CZ944585A3 - Process for preparing novel tetraploid chamomile rich in bisabolol - Google Patents

Abstract

A process for extracting camomile together with bisabol uses as the source plant a specially selected strain of Matrica Chamomille of which the flowers dried at 40 DEG C contain at least 100 mg% of chamazulene and at least 200 mg% of (-)-???-bisabolol, the content of the other bisaboloids being below 50 mg%, wherein an initially tetraploidized diploid camomile or a tetraploid camomile, in which (-)-???-bisabolol is the main component of the essential oil, is subjected to various selection and propagation steps.

Description

Technical field

The present invention relates to a process for the preparation of a novel bisabolol-rich tetraploid chamomile of the camomile (Chamonilla recutita L., synonymous with Matricaria chamonilla L., Asteraceae) plant, wherein the dried flowers at 40 ° C contain at least 100 mg% chamazulene, at least 200 mg% of (-) - α-bisabolol and less than 50 mg% of other bisaboloids, with the exception of the Manzanian chamomile variety.

BACKGROUND OF THE INVENTION

Preparations of chamomile inflorescence [Chamonilla recutita (L.) Rauschert, synonymous with Matricaria chamonilla,

L.], due to their antiphlogistic and spasmolytic effect, find a wide therapeutic use and form an important part of the plant medicinal raw material assortment.

Particular therapeutic importance is attributed to the active ingredients of (-) - alpha-bisabolol and chamazulene. Therefore, a suitable camomile drug should have a high content of both.

The naturally occurring diploid chamomiles are very heterogeneous with respect to the active ingredient content. In particular, only one of the important active substances of chamazulene and (-) - alpha-bisabolol is available, or the active substances are absent or very low, in which case the main constituent of the essential oil consists of less active bisaboloids (bisabolonoxide A) or B, bisabololoxide A).

DEGUMILL (a camomile variety protected and listed in West German Patent 24 02 802 - a protected variety registered in the former GDR under the name Degumill; Italian Patent No. 1,035,096) is known to have a camomile variety which also contains a high content of (-) - alpha-bisabolol and chamazulene. The chamazulene content of this chamomile variety DEGUMILL is stable only if accidental crosses or foreign pollination with other camomile varieties, whose bisabolol and chamazulene content is substantially lower and the generally active, low-active camomile content is avoided. The risk of cross-pollination is almost everywhere, as wild camomile occurs practically in general. In addition, various tetraploid camomile forms are known (for example, BODEGOLD, GDR; POHORELICKÝ, CZ and SR; GOLD LAN, Poland; BK-2, Hungary). Although these tetraploid forms have a satisfactory chamazulene content, the important active ingredient (-) - alpha-bisabolol is only very small in these tetraploid species, and the other bisaboloids (bisabolol A and B as well as bisabolonoxide), which account for more than half essential oils in known tetraploid camomile species.

SUMMARY OF THE INVENTION

It has now been found that, quite generally, from naturally occurring chamomile populations, such as diploid or tetraploid cultivated chamomile, bisabolic-rich tetraploid chamomile plants, which on the one hand are no longer susceptible to foreign pollination of naturally occurring chamomile On the other hand, they have a high content of chamazulene and (-) - alphabisabolol, whereas the content of (-) - alpha-bisabolol significantly exceeds the content of chamazulene and at the same time other bisaboloids are virtually absent, or only to a small extent. It is also important that the high content of the main active ingredients (-) - alpha-bisabolol and chamazulene in the chamomile according to the invention is permanent, that is to say, it is maintained at every propagation.

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of a novel bisabolol-rich tetraploid chamomile of the camomile (Chamonilla recutita L., synonymous with Maquarius and chamonilla L., Asteraceae) of which the dried flowers contain at least 100% dry matter. mg of chamazulene, at least 200 mg% of (-) - α-bisabolol and less than 50 mg% of other bisaboloids, with the exception of the Manzana chamomile variety, characterized in that between 1,000 and 10,000 individual plants have a diploid or tetraploid population chamomile determines (-) - .alpha.-bisabolol in the essential oil and selected those chamomile plants for which (-) - .alpha.-bisabolol is the major constituent of essential oil and wherein the flowers dried at 40 DEG C. contain at least 100 mg% chamazulene; up to 100 mg% of (-) - α-bisabolol and less than 50 mg% of other bisaboloids, these plants are separated from the parent population, multiplied and subjected to further selection and and optionally, the propagation seed and / or the camomile drug are obtained from the chamomile plants obtained, and in the case where a diploid chamomile, which excludes the Degumill variety, is used as the starting chamomile, tetraploidization by means of chemicals is carried out after all selection steps at temperatures ranging from 0 to 35 ° C, by means of garama-rays, X-rays, or UV rays at temperatures ranging from 0 to 35 ° C, by elevated temperatures ranging from 33 to 50 ° C, by reduced temperatures ranging up to 0 up to 5 ° C, using the decapitation-callus method or using anther cultures.

Selection and multiplication in the method of the invention consists of the following steps:

(a) selection of at least 10 individuals of tetraploid plants according to the active substance content, ie at least 100 mg% chamazulene, at least 200 mg% bisabolol and less than 50 mg% other bisaboloids, all based on dry substance, current flowering time, according to uniformity of basic branching and, depending on the flower size from 20 to 40 mm, the cloning of the plants thus selected and the seed obtained from the plants obtained by cloning,

(b) growing offspring from the seed obtained in accordance with paragraph (a), the subsequent selection referred to in paragraph (a) and obtaining seed from the plants so selected;

(c) repeating the measures referred to in paragraph three to five times

b),

(d) growing progeny from the seed obtained under (c), selecting said progeny from (a), cloning the plants thus selected and obtaining seed from the plants obtained by cloning.

Surprisingly, the novel tetraploid camomile according to the invention is characterized by a number of novel advantageous properties in comparison with known camomiles or possible camomile varieties. In particular, it is no longer susceptible to foreign pollination by naturally occurring camomile populations (wild camomile) as the known camomile variety DEGUMILL. Compared to the diploid and tetraploid varieties of chamomile, the chamomile according to the invention differs by a surprisingly high content of the active ingredient (-) - alpha-bisabolol (at least 200 mg%), while the content of other bisaboloids (e.g. bisabololoxides) is very low and less than 50 mg%. .

The known camomile species described in the literature all exhibit a very high content of bisaboloids and partly bisabolonoxides, which generally make up 50% of the essential oil (i.e., about 1% essential oil contains about 500 mg per 100 g of drug).

Furthermore, the camomile according to the invention, in contrast to the known tetraploid camomile varieties, is homogeneous in terms of the substances contained, the properties of chamazulene and (-) - alpha-bisabolol being constant (i.e. homozygous).

On the other hand, the evaluation of individual plants of known tetraploid camomile varieties shows that individual individuals belong to different types in terms of the substances contained, which means that a single random collection of the drug produced therefrom shows both qualitatively and quantitatively different inhomogeneous results.

In addition, the camomile of the invention surprisingly differs from known tetraploid camomile varieties, for example, by one or more of the following properties: improved seed germination, less useless green matter (i.e., higher flower yield); lower water content in flowers (higher yield of dried flowers and shorter drying time); more suitable for mechanized harvesting because the flowers are significantly localized in one plane and most of them bloom at the same time (thus allowing a uniform harvesting date); better drug shelf life (lower propensity for disintegration and waste generation); especially aromatic typical camomile fragrance. The aforementioned advantages, in particular, arise when, in addition to the selection for active ingredients, one or more of the aforementioned traits are selected, seed is obtained from the selected plants, a new selection is made from the offspring thus obtained and these measures are carried out three to five times.

By default, diploid chamomiles come

II, for example, consider Chamomile of Argentine origin

- 8 / see L.Z. Padula, R.V.D. Rondina and J.D.Coussio, Quantitative evaluation of essential essential oil, total: azulenes and chamazulene in German chamomile grown in Argentina; Planta med. 30, pp. 273-280 (1976), as well as all camomiles which exhibit measurable concentrations of (-) - alpha-bisabolol / in essential oil as a rule more than 5% in essential oil. Possible diploid chamomiles are described in the following literature: H. Schilcher, "Recent findings in qualitative determination of chamomile essential oil or chamomile flowers", Planta med. 23, 132-144 (1973), 0. Motl, M. Pelklová, V. Lukeš and M. Jasicová "To Gas Chromatography Analysis and to Chemical Types of Camomile Oil". Sheet. Pharm. 310,

210-215 (1977); Ch. Franz, J. Holzl and A. Vomel "Preliminary morphological and chemical characteristics of several populations and variations of Matricaria chamomile L.", Acta Hort., 73, 109-114 (1978).

By default, the camomile also comes into consideration as the diploid form of Degumill camomile. In this case too, for example, a tetraploid chamomile having a chamazulene content of at least 150 mg% and a bisabolol content of at least 200 mg%, when carried out, is harvesting the inflorescence of the chamazulene in the vegetation stage at which it is heated.

- 9 30 to 70% of tubular inflorescence flowers are open and drying is carried out at an air temperature of maximum ° C.

Subsequent inclusion of the selection and propagation steps of the present invention can further increase said bisabolol and chamazulene values.

Camomile essential oil generally consists of the main constituents of pharaesene, spathulenol, chamazulene, one of the 4 bisaboloids of [alpha] - / - alpha-bisabolol, bisabololoxide A, bisabololoxide B or bisabolohoxide as far as diploid individual plants are concerned; in a mixed sample of population: as well as in tetraploid ✓

Individual bisabolides may be contained side by side as well as spiroether. Together, these substances make up 70 to 80% of the essential oil. Therefore, particularly preferred are starting diploid chamomiles, wherein [alpha] - [alpha] -bisabolol represents the predominant component / more than half the amount of the above-mentioned substances.

Particularly suitable are diploid camomiles as starting materials, in which, for example, alphabisabolol constitutes at least 40% of the essential oil or at least 50% of the bisaboloids.

Examples of suitable tetraploid chamomile are: chamomile types Bodegold / GDR / _f .

- 10 Pohorelicky / Czechoslovakia /, Zloty Lan / Poland /, BK-2 / Mačíarsko / · (these species are described in the following literature: M. Chládek and V. Kosová, Pharmazie 13, 712-713 (1958), W.Czabajska Poznań (1963), W. Poethkft and P. Bftlin Pharm. Z. Halle, 108, 813-823 (1969)} I. Sárkány, Herb Hangar, 4 (1), 125-169 (1965). Basically, the same applies to tetraploid starting chamomiles due to the content of / - / - alpha-bisabol as to diploid starting chamomiles.

Suitable diploids are particularly suitable. and tetraploid chamomile populations containing as a rule at least 100 mg% chamazulene in the pooled sample /

and 50 to 100 mg% (-) - alpha-bisabolol (based on the dry weight of the flowers dried at 40 ° C), i.e. seed of such chamomile populations is obtained in the case of diploid chamomile seed and the seed is tetraploidized and / or knowledge of the bisabolol content of the parent plants. In the case of the starting tetraploid camomile, the seed described above is then immediately selected and propagated.

Suitable diploid and tetraploid starting camomiles are selected by screening individual plants. Generally, the .w

Investigate between 1,000 and 10,000 individuals and find a few individuals who have a high content of / - / - alpha-bisabolol according to the given categories and are useful as a starting camomile. Seeds are obtained from such selected individuals in a conventional manner and this is tetraploidized. Upon completion of this process, the tetraploid plants are selected from the remaining diploid plants. From the tetraploid plants thus obtained, optionally after previous propagation, all plants having at least 100 mg of chamazulene, at least 200 mg% of - / - alpha-bisabolol and less than 50 mg in flowers dried at 40 ° C are now selected. % of other bisaboloids. These flowers are harvested at the stage of development where 30 to 70% of all tubular inflorescences are open. Optionally, further selection and multiplication steps may follow, for example affecting the following characteristics or optional properties: the term of simultaneous flowering, uniform base branching and narrow flowering zone (i.e. better suitability for mechanized harvesting), large inflorescences, better shelf life of the drug, especially aromatic odor .

When starting from tetraploid chamomile, only the previously mentioned selection shall be made for those plants whose flowers dried at 40 ° C contain at least 100 mg% chamazulene, at least 200 mg% / - / - alpha-bisabolol and less than 50 mg% of other bisaboloids, the harvest of these flowers also being carried out at the developmental stage, with 30 to 70% of all tubular inflorescences open. Also in this case, further selection and propagation steps may follow, leading to further improvements (as mentioned above).

A particularly preferred camomile is obtained when selecting, according to the content of chamazulene and / - / - alphabisabolol, those plants (known or prepared according to the invention) whose chamazulene content is at least 200 mg%, preferably 250 mg%, and whose content () The alpha-bisabolol is at least 300 mg%, preferably 400 mg (the content of other bisaboloids is still below 50 mg%).

Tetraploidization can be carried out in a manner known per se by affecting plant parts (seeds, roots), shoot ends, germs, arm buds or tissues of diploid chamomile starting plants / chemicals, X-rays, gamma rays or UP-rays. It can also be carried out using callus decapitation, anthropogenic culture, or by applying high or even low temperatures to chamomile plants, plant parts or chamomile plant tissues. For example, reference may be made to Werner Gottschalk's book Die Bedeutung der Polyploidie fur die Bvolution der Pflanzen, Gustav Fischer Verlag, Stuttgart 1976, in particular pages 13 to 22.

Tetraploidization using chemicals:

Examples of suitable tetraploidization chemicals are: colchicine, acenaphthene, alkaloids such as atropine, veratrine, nicotine, sanguinarine benzene, diphenyl and phenathrene derivatives, naphthaline and naphthaline derivatives, diphenylamine, tribromaniline, p-dichlorobenzene, methylnaphthtoquinone, methylnaphthtoquinone, methylnaphthtoquinone, and related substances, hexachlorhexane, methamphetamine hydrochloride, alkali metal alkyl carbohydrates such as sodium isopropylcarbamate, phenylurethane, cacodylic acid salts (e.g. sodium salt), lily of the valley lily such as convallarin, convallatoxin and convallamarin, heteroauxin, germisane / phenylmercuric phosphate tetrisulfate) mercury (ethyl chloride, phenyl hydroxide and phenyldinaphthyl methanedisulfonate), chloroform, gas (NgO) and mixtures thereof. Pellets and compost are also suitable

- 14 and cow manure.

The action of the substances mentioned herein is carried out, for example, at temperature limits. 0 and 35 ° C, preferably between 12 and 30 ° C, especially at temperatures of 15 to 25 ° C.

The application is carried out, for example, by treating the seeds, the tips of the shoots, the roots / in particular the roots or germinating seedlings on the stalks, the cut areas on leaves or stems, on cell suspensions of meristem tissue, kallus culture. buds. Chemicals are usually used in the form of solutions in water, weakly alcoholic solutions / alcohol content generally below 5 or weakly acidic solutions. The pH of the weakly acidic solution is, for example, between 5.5 and 6.5, the acidification being carried out with lower aliphatic acids, such as acetic acid. If alcoholic solutions are used, they may also be weakly acidic. The concentration of chemicals in these solutions may be, for example, 0.01 to 0.5 S, preferably 0.02 to 0.2 S, particularly 0.05 to 0.1 S. The gaseous substances are used as such, optionally under pressure (e.g. 1 to 100 kPa). The duration of action is, for example, 1 to 36, preferably 2 to 12, in particular 4 to 6 hours.

- 15 The most effective concentrations as well as the duration of action should be suitably tested in the trials.

Particularly favorable is, for example, the action of colchicine at temperatures between 0 and 35 ° C, preferably at 12 to 30 ° C, in particular at 15 to 25 ° C. This can be done, for example, by allowing diploid chamomile seed to swell in 0.01 to 0.2%, in particular 0.02 to 0.1%, preferably in a 0.0% colchicine solution, or germinated 5 to 7 days old The well-developed germinating seedling of the diploid starting chamomile (with the collar leaves down) is immersed in 0.01 to 0.2%, in particular 0.02 to 0.1%, preferably 0.0 colchicine solution. In the latter process, the ambient atmosphere should have 100% relative humidity. For example, the duration of colchicine treatment is 4 to 10 hours. When germinating seedlings are used, an exposure time of up to 10 hours is generally sufficient. If seed is used, the exposure time may possibly extend up to 36 hours.

After exposure to chemicals, the swollen seeds, germinating seedlings or other plant parts are washed several times with water. The swollen seeds are then sown, for example. Plants with or without treated roots Parts or treated germinated seedlings are applied

- 16 example, they crop into the hazel for plants. From such treated seeds or germinating seedlings the plants are grown / for example in a greenhouse; at temperatures between 18-25 ° C by day and 10-16 ° C at night), plants whose pollen is about 1.5 times greater than the pollen of the starting material, optionally having a chromosome number of 36 in somatic cells, are selected.

If other plant parts (aboveground or underground) are treated with chemicals, later shoots and roots or flowers / seeds resulting from such chemical treated parts are subjected to later tetraploidization _{to be} carried out. only from this armpit, or from this bud the sprout and the flowers created on it, or the seeds on the number of chromosomes.

For example, pgclu size measurement and chromosome counting can be performed as shown in the example.

- 17 Irradiation amount: 5 50 Steal. Preferably, gamma rays and X-rays are suitable.

Examples of suitable UP-rays are those having a wavelength of 400 to 30 nm, preferably 350 nm.

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

Use of high and low temperatures:

Suitable temperatures are, for example, temperatures between 33 and 45 ° C. For example, swollen seeds, germinating seedlings, shoot tips and meristematic tissue are exposed to these temperatures. Treatment time: for example 1 to 48 hours, preferably 12 to 24 hours.

The following are low temperatures:

to 5 ° C, preferably 0.5 to 4 ° C, especially 2 ° C,

For example, swollen seeds, germinating seedlings, shoots and meristematic tissue are exposed to these temperatures. Treatment time: for example 1 to 100, preferably 20 to 40 days. Plants thus treated, or parts thereof, are further treated as well as after exposure to chemicals.

K38

Decapitation callus method (decapitation = clipping of the head or tip / tip / or back cut). Decapitation is carried out, for example, in young plants on the stem, preferably at the end of the vegetation cone, after forming 4-6 leaves or even petioles · The buds formed on the injured tissue Aallus tissue /, or the shoots are separated, brought to rooting are grown further in pots and the tetraploid form of the transferred plant is selected in the same way as chemicals,

Anther culture / preparation of plants with a single number of pollen vesicle chromosomes with the following. spontaneous or artificial tetraploidization / ·

Closed flowering tubular flowers are harvested from flowering plants whose anthers (pollen sacs) are in the first stage of pollen mitosis. The roasters are removed using a micromanipulator and transferred to Petri dishes, which are filled, for example, with Nitsch and Nitshhe broth (Table 1). The petri dishes are then stored in the culture chamber for 16 hours per day at 28 ° C, at night. at 20 ° C. After about 4 weeks, the anthers begin

- 19 buds and plantlets to grow up. These are haploid in the diploid parental share with the tetraploid parental share: dihaploid: after the root system has been established, for example, they are transplanted into the garden soil and brought to bloom in the greenhouse. These (di) haploid plants are sterile, but can be converted to polyploid forms by treatment of the ends of the shoots, roots or chemicals such as colchicine to form homozygous plants which can then be further propagated by seeds. For further procedures see treatment with chemicals such as colchicine.

Table 1

Culture medium according to Nitsch and Nitsch / Science, 1969 / mg / l

Π0 ₃ 950 NH ₄ NO ₃ 720 MgSO ₄ x 7 HgO 185 CaCl ₂ 166 KHgPC ^ 68 MnSO 4 x _H-2 0 25 H _o B0 _n 10

 »T» t * KW ??

Continuation of Table 1. mg / l - ZnSO4 * 7 HgO · ~ * 10

NagMoO 4 x 2 HgO 0.25

CuSO 4 x 5 HgO 0.025 ml solution of 7.45 & disodium ethylenediaminotetraacetic acid and 5.57 g PeSO 4 x 7 HgO per 1000 ml

Myo-Inosit 100

Glycine 2

Nicotinic acid 5

Pyridoxin-HCl 0.5

Thiamin-HCl 0.5 folic acid 0.5

Biotin 0.05

Sucrose 20 g

Nutrient broth prepared ZDIPCO-Bacto-Agar / 8 g Indolylacetic acid 0.1 pH of the medium is adjusted to 5.5

By measuring the pollen size and / or by counting the chromosomes of the selected tetraploid chamomile plants which can be obtained by the above options, plants having a minimum chamazulene content of 100 mg% and a minimum content of bisabolol of 200 mg% are then selected, of the bisabololoids (especially bisabololoxides) should be below 50 mg% (based on dried flowers, see Example 1).

4 the plants, after removal of the already blooming inflorescences, are allowed to bloom in a greenhouse at a temperature of 18 to 24 ° C by day and 12 to 14 ° C at night and for a day of at least 14 hours, all seeds being harvested for 4 weeks that are blooming or before disintegration. After drying at an air temperature between 20 and 35 ° C, the camomile propagation seed of the invention is obtained.

In addition, the following may be considered as further criteria for this selection: a / approximately the present flowering time, b / uniform base branching and a narrow flowering zone of about 10, in particular 5 cm,

(C) large inflorescences with an outside diameter of about 30 mm (20 to 40 mm),. in particular 25 to 35 mm.

The use of these additional criteria a /, b / e / or c) leads, for example, to high flower and drug yields and is more suitable for machine harvesting.

If starting from the known tetraploid camomile, the selection described above, as well as, optionally, further selection steps and propagation, is carried out.

In order to obtain a camomile which, in addition to a content of at least 100 mg% of chamazulene and at least 200 mg of $ / - / - alpha-bisabolol (based on dried inflorescences) providing high flower yield and better suited for machine harvesting, the following procedure is recommended: the plants (as described above) are propagated vegetatively by hybrids and monitored for 3 to 5 generations while still being selected according to the above criteria and possibly additional criteria (a) to (c). Plants grown according to these criteria are cloned, seed is obtained from cloned e plants, the plants derived therefrom are again selected according to the stated minimum chamazulene and bisabolol content, as well as other criteria a / to c /, and seed is again recovered from the latter.

Sequence - selection according to the above-mentioned minimum chamazulene content of 100 mg% and / - / - alpha-bisabolol of at least 200 mg% (other bisaboloids below 50 mg%), as well as other criteria and / to c / up to 5 times.

This is followed by another sequence of sowing selection, as described previously (possibly cloning / seed harvesting).

The seed obtained thereafter is the camomile propagation seed of the invention.

The propagation by crossbreeding is carried out in the following manner: To propagate the crossbreeding, under the conditions of a short day, the plant outgrowth (clone mother plant) must produce short bud-free shoots. This occurs in the winter of the year in a greenhouse without additional illumination or in air-conditioning chambers for a length of day of 6 to 10, preferably 8 hours, and temperatures of 10 to 15 ° C, preferably 12 ° C. Suitable for cloning or multiplication are hybrids of shoot leaves and especially short shoots (side shoots). They are rooted at temperatures of 12 to 18 ° C, preferably 15 ° C, and for a day length of 12 to 16, preferably 14 hours, in a saturated atmosphere (about 100% relative humidity).

i & amass

For example, a 1: 1 peat sand mixture may be used as a substrate; pure quartz sand, asbestos cubes for crisps, peat cubes for crisps, etc. are also suitable.

For example, the following soils are suitable for sowing: garden soil, humous medium-heavy clay soil, clay or humous sandy soils.

Sowing can take place in greenhouses and in the field. The germination and growth temperatures of the plants are between 12 and 24 ° C, in particular 18 to 20 ° C. If sowing is carried out in the open field, it is preferably sown in autumn / September / only / or early in spring / March / April /. These terms apply to all territories eligible for cultivation (eg Northern Hemisphere, temperate to subtropical).

The new camomile according to the invention belongs to the species of the plant camomile plant, the botanical designation Matricaria chamomilla L. (synonym Chamomilla recutita / 1)., Rauschert, and is defined by the active substance data in point 1 of the definition of the subject matter of the invention.

The stated values of the active substances of chamazulene, / - / alpha-bisabolol and bisabololoxides always refer to the developmental stage of flowers, where 30 to 70% of

- 25 40 to 60% of all tubular flora of your flower is open, that is to say, the flowers used to determine the active substance content have been harvested by that date and then dried in a box dryer at 40 ° C for 72 hours.

When the chamomile flowers are harvested at a time when the flower development stage has progressed, that is to say, for example, 100% to or up to 100% of all tubular inflorescence flowers are open (e.g., in full bloom) and / or harvested flowers are dried at temperature higher than 40 ° C, the content of the active substances / - / - alpha-bisabolol and chamazulene may be lower, since at higher temperatures and / or at a later harvest, more significant decomposition of azulene and bisabolol may occur.

The term "other bisaboloids in point 1 of the definition of the subject matter of the invention" means in particular: / - / - alpha-bisabololoxide A and B; - / - alpha-bisabolonoxide A; the other constituents of the essential oil of the camomile variety according to the invention are Eze-dicycloether, farnesene, spathulenol and, in moderate concentrations, various volatile terpenic hydrocarbons.

For example, chamomile flowers of the present invention are oven dried (box) at 40 ° C / harvest.

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26 as defined above / between 100 to 200 mg% of chamazulene, between 200 to 450 mg% of - / - alpha-bisabolol and very little, that is to say 5 to 50 mg% of other bis-bolides based on dry matter , which means relative to the absolute dry weight of the flowers. This dry weight is determined by drying with a separate?

a sample of chamomile flowers in a drying cabinet at 105 ° C up to a constant weight of 72 to 96 hours | din /. The active ingredient content of the flowers dried, for example, at a temperature of 35 to 50 ° C (drug) is converted to the dry weight of the flowers determined at 105 ° C.

In its phenotype, the camomile obtained according to the invention is similar to the known tetraploid camomile varieties / e.g. Bodegold, Zloty Lan,

BK-2 ,, Pohorelicky velkokvetý /; they differ from them.

In particular, in the camomile according to the invention, the (-) alpha-bisabolol is a major component of the flower oils and, moreover, the content of the other bisaboloids (bisabololoxides A and B as well as bisabolonoxides) is clearly lower. The other constituents of camomile essential oil are farnesene, spathulenol and Ennln-dicycloether.

The camomile obtained according to the invention can be grown on all soils except those containing more than 20% organic matter (humus and soil organisms); they are not

- 27 no agrotechnical and cultivation measures are needed; only a long day with more than 13 hours of maximum length of day is necessary for cultivation, especially temperate zones and subtropical areas are considered.

The chamomile obtained according to the invention usually has still further advantages: higher yield, medium late harvest, uniform height, narrow flower zones and large inflorescences; hence the particular suitability for mechanized harvesting. In addition to this, the fact that plants sown at the same time usually bloom practically together at the same time

greatly simplifies and facilitates harvesting.

Finally, the above advantages will be exemplified when the following process conditions are applied: from tetraploid chamomile plants having a chamazulene content of at least 100 mg% and / - / - alpha-bisabolol of 200 mg%, while the content of other bisaboloids (especially bisabololoxides) is below 50 mg % (all related to dried inflorescences) only those plants that are selected are selected

- flowering at about the same time,

have a uniform base branch and a narrow flower zone of about 10 cm, and

- large inflorescences with an outside diameter of about 30 Wt

The selected plants are propagated vegetatively by hybrids and monitored for 3 to 5 generations, the selection being made according to the above criteria. Selected plants are propagated vegetatively / cloned / allowed to bloom together and seed is harvested from them. The seeds obtained from the seeds are again selected according to the above criteria and the sequence of sowing - selection - seed yielding is 3-5 to cover the opposite.

The drug prepared from chamomile obtained according to the invention has a maximum active ingredient content of chamazulene and / - / - alpha-bisabolol when harvesting the chamomile inflorescence at the vegetation stage where, for example, 30-70% of tubular flowers, preferably 40-60, i.e. In general, 50% of tubular inflorescence flowers are open and drying is carried out at an air temperature of at most 90 ° C, for example 35 to 50 ° C, in particular 40 ° C.

Drying can be carried out either by supply air or by shading, but it must be ensured that the heat supplied does not exceed the amount required for complete drying. It is also advantageous to check with constant weighing by means of checkweighing.

Drying may be performed spontaneously or artificially (for example, artificially heated warm air). The yield of active substances is greatest in spontaneous drying with the exclusion of sunlight, preferably at 40 to 60 ° C, in particular at 40 to 50 ° C. The drying process should be carried out as soon as possible after harvesting. The drying should take place in thin layers, for example 5 to 20 cm, preferably 10 cm. Drying can also be carried out in well ventilated halls at temperatures between 20 and 30 ° C. The air temperature for drying should not exceed 60 ° C. For example, a temperature between 35 and 50 ° C is favorable.

The content of active ingredients in the drug prepared from chamomile obtained according to the invention, in particular the main active ingredients, chamazulene and / - / - alpha-bisabolol, depends on the vegetation stage at which the flowers are found at the time of harvest and before drying the harvested flowers. The higher the drying temperature, the more! the decomposition of the active ingredients takes place, i.e. the lower the content of active ingredients in the dried flowers. For the same reason, direct sunlight is unfavorable to the wolf and should be avoided wherever possible. The vegetation stage of the flowers is characterized by how many% of tubular flowers of the inflorescence are open at a certain time (here, the harvest time). You can also harvest flowers, where it is open

30 to 50%, 30 to 70%, 40 to 60%, 60 to 100% or 90 to 100% tubular inflorescence. The content of the active substances depends on at which stage

- flowers are always found, and is highest when it is. open 40 to 60% of all tubular flowers, and is lower for both less and more open tubular flowers inflorescences.

The great advantage of the chamomile obtained according to the invention is therefore that it is evenly flowering, i.e. the predominant number of plants after sowing has the same flowering stage, which means that, for example, in most plants 40 to 60% of all tubular of flowers. Thus, a perfect harvest of inflorescences at the optimum time is possible with the chamomile obtained according to the invention and this chamomile is suitable for mechanized harvesting.

The relationship of the chamazulene content and / - / - alpha-bisabolol to the flowering stage at harvest time and to the drying temperature of the drug is shown, for example, in the following table (the content of other bisaboloids in all cases being less than 50%).

- 31 Table

- Temperature - -Vegetation stage of flowers at harvest time

suěení /temperature air / 30 to 70% of all tubular inflorescence flowers open 70 to 100 # of all tubular flowers inflorescence open Content of active substances in dried material Content of active substances in dried material maximum Deň: 32 ° C Chamazulen ^ at least 100 mg / - / - Alpha-bisabolol: at least 200 mg Chamazulen: at least 40 mg / - / - Alpha-bisabolol: at least 120 mg • Content of active substances in alcoholic extract: Content of active substances in alcoholic extract: maximum Deň: 32 ° C Chamazulen: at least 5.0 mg # / - / - alpha-bi-sabolol: at least 10.0 mg # Chamazulen: at least 2.0 mg # / - / - Alpha-bisabolol: at least 6.0 mg # Content of active substances in dried material: Content of active substances in dried material: between 50 to 70 ° C Chamazulen ^ at least 50 mg # Chamazulen: at least 30 mg

- 32 Continued table

Temperature Vegetation stage of flowers at a time. harvest drying 20 to. 70% of all tubes- 70 to 100% of all tubes / temperature of inflorescence metal flowers inflorescence air / open open ..........

/ - / - Alpha-bisabolol: / - / - Alpha-bisabolol:

at least 150 mg% at least 100 mg%

Content of active substances Content of active substances in alcoholic in alcoholic between 50 _Λ extract extract up to 70 ^W C ____________________

Chamazulen: not less than 2,5 mg% / - / - alpha a-rbi with abolo 1 not less than 7,5 mg%

Chamazulen: at least 1.5 mg & / - / - alpha-bisabolol: at least 10.0 mg%

Consequently, the chamomile used to produce the compositions of the invention, for example, in the harvesting stage of the vegetation stage when 30 to 100% of tubular inflorescences are open, and drying can be carried out at atmospheric temperatures of up to 70 ° C, a dry material is obtained whose chamazulene content is in any case at least 30 mg% and whose content of alpha - / - alphabisabolol is at least 100 mg%, while the content of other bisaboloids is less than 50 mg%.

- 33 The content of other bisaboloids is still less than 10 mg% in alcoholic extracts.

The content of essential oil, which is produced from a dry material under the conditions in the above table, is still at least 3.5% chamazulene, at least 10% / - / - alpha-bisabolol and less than 10% other bisaboloids.

The determination of [alpha] - [alpha] -bisabolol, as well as other ingredients for camomile, is carried out by the usual gas-chromatographic method.

The exact method of analytical determination is contained in Annex A / after the text of Example 4 /.

The active substance chamazulene is found not in the chamomile flowers as such, but in the form of the sesquiterpenic lactone matrix.

Matricin exhibits pharmacological activity close to chamazulene. From this precursor, matrixin is formed, for example, by heating (e.g., steam distillation to prepare a tea infusion), immediately chamazulene. In camomile, it is therefore customary not to report the content of matrix, but the content of the chamazulene which forms therefrom.

Possibilities of using chamomile obtained

34 according to the invention are as follows: obtaining a drug of camomile, camomile essential oil, camomile extracts as well as / - / - alpha-bisabolol, chamazulene and other camomile ingredients. For example, camomile yields or extracts can be obtained from the drug obtained from chamomile according to the invention, for example, by extraction with an alcohol or an aqueous-alcoholic mixture or by supercritical gas extraction. Furthermore, camomile essential oil, / - / - alpha-bisabolol, chamazulen and other camomile ingredients can be obtained from the drug.

The alcoholic extracts of the drug contain, for example, at least 1.5 mg%, preferably 5.0 mg% chamazulene /

and at least 5.0 mg%, preferably at least 10 mg% of - / - alphabisabolol in the alcoholic extract of the drug and less than 8 mg% of the other bisaboloids. The preparation of such extracts is carried out in a conventional manner.

For example, instrumentation, such as so-called trough mixers, percolators and other suitable extraction devices can be used for extraction. The temperature during the extraction is, for example, 10 to 50 ° C. Cooling is not required.

Suitable solvents are, in particular, straight-chain or branched aliphatic mono- or polyhydric alcohols having 1 to 6 carbon atoms, such as e.g.

methanol, ethanol, 2-propanol, butanol, glycerol, isopropylidene glycerol and others, as well as a mixture of these solvents with water. _ _ _ _

Mixtures of these solvents may also be used. The minimum amount of solvent is 2 parts solvent per 1 part of dried material. Usually 2 to 20 parts of solvent per part of dried material are used, in particular 3 to 10 parts of solvent per part of dried material.

Fresh chamomile flowers or frozen chamomile flowers can also be used for the production of extracts, especially alcoholic extracts (if the fresh flowers were frozen).

The oil prepared from the drug contains at least 3-5% of chamazulene, preferably at least 5% of chamazulene and at least 10% of .alpha.-bisabolol and less than 10% of other bisaboloids.

The production of such essential oil is usually carried out by heating the drug with water, optionally in the presence of ascorbic acid (e.g. in the form of a salt, in particular sodium ascorbic acid) to you at a pH between 4 and 6, preferably 5 to 5.5. The pH is adjusted with an acid, for example a slurry

- 36 hydrochloric acid. For example, 10 to 50 parts by weight of water and optionally 0.1 to 1 part by weight of ascorbic acid are used per part by weight of the dried material.

Heating is usually carried out for 2 to 8 hours.

The aqueous distillate obtained is repeatedly shaken with a lower aliphatic liquid hydrocarbon such as petroleum ether (e.g. boiling point 35-60 ° C), pentane, xylene, decalin, the organic phase is dried (e.g. over sodium sulfate), and the organic solvent is removed gently (e.g. by distillation on a rotary evaporator or by distillation at 40 to 70 ° C, preferably at 50 to 60 ° C. The above-boiling solvents are vacuum distilled off.

The determination of the constituents is carried out as previously stated.

When chamomile flowers are harvested at the vegetation stage when 30 to 100% of tubular inflorescence is open and the flowers are dried at an air temperature of up to 70 ° C, then the drug obtained contains at least 30 mg% chamazulene, at least 100 mg% / - / - alpha -bisabolol and less

- 37 than 50 mg # of other bisaboloids, and the steam-derived oil distillation of this drug contained at least 3,5% chamazulene, at least 10% / - / - alphabisabolol and less than 10 mg # of other bisaboloids and alcoholic extract prepared by extraction, with The lower alkanols contain at least 1.5 # chamazulene, at least 5.0 mg of # / - alpha-bisabolol and less than 10 mg # of other bisaboloids.

When chamomile flowers are harvested at the vegetation stage when 30-70 # tubular flowers are opened and the flowers are dried at a maximum temperature of 50 ° C, then such a drug contains, for example, 100 mg of chamazulene, at least 200 mg of [- / - alpha-bisabolol and less than 50 mg # of other bisaboloids and% essential oil thereof containing at least 3.5% chamazulene, at least 10 µg of # other bisaboloids, and chamomile alcoholic extract produced by lower alcohols contains

1.5 mg # chamazulene, 5.0 mg # / - / - alpha-bisabolol 10 10 mg # other bisaboloids.

When chamomile flowers are harvested in the vegetation stage, when 30-70 # tubular inflorescence flowers are opened and the flowers are dried at an air temperature between 50-70 ° C, then the drug obtained contains, for example, at least 50 mg # chamazulene, at least

- 38 150 mg% of - / - alpha-bisabolol and less than 50 mg% of others; bisaboloids, and the essential oil thereof contains at least 3,5% chamazulene, at least 10% (alpha) - alphabisabolol and less than 10% other bisaboloids and ♦ of which the alcoholic extract obtained contains at least 2,5 mg% chamazulene, at least 7,5 / - / - alphabisabolol and less than 10.0 ipg% of other bisaboloids.

When chamomile flowers are harvested in the vegetation stage, when 70-100% of tubular inflorescence flowers are opened and the flowers are dried at an air temperature between 50-70 ° C, then the drug thus obtained contains at least 30 mg% chamazulene, not 100 mg% / - / - alpha-rbisabolol and less than 50 mg% of other bisabolides, or of essential oil thereof, containing at least 3,5% of chamazulene, at least 10% of - / - alpha-bisabolol and less than 10% of other bisaboloids and alcoholic extract prepared of this drug it contains at least 1.5% chamazulene, at least 10 mg% of - / - alpha-bisabolol and less. than 10.0 mg% of other bisaboloids.

- 39 Examples

Example - - --- / Default chamomile is tetraploid /

Individual plant variability test of 10,000 plants of the tetraploid chamomile variety described by I. Sárkány / Herba Hungar. 4/1 /,

125-169 (1965), it was found that about. per 1000 individuals comes one plant has a high content of chamazulene, which is 20% by weight, as well as a high content of / - / - alpha-bisabolol, while the content of other bisaboloids, especially / - / - alpha-bisabolonoxide / is very low (less than 5 weight percent). Individuals were selected and subjected to the following stages:

Stage 1:

From the progeny (as described above) the selected tetraploid chamomile plants were selected those individuals;

A / they bloom at about the same time,

B / have a uniform base branch and a narrow zone. 10 cm, preferably 5 cm,

. π, '· *>

- 40 C / have large inflorescences with an external diameter of about 30 mm, preferably 25 to 35 µm,

D / reach or exceed the minimum content of chamazulene 150 mg # and bisabolol 300 mg #, and their content of other bisaboloids (especially bisabololoxides) is clearly below 50 mg. / All values refer to inflorescences that have been dried at 40 ° C and harvested at a stage where 30 to 70 # of all tubular inflorescences are open.

These plants were cloned. To this end, the plants (clone mother plants) were cut to approximately 15 cm of shoot length, at 8-10 hours, day length and at temperatures of 12-14 ° C, to produce new shoots (short side shoots). These short shoots were trimmed and planted in a peat sand mixture. Rooting of seedlings / hybrids / at 100 # relative humidity and day length of 14 hours took 7-14 days.

Instead of said cloning (propagation by seedlings), in vitro multiplication of the tissue sections of the plant capable of dividing (the so-called meristem division) was also used. Various plant parts, preferably the ends of the plants, are suitable for establishing a camomile culture

- 41 hounds or arm buds.

After rinsing the plants with H ₂ ° 2 ^{, under} aseptic conditions in a laminar flow / low power turbulence displacement filter, the tips of the humerus leaf buds, buds are removed and transferred to nutrient reagent containers such as Murashige and Skooga / Physiol. Plant. 15, 473-497 (1962)]. The reagent containers are placed in an air-conditioned space at 12-18, preferably 16 hours of day length (achieved by fluorescent lamps) at a light intensity of 500 to 1000 lux, preferably 1000 to 3000 lux and a temperature of 15 to 30 ° C, preferably 22 to 27 Deň: 32 ° C. Once it is clear that the explanations are growing well, they are transferred to the above-mentioned nutrient medium, but with a higher cytokinin concentration ^with / for example 30 mg (16-isopentenyladenine) and little or no auxin (0 to 0.3 mg / l acid) indolylacetic. This is followed by the growth of the stems and the advent organs as well as the bud buds are formed. This can be removed and grown as described above.

For the multiplication of plants in a larger number of explained explanations / third passages, ie the third breeding

generation / are transferred, after growth and formation of leaves in the former nutrient medium, to a culture medium containing 10 mg / l of indolylacetic acid, or

-3-indolylbutyric acid or 0.1 to 0.3 mg / l of alphanaphthylacetic acid. In this case, the plants are rooted after about 4 weeks and can be grown further in sterilized garden soil (steamed for 12 hours at 120 ° C) in pots: and in a greenhouse (under normal conditions for seedlings).

Múrshige and Skoog environment:

mg / l

NH.NO,

3

Ca / N0 _3/2 »-4 HgO CN ° ₃

400

144

MgSO ₄ .7 H ₂ 0 KCl

12.5

and.

NaFe-EDTA

MnSO ^ .4 H ₂ 0 ZnSO ^ .7 H ₂ 0 KJ

1.6

6.5

2.7

0.75

2.0 Indolylacetic acid

- 43 mg / l furfurylade nin 0.1

- thiamine 0,1 nicotinic acid 0,5 pyridoxine 0,5 glycine 2,0 myo-inosit 100 acid hydrolyzate 1 000 sucrose 2% purified agar powder 1%

Note to the above interpretation:

/ x Cytokinins are phytohormones that promote cell division.

Stage 2:

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

From the total of selected individuals in the time range

- 44. For 4 weeks, inflorescences that bloomed and were shortly before disintegration were continuously harvested to obtain seed. The drying was carried out in a well ventilated hall at an air temperature between 20-30 ° C; the seed was then sieved (5 x 0.4 µm) and purified; using a screening machine.

Stage 3:

A random sample was taken from the seed obtained in Stage 2 and about 2,000 progeny were grown from it (ecological conditions) as in Stage 2 / and selected according to the same criteria A / D / Stage 1. The individuals selected in this way were followed by the procedure of step 2.

Stage 4:

The seed from grade 3 was sown in autumn at two different sites.

Stand 1 /

450 m NN, 48.5 ° N / 11.5 ° 0,

750 mm total annual rainfall, humid mild climate, January -10 to 0 ° C, July +10 to 20 ° C;

ϊλ ^: Έ

- 45 explanations of abbreviations:

NN = North height above sea level

O »T« ^w - north latitude / n-degree / ° O - east longitude / n-degree /

Habitat II /

200m NN, 42 ° N / 1 ° N,

400 mm. total annual precipitation Mediterranean climate, January 0 to +10 ° G i

July +20 to +30 ° C.

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

The results of the field trial thus obtained were tested and evaluated for homogeneous growth, flower size and harvest date, in addition, random flower samples were examined for the active substance content. From the field trial, individuals were selected again which corresponded to the above parameters at step 1. From these, seed corresponding to step 2 was obtained.

Stage 5i

With the seed of step 4, steps 3 and 4 were repeated in the sequence and procedure indicated.

IN

- 46 Stage 6:

About 1,500 individuals were grown from the seed obtained in step 5 (ecological conditions as in step 2) and selected according to the same principle as in step 1. From the selected plants, 34 individuals were selected and cloned according to step 1. Ten plants of each clone were grown in random distribution at a distance of 40 x 30 cm freely in field (site I, see step 4) in an isolated location.

A soil permeable clay having a pH of 7.0; cultivation was carried out in early June, the first harvest in mid-July, then the plants were cut, bloomed again and provided a second seed harvest from Wednesday to the end of August.

The seed obtained from this material was carried out as in step 2.

The seed obtained according to step 6 is the camomile propagation seed of the invention.

Plant flowers from this seed for propagation (sowing September / October, harvest beginning June of the following year), which are harvested when 30 to 70% of tubular flowers are opened and then immediately dried at 40 ° C in a box dryer for 72 hours, content 'λ ·' y *.

-Μ eats, based on the weight of the dried flowers (dry matter), for example at least: 150 mg% chamazulene, 300 mg% / - / * alpha-bisabolol and a maximum of 50 mg% of other bisaboloids. /

Further exemplary data for plants obtained according to the invention:

1. Growth

Stem: straight, little branched

2. Olistěni

Sheet: 2 to 3x pinnate Power: Medium /

pinnate leaf, medium green pinnate leaf / stem center /; pinching: moderately to severely pinched.

3 · Inflorescence

Flower heads: about 30 mm outside diameter, about 15 mm inside diameter;

Weight of single inflorescence (dried): about 45 ^m g;

Flower head, length (mm): about 700 but dependent on the growing area (climate), sowing time, soil, fertilization, plant treatment, weather;

22JB23ffiSa5S2S2 *]

- 48 Beginning of flowering / calculated from 1 January /:

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

Flowers: Middle of June / see above /;

Inflorescence without stem, essential oil content (# dry matter): about 1.0 #; azulene content in essential oil: at least 15 #

Disintegration of dried inflorescences, flower drug: slight, if harvested - carried out before opening the last tubular flowers;

Pollen diameter: about 30yum;

Seed length: about 1.25 mm;

Number of chromosomes in somatic cells: 4n = 36 ·

4 · Fetus weight of one thousand seeds (TKM): 0,06 to 0,13 g:

5.KKF germination / about 75%

6. Purity up to 95% · ^of - ^:

7 · Additional characters

Dry flower ratio of fresh, dry = 5.5 to 6: 1, characteristic aromatic aroma of the drug, mildly aromatic, typical taste of tea infusion.

- 49 In addition, the following characteristics may exist:

Uniform height growth (balance) with a narrow flowering zone, therefore particularly suitable for mechanical harvesting, large flower baskets (inflorescences), medium yield; base branched form (3 to 5 times).

Obtaining Drugs:

The camomile obtained according to this example is sown in a conventional field. Once the plants have evolved and the inflorescence is in the vegetation stage, when about 50% of the tubular flowers are open, the chamomile is harvested with a chamomile harvesting machine, with the comb spacing of the harvesting machine being adjusted to harvest only the inflorescence. 50% · The harvest is transported as quickly as possible to a shady spot and spread there in a thin layer until further processing. Thereafter, the inflorescence is removed from the stems by a sieve device and dried to a constant weight. The weight loss is about 80%. The drying is carried out in the shade in a well-ventilated place in a layer about 10 cm thick.

After about 2-3 days, drying is complete. To remove stalk particles and flower waste, the camomile drug is screened and then compressed into bales.

- 50 Analysis

Essential oil: 960 mg% chamazulen: - 162 mg% / - / - alpha-bisabolol 330 mg%

If the drying is carried out, for example, in a stationary belt dryer using artificially heated air at a temperature between 50 and 70 ° C, the drying is completed in about 4 to 5 hours. To remove parts of stems and flower waste, the drug is screened and then compressed into bales. The analysis values for such a drug are as follows:

essential oil: 870 mg% / - / - alpha-bisabolol: 197 mg% chamazulen: 94 mg%

Example 2 / The default chamomile is diploid /

Variable test of individual plants performed on 10,000 plants of tetraploid camomile variety, according to I. Sarkány / Herb Hungar. 4, 1, 125-169, 1965], it has been found that about 1,000 individuals have a single plant having a high chamazulene content of 20 wt. % as well as a high content of [alpha] - [alpha] -bisabolol

- 51 50 wt. %, while at the same time the content of other bisaboloids, in particular / - / - alpha-bisabolonoxide, is very low / less than 5 wt. % /,

These individuals were selected and subjected to the following stages:

Stage 1:

Individuals having a minimum chamazulene content of 100 mg% and / - / - alpha-bisabolol of 200 mg% or more and having a content of other bisaboloids (especially bisabololoxides) below 50 mg% were selected from the progeny of the above-described selected tetraploid chamomile plants. . All values refer to inflorescences that have been dried at about 40 ° C and harvested at a stage where about 30 to 70% of all tubular inflorescences have been opened.

Stage 2

From the plants obtained according to step 1, all blooming flower heads were removed, then the plants arrived in the greenhouse and bloomed together under isolated conditions. The plants stood in 11-inch pots filled with garden ground, at a daily temperature of 18 to 24 ° C and at a night temperature

- 52 12-14 ° C. The length of the day was at least 14 hours and was achieved by additional lighting 2 From W / m in the winter half-year. Water was supplied as needed. «

From the total of selected individuals, the four-weekly were held for a period of four weeks in order to obtain seed that had bloomed shortly before disintegration. Drying was performed in a well ventilated hall at an air temperature between 20 and 30 ° C; then the seeds were sieved using a sieve screen (5 x 0.4 mm) and further screened on a shaker screen.

Step 2 provides seed which is a camomile propagation seed for the methods of the invention.

Plant flowers from this reproductive seed (sowing September-October, harvest at the beginning of June of the following year), which are harvested at that time, 30 to 70% of tubular flowers are opened and then immediately dried in a box dryer at 40 ° C for 72 hours, containing by weight. dried flowers, not less than 100 mg% chamazulene, not less than 200 mg% / - / - alpha-bisabolol and not more than 50 mg% of other bisaboloids ·

Obtaining Drugs:

The camomile obtained according to this example is yours

- 53 sow in the usual way in the field. Once the plants have evolved and the inflorescences are in the vegetation stage, when gsi 50% of tubular flowers are open, the inflorescences are harvested using a chamomile harvester, with the ridge distances of the harvester being adjusted to tear off only those flower heads. their tubular flowers are open to fifty percent. The harvested material is conveyed as quickly as possible to a shaded area and spread there in a thin layer until further processing. In a subsequent operation, the flower heads (inflorescences) in the sifter are freed from the stems and dried to a steady weight. The weight loss is about 80%. Drying is carried out in the shade in a well ventilated place on platforms with a thickness of about 10 cm.

After about 2-3 days, drying is complete. To remove stem parts and flower waste, the camomile drug is sieved on a sieve and pressed in the next. into packages.

Analysis:

essential oil: 940 mg% chamazulene: 132 mg% / - / - alpha-bisabolol: 243 mg%

If drying is carried out, for example, in a stationary

- 54 mm belt dryer using artificially heated air, at a temperature between 50 and 70 ° C, then drying is completed after about four to five hours. · To remove part of the stalks and flower dust, the sieves are screened on the sieves immediately thereafter. pressed into bales.

For example, the values obtained by analyzing such a drug are as follows:

essential oil: 775 mg # chamazulen: 65 mg # / - / - alpha-bisabolol: 163 mg #.

Example 3 / The default camomile is diploid /

Using the variability tests of individual diploid, low bisabolol-containing but not camomile-free camomile plants described by CH.Pranz and J. HOlzl and A. VBmelea in Acta Horticulturae 73, 109-114 (1978), it was found that up to 20 # All plants have an essential oil content of about 20% by weight and / or a high content of 50% by weight of (-) - alpha-bisabolol, while at the same time the content of other bisaboloids (especially - -) - alpha-bisabolonoxide is very low. These individuals were above

- 55 lectured and tetraploidized as follows:

The seeds of the chamomile plants thus selected were applied to filter paper attached with 0.05% b

aqueous colchicine solution and allowed to swell for 6 hours at room temperature (20 ° C). It was then removed from the filter paper, washed several times with water and sown in a greenhouse. Soil: 1: 1 peat + sand mixture, temperature: 18 to 20 ° C, relative humidity: about 1 60%, daytime under artificial light: 14 hours.

Germinating plants were observed until flowering, the success of polyploidization was evaluated by comparative measurement of pollen and seed size, as well as by the number of plant chromosomes (β 1 -pomycin = first seed-grown progeny, confirmed as tetraploid colchicinated plants).

Tetraploidization can also be performed as follows:

On water-moistened filter paper, germinated 5 to 7 days old, well-developed chamomile plants were plated at room temperature (20 ° C) for 4 to 6 hours, germinal downwards, in a 0.05% colchicine solution. The sensitive loop roots were spared; In order to avoid drying damage, the surrounding atmosphere must contain approximately 100%

- 56 relative atmospheric humidity. After treatment, the germinating seedlings were washed several times with water and pinned into a hazel. Further seed-like treatments followed.

Pollen size measurements were performed using a Leitz binocular microscope with a micrometric measuring eyepiece and micrometric carrier: object.

Chromosome counting is done at the root tips: Fresh green root tips 1 to 2 cm long were taken from young greenhouse or crossbreed plants and immersed in a 0.002 molar solution of hydroxyquinoline for 5 hours and then placed in IN HCl for 15 minutes. For examination, about 1 mm /

stain the root apex with a 2% orcein acetic acid solution and examine by oil microscopy.

Thus, in cells found in mitosis, the number of chromosomes (somatic cells 4 times 4n = 36) can be determined four times.

Those plants whose pollen diameter is about 50% higher than that of the diploid starting material (about 30 instead of 20 [mu] m) and those where the chromosome number has doubled to 36 (the diploid starting material is corresponding to 18) are tetraploid. (These were selected. Approximately 0.1 to 0.5% of the seeds or germinating seedlings were tetraploidized as described above and developed flowering intact plants. Now steps 1 and 2 corresponding to Example 2 are followed.

The chamomile flowers thus obtained contain, for example, at least 100 mg of # chamazulene, 200 mg of # / - / - alpha-bisabolol and at most 50 mg of # other bisaboloids / harvesting while 30 to 70% of all tubular flowers are opened and dried for 72 hours oven at 40 ° C /.

Obtaining Drugs:

The camomile obtained according to this example is sown in a conventional field. When the plants have developed and the inflorescences are in the vegetation stage, when about 50% of the tubular flowers are open, the inflorescences are harvested mechanically, with the ridge distances selected so that only those inflorescences whose tubular flowers are 50% open . The harvested material is transferred as quickly as possible to the shade and spread there in a thin layer until further processing. The inflorescence is then de-crosslinked and dried until a constant weight is reached. The drying is carried out in the shade in a well ventilated place in a layer of about 10 cm thickness.

Drying is complete after about 2 to 3 days.

- 58 No (·, 'x

5 '

In order to remove the remnants of the stems and flower waste,

the drug sieves and then squeezes into bales. | · $

with,

Analysis: - - _ _ _ - ..

essential oil: 910 mg% chamazulen: 117 mg% / - / - alpha-bisabolol: 252 mg%

Example 4 (default chamomile is diploid)

By means of the variability tests of individual diploid / camomile, only a few bisabolols containing but not free of bisanolol described by CH. Pranzem,

J. Hollzle and A. Vomel in Acta Horticulturae 73, 109114 (1978), found that up to 20% of all individuals had an essential oil content of about 20% by weight of chamazulene and / or a high content of (-) - alpha-bisabolol 50 % by weight, at the same time the content of other bisaboloids (in particular / - / - alpha-bisabolonoxide) is'; very low. These individuals are selected and, exactly as described in Example 3, are tetraploidized and the tetraploid individuals are selected. About 0.1 to 0.5% of the seeds or seedlings were tetraploidized and developed flowering intact plants.

Next, steps 1 to 6, corresponding to example 1, follow.

The chamomile flowers thus obtained contain, for example, at least 150 mg of # chamazulene, 300 mg of # / - / - alpha-bisabolol and at most 50 mg of # other bisaboloids / harvest when 30 to 70 # of tubular flowers are open and 72 hours of box drying oven at 40 ^o C /.

The other properties correspond to the camomile obtained according to Example 1.

Obtaining Drugs:

The camomile obtained in this example is sucked in a conventional field. Once the plants have evolved and the inflorescences are in the vegetation stage when about 50 # of the tubular flowers are open, these inflorescences are harvested with a harvesting machine, with the ridge distances adjusted so that only those inflorescences whose tubular flowers are 50 # open are torn. The harvest is transported as quickly as possible to a shady spot and spread there in a thin layer until further processing. Thereafter, the inflorescences are de-stalked by sieving and dried until a constant weight is reached. The weight loss is about 80 #.

Drying is carried out in the shade at a well aerated point in a layer of about 10 cm thickness.

* It takes about 2 to 3 days for drying to finish. in order to remove the remnants of the stems and flower waste, the camomile drug is screened and then compressed into bales.

Essential oil analysis: 1020 mg $ chamazulene: 173 mg # / - / - alpha-bisabolol: 418 mg #

Annex A

The starting material is the camomile drug obtained according to the invention. Only those inflorescences in which 30 to 70, in particular 40 to 60, tubular flowers are open are used in the manufacture of the drug.

Drying is carried out in a box dryer for 72 hours at 40 ° C. Essential oil of camomile flowers is obtained as described below by two-hour steam distillation of the drug:

2.0 g of the uncrumbled drug were mixed in a 1 liter round-bottomed flask with 250 ml of desalinated water and subjected to distillation on a Cleyenger reflux apparatus (steam distillation apparatus) for titration of small amounts of essential oils. 1 ml of analytical grade pentane is used as a template.

The reflux / reflux rate is 40 and 4 drops per minute. After the distillation is complete, the dissolved essential oil, preferably anhydrous, in the pentane is discharged into test tubes and the eventually adhering essential oil is rinsed into further pentane. To remove any residual water, add a small amount (per tip of the spoon) of anhydrous sodium sulfate to the solution and then filter through a glass porosity frit or after evaporating the pentane at 40 ° C in a water bath and drying in a desiccator three cky.

In this essential oil (about 20 mg), chamazulene and bisabolol are then determined.

Spectral photometric determination of chamazulene

Specific solution: All of the 2 g of drug obtained by the essential oil (about 20 mg), obtained as described above, is dissolved in 25 ml of n-hexane or cyclohexane

Measuring device - Photometer with filter (eg Eppendorf).

Wavelength: 578 nm ¢ V.

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AND·

- 62 Cuvette: 1 cm

Specific extinction of chamazulene / 1 g / 100 ml; 1 cm /:

20.8

Compensation fluid: n-hexane or cyclohexane

Found chamazulene content in mg / 100 g: 120 E ^ _s

If a photometer with a filter is not available for measurement, this measurement can also be performed using a spectral photometer as follows:

Measuring device: Spectral photometer / for example

PM QII or PM Q III Zeiss /

Wavelength: 605 nm

Cuvette: 1 cm

Specific extinction of chamazulene. (1 g) 100 ml;

cm /: 24.5

Compensation fluid:

n-hexane or cyclohexane

Chamazulene content found in mg / 100 g: 102 E ^

Bisabolol is then determined in the measured solution

Gas chromatographic determination of bisabolol and other bisaboloids

- 63 Gas-chromatograph:

Detector:

Carrier gas: Column:

Column content:

Detector temperature:

Hewlett Packard model 5750, Erba Fractovap 2350 or similar helium flame ionization detector

3.175 mm / = 1/8 inch /;

200 cm; steel

3% nitrilsilicone and rubber ”XE 60 on silane kieselguhr chromosorb WAW HP

125 to 150fxn. as a 320 ° C injection block carrier:

columns:

Temperature programming: Sample solution:,

Reference solution:

220 ° C to 220 ° C / minute solution used to measure chamazulene about 15 mg of standard bis-bolol are dissolved in cyclohexane to a volume of 25 ml

Injection amount: 5 µl of sample solution and compare

solution Evaluation: Evaluation is performed by comparison maxim / peak areas / Bisabolol content

in mg per 100 g of drug: 10 x weight / comparison / / ssig / x

Area / sample /

Area / comparison /

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

Device: Packard, model 7721, serie 800,

or Erba Fractovap serie 2350 Columns: glass columns 3m / 2 mm in diameter; 2 m / 2 mm. on average Cartridge: 3% methylphenyl silicone OV 1 ”rubber for silane Gaschrom Q 125 to 150 yum as carrier Carrier gas: 30 ml / minute Ng Temperature program: Injector temperature / 80 to 180 ° C, 2.5 / 3 / ° C / minute detector: 200 ° C Detector: flame ionization detector

- 65 Injection amount: about 2yul approx. 1:50 diluted oil

The evaluation is partly without, partly with internal standard. As the internal standard, methyl laurate or hexadecane is suitable. for chamazulene poor or chamazulene essential oil. For oils with a content of more than 5%, this should be preferred as an internal standard, the determination being carried out photometrically (at 578 nm).

Example 5

Production of chamomile drug extract

The chamomile flowers for the drug used are harvested according to Example 1 at a time when 30-70 # tubular flowers are opened and dried at a temperature not exceeding 50 ° C.

400 g of the camomile drug thus obtained, containing 105 mg of # chamazulene and 212 mg of [- / - alpha-bisabolol, were extracted with 2100 g of aqueous ethanol (40% ethanol by weight) in a trough mixer at 30 rpm for 3 hours. The drug material is then compressed and the extract filtered.

The content of the active substance in the extract is determined in a known manner:

' ⁷

- 66 Chamazulen: 3.7 mg% / - / - alpha-bisabolol: 10.2 mg%

Example 6

Preparation of fresh chamomile extract

758 g of fresh chamomile flowers harvested according to Example 1 at a time when 30 to 70% of tubular flowers are open (water content 74%) is extracted in a trough mixer at a speed of 65 rpm for 30 minutes with 510 g of ethanol (84 weight percent). The remainder of the drug is compressed and the extract is filtered.

The extract is determined in a manner known per se

active substances:

Chama zulen: 11.7 mg% / - / - alpha-bisabolol: 16.3 mg%

Claims (2)

PATENTOVÉ N Á R A process for the preparation of a novel bisabolol-rich tetraploid camomile of the plant camomile (Chamonilla recutita L., synonymous with Matricaria chamonilla L., Asteraceae), whose dried flowers contain at least 100 mg% chamazulene on a dry matter basis, at least 200 mg% of (-) - α-bisabolol and less than 50 mg% of other bisaboloids, with the exception of the Manzana chamomile variety, characterized in that from 1 000 to 10 000 individual plants the camomile diploid or tetraploid population is determined -) - α-bisabolol and select those chamomile plants in which (-) - α-bisabolol is the major component of essential oil and in which the flowers dried at 40 ° C contain at least 100 mg% chamazulene, at least 50 to 100 mg% (- ) -o-bisabolol and less than 50 mg% of other bisaboloids, these plants are separated from the initial population, multiplied and subjected to further selection and multiplication steps and optionally The camomile obtained in this way obtains the seed for propagation and / or the camomile drug, where, in the case where the diploid camomile from which the Degumill variety is excluded is used as the starting camomile, tetraploidisation with chemicals is carried out at temperatures up to 0 up to 35 ° C, using gamma-rays, X-rays, or UV at 0 to 35 ° C, using elevated temperatures ranging from 33 to 50 ° C, using reduced temperatures ranging from 0 to 5 ° C, using decapitation-callus methods or using anthracite cultures. 2. The method of claim 1, wherein the selection or propagation comprises the following steps: (a) selection of at least 10 individuals of tetraploid plants according to the active substance content, ie at least 100 mg% chamazulene, at least 200 mg% bisabolol and less than 50 mg% other bisaboloids, all based on dry matter, current flowering time, by uniformity of basic branching and, depending on the flower size from 20 to 40 mm, the cloning of the plants thus selected and the seed obtained from the plants obtained by cloning, (b) growing progeny from the seed obtained under paragraph (a), 'the subsequent selection referred to in paragraph (a) and obtaining seed from the plants so selected', (c) three to five times the repetition of the measures referred to in paragraph (b); (d) growing progeny from the seed obtained under (c), selecting said progeny from (a), cloning the plants thus selected and obtaining seed from the plants obtained by cloning.