FR2547982A1 - PROCESS FOR THE PRODUCTION OF A NEW SPECIES OF CAMOMILE (MANZANA NAME) - Google Patents

Abstract

PROCESS FOR THE PRODUCTION OF A NEW SPECIES OF CAMOMILE (MANZANA DESIGNATION) WHOSE FLOWERS, DRY AT 40C, CONTAIN AT LEAST 150MG OF CHAMAZULENE AND AT LEAST 300MG OF (-) - A-BISABOLOLE, THE CONTENT OF OTHER BISABOLOIDS BEING -50MG LOAD, BY TETRAPLOIDIZATION OF THE DEGUMILL DIPLOID CAMOMILE SPECIES, FOLLOWED BY SELECTION AND MULTIPLICATION PHASES.

Description

Preparations of capitulas of true chamomile (Chamomilla recutica

  (L) Rauschert, synonym of Matricaria chamomilla, L.) find, because of their antiphlogistic and spasmolytic action, a wide therapeutic use and constitute an important element of the arsenal of phytotherapy It is important to attribute in this respect an importance particular to active constituents (-) - C-bisabolole and chamazulene For this reason, a good drug derived from chamomile must have a content

  as high as possible in these two substances.

  Under the designation DEGUMILL (this is the species of chamomile mentioned in the German patent application 24 02 802, industrial property of the species in the German Democratic Republic, Degumill, Italian patent 1,035,096), a species of chamomile which has both a strong (-) bisabolole and chamazulene content. However, the chamazulene and bisabolol content of this DEGUMILL chamomile species is stable only if any crossbreeding or foreign fertilization with d other chamomile species with significantly lower levels of bisabolole and chamazulene, or with wild camomile, low in active ingredients, which is present everywhere This risk of crossbreeding by foreign fertilization is almost always present, because wild chamomile

grows virtually anywhere.

  Also known are various species of chamomile tetra dloldes (eg BODEGOLD, GDR, POHORELICKY, CSSR, ZLOTY IAN, Poland, BK-2, Hungary) .These tetraploid chamomile species have a chamazulene content which is not much higher than below that of the new species of chamomile Manzala But by cons, the important active substance

  (-) e-bisabolone is present only in extremely small amounts in these tetraplol chamomile species.

  of known and, instead, other bisaboloids (bisabolol oxide A and B, as well as bisabolone oxide) are predominant and together make up more than half of the essential oils in the known tetraploid chamomile species. However, it has been found a method for the production of a new species of chamomile tetraploid with advantageous properties. The invention thus relates to a new species of tetraploid camomile and a material for its propagation, a process for its production and for of this material as well

  that a drug derived from this plant and a process for its preparation.

  The new species of tetraploid chamomile in question, bearing the designation Manzana, is superbly distinguished from the known chamomile species DEGUMILL, as well as from other known tetraploid chamomile species, by a series of new and advantageous properties. DEGUMILI, it is no longer subject to foreign fertilization with populations of chamomile that grow naturally (camomil20 the wild) In addition, it has a significantly higher content of important active substance, (-) - O-bisabolole, DEGUMIIL species The Manzana species in question is surprisingly different from the known tetraploid camomile species by an incomparably higher content of the important active ingredient of (-) - o, -bisabolole, as well as by

  very low in other bisabololts.

  Moreover, the Manzana species in question is still surprisingly different from the known tetraploid camomile species by the following characteristics: improved native germination of the seeds; lower production of non-useable hardwood stems (ie, higher yield of flowers); lower water content of the flowers (which results in better yield of dried flowers and shorter drying times); better ability to mechanized harvest, 35 because the flowers are located to a large extent in the same plane and most of them flower at the same time (only one harvest date is possible); better preservability of the drug (less tendency to decompose

  and the formation of dust); Chamomile smell particularly aromatic and typical.

  In addition, the manzana chamomile species in question is homogeneous in terms of its constituents in comparison with the 5 so far known chamomile tetraploid species, its characteristics of content of chamazulene and (-) (-bisabolole (it is ie, homozygosity) being stable On the other hand, the individual planting trials of known chamomile tetraploads species show that different types of constituents must be assigned to different individuals, ie each chance of a drug prepared from them gives a non-homogeneous result, also different

  qualitatively rather than quantitatively.

  The object of the invention is the production of a new species of tetraploid chamomile having improved properties,

  in particular a higher content of (-) c-bisabolole.

  production of the new species of chamomile tetraploid in question is carried out by tetraploidisation of the chamomile species DEGUMILL (gene mutation), followed by new selection and multiplication phases (pedigree line culture for the vegetatively reproducible foreign fecundator) ) The Manzana species consists of 34 strains or branches which

  were treated separately in conservation culture.

  The tetraploidization can be carried out for example in a manner known per se by treating elements of the plant (seeds, roots, shoot tips, sprouts, axillary buds) or fabrics of the camomile plant DUGUMILI by chemicals, rays X, gamma rays or UV rays It may be carried out additionally by the decapitation method, by anther culture or by the application of high or even low temperatures to the chamomile plant DEGUMILL or to elements or Tissues of the chamomile plant DEGUMILL In this regard reference may be made to the work of Werner Gottschalk, "Die Bedeutung des Polyploidie f Ur die Evolution der Pflanze", Gustav Fischer Verlag, Stuttgart, 1976,

especially on pages 13 to 22.

  Tetraploidization by chemicals.

  For example, the following substances are used as chemicals for tetraploidisation: colchicine, acenaphthene, alkaloids such as atropine, veratrine, nicotine, sanguinarine, derivatives of benzene, diphenyl and phenanthrene. , naphthalene and naphthalene derivatives, diphenylamine, tribromaniline, paradichlorobenzene, methylnaphthoquinone, methylnaphthohydroquinone, salicylic acid and related substances, hexachlorhexane, pervitin (hydrochloride), alkyl-alkali carbamates such as isopropyl-sodium carbamate, phenylurethane, salts cacodylic acid (e.g. sodium salt), convallaria glucosides such as convallarin, convallatoxin and convallamarin, heteroauxine, germisan (phenylmercuripyrocatechol), organic mercury compounds such as ethyl mercury phosphate, ethyl mercury chloride, phenyl mercury hydroxide, phenyl dinaphthyl 15 methane disulfonate 1-mercury, chloroform, laughing gas (N 20), as well as mixtures of these substances also include oilseed cakes,

composts and cow manure.

  The treatment with the substances mentioned here is carried out for example at temperatures between 0 ° and C, preferably between 12 ° and 0 ° C., in particular between 15 ° and 2500 ° C. The application is carried out by treatment of seeds, spearheads , roots (especially root tips or seedling roots), ovaries, cutting surfaces on leaves or stems, suspensions of meristem cells, callus cultures, or even injection into the basal region of the stem or in the region of the axillary buds The chemical products are generally used in the form of solutions in water, weakly alkaline solutions (alcohol content below 5% as a rule) or weakly For example, acidic solutions of weakly acidic solutions are in the region of 5.5 to 6.5, the acidification being carried out for example by means of lower aliphatic organic acids, such as acetic acid. If alcoholic solutions are used, they may also be weakly acidic. The concentration of the chemicals in these solutions may, for example, be 0.01.

  0.5%, preferably 0.02 to 0.2% and in particular 0.05 to 0.1%.

  the gaseous substances can be used as such, if necessary under pressure (for example from 1 to 10 bar), the treatment time is, for example, from 1 to 36 hours, preferably from 2 to 1 hour, and in particular from 4 to 6 h The most active concentrations, as well as the duration of action, will be conveniently determined each Law by

preliminary tests.

  For example, particularly favorable results are obtained by treating with colchicine at temperatures of between 0 ° C. and 35 ° C., preferably between 12 ° C. and 30 ° C., in particular between 15 ° C. and 25 ° C., for example by swelling seeds of the DEGUMILL chamomile species in a 0.01 0.2% colchicine solution, in particular 0.02 0.1%, preferably 0.05%, or by immersing germinated and well-developed seedlings of DEGUMILL chamomile species, aged from 5 to 7 days (with the cotyledons downwards) in a 0.01 0.2% solution of colchicine, in particular 0.02 0.1%, preferably 0.05% When operating in the latter manner, the ambient atmosphere must have a relative humidity of the air of almost 100%. The duration of the treatment with colchicine is, for example, from 3 to 36 hours, and in particular from 4 to hours. If seedlings are used, a maximum duration of action of 10 hours is sufficient in general when

  uses seeds, the duration of action may extend up to 36 hours if any.

  Following treatment with the chemicals, the swollen seeds, seedlings or other elements of the plant are rinsed with water several times. The swollen seeds are, for example, cut with a saw. Other elements have been treated where the seedlings grown are, for example, transplanted into plant containers. With seeds or seedlings thus treated, the plants are grown (for example in the greenhouse: temperature between 18 and 25 ° C and between 10 and 16 C at night) and selects those in the pollen grains about 1/2-fold larger than the de-art material, that is to say that they contain a number of chromosomes of 36 cells. In cases where other parts of the plant (above or below ground) are subjected to treatment with B chemicals, only the shoots, roots or flowers / seeds from these treated elements will be subjected to treatment. Subsequent control of successful tetraploidization When, for example, treatment of the offspring or underarm treatment was carried out, the number of chromosomes was examined only when new shoots formed from these axillary buds or

  these reonons and flowers or seeds formed on these shoots.

  Measurements of pollen size and chromosome counting can be carried out for example as indicated

in example 1.

Tétraploidisation by irradiation.

  The intervention is carried out for example on seeds or root tips at a temperature of 0 to 35 C, preferably 10 to 3000 C, and in particular 15 to 250 C Total irradiation dose: 5 to 50 krads On will preferably use gamma or X-rays. As UV rays, it is possible to use, for example, those whose wavelength is from 400 to 30 μm,

preferably 350 nm.

  The plants or elements of plants thus irradiated are then also subjected to the same subsequent treatment as in

  case of use of chemicals.

  Application of high and low temperatures.

  For example, in the case of high temperatures, the temperatures are between 33 and 50 ° C., preferably between 42 and 4500 ° C. These temperatures are, for example, exposed swollen seeds, seedlings, shoot tips and Meristematic tissues Duration of treatment: for example 1 to 48 hours, preferably 12 to 24 hours. For example, the low-temperature temperatures used are from 0 to 5 ° C., preferably from 0.5 to 4 ° C. and especially from 2 ° C. These temperatures are, for example, exposed swollen seeds, seedlings, shoots and meristematic tissues of the treatment: for example from 1 to 100 and, preferably, from 20 to 40 days The plants or elements of plants thus treated are also subjected to the same treatment.

  subsequent treatment only when using chemicals.

  Decapitation method (decapitation = topping, blunt or back cut) decapitation is carried out for example on young plants at the stem, preferably at the apical vegetative c 8, after the formation of 4 to 6 leaves or even petioles or lateral shoots The buds or shoots that form from the injured tissue (callus tissue) are cut, rooted, raised in pots and

  Tetraploized plants are selected in the same way as in the case of treatment with chemicals.

  Anther culture (production of single garnish plants

  chromosome from the anthers, followed by spontaneous or artificial tetraploidisation).

  On flowering plants, closed tubular flowers are collected, the anthers (pollen sacs) are in the stage preceding the first pollen mitosis. Using a micromanipulator, the anthers are taken from the flower buds and transferred to Petri dishes which are filled for example with nutrient medium of Nitsch and Nitsch (Table 1). Then the Petri dishes are kept in a culture chamber under light for 16 hours a day, at a day temperature of 28 C and a night time of 20 C. After about four weeks, the anthers begin to open and seedlings begin to grow. These are haploid when the parent element is diploid and they are diploid with a tetraploid parent element; after formation of roots, they are potted, for example in garden soil, and brought to flowering in a greenhouse These (di) haploid plants are sterile, but they can be polyploidy by the treatment of spearheads, roots or stems with chemicals such as colchicine, in which case homozygous plants are formed which can then be propagated by means of seeds. For the remainder of the procedure, reference is made to the indications given in connection with treatment with products

  chemicals (eg treatment with colchicine).

Table 1

Middle of

  Nitsch and Nitsch culture (Science, 1969).

  mg / 1 KNO 3 NH 4 NO 3 Mg SO 4 x 7 H 20 Ca C 12

KH 2 PO 4

  Mn SO 4 x 4 H 20 10 H 3 BO 3 Zn SO 4 x 7 H 20 Na 2 Mo O 4 x 2 H 20 Cu SO 4 x 5 H 20 ml of a solution of 7.45 g of sodium disodium salt ethylenediamine tetraacetic acid and 5.57 g of Fe SO 4 x 7H 20 in 1000 ml myo-inosite Glycocol Nicotinic acid Pyridoxine-H Cl Thiamine-H Cl Folic acid Biotin Sucrose Nutrient medium base (Bacto-Agar DIFCO ) Indole-acetic acid

950 720 185 166 68 25

0.25 0.025

  0.5 0.5 0.5 0.05 20 g 8 g 0.1 μH of the medium set at 5.5 Among the tetraploid chamomile plants that can be obtained according to the different possibilities described above and which were selected by measuring pollen size and / or counting chromosomes, plants that meet the following conditions are then re-selected: a) Nearly simultaneous bloom, b) Regular branching at ground level and narrow zone flowers, ca. 5 cm, c) Large flower heads, with an outside diameter of about 25 mm (mm) to 25 mm, (d) Minimum chamazulene content of 150 mg O and bisabolol

  300 mg% and other bisaboloids (especially bisabolol oxides) content of less than 50 mg% (based on dried flowers, see example).

  The plants thus selected are then propagated vegetatively by cuttings (cloned) and sorted over 3 to 5 generations, the selection always taking place according to criteria a) d) indicated above. This means that the plants selected according to criteria a) d) are cloned, seeds are recovered from cloned plants,

  The resulting plants are selected according to criteria (a) (d) and seeds are taken from them again. The seed sequence selected from (a) (d) seed replenishment can be repeated 2-4 times.

  This is followed again by a sequence L seeding selection according to a) d) cloning recovery of seeds 7 The seeds obtained last then constitute the

  propagating material of the Manzana 30 chamomile species in question.

  Propagation by cuttings is carried out as follows: For propagation by cutting, the starting plants (cloned mother plants) must form short shoots, without buds, under short day conditions. in the winter semester at the greenhouse without additional lighting, or in rooms

  conditioned with a day time of 6 to 10, preferably 8 hours and at temperatures of 10 to 15, preferably 12 C.

  Suitable for cloning or propagation are cuttings of leaves, shoots and in particular short shoots (lateral shoots). They are rooted at a temperature of 12 to 18 ° C., preferably 15 ° C., and with a duration of 5 ° C. 12 to 16 days, preferably 14 hours, in a closed atmosphere (100% relative atmospheric humidity) As a substrate, it is possible to use, for example, a 1: 1 mixture of peat and sand. ; but pure siliceous sand,

  mineral wool cuttings cubes, peat cutting cubes or the like are also suitable.

  For seeding, for example, the following grounds will be used: garden soil; medium-weight clay soils

  humic; sandy, clay or humic soils.

  It is possible to sow in a greenhouse or even in the open field. Temperatures for germination and growth of plants are, for example, between 12 and 24 ° C., in particular between 18 ° and 20 ° C. In the case where seeding is carried out in the open field, sown preferably in autumn (September / October) or spring (March / April) These periods are suitable for all crop regions (eg hemisphere

  North, regions of temperate to subtropical climate).

  The new species of Manzana chamomile in question belongs to the genus of true chamomile cultivation plants bearing the botanical designation Matricaria chamomilla L. (synonym of Chamomilla recutica (L), Rauschert) and is defined by the following levels of active substances of its flowers dried at 40 ° C: at least 150 mg% chamazulene, at least 300 mg% (-) - bisebolol and less than 50 mg% other bisaboloids, on the basis of dry matter levels of active substances relate to the stage of flower development which is reached when 70%, and in particular 40 to 60% of all tubular flowers of a flower head are open (which means that the flowers used for the determination of substances The active ingredients were stored at this point and then dried for 72 hours at 40 ° C.

in the dryer cabinet).

  In the case where the harvest of chamomile flowers is carried out at a time when the flowering stage of these flowers is more advanced, that is to say at which 100% or nearly 100% of all tubular flowers of For example, a flower head is open (for example at the stage of full flowering) and / or in the case where the drying of the harvested flowers is carried out at a temperature higher than 40 ° C., the content of the active substances (-) " -bisabolole and chamazulene is weaker So if the harvest takes place at a stage of vegetation 10 o 30 to 100% of the tubular flowers of a capitulum are open and if the drying is carried out at a temperature up to 60 C (preferably protected from sunlight, for example in the drying conditions which are indicated a few pages later), the chamomile drug which is thus obtained contains at least 120 mg% of chamazulene and at least 200 mg% of (-) - o-bisabolole, while te neur in other bisabololdes

  remains below 50 mg% after as before.

  By-designation "other bisaboloids" means in particular: (-) - t-bisabolole oxide A and B, (-) O-bisabololeo oxide The essential oil of the species of chamomile in Question a for other constituents of en-in-dicycloethers, farnesol, spathulenol and, in lower concentrations, various terpene hydrocarbons very volatile For example, the flowers of the species Manzana in question, harvested at the stage 25 of flower development o 30 to 70% of tubular flowers of a flower head are opened and dried at 40 C in the dryer cabinet, contain between 150 and 200 mg% of chamazulene, between 300 and 450 mg% of (-) c-bisabolole and a little only, ie between 5 and 50 mg% other bisaboloids, based on the dry substance (ie on the basis of the absolute dry weight of the flowers) This dry weight is determined by drying of a separate sample of chamomile flowers of the species Manzana in question, in the dryer cabinet at 1050 C until the weight is constant (72 to 96 hours) The active substance content of the dried flowers for example between 35 and 10 50 C (drug) is then calculated with respect to the dry weight of the flowers, determined at 1050 C. In its phenotype, the Manzana species in question is similar to the previously known tetraploid chamomile species (eg Bodegold, Zloty Ian, BK-2, Pohorelicky Velkokvety); 15 it differs in particular from the fact that in the case of the species Manzana in question, (-) - X-bisabolone is the main constituent of the essential oil of flowers, that in addition the content of chamazulene is higher and the content of other bisaboloids (bisabolole oxides A and B, bisabolone oxide) is much lower.

  The other constituents of Manzana are farnesol, spathulenol and en-in-dicycloethers.

  The chamomile species in question can be cultivated successfully on all soils, with the exception of soils having a content of more than 20% in organic substances (humic substances and microorganisms of the earth); it does not require special agrotechnical procedures or special cultural measures; it suffices simply for the cultivation of a terrestrial day of more than 13 hours maximum duration, which means that the temperate zones and the subtropical regions are suitable

especially for its culture.

  The Manzana chamomile species in question has a late harvest period, a uniform growth height with a narrow flowering zone and large flower heads and, for these reasons, is particularly suitable for harvesting.

  In addition, plants of the species Manzana 5 which have been sown at the same time generally flake almost simultaneously, which simplifies and considerably facilitates harvesting.

  The Manzana chamomile species in question provides a high ratio. As examples of possibilities of use, mention will be made of the preparation of chamomile-based drugs, of oil

  chamomile, chamomile extracts, as well as (-) - natural abisabolole.

  A drug prepared from the Manzana chamomile species in question has the maximum chamazulene and bisabolole active substance content when the chamomile flower heads are harvested at the vegetation stage, for example 30 to 70%, preferably 40 to 70%, preferably 40 to 70%. at 60%, that is to say generally 50% of the tubular flowers of a capitulum are open and when the drying is carried out at an air temperature of 50 C maximum, for example 35 to 50 C Drying can be carried out either by artificial air supply, or by drying in the shade and even, if necessary, in the sun, making sure, however, that the heat input does not exceed the necessary extent Thus, it is advantageous to ensure that the weight constancy is reached by control weighing. The drying can be carried out spontaneously or artificially (for example with artificial hot air). The yield of substances The drying process should be carried out as soon as possible after harvesting. The drying should be carried out in thin layers, for example 5 to 20 cm. and, preferably,

10 cm thick.

  The measurement of chamazulene is carried out spectrophotometrically, in a manner analogous to the procedure usually followed with extracts of chamomile. The dosage of (-) - cw-bisabolole and the other constituents of the essence of chamomile is carried out according to gas chromatography methods normally applied for this purpose. In Annex A,

  precise description of analytical methods of assay.

  The active substance chamazulene is present in chamomile flowers, not as such, but in the form of its sesquiterpene-lactone, matricin Ia matricin has a pharmacological activity which corresponds to that of chamazulene from this progenitor matricin, chamazulene is formed immediately, for example on heating (for example by steaming, during tea infusion). This is why it is customary for chamomile to indicate not the content of matricin, but the content of the chamazulene that is formed from it, which may be

to be determined analytically.

Example 1

  Seeds of the chamomile species DEGUMIIL were placed on a sheet of filter paper impregnated with aqueous 0.05% colchicine solution and allowed to swell for 6 hours at room temperature (20 C). then removed from the filter paper, rinsed with water several times and sown in planting trays (in a greenhouse). Earth: peat and sand mixture 1: 1; temperature: 18 to 20 C; relative humidity of the air: approximately 60%;

  duration of the day in artificial lighting: 14 hours.

  The germinated plants were monitored until flowering, the success of the polyplo Idisation was found by comparative measurement of the size of pollen grains and seeds, as well as by counting the chromosomes of plants (F 1 = first generation , taken from seeds,

  colchicinated plants whose tetraploidy has been established).

  Tetraplocdization can also be carried out in the following way: Well-developed 5- to 7-day-old DEGUMIIT chamomile seedlings, germinated on water-impregnated filter paper, were placed with the cotyledons facing downwards. in a 0.05 / colchicine solution at room temperature (20 C) for 4 to 6 hours The roots of the germs, which are vulnerable, were thus formed: to avoid damaging them by drying, the ambient atmosphere must have a relative air humidity of almost% As a result of the treatment, the seedlings were rinsed with water several times and transplanted into planting boxes The subsequent treatment was the same as in the

case of the seeds.

  The pollen grain size measurements are made with a Ieitz binocular search microscope equipped with a micrometer eyepiece and a micrometer slide. Chromosome counting is done at the tips of the roots: on young plants or seedlings grown in a greenhouse, fresh root pieces of 1 to 2 cm are collected and then placed for 5 hours in a solution of 0.002 molar. In the examination, about 1 mm of the root tips are stained with orcein, 2% acetic acid, to be observed under a microscope immersed in the oil. mitosis, one can thus determine a chromoson stoc 1 four times

  higher than that of somatic cells (4 N = 36).

  Plants whose pollen grains are about 50% larger in diameter than the diploid starting material (about 30 microns instead of about 20 microns) and in which the somatic cell chromosome stock has been doubled to 36 ( in the diploid starting material, the number

  Corresponding is 18) are tetraploids These were selected about 0.1 to 0.5% seeds or seedlings were tetraploidized with the method indicated above and gave intact plants able to flower.

  We then move on to the next phases.

  Phase 1 Among the selected tetraploid chamomile plants (as described above), individuals were selected which a) flowered at about the same time, b) had regular branching at ground level and a narrow zone of flowers, approximately 5 cm, c) had large flower heads with an outer diameter of approximately 30 mm, d) reached or exceeded a minimum content of 150 mg% chamazulene and 300 mg% bisabolol, and bisabololites (in particular the bisabolol oxides being significantly lower than 50 mg% (all these values are related to heads that have been dried at 40 C and whose harvest was carried out at the stage where it had flowered only about 50 c (40 to 60%) of

tubular flowers of a capitulum).

  These plants were cloned For this purpose, the plants (clone mother plants) were first cut at about 15 cm of shoot length, then pushed back (short 10 side shoots) with a day length of 8 to 10 hours and at a temperature of 12 to 14 C The short shoots were

  cut and planted in a mixture of peat and sand With a relative air humidity of about 100%, an air temperature of 15 C and a day time of 14 hours, the rooting of the cuttings takes 7 to 14 days.

  Instead of the conventional cloning indicated (multiplication by cutting), one can also practice the in vitro multiplication of divisible tissue parts of the plant (so-called meristematic multiplication). Different parts of the plant, from

  preferably the tips of the offspring or the axillary buds, are suitable for the establishment of a culture of chamomile.

  After rinsing the plants with H 202, under the aseptic conditions, the "Iaminar Flow" (high-capacity filter for suspended material in the air, with turbulence free flow) is taken, axillary bud shoot tips of the leaves and transfer them to test tubes which are filled with a nutritious

  Murashige and Skoog medium (Pfsiol Plant 15, 473-497, 1962).

  The reaction tubes are placed in a climatic chamber where the daytime is 12 to 18 hours, preferably 16 hours (obtained by fluorescent tubes), the intensity of the light is

  500 to 10 000 lux, preferably 1000 to 3000 lux, and the temperature is 15 to 300 C, preferably 22 to 27 C.

  As soon as the explants show good development, they are transferred to the nutrient medium defined above, but having a higher concentration of cytokinin (30 mg / l N 6 -isopentenyladenine) and less or not at all. auxin (O 0.3 mg / l indole-acetic acid) Subsequently, the axes extend and it is formed of adventitious organs, as well as multiplied axillary buds These can be collected

  and put to push in the manner indicated above.

  Explants intended in large numbers for the multiplication of plants (3rd passage = 3rd generation of multiplication) are transferred, after having taken root and having formed leaves on the nutrient medium mentioned first, on a feeder layer which contains 10 mg / l of indole-acetyl acid or 3-indole-butyric acid or 0.1 to 0.3 mg / l of naphthylacetic acid. Under these conditions, the plants take root and can be planted after about 4 weeks, in pots filled with sterilized garden soil (steam treated at 120 C for 12 hours), to be grown in a greenhouse later (under conditions similar to those

  usually applied for propagation by conventional cutting).

  Nutrient Media of Murashige and Skoog NH 4 NO 3 400 (mg / l) Indoleacetic acid 2.0 (mg / l) Ca (N 03) 2 4 H 20 144 Furfuryladenine 0.1 KN 03 80 Thiamine 0.1 KH 2 PO 4 12.5 Nicotinic acid 0.5 Mg SO 4 7 H 20 72 Pyridoxine 0.5 K Cl 65 Glycocolle 2.0 Na Fe-EDTA 25 Myo-inosite 100 H 3 B 03 1.6 Hydro lysate 1000 Mn SO 4 4 H 20 6.5 Sucrose 2% Znso 4 7 H 20 2.7 Agar-agar powder Ki 0.75 purified 1% Phase 2 The plants obtained in phase 1 fade together in isolated greenhouse conditions. plants were in 11 cm pots, filled with garden soil, at a day temperature of 18 to 24 C and at night from 12 to 14 C Day duration

  was at least 14 hours and was reached in winter semester 35 by additional illumination (200 W / m 2) Water supply was on demand.

  Of all the selected individuals, the flower heads that had flowered and were about to fall were harvested for seed recovery over a period of 4 weeks. After drying the harvested flowers at room temperature, the seeds were harvested. have been recovered by blowing

lighter components of the flowers.

  Phase 3 On the seeds obtained in phase 2, a random sample was taken, from which about 2000 offspring were grown (ecological conditions similar to those in phase 2) and subjected to selection according to the same criteria. a) to d) that those of phase 1 With the selected individuals, we then proceeded in the same way

than in phase 2.

Phase 4

  Part of the Phase 3 seeds were sown in autumn at two different locations.

Location A)

  Altitude 450 m, 48.5 north latitude, 11.5 east longitude.

  Total Annual Precipitation, 750 mm Temperate-humid January, 10 to 0 C

July, + 10 to + 20 C.

Location B)

  Altitude 200 m, 42 north latitude, 1 east longitude.

  Total annual rainfall, 400 mm. 25 Mediterranean climate.

January, O to + 10 C

July, + 20 to + 30 C.

  At both locations, seeding was performed

late September / early October.

  Field experimental plantations thus obtained were examined and evaluated (quality control) for growth homogeneity, flower size and harvest period; in addition, samples of the random flowers were tested for active substances. In these field plantations, the individuals that corresponded to the parameters set for phase 1 were again selected from them. seeds were obtained in the manner indicated in the last paragraph of the

phase 2.

  Phase 5 With the seeds of phase 4, the phases 3 were repeated

  and 4 in the order and in the manner indicated.

  Phase From the seeds obtained in phase 5, about 1,500 individuals were grown (same ecological conditions as in phase 2) and were selected according to the same principle as was indicated for phase 1. plants so selected, 34 individuals were selected and cloned as in phase 1 The plants of each clone were planted tenfold in a random distribution, at a distance of 40 x 30 cm in the open field (location A , see phase 4), at isolated points The earth was a loess, 15 p H 7.0; the planting was carried out at the beginning of June, the first harvest of the seeds took place in the middle of July, then the plants were cut, bloomed again and gave,

  between the middle and the end of August, a second crop of seeds.

  Seed recovery of this material was performed

  in the same way as in phase 2.

  The seeds obtained in phase 6 are the propagating material of the Manzana chamomile species in question. The flowers of plants from this propagating material (seeding in september / October, harvest beginning of June of the following year), which are picked at the time when about 50% (40 to 60%) of the tubular flowers of a flower head are open, and which are dried immediately after in the drying cabinet at 40 C for 72 hours, contain for example, on the basis of dried flower weight (dry substance): 150 mg% chamazulene, 300 mg% (-) - ot-bisabolole and at most

mg% other bisaboloids.

  Other data, provided by way of example, concerning

  plants of the chamomile species in question, obtained according to Example 1.

  1 Growth Homogeneous growth height (alignment) with a narrow flower zone, therefore particularly suitable for

  mechanical harvest, large heads, average yield.

  Shape: branched at ground level (3 to 5 times)

Stem: vertical, little branched.

  2 Foliage Leaf: pinnate, 2 to 3 times Thickness: medium Pinnate leaf, medium green color

  Pinnate leaf (median stem); moderately to strongly pinnate.

  3 Inflorescence Flowers arranged in small basket (capitulum):

  outer diameter, about 30 mm inside diameter, about 15 mm.

  Weight of the individual heads (dry): 45 mg approximately.

  Flowering shoot, length (mm): about 700, but depending on the place of cultivation (climate), the seeding period, the

  soil, smoking, plant treatment, time.

  Start of flowering (days from 1 January): 160 th day (seeding in September, location, Freising, Federal Republic of Germany, otherwise depending on the factors mentioned above).

  Flowering: mid-June (see above).

  Inflorescences without stem: Content in essential oils (% of the dry substance): about 1.0%; azulene content in

essential oils: at least 15%.

  Disintegration of the dried inflorescences, drugs: weak in case of harvest before the opening of the last tubular flowers. Pollen diameter: about 30 micron seeds thickness: about 1.25 mm

  Number of somatic cell chromosomes: 4 N = 36.

4 Fruit

  1000 seeds weight of seeds: 0.06 to 0.13 g.

Germination capacity

, 75%.

6 Purity

94 95%

  7 Other features Dry / dry drying ratio (flowers) = 5.5 to 6: 1; odour

  aromatic characteristic of the drug; finely aromatic taste, typical of tea infusion.

  Annex A Production of the essential oil The starting material is a drug derived from the Manzana chamomile species For the preparation of the drug, only 30 to 70%, especially 40 to 60, flower heads are used. % of tubular flowers are open Drying takes place

  in a drying cabinet at 40 C for 72 hours.

  The essential oil of chamomile flowers is obtained,

  as described below, by steaming for two hours of the drug.

  2.0 g of unmilled drug are mixed in a one-liter round bottom flask with 250 ml of demineralized water and subjected to a two-hour liquid return distillation in a Clevenger apparatus (reflux apparatus). For pre-loading, 1 ml of pentane is used per analysis. The reflux rate is +4 drops / min. After the completion of the distillation. The essential oil dissolved in the pentane is emptied into reaction tubes in as anhydrous a state as possible and any residual essential oil which adheres to the equipment is rinsed with pentane. water residues, a spatula tip of Na 2 SO 4 is added to the solution, which is then filtered through a glass filter funnel of porosity D 3 or D 4, in neck flasks After pentane evaporation 40 C in a water bath and drying in a desiccator, the amount of oil

  is determined gravimetrically.

  In the oil thus obtained (about 20 mg), the chamazulene and the bisabolole are then added.

  D 2 sag 2 _e 22 E 2 chamazulene chamazetone Measuring solution: the whole of the essential oil, obtained from 2 g of drug (approximately 20 mg) (obtained

  as described above) is dissolved in 25 ml of n-hexane or cyclohexane.

  Measuring device: Filter photometer (eg Eppendorf).

Wavelength: 578 nm.

23 2547982 23

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

  Compensation liquid: n-hexane or cyclohexane.

  Content of chamazulene found, in mg / 100 g: 120 E 578 If a filter photometer is not available for measurement, the assay can also be carried out with a spectrophotometer: Measuring device: Spectrophotometer (eg PM Q II or 10 PM Q III "ZEISS") Wavelength: 605 nm Cuvette: 1 cm Specific extinction of chamazulene (1 g / 100 ml, 1 cm): 24.5 Compensation liquid: n-hexane or cyclohexane Chamazulene content found, in mg / 100 g: 102 E 605

  The bisabolole is then measured in the measurement solution.

  Assay by Chromatography in Bisabolol and Other Bisaboloides Beam Gas Chromatograph: Hewlett Packard Model 5750,

  Erba Fractovap 2350 or similar device.

  Detector: Ionization-flame detector.

Carrier gas: Helium

  Column: 1/8 of an inch; 200 cm; steel.

  Column Filling: 3% Nitrile Silicone Rubber "XE 60" On Silanized Infusoria Ground "Chromosorb WAW HP" 125 to

microns serving as support.

  Temperature Detector: 3200 C Injection block: 220 o C

Column: 85 220 C.

  Temperature programming: 40 / min Sample solution: We used the measurement solution for

chamazulene.

  Typical solution: About 15 mg of standard bisabolole were

  dissolved at 25 ml in cyclohexane.

  Injection quantity: 5 Fl of each sample solution

and typical solution.

  Evaluation: The evaluation is carried out by comparison of surfaces

peak.

  Bisabolol content in mg per 100 g of drug: x Quantity weighed (control) / -mg_ 7 x Surfae-hatillonmg_ 7 urace sample 7 The determination of other bisabololes can also be carried out by gas chromatography, for example under the operating conditions the following: Device: Packard, Model 7721, Series 800 or Erba Fractovap,

2350 series.

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

of diameter.

  Filling: 3% methylphenylsilicone rubber "OV 1" 1 on silanized infusoria "Gaschrom Q" I ', 125 to 150

serving as a support.

  Carrier gas: 30 ml / min of N 2 Temperature program: 80 to 180 C, 2.5 (3) C / min / Temperature of the injector / detector: 200 C

  Detector: Flame ionization detector.

  Amount injected: about 2 el of the essential oil diluted to about 1:50.

  The evaluation is carried out partly and partly with internal standard. As an internal standard, lauric acid methyl ester or hexadecane are suitable for chamazulene or camazulene-free oils. Oils with a chamazulene content greater than 1%, the ester is preferred to the internal standard, the determination of the content being made photometrically (at 578 nn).

Claims (12)

  1 Chamomile tetraplo Xde of the genus of true chamomile cultivation plants (Chamomilla recutica (I) Rauschert, synonym of Matricaria chamomilla L, asteraceae), whose flowers, dried at 40 C, contain at least 150 mg% of chamazulene; at least 300 mg% (-) "-bisabolole and less than 50 mg% other   bisabololdes, on the basis of the dry substance.   2 Camomile multiplication material according to claim 1.   3 Process for the production of a new species of tetraploid chamomile (designation Manzana) of the genus of true chamomile cultivation plants (Chamomilla recutica (L) Rauschert, synonym of Matricaria chamomilla L, asteraceae), whose flowers, dried at 400 C contain at least 150 mg% of chamazulene, at least 300 mg% of (-) oc-bisabolole-and less than 50 mg% of other bisabololes based on the dry substance, characterized in that the species of diploid chamomile DEGUMILL is tetraplo Ydisated in a manner known per se, the selected plants 6 traploles are subjected to new phases of selection and multiplication and, where appropriate, a multiplication material and / or a camom 2 drug They are prepared from plants   of chamomile that are obtained in this way.   Process according to Claim 3, characterized in that the tetraploidisation is carried out by means of chemicals at temperatures between 0 and 35 C, using gamma rays, X-rays or UV rays at temperatures between 0 and 35 C, by means of high temperatures of 33 to 50 C,   by means of low temperatures of 0 to 5 C, by means of decapitation method or by anther culture.   Process according to claim 3 or 4, characterized in that following tetraploidisation: 26 2547982   a) a selection of tetraplo Xdes plants is carried out according to their content of active substances (at least 150 mg% of chamazulene, at least 300 mg% of bisabolole and less than 50 mg% of other bisaboloids, all on the basis of of the dry substance), based on the simultaneous flowering date, the uniformity of the branching at ground level and the size of the flower heads, which should be between 25 and mm, the plants thus selected are cloned and seeds are recovered from the plants obtained by cloning, b) descendants from the seeds thus obtained are cultured, then a selection following a) is carried out and seeds are again recovered from the plants selected, c) the operations according to b) are repeated 2 to 4 times, d) descendants from seeds obtained in c) are cultivated, a selection is made according to a), the plants thus selected are cloned and seeds are recovered   from plants obtained by cloning.   Chamomile or camomile multiplication material obtained according to any one of claims 3 to 5.   Use of chamomile according to any one of claims 1 to 6 for the production of propagating material.   Process for the preparation of propagating material, characterized in that seeds or cuttings are recovered, in a manner known per se, from chamomile plants according to   any of claims 1 to 6.   9 Use of a chamomile according to any one of claims 1 to 6 for the preparation of a drug in the   chamomile. Process for the preparation of a drug with chamomile   from chamomile flowers according to any one of claims 1 to 6, which contains at least 150 mg% of   chamazulene and at least 300 mg% of (-) - o <-bisabolole, the   other bisaboloids below 50 mg%.   11 Process for the preparation of a chamomile drug from chamomile flowers according to any one of   claims 1 to 6, which contains at least 150 mg% of   chamazulene, at least 300 mg% of (-) "-bisabolole and less than 27 2547982   mg% other bisaboloids, characterized in that the flowers are harvested at the vegetative stage or only 70% of the tubular flowers of a flower head to be opened and in that the flowers are dried at a temperature air not exceeding 500 C. 12 Chamomile drug containing at least 150 mg% chamazulene, at least 300 mg% (-) 1-bisabolole and less than 50 mg% other bisaboloids, characterized in that we use,   for its preparation, chamomile flowers according to any one of claims 1 to 6.