, a synonym

Matricaria chamomilla L., Asteraceae) for the production of a new variety of tetraploid chamomile (known as Manzana) with flowers dried at 40 ° C containing at least 150 mg of camatsulene, at least 300 10 mg of (-) - α-bisabolol and less than 50 mg% of other bis-aboloids.

The invention also relates to methods for preparing chamomile herb, extract and essential oil using new chamomile, and to the use of new chamomile for the preparation of extracts or essential oil.

Due to their anti-inflammatory and antispasmodic activity, preparations derived from the flowers of true chamomile [Chamomilla recutita (L.) Rauschert, synonymous with Matricaria chamomilla (L.)] have a wide therapeutic use and form an important part of herbal medicines. Of particular therapeutic importance are the active ingredients (-) - α-bisabolol and camazulene. A good chamomile herb should therefore have the highest possible concentration of both of these substances.

The name DEGUMILL (in this case the chamomile variety mentioned in claim 2 402 802 of the DE patent; the DDR species protection right Degumlll; IT patent 1 035 096) is a chamomile variety which has both a high concentration of (-) - α-bisabolol and camatsulate -30 nia. However, the content of camazulene and bisabolol in this chamomile variety, DEGUMILL, is stable only when all crossing and, respectively, cross-pollination with other chamomile varieties with significantly lower bisabolol and kamatsuene content and with the ubiquitous active ingredient-poor wildflower are prevented. Through this cross-pollination

_____ · - - I

2 80932 The risk of cross-breeding is almost always present because wild moss is found virtually everywhere.

Various tetraploid kamo-5 millal varieties are further known (e.g. BODEGOLD, DDR; POHORELICKY, CSSR; ZLOTY LAN, Poland and BK-2, Hungary). These tetraploid chamomile varieties have a camatsulene content that is not substantially lower than that of the new chamomile variety Manzana. On the other hand, however, these known 10 tetraploid chamomile varieties contain only an extremely small amount of the active ingredient (-) - α-bisabolol and are replaced by other bisaboloids (bisabolol oxide A and B and bisabolonoxide) which account for more than half of the known tetraploid chamomile species. essential oil.

A method has now been invented for producing a new variety of chamomile with advantageous properties. The invention is characterized by the objects defined in the claims and the situations, respectively.

The new tetraploid chamomile variety according to the method, known as Manzana, is surprisingly characterized by a number of new and advantageous properties over the known chamomile variety DEGUMILL and other known tetraploid chamomile varieties. Unlike DEGUMILL, it is no longer susceptible to cross-pollination with naturally occurring chamomile plants (wild chamomile). In addition, it contains a significantly higher concentration of the important active ingredient (-) - α-bisabolol than the DEGUMILL variety.

30 Of the known tetraploid chamomile varieties, the Manzana variety of the method differs surprisingly in that it has an incomparably higher content of the important active ingredient (-) - α-bisabolol and a very low content of other bisabolites.

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In addition, the Manzana variety according to the method differs from the known tetraploid chamomile varieties in terms of the following characteristics: better seed germination; a smaller proportion of unusable grass mass (i.e.

5 higher flower yield); lower humidity of inflorescences (ie.

better yield of dried inflorescences and shorter drying times); better suitability for mechanical harvesting, as the inflorescences are largely in the same plane and most of the inflorescences bloom simultaneously 10 (thus also enabling a uniform harvest season); better herbal stability (less tendency to disintegrate and crush); and a particularly aromatic and typical chamomile aroma.

Furthermore, the chamomile variety Man-15 Zana according to the method is, in contrast to the previously known tetraploid chamomile varieties, homogeneous in terms of the ingredients it contains, whereby the properties are stable (i.e. homozygous) in terms of camatsulene and (-) - α-bisabolol content. Individual individuals of the known tetraploid chamomile-20 varieties have different types of constituents, which means that each incremental sample taken from these prepared herbs gives a different, both qualitatively and quantitatively, inhomogeneous result.

It is an object of the invention to provide a new tetraploid chamomile variety with better properties, in particular a higher concentration of (-) - α-bisabolol.

The validation of the chamomile variety according to the new method takes place through tetraploidation (gene mutation) of the chamomile variety DEGUMILL and other related selection and propagation steps (breeding of the strain into vegetatively propagating cross-breeding individuals). The Manzana variety consists of 34 strains or 35 lines which have been treated separately in livestock breeding.

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Tetraploidization can take place, for example, in a manner known per se, by treating parts of the DEGUMILL chamomile plant (seeds, roots, shoot tips, sprouts or leaf-rubbing buds) or tissue with chemicals, X-rays, ganas rays or UV rays. It can further take place by the decapitation-call method, by means of pollen culture or by using high or low temperatures to the DEGUMILL chamomile plant or to parts or tissues of the DEGUMILL chamomile plant, respectively. In this connection, reference is made, for example, to Werner Gott-Schalk, "Die Bedeutung der Polyploidie fur die Evolution der Pflanzen"; Gustav Fischer Verlag, Stuttgart, 1976; in particular pages 13 to 22.

Manufacture of tetraploids by chemicals.

Suitable chemicals for the preparation of tetraploids are, for example, colchicine, acenaphthene, alkaloids, such as atropine, veratrine, nicotine, sanguinarine, benzene, diphenyl and phenanthrene derivatives, naphthalene and naphthalene derivatives, tribromine, triphenylamine, diphenylamine ribbenzene, methylnaphthoquinone, methylnaphthohydroquinone, salicylic acid and related compounds, hexachlorhexane, methamphetamine (hydrochloride), alkyl alkali carbamate such as isopropyl sodium carbamate, phenyluretane, phenyluretane, cacodyl acid convallarin, convvalloxin and convallamarine, heterouxin, germisane (phenylmercuric brentcatechin), organic mercury compounds such as ethylmercury phosphate, ethylmercuric chloride, phenylmercuric acid oxide, phenylamine mercuric acid, phenylelinophen (N20) and their substances n mixtures. Oilcakes, compost and cow dung are still possible.

The treatment with the substances mentioned here takes place, for example, at temperatures of 0 to 35 ° C, in particular at 12 to 30 ° C, in particular at 15 to 25 ° C.

5 80982 Use is made, for example, by treating seeds, shoot shoots, roots (especially root tips or roots of cotyledons), porcine seedlings, leaf or stem cross-sections, cell suspensions of growth cell cultures 5 or wound cork cultures, or by spraying the base of the stem into a part or area of truncated buds. Chemicals are usually used in the form of aqueous solutions, low-alcohol (usually less than 5% alcohol) or weakly acidic solutions. Weakly acidic solutions have a pH of, for example, 5.5 to 5.5, in which case the acidification takes place by means of lower, organic, aliphatic acids, such as acetic acid. In the event that alcoholic solutions are used, they may also be weakly acidic. The concentrations of the chemicals in these solutions can be, for example, 0.01 to 0.5%, in particular 0.02 to 0.2%, in particular 0.05 to 0.1%. Substances in gaseous form are used as such, possibly under pressure (for example 100 to 1000 kPa). The processing time is, for example, 1 to 36 hours, in particular 2 to 12 hours, in particular 4-6 hours.

The most effective concentrations, as well as the exposure time, would be expedient to be examined in each case by means of preliminary experiments.

Particularly preferred is, for example, treatment with colchicine 25 at 0 to 35 ° C, in particular at 12 to 30 ° C, in particular at 15 to 25 ° C. This can be done, for example, by allowing the seeds of the DEGUM11 chamomile variety to swell in a solution of 0.01 to 0.2%, in particular 0.02 to 0.1%, mainly 0.05% of colchicine. , or that germinated, 5-7-day-old, well-developed cotyledons of the DEGUM11 chamomile variety (cotyledons downwards) are immersed in 0.01 to 0.2%, in particular 0.02 to 0.1%, mainly 0.05% colchicine solution. In the latter method 35, the relative humidity of the ambient air should be close to 100%. The length of the colchicine treatment is, for example, 3-36 6 80982 hours, in particular 4-10 hours. When using locust seedlings, the exposure time up to 10 hours is usually sufficient. When using seeds, the exposure time can possibly be extended up to 36 hours.

5 After chemical treatment, the swollen seeds, cotyledons or other parts of the plant are rinsed several times with water. For example, swollen seeds are sown. Plants whose roots or other plant parts have been treated, for example, are replanted in 10 plant boxes. The plants are grown from seeds or cotyledons treated in this way (for example in a greenhouse; temperature 18-25 ° C during the day and 10-16 ° C at night), and plants with pollen about 1 1/2 times higher than the pollen of the starting material or with somatic 15 the number of cells has a chromosome number of 36. In the case of chemical treatment of other parts of plants (above-ground or underground parts of plants), tumors, roots or flowers / seeds originating exclusively from the treated parts are examined for later formed tetraploids. When the shoot or leaf blight treatment is carried out, only the new plant resulting from this leaf blight or this shoot and the flowers or seeds formed in it shall be examined for chromosome number.

The measurement of pollen size and the counting of chromosomes can be performed, for example, as described in Example 1.

Manufacture of tetraploids by irradiation.

The effect is applied, for example, to the seeds or the root tips at a temperature of 0 to 35 ° C, in particular 10 to 30 ° C, in particular 15 to 25 ° C. Sum of radiation: 5 - 50 Krad. These are mainly gamma and X-rays. Suitable UV rays are, for example, rays with a wavelength of 400 to 30 nm, in particular 350 nm.

35 Plants or parts of plants so irradiated are then treated 7 80982 in the same way as in the case of chemical treatments.

Use of high and low temperatures.

Examples of high temperatures are temperatures from 35 to 50 ° C, in particular from 42 to 45 ° C. For example, swollen seeds, cotyledons, tumor canopies and growth cell tissue are placed at these temperatures. The duration of the treatment is, for example, 1-48, mainly 12-24 hours.

10 Examples of suitable low temperatures are 0 to 5 ° C, in particular 0.5 to 4 ° C, in particular 2 ° C. For example, swollen seeds, cotyledons, cotyledons and growth cell tissue are placed at these temperatures. The duration of the treatment is, for example, 1 to 100, mainly 20 to 40 to 40 days.

The plants or parts of plants thus treated are then further treated in the same way as in the case of chemical treatment.

Decapitation-call method (decapitation = lat-20 vomiting or tip removal or tip cutting).

The decapitation is carried out, for example, on the stems of young plants, preferably on the growth points of the tips, after the formation of 4-6 leaves or also on leaf stalks or side shoots. Buds or shoots formed from a wound cell (callus cell-25 revenge) are cut off, brought to rooting, further cultured in pots, and tetraploidized plants are selected in the same manner as when treated with chemicals.

Ponchi cultures (production of simple chromosomal native plants from stamen ponies followed by spontaneous or artificial tetraploidation)

From the flowering plants, closed horn flowers are collected, the stalks of the stamens of which are in the stage before the first pollen size. The stems of the stools are taken from the flower buds by means of a micromanipulator and transferred to petri-8 80982 plates filled with Nitsoh and Nitschiri medium (Table 1). The Petri dishes are then stored in a culture room with 16-hour days at 28 ° C at day temperature and 20 ° C at night-5. After about 4 weeks, the petioles begin to open and the plants grow. In the case of diploid plants these are haploid and in the case of tetraploid plants these are dihaploid; after root formation, they are transferred, for example, to the garden soil and flowered in a greenhouse. These (di) haploid plants are sterile; however, polyploid plants can be formed by treating shoot shoots, roots, and stems with chemicals such as colchicine to form homozygous plants, which can then be propagated by seed. See further processing in connection with chemical treatment (eg colchicine treatment).

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table 1

Nitsch and Nltsch medium (Science, 1969) mg / l KN03 950 NH4NO3 720

MgSO 4 x 7 H 2 O 185

CaCl2 166 KH2PO4 68

MnSO 4 x 4 H 2 O 25 h 3bo3 10

ZnSO 4 x 7 H 2 O 10

Na 2 MoO 4 x 2 H 2 O 0.25

CuSO 4 x 5 H 2 O 0.025 5 ml of a solution of 7.45 g of the disodium salt of ethylenediaminetetraacetic acid and 5.57 g of FeSO 4 x 7 H 2 O filled to 1000 ml with myo-inositol 100

Glycine 2

Nicotinic acid 5

Pyridoxine HCl 0.5

Thiamine HCl 0.5

Folic acid 0.5

Biotin 0.05

Sucrose 20 g

Finished medium (DIFCO-Bacto-agar) 8 g

Indoleacetic acid 0.1

The pH of the medium was adjusted to 5.5 10 80982

From the tetraploid chamomile plants selected by measuring the size of the pollen particles and / or counting the chromosomes, which can be obtained according to the alternatives described above, the plants that meet the following conditions are then selected: a) approximately simultaneous flowering, b) uniform root-derived branching and a narrow flower zone of about 5 cm, c) large flower clusters with an outer diameter of 10 about 30 mm (25-35 mm), d) a minimum content of camazulene 150 mg% and bisabolol 300 mg% and other bisaboloids (especially bisabolol oxides) less than 50 mg% (calculated on dried flowers; see example).

15 The plants thus selected are now propagated by cuttings (cloned) and selected over 3-5 generations, in which case the selection is always based on the criteria a) to d) above, the plants selected are cloned, the cloned plants are seeded, and the 20 plants obtained are selected according to criteria a). ) to (d) and the latter shall again be seeded.

The period of sowing - selection according to a) to d) - seed intake can be repeated 2-4 times.

It is once again accompanied by the section on selection - cloning - seed production according to sowing points a) to d) 25.

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

The plug is then added as follows:

For cuttings, stem plants (clone-30 plants) must form under conditions where the time of day is short, flower-free shoots. This takes place in the winter half of the year in a greenhouse without additional lighting or in a weather chamber for a day length of 6-10, preferably 8 hours and at a temperature of 10-15 ° C, preferably at 12 ° C. Leaf, shoot and especially short (side shoot) cuttings are suitable for cloning or propagation, respectively. They take root

II

11,80982 at 12-18 ° C, preferably 15 ° C and a day length of 12-16, preferably 14 hours in a relative atmosphere (about 100% relative humidity). As the growing medium, for example, a peat-sand mixture in a ratio of 1: 1 can be used; therefore, pure quartz sand, rock wool cubes, peat cuttings and the like are also suitable.

For sowing, the following substrates are suitable for sowing in this context: garden soil, humus-rich, 10 medium-heavy clay soils, clayey or humus-rich sandy soils.

Sowing can be done in a greenhouse or also in the open. The germination and growth of the plants take place, for example, at a temperature of 12-24 ° C, in particular 18-20 ° C. In the case of sowing in the open, sow 15 mainly in autumn (September / October) or spring (March-month / April). These deadlines refer to all relevant crop areas (eg northern hemisphere, temperate to subtropical climates).

The new chamomile variety Manzana 20 according to the method belongs to the genus of chamomile cultivars with the botanical name Matricaria Chamomilla L. (synonym Chamomilla recutita (L.), Rauschert) and is defined by the data on active ingredients in claim 1. The active substance concentrations given in it are calculated from the flower development stage reached when 30-70%, in particular 40-60% of all the horn flowers of the flower buds have opened (i.e. the flowers used in the active substance assay were picked at this time and then dried for 72 hours at 40 ° C). in the oven).

30 If the chamomile flowers are harvested at a time when the development stage of the flower buds has passed the above stage, ie eg 100% or almost 100% of all the horn flowers of the flower buds have opened (eg the full flowering stage) and / or if the harvested flower buds are dried above 40 ° C, the concentration of the active substances, camazulene and (-) -? - bisabolol, remains lower. Thus, if the harvest is carried out at a growth stage in which 30-100% of the horn flowers have opened and drying is carried out at a temperature up to 60 ° G (preferably avoiding sunlight, for example under drying conditions described below), the chamomile drug thus obtained contains at least 120 mg % of camatsulene and at least 200 mg% of (-) - .beta.-bisabolol, while the concentration of other bisaboloids remains the same, less than 50 mg.

The term "other bisaboloids" in claim 1 refers in particular to (-) - «/ - bisabolol oxide A and B; (-) - (X-bisabolonoxide A, other constituents of the essential oil of the chamomile variety according to the process are en-in-dicycloether, farnesene, spatulenol and, in low concentrations, various volatile terpene hydrocarbons. In an oven at 40 ° C dried Manzana variety according to the method (picked during the inflorescence development stage, in which 30-70% of the horn flowers of the mycorrhizae have opened) contain, for example, 150-200 mg% of camazulene, 300-450 mg% of (-) - ^ - bisabolol 20 and only a little, i.e. 5-50 mg% of other bisabolites, calculated on the dry matter (ie calculated 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 an oven at 105 ° C to 25 constant weight (72-96 hours). For example, the active ingredient content of flowers (herbs) dried at 35-50 ° C is then calculated from the dry weight of the flowers at 105 ° C.

In appearance, the Manzana variety is 30 varieties of known tetraploid chamomile varieties (e.g. Bodegold, Zloty Lan, BK-2, Phorelicky

Velvet hydrogen); it differs from these in particular in that the Manzana variety according to the method contains (-) - V-bisabolol, the main component of the essential oil of flowers, that it also has a higher content of camazulene and 35 other bisaboloids (bisabolol oxides A and B; bisabolol oxide) less.

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The other ingredients in the essential oil of the Manzana variety are farnesene, spatulenol and en-in-dicycloether.

The chamomile variety according to the method can be successfully cultivated in all soil types except those with an organic matter content of more than 20% (humic substances and soil organisms); no specific agricultural techniques or cultivation measures are required; only a long time of day is needed for cultivation, with a maximum daily length of 13 10 hours, which means that temperate and subtropical zones are particularly relevant for cultivation.

The Manzana chamomile variety according to the method has a medium-late harvest time, a uniform growth height, a narrow flower zone and large inflorescences and is therefore particularly suitable for mechanical harvesting. In addition to this, plants sown at the same time as Manzana usually burst into the flower practically at the same time, so that this also greatly simplifies and facilitates the harvest. The Manzana-ka-20 momilla variety according to the method produces a high yield. Applications include the preparation of chamomile herb, chamomile oil, chamomile extracts and natural (-) - β-bisabolol.

The herb made from the Manzana chamomile variety according to the method has a maximum content of active ingredients, camazulene and bi-25 sabolol when the chamomile blossoms are harvested at a growth stage where, for example, 30-70%, preferably 40-60%, i.e. generally 50% of the mycelium the drying takes place at an air temperature of at most 50 ° C, for example 35-50 ° C.

30 Drying can then take place either by means of an artificially directed air stream or also by drying in the shade, possibly also in the sun, provided that care is taken to ensure that the amount of heat introduced into the product does not exceed what is necessary to achieve complete drying. It is therefore advantageous that the constant weight obtained is determined in each case by reference weighings. Drying can take place spontaneously or artificially (for example with heated air). The active ingredient yield is at its highest in spontaneous drying protected from sunlight. The drying process must take place as soon as possible or immediately after harvest. Drying should be performed using thin layers of drying material, for example 5-20 cm, preferably 10 cm thick.

Camazulene is determined spectrophotometrically similar to the conventional method for chamomile extracts. The (-) .beta.-bisabolol and the other constituents of chamomile oil are determined for this purpose by a conventional gas chromatographic method. A detailed description of the analytical methods is given in Annex A.

The active substance camatsulene is not present in chamomile flowers as such but as sesquiterpene lactone, matrix. Matrix has a pharmacological effect similar to that of matsulsulene. This precursor, matrix, is immediately formed, for example, by heating (e.g. water-steam distillation, tea extraction) camazulene. Therefore, it is customary not to give the matricin content of the chamomile evening, but the analytically determinable content of the resulting kamatsulene.

Example 1 Seeds of the DEGUMILL chamomile variety were transferred to filter paper dipped in 0.05% aqueous colchicine and allowed to swell at room temperature (20 ° C) for 6 hours. They were then removed from the filter paper, rinsed several times with water and seeded in seed boxes (in a greenhouse): soil type: peat-sand mixture 1: 1, temperature 18-20 ° C, relative humidity about 60%, daytime in artificial light for 14 hours.

Germinated plants were monitored until flowering, the success of polyploidization was confirmed by comparative measurements of pollen clusters and seed size, and the chromosomes of the plant were counted (F ^ progeny = first, tetraploid-derived) seed of colchicine-treated plants.

Tetraploidization can also take place as follows: 5 5-7 day old, well-developed chamomile seedlings of the DEGUMILL variety germinated with water-soaked filter paper were placed in a 0.05% colchicine solution with cotyledons downwards for 4-6 hours at room temperature (20 ° C). In doing so, care was taken with delicate locust roots; in order to avoid drying damage to them 10, the relative humidity of the surrounding atmosphere must be close to 100%. After treatment, the seedlings were rinsed several times with water and planted in plant boxes. Further processing took place as in the case of seeds.

Pollen size measurements are performed with a Leitz binocular examination microscope with a micrometer-measuring eyepiece and a micrometer slide.

Chromosome counting is performed from the root tips.

From the seedlings or cuttings-20 grown in the greenhouse, fresh root ends 1-2 cm long are collected and placed in 0.002 molar hydroxyquinoline solution for 5 hours and then in 1N HCl for 15 minutes. For examination, approximately 1 mm of the roots are stained with 2% orsein-acetic acid and examined under immer-25 pig oil microscopy. Thus, a 4-fold chromosome of somatic cells (4n = 36) can be determined from cells in the mitotic stage.

Plants with a pollen particle diameter about 50% larger than the diploid starting material -30a (about 30 μm instead of about 20 μm) and with a somatic cell chromosome that has doubled to 36 (the corresponding number for the diploid starting material is 18) are tetraploid. These were selected separately. About 0.1 to 0.5% of the seeds or seedlings, respectively, were tetraploidized by the method described above and developed flowering intact plants. This was followed by the following steps: 809-82

Phase 1:

From the selected (as described above) tetra-ploid chamomile plants, those individuals were selected that 5 A) flowered at approximately the same time, B) branched evenly, from the root and had a narrow flower zone, about 5 cm, C) with large mycorrhiza, about 3Q mm, 10 D) having a minimum concentration of 150 mg% for camatsulene and 300 mg% for bisabolol or exceeding said values and a concentration well below 50 mg% for other bisaboloids (especially bisabolol oxides). (All values apply to mycera-15, which were dried at 40 ° C and collected at a time when only about 50% (40-60%) of all mycorrhizal horn flowers had opened.

These plants were cloned. In addition, the plants (parent plants used for cloning) were cut so that the length of the shoot 20 became about 15 cm, they were brought to a day length of 8-10 hours and 12-14 ° C to form new shoots (to form short side tumors). The dwarf shoots were cut and placed in a peat-sand mixture. At approximately 100% relative humidity, at an air temperature of 15 ° C for 25 and 14 hours of daytime, cuttings took 7-14 days to take root.

Instead of the described conventional cloning (plug propagation), in vitro propagation of plant-divisible tissue parts (so-called growth cell-30 propagation) can also be used. Different parts of the plant are suitable for setting up a chamomile sauna plantation, mainly head tumors or difficult to grow.

After rinsing the plants with I 2 Cl 2 under aseptic conditions in a "laminar flow" (high power 35 fluid filter with low turbulence exhaust thrust flows), the shoot peaks of the leaf sludge are removed and transferred to test tubes filled with medium prepared, for example, from Murash. (Physiol. Plant. 51, 473-497, 1962). The test tubes are placed in an aerated room with a day length of 12-18, mainly for 16 hours (achieved by fluorescent-fluorescent tubes), a light intensity of 500-10,000 lux, mainly 1000-3000 lux and a temperature of 15-30 ° C. , mainly 22-27 ° C. 5 As soon as the grafts grow well, they are transferred to the above-mentioned medium, which, however, has a higher concentration of cytokinins (cytokinins are plant hormones that promote cell division) (e.g.

30 ml / l N, N-isopentenyladenine) and little or no auxin (0-0.3 mg / l indoleacetic acid). In the future, the spines will stretch and form late organs as well as more awkward buds. These can be removed and grown as mentioned above.

In order to propagate the plants to a greater extent, certain grafts (stage 3 = 3rd generation) are transferred after rooting and growth with the first medium to a medium containing 10 mg / l indoleacetic acid or 3-indolebutyric acid or 0.1-0, 3 mg / l-naphthylacetic acid. In this case, the plants 20 take root and, after about 4 weeks, can be planted in pots filled with sterilized garden soil (steamed for 12 hours at about 120 ° C) and further grown in a greenhouse (under conditions suitable for conventional cuttings).

25 Medium according to Murashigen & Skoog: mg / 1 mg / l NH 2 NO 4 400 Indoleacetic acid 2.0

Ca (NO-j) 2'4H2O 144 Furfuryladenine 0.1 KNO ^ 80 Thiamine 0.1 30 KI ^ PO ^ 12.5 Nicotinic acid 0.5

MgSO 4 '7H 2 O 7 2 Pyridoxine 0.5 KCl 65 Glycine 2.0

NaFe-EDTA 25 myo-inositol 100 H ^ BO ^ 1.6 Casein hydroly- 1000 33 gave 3 5

MnSO 0. * 4H-, 0 6.5 Sucrose 2% 4 2

ZnSO. * 7H ~ C> 2.7 Purified agar powder 1% CI 0.75 18 80982

Step 2:

The plants obtained in step 1 bloom under isolated weather conditions, in the greenhouse at the same time. The plants were then in 11 cm pots filled with garden soil at a daytime temperature of 18-24 ° C and a nighttime temperature of 12-14 ° C. The length of the day was at least 14 hours, and in the winter half of the year it was achieved with additional lighting 2 (200 watts / m). Irrigation was performed as needed.

10 Among the selected individuals, for a period of 4 weeks, the mycelium that had flowered and had just fallen into Haan was continuously collected for seed intake. Drying was performed in a well-ventilated hall at an air temperature of 20-30 ° C; then the seeds were separated with a sieve (5 x 0.4 mm) and subsequently cleaned with a whisk.

Step 3:

From the seed yield obtained from step 2, a random test was taken, and from this about 2000 offspring were grown (ecological conditions as in step 2) and selected according to the similar criteria of step 1 A) -D). For individuals so selected, the procedure was as in step 2.

Step 4; 25 Part of the Stage 3 seed crop was sown in the autumn in two different growing areas.

Habitat I

450 m NN, 48.5 ° north latitude / 11.5 ° east longitude, 750 mm annual rainfall, humid-temperate 30 climate, January -10 to 0 ° C, June +10 to + 20 ° C.

NN = north at normal zero = altitude ° N = north latitude (degrees) ° 0 = east longitude (degrees) 19 80982

Habitat II

200 m NN, 42 ° N / longitude, 400 min annual rainfall, Mediterranean climate, January 0 - + 10 ° C, June +20 - + 30 ° C.

5 Sowing was carried out in both growing areas at the end of September / beginning of October.

The field test conditions thus obtained were retested and evaluated for homogeneous growth, flower size, and date of harvest, in addition, flower test-10 samples were examined for active ingredient concentration. Individuals that met the parameters mentioned in step 1 were re-selected from the field growth. From these, seeds are obtained according to the last sentence of step 2.

Step 5: 15 Using the seeds of Step 4, Steps 3 and 4 were repeated in the above order and as shown.

Step 6:

From the seeds obtained from step 5, about 20,500 individuals were grown (ecological conditions as in step 2) and selected according to the same principle as described in step 1. From the plants thus selected, 34 individuals were selected and cloned according to step 1. Every tenth plant of each clone was planted by random distribution of them at a distance of 40 x 30 cm to open ground (site I, see step 4) in an isolated site. The soil was loamy, pH 7, Q. Planting was carried out in early June, the first seed harvest was obtained in mid-July, after which the plants were cut, they bloomed again 30 and produced a second seed harvest from mid-August to the end of August.

Seed harvesting of this material was performed according to step 2.

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The seeds of step 6 are a propagation product of the chamomile variety Manzana according to the invention.

Flowers of plants obtained from this propagation product (sowing in September / October, harvest at the beginning of 5 June of the following year), picked at the time when about 50% (40 to 60%) of the mycorrhizal horn flowers have opened, and. immediately after drying at 40 ° C for 72 hours, contain, for example, 150 mg% of camazulene, 300 mg% of (-) - Ot-bisaboΙοί and not more than 50 mg% of other bisabolites, calculated on the weight (dry matter) of the dried flowers.

Other exemplary information on plants of the chamomile variety according to the method obtained according to the example: 1. Growth

Uniform growth height (uniformity) with 15 narrow flower zones, therefore particularly suitable for mechanical harvesting, large flower buds, medium yield.

Shape: branched from the root (3 to 5 times);

Stem: erect, slightly branched.

2. Press 20 Magazine: double, 2-3 fold;

Strength: medium;

Double-striped leaf, color medium green;

Double-striped leaf (stalk center); sloppiness:. from medium to strong.

25 3. Inflorescence

Mycelium (inflorescence): about 30 mm outside diameter, about 15 mm inside diameter;

Single inflorescence weight (dry): about 45 mg; 30 Flower stalk, length (mm): about 700, but depending on the growing area (climate), sowing season, soil, fertilization, plant treatment and weather conditions.

Beginning of flowering (days from 1 January): about 160 days (sowing in September, growing place-35 ka Freising, Federal Republic of Germany, otherwise dependent on the above factors).

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Flowering:

In mid-June (see above);

Inflorescences without stem, essential oil content (% of dry matter): about 1.0%; azulene content in essential oil: at least 15%;

Degradation of dried inflorescences, flower buds, is negligible if the harvest is completed before the last horn flowers open;

Pollen particle diameter: about 30 Jim; 10 seed length: about 1.25 mm; chromosome number of somatic cells: 4n: 36; 4. Sato

Thousand seed mass (TKM): 0.06 to 0.13 g; 5. Germination (KF) 15 75% 6. Purity 94 - 95% 7. Other characteristics

Drying ratio fresh: dry (flowers) = 5.5 - 6: 1, aromatic aroma characteristic of roh-20 dox, nicely aromatic typical tea extract taste.

Manufacture of herbs

The chamomile obtained as described above is sown in the usual manner. Once the plants have developed and the mycelium 25 is in a growth phase in which about 50% of the horn flowers have opened, the mycorrhizae are collected with a chamomile picker, with the comb comb being adjusted so that only those mycorrhizae with 50% horn flowers are picked. The crop is taken to the shade as soon as possible, where it is applied in thin 30 layers. Finally, the mycorrhizae are separated from the stems in a strainer and dried until the weight remains constant.

About 80% of the weight is lost. Drying takes place in the shade, in a well-ventilated place, in a loft, in layers of about 10 cm.

35 After about 2-3 days, stop drying. To separate the stem parts and the crushed flower, the dried material is sieved and finally baled.

22 8 o y 8 2

Analytical values: essential oil 950 mg% camatsulene 150 mg% (-) - od-bisabolol 300 mg% 5 If drying is carried out, for example, in a stationary belt dryer with artificially heated air at a temperature between 50 ° C and 60 ° C, stop drying for about 4 hours after. To separate the stem parts and the flower crush, the dried material is sieved and the pu-10 is finally crossed into bales. Examples of analytical values for such an herb are: essential oil 850 mg% camatsulene 80 mg% (-) - oi.-bisabolol 200 mg% 15

Appendix A

Production of essential oil The starting material is a herb obtained from the Manzana chamomile variety. Only flower-20 mycelium with 30-70% open horn flowers, especially 40-60%, is used to make the herb. Drying takes place in an oven at 40 ° C for 72 hours. The essential oil of chamomile flowers is obtained as described below by two-hour steam distillation of the herb.

2.0 g of the uncomminuted herb are placed in 1 liter round-bottom birches together with 250 ml of desalinated water and subjected to a two-hour reflux distillation on a Clevenger (steam distillation reflux apparatus for determining small amounts of essential oil).

30 1 ml of pentane is used as collector for analysis. The recovery rate is 40 +4 drops / minute. At the end of the distillation, the essential oil dissolved in the pentane is poured into the test tube as anhydrously as possible and any remaining essential oil adhering to the apparatus is rinsed with pentane. To remove any residual water, a spatula tip of dried Na 2 SO 4 is added to the solution and the solution is then filtered through sintered glass with a porosity of D 3 or D 4 into a round-bottomed flask. After evaporation of the pentane at 40 [deg.] C. in a water bath and subsequent drying in a desiccator, the amount of oil is determined gravimetrically.

The oil thus obtained (about 20 mg) is then assayed for camatsulene and bisabolol.

5 Spectrophotometric determination of camatsulene

Measuring solution: The essential oil obtained from 2 g of the herb (obtained as described above) is completely dissolved (approx. 20 mg) in 25 ml of n-hexane 10 or cyclohexane.

Measuring device: Filter photometer (eg

Eppendorf).

Wavelength: 578 nm

Cuvette: 1 cm 15 Specific extinction of camatsulene (1 g / 100 ml; 1 cm): 20.8

Compensating fluid: n-hexane or cyclohexane 20 Determined concentration of camatsulene, mg / 100 g 120jE ^ 7g

If a filter photometer is not available for the measurement, the determination may also be performed with a spectrophotometer:

Measuring device: Spectrophotometer (eg PM Q II / or PM Q III "ZEISS") 30 Wavelength: 605 nm

Cuvette: 1 cm

Specific extinction of camatsulene (1 g / 100 ml; 1 cm): 24.5 35 Compensating fluid: n-hexane or cyclohexane

Determined concentration of camatsulene, mg / 100 g: 102; EgQg

The standard solution is then assayed for bisabolol.

24 80982

Gas chromatographic determination of bisabolol and other bisaboloids

Gas chromatograph: Hewlett Packard Modell 5750, 5 Erba Fractovap 2350 or similar device

Detectors: Flame ionization detector

Carrier gas: Helium

Column: <· ν0.32 cm (l / 8 "); 200 cm; steel 10 Column packing: 3% nitrile silicone rubber" XE 60 "with silica gel supported on silanized" Chromosorb WAW HP "125 - 150 pn Temperature:

Detectors 320 ° C

Feed nozzle: 220 ° C

Column: 85-220 ° C

Temperature programming: 4 ° / minute Sample solution: A measuring solution of camatsulene is used.

Reference solution: Dissolve about 15 mg of standard bisabolol in cyclohexane to 25 ml

Feed rate: 5 μΐ of each of the sample solution and reference solution

Judging: Judging is done by comparing the area of the peaks

Bisabolol content in milligrams per 30 100 g of herb: 10 x weighed amount (comparison)

Cm C x

Surface Sub-sample

When compared per area

II

25 80982

The determination of other bisaboloids can likewise be carried out by gas chromatography, for example under the following experimental conditions:

Device: Packard, Model 7721, 5 Serie 800, or equivalent ti Erba Fractovap Serie 2350

Columns: Glass columns, 3 m / 2 mm in diameter; 2 m / 2 mm diameter 10-gauge Filling: 3% methylphenylsilicone rubber "OV 1" with silica gel silanized "Gashrom Q" 125 - 150 pm 15 Carrier gas: 30 ml / minute ^ Temperature programming: from 80 ° C to 180 ° C: 2.5 (3) ° C per minute

The injection / of detectors

temperature: 200 ° C

20 Detectors: flame ionizer detector

Feed rate: approx. 2 μl of essential oil diluted approximately 1:50 25

The review took place partly without, partly using an internal standard. Lauric acid methyl ester or hexadecane are suitable as internal standards for samples of camatsulene-poor, for oils with a camatsulene content of more than 5%, this should be considered as a better internal standard, where the concentration is determined photometrically (at 578 nm).

Claims (5)

A process for the preparation of a tetraploid chamomile variety (called Manzana), whose flowers dried at 40 ° C contain at least 150 mg% of the camazule, at least 300 mg% (-) - a-bisabolol and less than 50 mg% of the other bisabolols calculated dry chamomile (Chamomilla recutita (L.) Rauschert, synonym Matricaria chamomilla (L.), Asteraceae), characterized in that the diploid chamomile species DEGUMILL is tetraploidized with chemicals at a temperature of 0 - 35 ° C. with gamma radiation, X-ray or UV radiation at a temperature of 0 - 35 ° C; using high temperatures 33-55 ° C; or using low temperatures 0 - 5 ° C; and after tetraploidization a) selection of tetraploid plants on the basis of active substance content (at least 150 mg% of camazule, at least 300 mg% bisabolol and less than 50 mg% of other bisaboloids, all based on dry weight), is carried out, coincident flowering time, even from the root outgoing branching and a size of 25 - 35 mm for the flower heads, the well-selected plants are cloned, and from the plants obtained by cloning, seeds are taken, b) are derived from a well-received seed, then selected in accordance with paragraph a) and from the selected plants are eaten seeds; (c) the measures in paragraph (b) are repeated 2-4 times, (d) from a seed obtained in paragraph (c) taken offspring which are selected according to paragraph (a), the thus selected plants are cloned and from the plants obtained by cloning, seeds are taken and from this kind of camomile plants obtained are possibly a propagating product and / or camomile drug is made. 2. A process for producing chamomile drug using the novel tetraploid chamomile variety (named Manzana) prepared according to claim 1, characterized in that chamomile balloons are harvested at a growing stage, where 30 to 70% of the flower heads of the flower heads are opened. themselves and the flowers are dried at an air temperature not exceeding 50 ° C. 5 Process for producing chamomile drug using the new tetraploid chamomile variety (named Manzana) prepared according to claim 1, characterized in that the chamomile plants obtained are further propagated and the flowers are harvested in a growing stage, where 30 - 100% of the flower heads tubular flowers opened and the flowers dried at an air temperature of not more than 60 ° C, yielding dry material containing at least 120 mg% of camazule, at least 200 mg% of (-) - α-bisabolol and less than 50 mg% of other bisaboloids. 15 Process for the production of alcoholic chamomile extract or essential chamomile oil using the chamomile variety prepared according to claim 1 or its dried balloons, characterized in that the extracts obtained by extraction with alcohol contain at least 5 mg% of the camazule, and at least 15 mg% ( -) - α-bisabolol and less than 8 mg% of other bisaboloids, and the ethereal oil obtained by destination in the presence of water contains at least 5% of camazulene and at least 15% (-) - α-bisabolol and less than 10% of other bisaboloids. 5. Use of the tetraploid chamomile variety prepared according to claim 1 or its dried balloons for the production of alcoholic chamomile extracts or essential chamomile oil.