CS244688B2 - New variety of amomile cultivating method - Google Patents

Abstract

Process for producing a pharmaceutical camomile product wherein flowers of the tetraploid camomile variety Manzana containing at least 150 mg % of chamazulene and at least 300 mg % of (-)- alpha -bisabolol, other bisaboloids amounting to less than 50 mg %, are harvested at a stage of growth when only 30 to 70% of the tubular florets of the flower head are open and dried at an air temperature not higher than 50 DEG C.

Description

A method for obtaining a new camomile variety (Manzana designation) whose flowers dried at 40 ° C contain 150 mg% chamazulene and at least 300 mg% (- (- alpha-bisabolol, with other bisaboloids below 50 mg%) by tetraploidization of the diploid camomile variety DEGUMILL and subsequent selection and propagation.

Method of preparation of a new variety of camomile (designation Manzana).

The chamomile flower head chamomile (Chamomilla recutita (L.) Rauschert, synonymous with Matricaria chamomilla L.) finds a wide therapeutic use due to its antiphlogistic and spasmolytic effect and forms an important part of the range of medicinal plants. The active substances (-) - alpha-bisabolol and chamazulen are of particular therapeutic importance. Therefore, a high-quality camomile drug should contain the highest possible content of both substances.

DEGUMILL (the type of chamomile mentioned in German Patent Specification No. 24 02 802; NDr - DEGUMILL type of protection; Italian Patent No. 1 035 096) is known to have a chamomile type which also has a high content (-) -alpha-bisabolol and chamazulene.

The content of chamazulene and bisanolol is permanent only if accidental crosses or foreign pollination with other camomile species whose content of bisabolol and chamazulene is substantially lower, possibly with a content-containing wild camomile present everywhere, is avoided.

This risk of cross-pollination is almost always a threat, as wild camomile occurs almost everywhere.

Tetraploid camomile species are also known (for example, BODEGOLD, GDR; POHORELICKÝ CSSR; LANGLES, Poland, BK-2, Maclarsko). These tetraploid camomile species have a chamazulene content which is not substantially lower than that of the Manzanian species.

On the other hand, in these known tetraploid species, the active ingredient (-) - rlfr-bisabolol is present in very small amounts and, as a result, other bisaboloids (bisaboloxide A and B as well as bisabolonoxide) predominate, producing more than half of the ether oil in known tetraploid chamomile species.

We have now found a process for the production of a novel tetraploid camomile species with advantageous properties.

The invention therefore relates to the points defined by the subject matter of the invention, or the fusion of things.

The novel tetraploid chamomile of the invention, designated Manzana, has surprisingly many new and advantageous properties compared to the known chamomile DEGUMILL as well as other known tetraploid chamomile.

Compared to DEGUMILL, it resists foreign pollination by naturally occurring camomile populations (wild camomile). In addition, it has a significantly higher content of the important active ingredient (-) - alpha-bisabolol than DEGUMILL.

Compared to the known tetraploid camomile species, the Manzana species of the invention are distinguished by a surprisingly incomparably higher content of the important active ingredient (-) - alpha-bisabolol as well as a very low content of other bisaboloids.

Surprisingly, Manzana also differs from known tetraploid chamomile species with other properties: improved seed germination; less waste of the unusable part of the plant (ie higher flower yield); lower water content in flowers (ie higher yield of dried flowers and shorter drying time); it is more suitable for machine harvesting because the flowers are liquidated in one plane and most flowers bloom at the same time (this allows for a uniform harvesting date); better shelf life of the drug (less tendency to disintegration and pulp formation); characterized by a particularly aromatic and typical aroma of chamomile.

Furthermore, the chamomile type Manzana according to the invention is homogeneous with respect to the substances as compared to the known tetraploid chamomile species, with the properties of the chamazulene and (a) -alpha-bisabolol (i.e. homozygous) content remaining permanent.

In contrast, tests with individual plants of known tetraploid species show that individuals have different levels of substances, i.e. each sample taken from the drug thus obtained differs both qualitatively and quantitatively, being inhomogeneous.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel tetraploid camomile species having improved properties, in particular a higher content of (-) - alpha-bisabolol.

The production of the new camomile species according to the invention is carried out by converting it to the tetraploid form of the camomile DEGUMILL (gene mutation) and the subsequent further steps of selection and propagation (cultivation of mother plants for vegetatively propagated foreign fertilizers). Manzana consists of strains or lines, which have been separately processed while maintaining cultivation.

Conversion to the tetraploid form can be accomplished, for example, by treating parts of plants (m (m names, roots, sprouts, sprouts, buds, raids)) or DEGUMILL chamomile tissue using chemicals, X-rays, gamma-rays, uf-rays. by the method of dakepitization of callus, using anthropogenic cultures, applying high as well as low temperatures to chamomile plants of the DEGUMILL species, possibly part of the plant or tissue of DEGUMILL plants.

For example, reference may be made to Werner Gottschalk's book Die Bedeutung der Polyploidie für Due Evikztuon der Pflanzen, Gustav Fischer Verlag, Stuttgart, 1976, in particular pages 13 to 22.

Conversion to tetraploid form using chemicals.

For example, the following chemicals can be converted to the tetraploid form: Colchicine, acnaften, alkaloids such as atropine, veratrine, nicotine, sanguinarine, benzene derivatives, diphenyl and phenathrene, naphthaline and naphthalene derivatives, diphenylamine, tribromaniline, p-dichlorobenzone, methylnaphthochine, methylnaphthochine salicylic acid and related substances, hexachlorhexane, pervitin (hydrochloride), alkali metal alkyl carbamates such as sodium isopropylcarbamate, phenylurethane, cacodylic acid salts (e.g. sodium salt), Konvallaria glycosides such as Convallarin, convallatoxin and convallamarin, heteroauxin, Germisrokatech (phenylurethmer) organic mercury derivatives such as ethyl mercury E-phosphate, ethyl mercury E-chloride, phenyl mercury E-hydroxide, phenyl mercury naphthyl methanesulfonate, chloroform, the so-called tomato gas (N ₂ 0), and mixtures thereof. Pellets, compost and cow manure are also possible.

P ^s with ^b en ^{e ye} l ^á said weave is prov ⁱ ^ i p ^Rikli farm is considerably ^during te ^p diisopropylamine, among. 0 and 35 DEG C. The advantage of the ou ^{d. 12,} and especially ³⁰ ° C and ^{25 s} at ²⁵ ° C.

Application is effected, for example, by treatment of seeds, shoot tops, roots (especially root endings or germ roots), fertility, leaf or stem cuts, cell suspensions from interstitial tissue, callus cultures or injection into the basal area of the stem or into field of buds in the armpits.

Chemicals are generally used in the form of solutions in water, weakly alcoholic solutions (alcohol content is generally less than 5%), or weakly acidic solutions are used. The pH of the weakly acidic solution is between 5.5 and 6.5, the acidification being carried out with low organic aliphatic acids such as acetic acid.

If alcoholic solutions are used, these may also be slightly acidic. The concentration of 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%. The gaseous substances are suitable for use under pressure (for example 1 to 10 bar). The duration of action is, for example, 1 to 36, preferably 2 to 12, in particular up to 6 hours.

The most effective concentrations as well as the duration of action had to be expediently evaluated each time in the experiments. It is especially advantageous as treatment with colchicine at temperatures between ⁰ and ³ 5 ° C, above Oda l ^{H 2 and} 30 ° C, especially ² to L ^{s and 25} ° C.

This can be done, for example, by introducing DEGUMILL chamomile seeds to swell in 0.01-0.2%, in particular 0.01-0.1%, preferably 0.05% colchicine solution, or germinated, 5-7%. The days-old, well-developed camomile DEGUMILL species (with the collar leaves down) are immersed in 0.01 to 0.1%, in particular 0.02 to 0.1%, preferably 0.05% colchicine solution. '

In the latter process, the ambient atmosphere should have about 100% relative humidity. The duration of the colchicine treatment is about 3 to 36 hours, in particular 4 to 10 hours. When using seedlings, an exposure time of up to 10 hours is generally sufficient. When seeds are used, the exposure time can be extended up to 36 hours.

After exposure to chemicals, the swollen seeds, seedlings or plant parts are washed repeatedly with water. The swollen seeds are sown for example. Plants with treated roots or parts of plants or treatment of a seedlings are pricked in a plant box.

The thus treated seeds optionally seedlings were grown plants, such as glass ^CART at ^at rate ^l OTE limit ^{and 18 and} 25 ° C during the day and ϋ0 and ^{from 16} ° C at night and ^Yb Iraj ^s is increased ^and n ^y a ^{i HZ} c pollen is about l, 5 times larger than the pollen source material, or the number of chromosomes of somatic cells is 36th

If individual parts of plants (aboveground or underground parts) are treated with chemicals, only shoots expelled from the treated parts, roots or flowers / seeds are subjected to later examination and conversion into tetraploid form. If, for example, the shoots or the treatment of the axils have been carried out, then only the shoots or the eye which has been expelled from this eye are examined and the flowers or seeds produced on it are examined for the number of chromosomes.

Pollen size measurements and chromosome counts can be performed, for example, as described in Example 1.

Conversion in tetraploid form by beams.

Treatment is carried out for example to the seeds or root ends at temperatures between 0 and 35 ° C, preferably 10 to 30 ° especially 3 to ^{15 s} and ²⁵ ° C. ^{Oz, and} rhenium amount: 5 x crouch. Preferably, gamma- and X-rays come in the livestock. Suitable U-rays are those having a wavelength of 400 to 300 nm, preferably 350 nm.

Irradiated plants, or parts thereof, are treated further, such as after exposure to chemicals.

Use of higher and lower temperatures.

As high temperatures are, for example, temperatures between 33 and 50 ° C, preferably bound ^{and 42-4} 5 ° C. ' T ^{e t h} e ^p MTO ^Lot m are ^{of ex} and ^d-TX point: seeds swell ^and, once ^the above ^h one ^To a meristematic tissue.

Duration of action: for example 1 to 48 hours, preferably 1.2 to 24 hours. As low temperatures are, ^ú weighting: 0 ^and 5 °, preferably 3 to ^4, especially from 0.5 ^s to ² ° C. Te to these ^pl Otama are subject ^{of ex} ad: swollen seeds, seedlings, shoots and meristematic tissue. Duration of action: for example 1 to 100, preferably 20 to 40 days. Plants thus treated, or parts thereof, are processed further as after treatment with chemicals.

Callus decapitation method (decapitation = removal of head or tip removal or back cut) The decapitation is carried out, for example, on young plants on the stem, preferably at the end of the vegetation cone, after the formation of 4-6 leaves or also on petioles or side shoots.

The buds are brought to the same as emerging from the injured tissue (callus tissue), eventually shoots are cut off, seasoned, cultivated further in pots and tetraploid plants are selected during processing by chemicals.

Anther cultures (preparation of plants with a single number of chromosomes from anther vesicles with subsequent spontaneous or artificial tetraploid transformation).

The flowering plants harvest closed tubular flowers whose anthers, anther sacs are found in the stage of the first pollen mitosis. The anthers are removed from the flower buds by means of a micromanipulator, transferred to petri dishes, which are filled, for example, with Nitsch and Nitsch broth (Table 1).

The Petri dishes uchovávájív culture chamber for 16 days at 28 ° C by day and ²⁰ ° C at night .. As the ^IP ujalynuU up ^{by 4 characterized d} N ^{for No} for either ^p ciliated ^{ue p} u ^k and ^t, and grows grow up. These are haploid in the diploid parent moiety, dihaploid in the tetraploid parent moiety; after the root system is established, they are transplanted to the garden soil in pots and brought to bloom in the greenhouse.

These dihaploid plants are sterile, but can be converted into polyploid forms by treatment of the ends of the shoots, roots, or stems with chemicals such as colchicine, producing homozygous plants that can be propagated further by seeds. For other procedures, see chemicals (for example, colchicine).

Table 1

Nitsch and Nitsch culture (Science, 1969)

7H2 ° ^4H 2 ° ^7H 2 ° x 2H ₂ ° ^t 2 ° mg / 1

950

720

185

166

0.25

0,025

KN ° ₃ nh ₄ no ₃ MgSO 4 x CaCl ₂

KH ₂ P ₄

MnS ° 4 x ^H 3B ° ³

ZnS ° 4 x

Na2Mo04

CuS ° 4 x ml solution from 7.24 g disodium ethylenediaminetetraacetic acid and 5.57 g FeS ° 4 x 7H ₂ ° per 1000 ml myoinosit glycine nicotinic acid pyridoxine hydrochloride thiamine hydrochloride folic acid biotin sucrose broth (DIFCO-Bacto- agar) indolylacetic acid pH of the medium adjusted to 5.5

100 ALIGN!

0.5

0.5

0.5

0,05 g s g 'From the measurement of the pollen size and / or number of chromosomes of the selected chamomile tetraploid plants obtained by the previously described possibilities, the following plants are selected which meet the following conditions:

a) Flowering at approximately the same time

b) Even, basic branching and narrow flowering zone about 5 cm

(c) Large flower heads of external diameter of about 30 mm (25 to 35 mm),

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

The plants thus sought are now propagated vegetatively by seedlings (by means of clones) and are monitored for 3 to 5 generations, the choice being carried out according to criteria (a) to (d) above. That is, the plants found under criteria (a) to (d) are cloned, seed is obtained from the cloned plants, the plants derived therefrom are selected according to criteria (a) to (d), and seed is recovered from the last.

Sequence sowing - selection according to a) to d) seed yield can be repeated 2 to 4 times. The final sequential sowing - selection according to a) to d) - cloning - seed recovery is then carried out.

The seed finally obtained is then a seed for propagation of the Manzana camomile according to the invention.

Propagation by seedlings is carried out as follows: In order to propagate by seedlings, the starting plants (clonal mother plants) must produce short shoots free from flower buds under short day conditions. This occurs v.zimní half of the greenhouse without additional illumination not ^b o ^kl ima ^those ionised Tomori daily d ^e l ^{6-10 alkyl, or o} n ^{e 8h} Odin ^t e ^p s ^{Lot 10-15} ° C preferably ¹² ° C.

The cloning or propagation are suitable leaf, shoot and especially seedlings with ^the caliber ^tk YM ^{ye H} on ^{alkyl (p} ostranními shoots). ^The E includes ^for the ^row ene ^with Doc ^{h-i Pr} I te ^pl rpm ^{of 12} to ¹⁸ ° C, above ^h Oda ^{at 15} ° C ad ^e LCE of ^12-16, preferably ¹⁴ hours ^and nvu ^p Raven Atmos ^fer e (about 100% relative air humidity).

For example, a 1: 1 peat-sand mixture may be used as a substrate, pure silica sand, crushed stone for seedlings, peat for seedlings, and the like are also suitable.

Suitable for sowing are: garden soil, humous medium-heavy loamy soils, loamy or humous sandy soils. They can be planted in a greenhouse or open ground. Temperatures key ^I, Ar en ^{No growth} is voltage between ^Rikli and ^{d. 12-24} ° C, especially 18 ^s and ²⁰ ° C. In at ^p and ^DE freely ^eh o ^y seeding is preferably sown in the autumn (September / October) or early spring (March / April). These terms apply to all eligible territories (eg northern hemisphere, mild to subtropical climate).

The new camomile species according to the invention belongs to the cultivated plant species of the true camomile of the botanical name Matricaria Chamomilla L. (synonym Chamomilla recutita / L. / Rauschert) and is derinated by the active substance data according to point 1.

The active substance content values given there refer to those developmental stages of the flower which are obtained when 30 to 70%, in particular 40 to 60%, of all tubular flowers of the flower head is open, meaning to determine (the active substances used were hair during this time). ^and NY ^p Otom dried and ^yp of a ^{d b} at ^{72 hours} at Odin. ^t ep ^l ot ⁴⁰ ° C oven).

If the chamomile flowers are harvested at a time when the development stage of the flowers has progressed, eg 100% or almost 100% of all tubular inflorescences have already opened (eg at full bloom) or / and if they occur drying the harvested flowers on that lot ^of the ^{p yšší} than ⁴⁰ ° C \ ^b SAH is employed ^diacrylate e ^k H ^ AHA satoloh and chamazu ^ nu her ^primary and.

Thus, when harvesting is carried out in the vegetation stage, where 30 to 100% of tubular inflorescence flowers are opened and dried at a temperature of up to 60 ° C (preferably with the exclusion of sunlight, e.g. under such drying conditions as described below). the camomile drug thus obtained contains at least 120 mg% of chamazulene and at least 200 mg% of (-) - alpha-bisabolol, while the content of the other bisaboloids remains, as in the previous explanation, below 50 mg%.

Other bisaboloids in the definition of the subject matter of the invention are in particular meant:

chamomile species traces easily (-) - alpha-bisaboloxide 1 and B; (-) - alpha-bisabolone oxide A; other proportions of the ether oil according to the invention are En-In-dicycloether, farnesene, spathulenol and small concentric terpenic hydrocarbons.

For example, kv ^ethyl SIMEN SU ^propenoate RNE ^EXAMPLE te ^pl ot ⁴⁰ ° C (free of damage in the herd-floral development ^which is apertured 30 to 70% of the tubular flowers of the inflorescence) type Manzana according to the invention contains between 150 to 200 mg% chamazulene , between 300 and 450 mg% of (-) - alpha-bisabolol and very little, i.e. between 5 and 50 mg% of other bisaboloids, based on dry drug (i.e., based on absolute dry flower weight).

The absolute dry weight was determined by drying a separate sample of camomile flowers ^p o ^dL e VYN ^s crawling type ^M Anzano SU ^{Arne at t} e ^pl OTE ¹⁰⁵ ° C to constant weight ^(p o dot »at ⁷² and ⁹⁶ hours) . ^B is O ^h U ^{c h} innýc ^la te ^{to, comm} graceful ^{e p} s ³⁵ to ⁵⁰ ° C are enýc ^{w h e} Tu q ^{(<DRO <JY),} then a flag on the dried flowers ^{of e p} s. te ^p lot ^{of 105} ° C.

In its phenotype, the Manzana species of the invention are similar to the hitherto known tetraploid, BK-2, Pohořelický Velkokvětý); other than (-) - alpha-bisabolol, the main constituent of ethericchamazulene is lower and the content of other bisoboloids is lower. Other ethereal oil components and En-In-dicycloether.

chamomile species (for example Bodegolg, Zloty Lan in particular by the fact that in the Manzana species according to the invention the flower oil that in addition has a higher content (bisobololoxide A and B; bisabolonoxide) substantially of manzana species is farnesene, spathulenol

The chamomile species of the invention may comprise more than 20% of organic matter (humus-like agrotechnical measure or cultivation day of more than 13 hours, i.e., tropics).

be cultivated successfully on all soils containing more substance and soil organisms) no special measure is required; Cultivation requires only the so-called long for cultivation are the most temperate zones and sub

The chamomile type of Manzana of the invention has a medium late harvest time, a uniform growth height with a narrow flower zone and large flower heads and is therefore particularly suitable for mechanical harvesting. Furthermore, at the same time planted Manzana plants bloom practically together at the same time, so harvesting is greatly simplified and facilitated.

The camomile of the Manzana species according to the invention yields a high yield. Possible uses include extracts, as well as an example: obtaining chamomile as a drug, chamomile oil, chamomile natural (-) - alpha-bisabolol.

Drug camomile species Manzana invention has a maximum content of nu and bisabolol when the flower heads are harvested in vegetation stage 70% preferably 40 to 60%, i.e., generally 50% of the tubular flowers of flower heads open, and ^p Roveda was dried ^with EN ^{even when} te mod ^p ture ^d uc u max ^h ^ 50 ^ cially ^{of ex} adn ^{3 of} 5 to 50 "C.

The active ingredients of the chamazule are, for example, from 30 to 30%

The drying process can be carried out either by artificial air supply or also by drying in the shade or even in the sun, taking care that the heat supply is not higher than necessary for perfect drying.

It is also advantageous to see from time to time the constant weight achieved. Dry1

be top tall early, up to 20 cm, spontaneous or artificial (for example, by artificial warm air). Yield of active ingredients after spontaneous drying with the exclusion of sunlight. The drying process, if necessary simultaneously, preferably at 10 cm is harvested. Drying should take place in thin layers, for example layer thickness.

The determination of chamazulene is carried out spectrophotometrically as with conventional camomile extracts. Determination of (-) * alpha-bisabolol as well as other camomile oil ingredients is carried out by conventional chromatographic methods.

A detailed account of the analytical procedures is given in Annex A.

The active substance chamazulene is present in camomile flowers not as such, but in the form of a secsviterpenic lactone matrix. Matricin is pharmacologically active, its effect corresponds to chamazulene. For example, chamalzulen is immediately formed from the precursor matrix by heating (e.g., steam distillation) by infusion of tea. It is therefore customary to report the chamazulene content of the chamomile, not the matrix content.

Examples

Example 1

DEGUMILL chamomile seeds are spread on filter paper with a 0.05% aqueous colchicine solution and allowed to swell at room temperature (20 ° C) for 6 hours. They are then removed from the filter paper, washed repeatedly with water and sown in a greenhouse: soil: peat-sand at a ratio of 1: 1, temperature: 18 to 20 ° C, relative air humidity of about 60%, length of day under artificial lighting : 14 hours.

Germinating plants were observed only until flowering, the result of the conversion to polyploid form was evaluated by comparative measurement of pollen and seed size and chromosome count (F-potency) = the first seed progeny that represents the first tetraploid plants after colchicine treatment.

Conversion to the tetraploid form can also be carried out as follows: On water-moistened filter paper, germinated, 5-7 days old DEGUMILL chamomile seedlings at room temperature (10 ° C) for 4 to 6 hours with the leaflets down, were transferred to 0, 05% colchicine solution. Looped roots were spared; In order to prevent drying damage, the surrounding atmosphere must have approximately 100% real air humidity.

After treatment, the seedlings were washed several times with water and plated in plant boxes. This was followed by the same treatment as the seeds.

Measurement of pollen size was performed by a Leitz binocular research microscope with a microscope equipped with a micrometer and a micrometer slide.

The chromosomes are counted at the root tips: Fresh green root tips 1 to 2 cm long were collected from young plants or seedlings grown in the greenhouse: 5 hours were loaded in 0.002 molar hydroxyquinoline solution and then for 15 minutes in 1N hydrochloric acid.

For microscopic examination, about 1 mm long root ends were stained with 2% orcein-acetic acid and microscopically examined for oil immersion. Thus, four times the number of chromosomes of somatic cells (4n = 36) can be determined in cells found in the mitosis stage.

Those plants in which the pollen diameter was about 50% greater than that of the diploid starting material (about 30 instead of about 20%) and in which the somatic cell chromosome number was doubled to 36 (the corresponding number of diploid material is 18) are tetraploid.

These were selected. About 0.1 to 0.5% of the seeds or germinate plants were transformed into the tetraploid form as described above to yield flowering, intact plants. The next steps are as follows:

Step 1:

From (as described above) selected chamomile tetraploid plants were selected those that

a) almost simultaneously bloom,

(b) have an even, basic branching and a narrow floral zone of about 5 cm;

(c) have large flower heads of an internal diameter of about 30 mm;

d) reach the minimum content of the chamazulene of 150 mg% and 300 mg% bisabolol or it crosses and the content of other bisaboloids (particularly bisabolol oxides) is clearly PDO 50 mg%) All values are based on flower heads which were dried ^{at te pl} of ^{i 40} C refrigerati ^HŽ skMzeň ^was provetona in stacliu and ^{when p} te as tearing ^and 5 ^0% (40-60%), tubular flowers blossomed headers.

These plants were cloned. For this purpose the plants (clone mother plants) initially cut to about 15 cm long and shoots at a length of 8 to 10 hours and at 12 to ¹⁴ ° C hilarious к new shoots (Cr ^and TK ^s side shoots). Kr ^and TK shoots ^{y s y} l b ^{y of} the OF ^and n ^y and placed in a mixture of peat-sand. At about 100% relative humidity, air temperature ¹⁵ ° ^C and no ^lon ce ^{d 14 h} of ^di n ^t tearing evolution ^to ořenového hundredth betting with ^y ^ ^d of elk after ^b u ^{t o} 7 ¹⁴ days.

Instead of the usual cloning (multiplication of seedlings) it is also possible to use multiplication of tissue parts capable of dividing in vitro (so-called meristem propagation). Various parts of the plant, preferably the ends of the shoots or buds in the axils, are suitable for establishing a camomile culture.

After rinsing the plants, under aseptic conditions in the laminar flow (high performance conditions in the laminar flow (high performance filter with displacement flow limiting turbulation)) the ends of the shoots are removed from the leaf axes and transferred to reagent containers filled with nutrient media such as Murashige and Skoop Physiol Plant 15: 473-497 (1962).

Reagent containers are removed in the air conditioning compartment for 12 to 18 preferably for 16 hours day length (obtained fluorescent lamp), light intensity from 500 to 10 000 lux with the above ^h Oda ¹ 00 ^{0-3 0} 00 ^l UX and te ^p lo ^{thee 15 and 30} ° C, above about ^{d h} ou ^{22 and 27} ° C.

Once the explanations achieve good growth, they are transferred to the above culture medium but with a higher cytokinin concentration (30 mg / l N ⁶ -isopentenyladenine) and with little or no auxin (0 to 0.3 mg / l indolylacetic acid). As a result, the axes stretch and adventurous organs are formed, as well as multiple buds in the armpits. These can be removed and grown as described above.

Explanates intended for the multiplication of plants (third passage = third generation of propagation) are transferred after growth and leaf formation in the initially mentioned culture medium to a culture medium containing 10 mg / l indolylacetic acid or 3-indolylbutyric acid or 0.1 up to 0.3 mg of alpha-naphthylacetic acid.

/

Plantlets rooted and after about 4 weeks, can be grown in pots with sterile for ^h r ^o n ^{i c} ou pMou (expiry ^p o ^d a ^b l ^{2 h} of ^di n ^@ te ^pl OTE l ²⁰ ° C) and ^{KL Cart} en u (for ^C o ^d m ^EQS common to the conventional propagation cuttings) grown further.

Murashige and Skog culture medium mg / 1 mg / l

nh ₄ no _s 400 Indolylacetic acid 2,0 Ca (NO ₃ ) ₂ .4H ₂ O 144 furfuryladenin 0.1 ^KN0 3 80 thiamine 0.1 kh ₂ Mon ₄ 12.5 nicotinic acid 0.5 MgSO ₄ 7H ₂ O 72 pyridoxine 0.5 KC1 65 glycine 2,0 NaFe-EDTA 25 myoinosit 100 ALIGN! ^H ₃ B ° ₃ 1/6 Casein-hydrol. 1 000 MnSO..4H _o 0 4 2 6.5 sucrose 2% ZnSO ₄ .7H ₂ O 2.7 purified powdered agar 1%

Stage 2:

The plants obtained according to step 1 bloomed together in a greenhouse under isolated conditions. The plants stood in 11-cm pots filled with garden soil at 18-24 ° C by day and 12-14 ° C at night. The day lasted at least 14 hours and was achieved by additional lighting (200 Watt / n ^) in the winter half of the year. Water was supplied as needed.

Of the selected individuals, for 4 weeks the heads that bloomed and were shortly before disintegration were continuously harvested on the seed. The seed was obtained after drying the harvested flowers at room temperature by blowing out lighter flower parts.

Stage 3:

A random sample was taken from the seed obtained in Grade 2 and about 2000 offspring were obtained (ecological conditions as in Stage 2) and selected according to the same criteria as in Stage 1 (a) to (d).

Stage 4:

A part of the seed of step 3 was sown at two different sites in the autumn.

Station A)

450 m NN, 48.5 ° N / 11.5 ° O

750 mm annual precipitation wet, mild climate, January -10 to 0 ° C, July +10 to +20 ° C,

NN = North bas. altitude plane = sea level ° N = north latitude (ngrad) ° O = east longitude (ngrad)

Station B)

200 m NN, 42 ° N / L ° O

400 mm annual rainfall Mediterranean climate, January 0 to +10 ° C,

July +20 to +30 ° C.

The sowing at both sites was done in late September / early October.

The results obtained in this field trial were tested and judged in terms of homogeneous growth, flower size, harvest time, and in addition, random flower samples were monitored for drug content. Again, those individuals corresponding to the parameters listed in step 1 were selected from the field result of the experiment. Seeds corresponding to step 2 were obtained from them.

Stage 5:

Steps 3 and 4 were repeated with the seed of step 4, respectively.

Stage 6:

About 1,500 individuals (ecological conditions as in step 2) were obtained from seed obtained in step 5 and selected according to the same principle as in step 1. 34 individuals were selected from the selected plants and cloned according to step 1.

In each case, 10 plants of each clone were grown in random distribution at a distance of 40 x 30 cm in a free field (site A, see step 4) in an isolated location. The soil was of permeable clay, pH 7.0; cultivation began in early June, the first seed harvest was in the middle of July, then the plants were mown, bloomed once more and provided another seed harvest in the middle and late August.

The seed of this material was obtained according to step 2.

The seed obtained according to step 6 is seed for the propagation of camomile of the species Manzana. Plant flowers from this propagating seed (September / October sowing, harvest in early June of the following year, which have been combed to the deadline when about 50% (40 to 60%) of tubular head flowers are open and which are then dried for 72 hours at For example, 150 mg% chamazulene, 300 mg% (-) - alpha-bisabolol and a maximum of 50 mg% other bisaboloids are included in the oven at a temperature of 40 ° C in the oven.

Further exemplary data for the plants obtained according to the example of the camomile species according to the invention:

1. Growth

Uniform growth height (balance) with a narrow flowering zone, therefore particularly suitable for mechanized harvesting, large flower heads, medium harvesting;

Shape: basic branched (three to five times)

Cone: straight, little branched

2. Olistění

Leaf: pinnate, two to three times

Strength: Medium;

Pinnate leaf, medium green

Pinnate leaf (center of cone); medium to severe constriction

3. State of inflorescence

Flower baskets (flower heads):

about 30 mm outer diameter, about 15 mm inner diameter;

Weight of single cup (dry): about 45 mg

Flower head, length (mm): about 700, but depends on the place of cultivation (climate), sowing time, soil, fertilization, plant treatment, ventilation;

Beginning of flowering (from 1 January):

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

Flowering: middle of June (see above);

Petioles, ethereal oil content (% dry matter); about 1.0%, azulene content in an ethereal oil; at least 15%;

Disintegration of dried flowers, flower drug: mild if harvested before opening the last tubular flowers;

Pollen diameter: about 30 µm;

Seed length: about 1.25 mm;

Number of chromosomes in somatic cells: 4 n: 36.

4. Fetus

Thousand seed mass (TCM) of seeds: 0.06 to 0.13

5. Germination (KF)

75.75 '%

6. Purity

- 95%

7. Other characters

Drying ratio of fresh: dry (flowers) = 5.5 to 6: 1, remarkable aromatic aroma of the drug, delicate aromatic typical taste of tea infusion.

Annex A

Obtaining an ethereal oil

The starting material is a drug from the type of chamomile Manzan. For the manufacture of the drug, tubular flowers are used where 30 to 70%, in particular 40 to 60%, of tubular flowers are open. Drying is carried out in an oven at 40 ° C for 72 hours.

Camomile essential oil is obtained as described in the following two-hour steam distillation of the drug:

The 2.0 hour non-comminuted drugs are mixed in a 1 liter round-bottomed flask and subjected to a two-hour distillation in a reflux apparatus (water distillation apparatus for gravimetric determination of small amounts of ethereal oils). 1 ml of pentane for analysis is used as a template. The reflux rate is 40 + 4 drops per minute.

After the distillation is complete, the etheric oil contained in the pentane is preferably separated from the water and is washed off with pentane in the apparatus, possibly containing the residual ether oil. To remove any residual water, a small amount of anhydrous sodium sulphate is added to the solution, and the solution is then filtered through a D 3 or D 4 glass frit into a ground glass flask.

After evaporation of the pentane at 40 ° C in a water bath and drying in a dessicator, the oil is evaluated gravimetrically.

The oil thus obtained (about 20 mg) is then determined for chamazulen and bisabolol.

Spectral photometric evaluation of chamazulene

Determined solution: A total of 2 g of the drug obtained, the ether oil (about 20 mg) obtained as described above, is dissolved in 25 ml of n-hexane or cyclohexane. Measuring apparatus: Photometer with filter (eg Eppendorf). Wavelength: 578 nm Cuvette: Specific extinction 1 cm chamazulene

(1 g / 100 ml; 1 cm): 20.8

Condensation liquid: n-hexane or cyclohexane

Content of chamazulene found in mg / 100 g: 12Ο.Ε ₅₇ θ

If a photometer with a filter is not available for measurement, the determination can also be carried out using a spectral photometer.

Measuring apparatus: Wavelength Spectral photometer (eg PM Q II) or PM Q III ”Zeiss) 605 nm Cuvette Specific extinction 1 cm chamazulene (1 g / 100 ml; 1 cm): 24.5

Compensation fluid: n-hexane or cyclohexane

Content of chamazulene found in mg / 100 g: 1Ο2.Ε ^ θ ₅

Bisabolol is then measured in the measured solution.

Gas chromatographic determination of bisabolol and by gas chromatography Hewlett

Detector:

Column:

Column content:

Temperature:

Detector:

Injection block:

Column:

Temperature programming

Sample solution:

Reference solution:

Injected amount:

Evaluation:

Bisamolol content in mg 100 mg drug:

Evaluation of other bisaboloids can be made from the following conditions:

Equipment:

Columns:

Cartridge:

Packard, Modell 7,721, Glass Columns 3 m / 2 mm 3% methylphenyl silicone Gaschrom Q 125 to 150 µm other bisaboloids

Packard Model 5 750, Erba Fractovap

350 or similar device

Flame ionization detector

0.031 cm; 200 cm; steel% silicone rubber XE 60 on siliconized diatomaceous earth Chromosorb WAW HP 125 to

150 µm as carrier

320 ° C

220 ° C to 220 ° C

4 ° / minute

A specific solution is used as in chamazulene

About 15 mg of standard bisabolol is dissolved in cyclohexane to a volume of 25 ml

From the sample solution and the reference solution always 5/11

The evaluation is performed by comparing the areas of maximum 10 ^x weight (control) ^x

Area / sample:

Area / control may be performed by gas chromatography, for example

Series 800 or Erba Fractovap Series 2,350 in diameter; 2 m / 2 mm diameter rubber 'OV 1 on siliconised diatomaceous earth as carrier material.

Carrier gas: Temperature program: 30 ml / minute N ₂ 80-180 ° C, 2.5 (3) ° C / minute Injector / detector temperature: 200 ° C Detector: Flame ionization detector

Sprayed

amount: about 2 / д1 about 1:50 diluted ethereal oil

The assessment is carried out in part without, in part with an internal standard. As the internal standard, methyl laurate or hexadecane is suitable for chamazulene-poor or chamazulene-free oil. For oils with a chamazulene content greater than 5%, chamazulene should be used

as an internal standard, the content determination is carried out photometrically (at 578 nm).

SUBJECT OF THE INVENTION

Claims (4)

1. Method for obtaining a new tetraploid variety of camomile (Manzana designation) of the plant species Chamomilla recutita L., synonymous with Matricaria chamomilla L., Asteraceae, whose flowers dried at 40 ° C contain at least 150 mg% chamazulene, at least 300 mg% of (-) - alpha-bisabolol and less than 50 mg% of other bisaboloids, characterized in that the diploid chamomile variety DEGUMILL is tetraploidized and the selected plants are subjected to further selections and propagation, optionally from chamomile plants obtained therefrom variety, obtains multiply and / or chamomile drug. Method according to claim 1, characterized in that the tetraploidization is carried out with chemicals at temperatures between 0 and 35 ° C by means of gamma-rays, X-rays or U-rays at temperatures between 0 and 35 ° C, by means of higher temperatures from 33 up to 50 ° C, using lower temperatures from 0 to 5 ° C, using a callus decapitation method or an anther culture. 3. The process according to claim 1, wherein the tetraploidization is carried out (a) selection of tetraploid plants according to the active substance content (at least 150 mg% chamazulene, at least 300 mg% bisabolol and less than 50 mg% other bisaboloids, all based on dry matter), by current flowering, uniform flowering time, uniform branching and size flower heads 25 to 35 mm, the plants thus selected are cloned and seed obtained from cloning, b) offspring are obtained from the seed thus obtained, followed by selection by step (a) seed shall be recovered from the selected plants, (c) the measures referred to in (b) are repeated two to four times; d) offspring are obtained from the seed obtained according to step c), this is selected according to step a), the plants thus selected are cloned and seed obtained from the cloning plants. 4. A method for producing a chamomile drug from chamomile flowers according to items 1 to 3, characterized in that the flowers are harvested at the vegetation stage at a time when only 30-70% of the tubular flowers in the inflorescence are open and the flowers are dried at air temperature not higher 50 ° C.