HU202034B - Method for producing novel camomile of improved feature rich in tetraploide and bisabalde - Google Patents

Description

The present invention relates to novel tetraploid and bisabol soluble chamomile populations with improved properties, which allows the production of a chamomile species rich in valuable active ingredients than is known in the art.

The flowers of true camomile (Chamomilla recutita (L.) Rauschert, related to Matricaria chamomilla L.) are widely used for medicinal purposes due to their antiflogistic and spasmolytic effects and form an important part of herbal medicine. The (-) -? -Bisabolol and camulene active ingredients are particularly important from a medical point of view, so a good chamomile drug should have as many of these active ingredients as possible.

The active ingredient content of naturally occurring diploid chamomile varies greatly. Thus, for example, only one of the important kamazulene and (-) -? -bisabolol may be present in the plant, in some cases none or only a very small amount, when the main component of the essential oil is the ineffective bisaboloids ( bisabolonoxide A or B, bisabololoxide A).

The designation "DEGUMILL" (German Patent No. 2,402,802), Degumill German Plant Variety Protection,

Italian Patent No. 035,096) is known as a chamomile species which has a high content of both (-) -? -Bisabolol and kamazulene. However, the chamazulene and bisabolol content of this DEGUMILL chamomile species is stable only if cross-breeding or foreign pollination with chamomile varieties with significantly lower chamazulene and bisabolol content is prevented. From this point of view, the danger of wild camomiles, their crossing and foreign pollination is almost always present, since wild camomile is practically everywhere. In addition, various tetraploid chamomile species are known (e.g., BODEGOLD, DDR; POHORELICKY, CSSR; ZLOTY LAN, Poland: BK-2, Hungary). These tetraploid chamomile species have satisfactory content of chamazulene but contain only a very small amount of the very important (-) -? -Bisabolol and instead contain the usual bisaboloids (bisabololoxide A and B and bisabolonoxide) in these chamomile species. more than half of the essential oil.

It is an object of the present invention to provide a process for the production of a novel tetraploid chamomile species having advantageous and improved properties.

It has now been found that certain treatments or procedures of naturally occurring chamomile populations and diploid or tetraploid cultured chamomiles result in bisabolically soluble tetraploid chamomile plants which are no longer susceptible to foreign pollination and -a-bisabolol content is shown such that (-) - a-bisabolol is significantly higher than the kamazulene content and at the same time there is practically no or very little other bisaboloid. It is also of great importance that the content of the main active ingredients, (-) - abisabolol and kamazulene in the chamomile obtained by the process, is constant, i.e. remains constant after each propagation.

The process according to the invention is characterized in that the (-) -? -Bisabolol content of a diploid or tetraploid chamomile population, with the exception of the diploid chamomile species of the species DEGUMILL, is always determined in the essential oil and selected from the chamomile plants containing at least 100 mg% kamazulene, at least 50-100 mg% (-) - a-bisabolol and less than 50 mg% other bisaboloids dried at 40 ° C, these chamomile plants are propagated separately from the initial population and

A. At least 10 individuals are selected from these plants which exhibit concurrent flowering time, flower head size of 20 to 40 mm and at least 100 mg% of kamazulene, at least 200 mg% of bisabolol and less than 50 mg% of additional bisaboloid active ingredient based on dry matter content and optionally subsequently cloning the diploid or tetraploid tetraploid plants so selected, which cloning or

(a) by cuttings propagated by cutting back plants, replanting at 12 to 14 ° C and rooting the resulting short shoots at high relative humidity and 1218 ° C in peat-sand soil, or

b) Meristem propagation is done by treating divisible tissue portions, such as shoot tips or underarms, at a temperature between 15 ° C and 30 ° C in a standard nutrient such that additional organs (buds, shoots, roots) are formed, repeating several times in medium or, if desired, in a single medium, and then cultivating and propagating the plants so obtained, as described above, at high relative humidity and 10-18 ° C,

B. allowing the plants obtained under A to bloom under set-aside conditions and continuously harvesting, for several weeks, seedlings that are in the vegetative stage when 30-70% of the total flower buds of a flowerhead are open, and the flowerheads obtained are harvested. dried at ambient temperature between 30 ° C and 30 ° C, the seed obtained is grown under isolated conditions at 18 to 24 ° C at day temperature and 12 to 14 'at night, optionally with additional lighting, and selected from the plants thus obtained. they bloom at the same time, have evenly leafy branches, have flower heads of between 2 and 40 mm and contain at least 100 mg% of kamazulene, at least 200 mg% of (-) - a-bisabolol and less than 50 mg% of other bisaboloids so selected planting seeds again from the plants and, where appropriate, cultivating the seeds in different places of production and selecting them according to the requirements described above,

C. repeat steps B to 3 to 5 times,

D. living conditions between 18 and 24'C under conditions isolated from seeds obtained under C.

HU 202034 Β and at night temperatures between 12 and 14 'C, plants are grown again and at the same time with flowering time, uniform leaf branching and flower head size of 20 to 40 mm, and at least 100 mg% kamazulene, at least 200 mg% bisabolol and less than Selected for 50 mg% of the other bisaboloid content, the resulting plants were cloned as in A. and the resulting plants were again seeded and, in the case of a diploid chamomile, at 0 to 25 ° C with chemicals 0 and It is tetraploidised at 35 ° C with gamma rays, X-rays and UV rays, high temperature between 33 and 50 ° C, low temperature between 0 and 5 ° C, or decapitated callus, or subjected to anther culture and subsequent tetraploidization. and optionally obtaining the drug and / or propagation material from the camomile plants thus obtained by harvesting the flowers at a vegetative stage when 30-100% of the tubular flowers of a flowerhead are open and the flowers are at an ambient temperature of 70 ° C. dried while crushing the resulting dry flower heads and sifting the propagation material (seeds) to obtain propagation material.

The novel tetraploid chamomile produced by the process surprisingly has a series of new advantageous properties compared to the known chamomile species. One of these characteristics is that, unlike the known DEGUMILL species, it is not prone to pollination with naturally occurring camomile populations (wild camomiles). The chamomile produced by the process of the present invention is surprisingly different from known diploid chamomile and tetraploid chamomile species because of its high content of active ingredient, (-) -? -Bisabolol (at least 200 mg%), while the usual bisaboloids (e.g. bisaboloxides) the amount is very small, less than 50 mg%. For example, the known and described chamomile varieties contain very high levels of bisaboloxides and partially bisabolone oxides, typically about 50% of the essential oil of the drug (i.e., about 500mg per 100g of drug in 100% drug).

In addition, the chamomile according to the invention is homogeneous with respect to the tetraploid chamomile species known so far, i.e. the content of kamazulene and (-) -? -Bisabolol is constant (homozygous).

In contrast, studies on the individual tetraploid chamomile species on individual plants show that they differ in content, with the result that each spot test on the drugs produced from them produces different in both quantity and quality, inhomogeneous.

In addition, the chamomile produced by the process of the present invention differs from the previously known tetraploid chamomile species in one or more of the following characteristics: improved germination capacity of seeds, reduced waste from untreated plant mass (i.e. higher flower / plant mass ratio, lower water content, higher yields of dried flowers and shorter drying times), better suited to machine harvesting because the flowers are largely flat and the vast majority of flowers open at the same time (allowing for near-optimal determination of harvest time), better durability of the product ( less susceptibility to disintegration and debris formation), particularly aromatic and distinctive chamomile odor. For example, the latter properties are obtained when, in addition to the active ingredient content, one or more of the above properties are selected, seed is selected from the selected plants, the offspring produced by rejecting them are re-selected and these steps are repeated 3-5 times.

Accordingly, it is an object of the present invention to provide a novel tetraploid chamomile species having improved properties, in particular higher (-) -? -Bisabolol content.

Initial diploid chamomile can be any chamomile species that has a significant measurable concentration of (-) -? -Bisabolol in its essential oil (generally more than 5% of the essential oil). For example, diploid chamomiles such as those described in Schilcher, H. "Neuere Erkenntnisse bei der Qualintatzbeurteilung von Kamillenöl beziehungsweise Kamillenblüten", Planta Med., 23, 132-144 (1973); Motl,,., M. Felklová, V. Lukes & M. Jasicová, "Liquid gaschromatograph analysis and chemistry typen von Kamillenöl", Arc. Pharm. 310: 210-215 (1977); Franz, Ch "J. Hölzl & A. Vöméi," Preliminary Morphological and Chemical Characterization of Somatic Populations and Varieties of Matricaria chamomilla L. ", Acta Hort. 73, 109-114 (1978).

For some diploid plants, the essential oil of chamomile is usually composed of the main components of wood mesh, spatuleol, kamazulene, one of the four bisaboloids, (-) - α-bisabolol, bisabololoxide A, bisabololoxide B, or bisabolonoxide. There may be several bisaboloids side by side in the mixed population sample and in tetraploid individuals. The ethereal oil may also contain spiroethers. Particularly suitable are diploid parent chamomiles in which (-) -? -Bisabolol is the predominant component, i.e., more than half of the substances listed above. The above materials together make up 70-80% of the essential oil. The starting chamomile is mainly diploid chamomile in which, for example, (-) -? - bisabolol is at least 40% of the essential oil or at least 90% of the bisaboloid.

For example, the initial tetraploid chamomile is Bodegold (German Democratic Republic), Pohorelicky (Czechoslovakia), ZLOTY Lan (Poland), BK-2 (Hungary). These species are also known in the art. Otherwise, the (-) -? - bisabolol, in terms of content, of the tetraploid parent chamomile has the same requirements and properties as the diploid parent chamomile.

Specifically, diploid and tertraploid chamomile populations are included which generally contain at least 100 mg% kamazulene and 50-100 mg% (-) -? -Bisabolol (relative to the dry matter of the flowers dried at 40 ° C) in the pharmaceutical composition sample, i.e. diploid starting 3

In the case of chamomile HU 202034 ilyen we obtain seeds from such a chamomile population and tetraploidize these seeds (without consideration and / or knowledge of the bisabolol content of the parent plants). In the case of a tetraploid starting chamomile, the seeds are then subjected directly to the following selection and propagation operations.

The appropriate diploid and tetraploid starting chamomiles are selected by examining each plant. Generally, for example, 10,000 to 10,000 individuals from an initial population should be screened to find some individuals that have a high content of (-) - abisabolol according to the above requirements, which makes them suitable for the role of the initial chamomile. Seeds thus searched are then harvested in the usual manner and tetraploidized. After tetraploidization is complete, tetraploid plants are selected from the remaining plants that remain diploid. From the tetraploid plants thus obtained, optionally, after pre-propagation, all plants having at least 40 mg dried flowers at 40 ° C, at least 100 mg% kamazulene, at least 200 mg% (-) - a-bisabolol and less than 50 mg% additional bisaboloid are selected. They contain. Harvesting of these flowers is carried out during the growing season, when 30-70% of all the flowerheads of the flowerhead are open. Where appropriate, further selection and propagation operations may subsequently be carried out to improve, for example, the following characteristics and characteristics: Simultaneous flowering time, uniform conifer branching, flower height between low altitudes (ie better harvesting capacity), large flower heads, low cost better shelf life, special aromatic scent.

When starting from a tetraploid chamomile species, we only select for plants whose flowers dried at 40 ° C contain at least 100 mg% of kamazulene, at least 200 mg% of (-) - a-bisabolol and less than the usual bisaboloids. They contain 50 mg%. Still, flower harvesting is carried out during the growing season, when 30-70% of the flowerhead tubular flowers are open. Again, further selection and propagation operations may be performed to achieve further improvements as described above.

A particularly advantageous camomile is obtained when the choice of kamazulene and (-) -? -Bisabolol is made on known plants or plants of the invention having a content of at least 200 mg%, preferably 250 mg%, and (-) -? - bisabolol. containing at least 300 mg%, preferably 400 mg%, and the usual bisaboloid content is below 50 mg%.

For example, tetraploidization is carried out in a manner known per se by treating parts of plants (seeds, roots, apexes, germs, axillary glands) or diploid parent chamomile plants with chemicals, X-rays, gamma rays or ultraviolet rays. It may also be accomplished by means of an end-call method, an anther culture, or high or even low temperatures that affect chamomile plants or parts or tissues thereof. In this connection, reference is made to Wemr Gottschalk's book "Die Bedeutung Polyploidie für die Evolution Pflazen" (Gustav Fischer Verlag, Stuttgart, 1976, mainly pages 13-22).

Chemical agents for tetraploidization with chemicals include colchicine, acenaphthene, alkaloids such as atropine, veratrin, nicotine, sangvinarine, benzene derivatives, diphenyls and phenanthrenes, naphthalene derivatives, diphenylamine, tribromoaniline, para-diclorone, , methylnaphthohydroquinone, salicylic acid and related substances, hexachlorhexane, methamphetamine, (hydrochloride), alkyl alkali carbamates such as isopropyl sodium carbamate, phenylurethane, salts of cacodylic acids (e.g., sodium salt), convallaria glyoxosin, , heteroauxin, germizan (phenylmercuropyrocatechin), organic mercury compounds such as ethylmercury phosphate, ethylmercury chloride, phenylmercuric hydroxide, phenylmercinodinaphthylmethane disulfonate, chloroform gas (N ₂ O) and mixtures of these substances . In addition, oil cake, compost and cow manure may be considered or applied.

The treatment with the listed materials can be carried out, for example, at a temperature between 0 and 35 'C, preferably at a temperature between 12' C and 30 ', in particular between 15' C and 25 'C.

The application can be carried out, for example, by treatment of seeds, ventricle tips, roots (especially roots of root tips or sprouts), fruit clumps, leaves or dry cut surfaces, cell suspensions of meristem tissue, callus cultures or injection of material into basal stamens, aqueous solution, in the form of a slightly alcoholic solution (alcohol content substantially below 5%) or in a slightly acidic solution. For example, the pH of weakly acidic solutions may range from 5.5 to 6.5, in which case the acidification is carried out with lower organic aliphatic acids such as acetic acid. If alcoholic solutions are used, they may also be slightly acidic. The concentration of the chemical in these solutions may be, for example, 0.01-0.5%, preferably 0.02-0.2%, especially 0.05-0.1%. Gaseous materials may also be used, for example at a pressure of 1 to 10 bar. The treatment duration may be, for example, 1-36 hours, preferably 212 hours, especially 4-6 hours.

Preliminary experiments should always be used to determine the most effective concentrations and times.

Treatment with colchicine is particularly advantageous, for example, at temperatures between 0 and 35 'C, preferably between 12 and 30' C, in particular between 15 and 25 'C. Preliminary experiments may, for example, be carried out by swelling the seeds of the starting diploid chamomile in a 0.01-0.2%, preferably 0.02-0.1%, mainly (7.05%) colchicine solution or germinated germinated 5-7 days old diploid starting camomile (with germinal leaves down) 0.01-0.2%, preferably 0.02-0.1%, mainly 0.05% For the latter process, the ambient atmosphere should be approximately 100% relative humidity. For example, the duration of the treatment with colchicine is 3 to 36 hours, preferably

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It can take 4-10 hours. In the case of germination, the exposure time may be up to 36 hours in some cases.

After treatment with the chemical agent, the swollen seeds, sprouts and other parts of the plant are rinsed several times with water. For example, the swollen seeds are drained. Treated root plants, other parts of plants, or treated germs, for example, are planted in planting boxes. For example, the plants formed from the seeds or germs so treated are kept in a greenhouse at 18-25 ° C during the day and at 10-16 ° C at night, and at the end of the growing season the plants with a pollen content of about 1.5 times higher are selected. in the case of pollen from the parent plants or having a somatic cell chromosome number of 36. If other parts of the plant (above or below ground) are chemically treated, only shoots, roots, flowers or seeds from treated parts shall be subjected to subsequent treatment. for tetraploidization. For example, if we are treating a sprout or a deciduous leaf, then only the new sprout from that sprout or deciduous leaf and the flowers or seeds formed therein are examined for chromosome number.

Pollen size measurements and chromosome counts may be performed, for example, as described in Example 3. For tetraploidization with rays, for example, the seeds or root tips are treated at a temperature between 0 and 35 'C, preferably between 10 and 30' C, in particular between 15 and 25 'C. The irradiation is 5-50 Krad. Preferably, gamma or X-rays are used.

For example, ultraviolet rays having a wavelength of 400-30 nm, in particular 350 nm, are preferred.

The plants or parts of plants irradiated in this way are treated in the same way as after treatment with chemicals.

When high temperatures are used, for example, temperatures of 33-50 ° C, preferably 42-45 ° C, may be used. These temperatures include, for example, swollen seeds, germs, shoots and tissue capable of proliferation. The duration of the treatment may be, for example, 1 to 48 hours, preferably 12 to 24 hours.

Low temperatures can be from 0 to 5 'C, preferably from 0.5 to 4' C, especially 2 'C. Exposure to these temperatures includes, for example, swollen seeds, germs, shoots, and tissue capable of proliferation. The duration of treatment is, for example, 1 to 100 days, preferably 20 to 40 days. The plants or parts of plants so treated are treated in the same way as after treatment with chemicals.

The decoupling callus procedure is, for example, on young plants on the stalk, preferably on the apex of a growing cone, shown in Figs. after the formation of leaves, or on the stems or side shoots. Buds and shoots from tissue (callus) tissue are harvested, rooted, grown in pots, and tetraploidized plants are selected in a manner similar to chemical treatments.

Anther cultivation (creation of plants from half anthers with a simple set of chromosomes and subsequent or subsequent spontaneous or artificial tetraploidization).

From flowering plants we harvest closed tubular flowers with anthers before the first pollen indirect nucleation state. The buds are removed from the anthers using a micromanipulator and placed in petri dishes, which are filled with, for example, Nitch and Nitch (Table 1). The petri dishes are then kept in a growing space at 16 ° C during the day and at 20 ° C overnight. After about four weeks, the anthers begin to crack and the plants grow blue. These are haploids for a diploid parent and, if one parent is a tetraploid, they are dihaploids. After the roots have formed, the new plants are placed in a garden pot, for example, and grown in a greenhouse until flowering. These (di) haploid plants are sterile but can be polyploidized by chemical treatments such as colchicine by apex, root or stem treatment, which results in homozygous plants which can then be further propagated by seeds. Further treatment is the same as chemical treatment, such as colchicine.

Table 1

Nitchh and Nitchh Nutrient (Scinece, 1969)

mg / 1 KNO3 950 NH4NO3 720 MgSO4x7H2O 185 CaCl2 166 KH2PO4 68 MnSO4x4H ₂ O 25 H3BO3 10 ZnSO4x7H ₂ O 10 Well ₂ MoO4x2H ₂ O 0.25 CuSO4x5H ₂ O 0,025 7.45 g per 1000 ml ethylenediaminetetraacetic tetraecet- acid disodium salt and 5.57 g FeSO4x7H ₂ O solution of 5 ml myo-inositol 100 glycine 2 nicotinic acid 5 pyridoxine-HCl 0.5 Thiamine HCl 0.5 acid 0.5 biotin 0.05 saccharose 20g ready medium (DIFCO-BACTO-Agar) 8 g indole acetic acid 0.1 The pH of the substance is 5.5 is set. Pollen size measurement and / or chromosomes Chamomile tetraploid selected by counting prescriptions - which are described above- can be obtained from opportunities - we select them a plants containing at least 100 mg% and bisabolol content not less than 200 mg%, however, the rest bisaboloids (mainly bisabolol oxide) <50 mg% (a with reference to dried flowers, Example 1). 5

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After removing all flowered flower heads, the plants so selected are allowed to flower at 18-24 ° C at day temperature and 12-14 ° C at night, and for at least 14 hours during the day, with all flowers blooming or shortly before disintegration for 4 weeks. the flower head is harvested to obtain seed. The plants are kept in a greenhouse for four weeks. After drying at a temperature of about 20-35 ° C, for example, chamomile propagation material of the present invention is obtained.

Additional criteria for this selection include:

a) Flowering at about the same time,

b) a smooth coniferous branch and a flower placement band of about 10 cm, preferably 5 cm,

c) large flower heads having an outer diameter of about 30 mm (20 to 40 mm), preferably 25 to 35 mm.

The use of the additional criteria a), b) and / or c), for example, results in greater flower and medicinal ingestion and makes chamomile more suitable for mechanical harvesting.

Starting from the already known tetraploid chamomile, the selection as described above, as well as, if necessary, further selection and propagation, are carried out in a similar manner.

If you want to produce chamomile that has a high flower yield in addition to at least 100 mg% kamazulene and at least 200 mg% (-) - α-bisabolol per dried flower head and is more suitable for machine harvesting, it is advisable to proceed as follows: The selected plants (as described above) are vegetatively propagated by cuttings and selected for 3-5 generations, which are always selected according to the criteria set forth above and, where appropriate, the additional criteria a) to c). Plants searched for by these criteria are cloned, seeded from the cloned plants, the plants obtained therefrom are again selected for the minimum content of kamazulene and bisabolol and, if appropriate, the additional criteria a) to c), and the latter are again seeded. Selection for sowing, for a content of at least 100 mg% of kamazulene and at least 200 mg% of (-) - a-bisabolol (for other bisaboloids below 50 mg) and, if appropriate, for the additional criteria a) - c) and for seed production. its series can be repeated 3-5 times.

This is followed by a series of sowing, selection (possibly cloning) and seed extraction as described above. The seed finally obtained then forms the chamomile propagation material of the present invention.

The cuttings are done as follows: For the cuttings, the parent plants (cloned mother plants) must be trained as short shoots without buds under short daylight conditions. This is carried out in the winter semester in a greenhouse, without additional lighting or in climate chambers for 6 to 10 hours, preferably 8 hours, and at temperatures of 10 to 15 ° C, preferably 12 ° C. Leaf, stem and especially short-stemmed cuttings are suitable for cloning or propagation. They are rooted at a temperature of 12-18 ° C, preferably 15 ° C and at 12-16 hours, preferably 14 hours, in an atmosphere having a relative humidity of about 100%. As soil, for example, a mixture of peat and sand in a ratio of 1: 1 can be used, but pure quartz sand, rock wool cube, peat cube and the like can also be used.

For example, the following soil types may be used: Ground soils, humus, moderately bound clay soils, clayey or humus sandy soils. Seeding can be done in a greenhouse, but it can also be done outdoors. The temperature required for germination and growth of the plants is between 12 and 14 'C, preferably between 18 and 20' C. If sowing is done outdoors, it is preferable to do it in autumn (September, October) or spring (March, April). These dates apply to all areas suitable for chamomile production (including the northern hemisphere, from temperate to subtropical landscapes).

The new chamomile of the present invention belongs to the group of cultivated true chamomile plants. The botanical designation is defined by the active ingredient data of Matricari chamomilla L. (synonym: Chamomilla recutita (L.), Rauschert) and claim 1. The values given for the active substances kamazulene, (-) - a-bisabolol and bisabolol oxide refer to the flower development state that the plant reaches when 30-70%, especially 40-60%, of all the flowers of a flower head opened. The flowers used to determine the active ingredients are harvested at this time and then dried at 40 ° C for 72 hours in an oven.

If the chamomile flowers are harvested at a time when the flower opening is in an advanced state, that is, almost 100% of the flowerhead of a flowerhead is open (for example, the flowerhead is fully open) and / or when the harvested flowers are drying 40 At temperatures higher than 'C, the active substance content of kamazulene and (-) -? -Bisabolol may be lower, since higher drying temperatures and / or higher yields of azulene and bisabolol may occur at an ideal harvest.

In the context of claim 1, the term "other bisaboloids" is understood to mean in particular (-) -? - bisabololoxide A and B and (-) -? - bisabololoxide A. Other essential oils of the chamomile variety of the present invention include end-dicycloether, wood mesh, spatulenol and, in lower concentrations, various readily volatile terpene hydrocarbons. The flowers of the camomile of the present invention harvested as described above at 40 ° C contain, for example, 100-200 mg% kamazulene, 200-450 mg% (-) - a-bisabolol and only a small amount of 5-50 mg% other bisaboloids. The data refer to the absolute dry weight of the flowers. This absolute dry mass is determined by drying a separate sample of chamomile flowers of the present invention, which is dried in an oven at 105'C until the plant is stable (72-96 hours). The active ingredient content of flowers (herbs), for example, dried at 35-50 ° C, is then converted to the flower weight dried at 105 ° C.

The tetraploid chamomile species known to date is 61 similar to the camomile phenotype of the present invention

However, it differs from these mainly because (-) - abisabolol is the major component of the essential oil of flowers in the camomile of the invention and that the other bisaboloids (bisabololoxide A and B, bisabolon oxide) is significantly lower. Other essential oils of chamomile are wood mesh, spatulenol and end-dicycloether.

Chamomile according to the invention can be successfully grown on all soils, except for soils having more than 20% organic matter (humic matter and soil organisms). No special egrotechnical procedures or cultivation rules are required. All that is needed is a maximum day length of more than 13 hours, which is mainly suitable for temperate and subtropical areas.

The chamomile of the present invention often has the following advantageous properties: High yield, medium late harvest time, and practically uniform height, placement of flower heads within low altitudes and large flower heads, and, consequently, a special ability for mechanical harvesting. In addition, the flowers of concurrently sown plants generally bloom at the same time, so that harvesting becomes simpler and easier as a result.

The latter advantages are obtained by using the following methods: From chamomile plants which in tetraploid chamomile plants contain at least 100 mg% of kamazulene and 200 mg% of (-) -? -Bisabolol per dried flower head and , the remaining bisaboloids (mainly bisaboloxides) are below 50 mg%, only those that are selected

- bloom approximately simultaneously,

- they have a uniform coniferous branch and the flower heads are narrow in width, about 10 cm wide, and

- their flower heads are large and have an external diameter of about 30 mm.

The selected plants are propagated vegetatively through cuttings and selected for 3-5 generations, which are always selected according to the above mentioned criteria. The selected plants are vegetatively propagated (cloned), left to blossom together and seeded. The plants obtained from the seeds are again sorted according to the above criteria and the procedure of seeding, selection, seeding and seeding is repeated 3-5 times.

The maximum content of the active substance kamazulene and (-) -? -Bisabolol in a medicinal product of the chamomile according to the invention is obtained when the chamomile flower heads are harvested in a culture state where, for example, 30-70%, preferably 4060% , in particular 50% is already open and drying is carried out at a maximum air temperature of 50 ° C, for example between 30 ° C and 50 ° C, especially 40 ° C.

Drying can be done either by artificial airflow, drying in the shade, or in the sun, as appropriate, but care must be taken to ensure that the amount of heat applied to the flowers is not greater than is necessary to achieve perfect drying. It is also advantageous to verify by control measurements the mass constant achieved. The drying may be spontaneous or artificial (for example, with artificially generated warm air). The greatest yield of active ingredient is achieved by spontaneous drying, with the exclusion of sunlight, preferably at temperatures of 40-60 ° C, especially 40-50 ° C. Drying should be carried out as soon as possible after harvest or as soon as possible. During drying the flower heads should be in thin layers, for example 5-20 cm, preferably 10 cm. Drying can also be carried out, for example, in well-ventilated halls at 20-30 ° C air temperatures. The drying air temperature should generally not be higher than 60 ° C. For example, an ambient temperature of 35-50 ° C is preferred.

The amount of one of the main active ingredients of camomile, (-) -? - bisabolol, obtained from chamomile according to the invention, depends on the vegetation state in which the flowers are at harvest and on the drying of the harvested flowers. The higher the drying temperature, the stronger the decomposition of the active ingredients, i.e. the lower the active ingredient content of the dried flowers. For the same reason, direct sunlight adversely affects the drying result and should be avoided wherever possible. The flowering state of a flower is characterized by the percentage of tuber flowers that are open at a particular time (here at the time of harvest). On this basis, flowers can be harvested where, for example, 30-50%, 3070%, 40-60%, 60-100% or 90-100% of the flowers of a flower head are open. The active ingredient content depends on the stage at which the flowers are growing, and the active ingredient content of the chamomile of the invention is highest when 4060% of the total tubular flowers are open. With less or greater amounts of open tubing, the active ingredient content will be lower. The great advantage of the camomile according to the invention is therefore that the flowers of each plant are uniformly flowered, which means that the majority of the plants from each shoot are in the same flowering state, i.e., in most plants 40-60% of the it opens in time. Therefore, the chamomile according to the invention can be harvested at optimum time in flower heads and consequently the chamomile according to the invention is particularly suitable for mechanical harvesting.

The following diet is used as an example of the relationship between the content of kamazulene and (-) -? - bisabolol at the time of harvest and the temperature at which the drug is taken (the remaining residual bisaboloids are in each case less than 50 mg%).

It follows that the chamomile used to produce the chamomile of the present invention, for example, is harvested in a vegetative state in which 30-100% of the flower buds are open and dried at an air temperature below 70 ° C. , a dry substance may be obtained which in each case has a content of at least 30 mg% in chamazulene and at least 100 mg% in (-) α-bisabolol, while the remaining remaining bisaboloids are always less than 50 mg%.

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drying temperature (air temperature) The vegetation state of the flowers at the time of harvest 30-70% of the flowers of a flower head are open 70-100% of the flowers of a flower head are open Active ingredient content in the dried material: Active ingredient content in the dried material: At 50 'C chamazulene: chamazulene: not higher not less than 100 mg% (-) - a-bisabolol: not less than 200 mg% Active ingredient in alcoholic extract: at least 40 mg% (-) - a-Bisabolol: Not less than 120 mg% Active ingredient in alcoholic extract: At 50 'C chamazulene: chamazulene: not higher not less than 5.0 mg% (-) - a-bisabolol: not less than 10 mg% Active ingredient in the dried material: at least 2.0 mg% (-) - a-Bisabolol: Not less than 6,0 mg% Active ingredient in the dried material: 50-70 ° C chamazulene: chamazulene: between Not less than 50 mg% (-) - α-bisabolol: Not less than 150 mg% Active ingredient in alcoholic extract: at least 30 mg% (-) - α-Bisabolol: not less than 100 mg% Active ingredient in alcoholic extract: 50-70 ° C chamazulene: chamazulene: between at least 2.5 mg% (-) - a-bisabolol: at least 7.5 mg% at least 1.5 mg% (-) - a-bisabolol: not less than 10,0 mg%

The remaining bisaboloids in the alcoholic extracts are always less than 10 mg%.

The essential oil prepared under the conditions given in the table above always contains at least 3.5% kamazulene, at least 10% (-) -? - bisabolol and less than 10% residual bisaboloid.

Chamazulene is determined spectrophotometrically in the same way as for chamomile extracts.

The determination of (-) -? - bisabolol and other substances in chamomile oil is carried out by the usual gas chromatography method. See Appendix A for a detailed description of the analytical determination methods.

In camomile flowers, kamazulene is not present as such, but as sesquiterpene lactone, i.e. in the form of matrix. Matrixin has a pharmacological action similar to that of kamazulene. From this matrix precursor, for example, heating (e.g., steam distillation, tea pouring) instantly produces kamazulene. Therefore, it has become customary for chamomile not to provide the matrix content, but the resulting kamazulene content.

The uses of chamomile according to the invention are, for example, for the production of chamomile, chamomile oil, chamomile extract and (-) -? - bisabolol, chamazulene and other substances in chamomile. Thus, chamomile extracts or chamomile extracts can be obtained from the medicinal product of the chamomile according to the invention, for example, by extraction with alcohols or alcohol mixtures or by extraction with hypercritical gases. In addition, chamomile oil, (-) - a-bisabolol, chamazulene and other substances in chamomile may be recovered from the recycled material.

For example, the alcoholic extracts of the pharmaceutical composition contain at least 35 1.5 mg%, preferably 5.0 mg% of kamazulene and at least 5.0 mg%, preferably at least 10 mg% of (-) - a-bisabolol, the remaining or other bisaboloids. less than 8 mg%. Such extracts are prepared in the usual manner.

For example, mixing devices such as trough mixers, percolators and other suitable extraction devices can be used for extraction. During extraction, the temperature may be 1050 ° C. No cooling required.

Suitable solvents are, in particular, straight or branched aliphatic aliphatic C 1-6 alcohols, such as methanol, ethanol,

2-propanol, butanol, glycerol, isopropylidene glycerol and the like, and mixtures of these solvents with water.

Mixtures of these solvents may also be used, the smallest amount of solvents being selected so that 2 parts of solvent are delivered to 1 part of dried material. Generally, 2 to 20 parts of solvent are added to 1 part of dried material, preferably 3 to 10 parts of solvent are added to 1 part of dried material. Fresh chamomile flowers and frozen chamomile flowers can also be used to make extracts, especially alcoholic extracts (frozen flowers are frozen in their fresh state).

An ethereal oil obtained from the pharmaceutical product contains at least 3.5% kamazulene, preferably at least 5% kamazulene and 65 at least 10% (-) -? -Bisabolol, preferably at least

HU 202034 Β

It contains 15% (-) - a-bisabolol and contains less than 10% residual bisaboloid.

Such an ethereal oil is generally prepared by heating the medicament to reflux with water, for example in the presence of ascorbic acid, preferably salt, in particular in the form of its sodium salt. The pH of the ascorbic acid mixture is between 4 and 6, preferably between 5 and 5.5. The pH is adjusted, for example, with an acid such as 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 added to 1 part by weight of the dried material. Heating and heating is usually carried out for 2-8 hours.

The resulting distillation product is shaken several times with a low-carbon aliphatic liquid hydrocarbon (e.g., 35-60 ° C in petroleum ether, pentane, xylene, deca), the organic phase is dried (e.g. with sodium sulfate) and the organic solvent is gently removed (e.g. by rotary evaporation or by distillation at 4070 ° C, preferably 50-60 ° C). For higher boiling solvents, this distillation can be carried out under vacuum.

The material content is determined as described above.

If the chamomile flowers of the present invention are harvested during a growing season in which 30-100% of the flower heads of a flower head are opened and the flowers are dried at an air temperature below 70 ° C, for example, at least 30 mg% of contains at least 100 mg% (-) - a-bisabolol and less than 50 mg% residual bisaboloid, and in the essential oil produced by the distillation of this pharmaceutical product in the presence of water at least 3.5% kamazulene, at least 10% (-) - a bisabolol and less than 10 mg% of the remaining bisaboloid. The alcoholic chamomile extract obtained by extraction with lower alcohols contains at least 1.5 mg% of kamazulene, at least 5.0 mg% of (-) -? - bisabolol and less than 10 mg% of residual bisaboloid.

If the chamomile flowers of the present invention are harvested in a vegetative state in which 30-70% of the flower heads of a flower head are opened and the flowers are dried at a temperature not higher than 50 ° C, such a medicinal product will for example contain at least 100 mg. containing at least 200 mg% (-) - a-bisabolol and less than 50 mg% residual bisaboloid, and optionally in an essential oil thereof, at least 3.5% kamazulene, at least 10% (-) - a-bisabolol and less than 10% residual bisaboloid. The alcoholic extract of this pharmaceutical composition contains at least 5.0 mg% of kamazulene, at least 10.0 mg% of (-) -? - bisabolol and less than 10.0 mg% of residual bisaboloid.

If the chamomile of the present invention is harvested in a vegetative state in which 30-70% of the flowers of a flower head are open and the flowers are dried at 50-70 ° C, the resulting medicinal product will, for example, contain at least 50 mg% of kamazulene, Contains at least 150 mg% (-) - a-bisabolol and less than 50 mg% of residual bisaboloid, and the resulting medicament contains at least 50 mg% of kamazulene, at least 150 mg% of (-) - a-bisabolol and 50 mg% there is less residual bisaboloid. Optionally, an essential oil prepared therefrom contains at least 3% by weight of kamazulene, at least 10% by weight of (-) abisabolol and less than 10% of residual bisaboloid. An alcoholic extract prepared from such a pharmaceutical composition contains at least 2 µmg% of camulene, at least Ifi mg% (-) - α-bisabol and less than 10.0 mg% of the remaining bisaboloid.

If the flowers are harvested in a vegatation state in which 70100% of the flowers of a flower head are opened and the flowers are dried at an air temperature of not more than 50 ° C, the resulting medicament is for example at least 40 mg% kamazulene, at least 120 mg% ) -is-bisabolol and less than 50 mg% of residual bisaboloid, and optionally an essential oil thereof contains at least 3% by weight of chamazulene, at least 10% of (-) -? -bisabolol and less than 10% of residual bisaboloid. An alcoholic extract of this pharmaceutical composition contains at least 2.0 mg% of kamazulene, at least 6.0 mg% of (-) -? - bisabolol and less than 10.0 mg% of residual bisaboloid.

If the chamomile flowers of the present invention are harvested in a vegetative state in which 70-100% of the flower heads of a flower head are opened and the flowers are dried at 50-70 ° C, the resulting product will for example contain at least 30 mg% of kamazulene. , contains at least 100 mg% (-) - a-bisabolol and less than 50 mg% residual bisaboloid, and optionally an essential oil thereof contains at least 3.5% kamazulene, at least 10% (-) - a-bisabolol and Contains less than% bisaboloid remaining. The alcoholic extract of this pharmaceutical composition contains at least 1.5 mg% of kamazulene, at least 10 mg% of (-) -? - bisabolol and less than 10.0 mg% of residual bisaboloid.

/. Example 1 (The initial chamomile tetraploid)

Described by Dragon I. [Herba Hungar. 4 (1), 125-169, 1965.] A study of 10,000 plant species of tetraploid chamomile species reveals that there is a plant with a high content of 20 wt. with a (-) -? - bisabolol content by weight, while the amount of residual bisaboloids (mainly (-) -? - bisabolone oxide) is very low (less than 5% by weight). These individuals are selected and subjected to the following operations:

Step 1:

From the offspring of tetraploid chamomile plants selected as described above, individuals which

A) bloom at about the same time,

B) have a smooth coniferous branch and the flowers have a horizontal horizontal band of about 10 cm, preferably 5 cm,

C) the flower heads are large and have an external diameter of about 30 mm, preferably 25 to 35 mm,

D) a minimum content of kamazulene of 150 mg% or more, a minimum content of bisabolol of 300 mg or more, and a residual amount of bisaboloids (mainly bisabololoxide) of significantly less than 50 mg%. (Vala9

HU 202034 Β is the value for flower heads that were dried at 40 ° C and harvested when 30-70% of all flower buds in a flower head were open.)

These plants are cloned. For starvation, the plants (cloned mother plants) are first cut back to a shoot length of about 15 cm, and new shoots (short side shoots) are allowed to grow at a temperature of 8 to 10 hours at 12-14 ° C. At about 100% relative humidity, 15 ° C, and 14 hours of day, the cut shoots are kept in a mixture of peat and sand and these cuttings are rooted for 7-14 days.

Instead of the usual cloning (cuttings propagation), the dividing tissue sections of the plant can be exposed to invitro propagation (so-called merismem propagation). Various parts of the plant are suitable for the establishment of a chamomile culture, in particular the shoot tips or the underarms.

After rinsing the plants under aseptic conditions with a H2O2 laminar flow (through a high performance float filter), the tips of the armpit buds are cut off and transferred to reagent bottles, such as Murashige and Ploog. , 1962). The reagent bottles are placed in a climatic chamber having a set day length of 12-18 hours, preferably 16 hours (with fluorescent fluorescent tubes), a luminous intensity of 500-10,000 lux, preferably 1000-3000 lux, and a temperature of 15-30 ° C, preferably 22-27 ° C. 'C.

When the transplanted plants show good growth, they are transferred to the medium described above, but this medium has a higher concentration of cytokinin (a phytohormone that aids in the decomposition) (e.g. 30 mg / l6-isopentenyladenine) and little or no auxin (0-0.3 mg / L indole acetic acid). Next, the axes elongate and produce additional organs, enlarged armpit buds. They can be harvested and cultivated in this manner.

A larger number of tissue cultures for plant propagation (3rd generation) are transferred to a medium containing 10 mg / L indole acetic acid or 3-indole butyric acid or 0.1-0.3 mg / L naphthyl after the leaves have grown and formed. - Contains acetic acid. Here, the plant blue roots and after about four weeks are transferred to sterilized (12 hours at 120 ° C) stuffed garden soil and grown in a greenhouse (under the usual conditions for propagation of cuttings).

Murashige and Skoog media contain the following substances:

mg / 1 mg / 1 NH4NO3 400 Indole acetic acid 2.0 Ca (NO3) 2.4H2O 144 Furfuryl Denmark 0.1 KNO3 80 thiamine 0.1 KH2PO4 12.5 nicotinic acid 0.5 MgSO4.7H2O 72 pyridoxine 0.5 KC1 65 glycine 2.0 Sethoxydim EDTA 25 Myo-inositol 100 H3BO3 1.6 Casein hydrolyzate 1000

mg / 1 mg / 1 MnSO ₄ .4H ₂ O 6.5 saccharose 2% ZnSO4.7H2O 2.7 cleaned WHO 0.75 agar powder 1%

Step 2:

The plants obtained in step 1 bloom together in a greenhouse under isolated conditions. Here the plants stand in 11 cm pots. The pots are filled with garden soil and have an ambient temperature of 18-24 'C during the day and 12-14' C at night. Daylight hours are at least 14 hours and are achieved with additional lighting (200 watts / m ² ) in the winter semester. Water is supplied to the plants as needed.

From the total number of selected individuals, flower heads that have already bloomed and shortly before disintegration are harvested continuously for 4 weeks to obtain seed. The drying is carried out in a well-ventilated hall at a temperature of 20-30 ° C, then the seeds are sifted through a sieve (5x0.4 mm) and subsequently cleaned on a vertical separator.

Step 3:

The seeds from Step 2 are randomly sampled and about 2000 offspring are grown (the ecological conditions are the same as in Step 2) and selected according to Step 1 A) to D). The individuals so selected are then subjected to Step 2.

Step 4:

One part of the seed obtained in step 3 was sown in two different places in the autumn.

I. area information:

450 η NN, 48,5 'N / 11,5' 0, annual rainfall 750 mm, humid temperate climate,

January -10-0 'C,

July + 10— + 20 'C.

The meanings of the symbols used above are as follows:

NN = northern standard zero - sea level,

N '= latitude,

0 '= longitude.

Π. site details:

200 m NN, 42 'N / l' 0, annual rainfall 400 mm, Mediterranean climate,

January 0- + 10 'C,

July + 20- + 30 'C.

Seeding was carried out at both sites in late September and early October.

The resulting test population is examined and certified for homogeneous growth, flower size, and harvest time, and tested for active substance content in a sample of flowers. Individuals were selected from the herd that met the parameters shown in step 1. Seeds are obtained from these in a manner consistent with the last part of step 2.

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Step 5:

With the seeds obtained in step 4, the processes described in steps 3 and 4 are repeated in that order.

Step 6:

Approximately 1500 specimens from the seeds obtained in step 5 (the ecological conditions are identical to those of step 2) and selected using principles similar to those used in step 1. From the plants so selected, 34 individuals were selected and cloned according to Step 1. Each of the 10 to 10 plants from cloning was planted at random (40x30 cm) in open field (Habitat I, step 4) in isolated or isolated locations. The soil was a loose material at pH 7.0. Seeding is carried out in early June, the first harvest of the seeds is carried out in mid-July, after which the plants are cut back, allowed to bloom again and the seeds are harvested for the second time between mid-August and the end of August.

The seed extraction of this material was carried out as described in step 2.

The seed obtained after step 6 forms the chamomile propagation material of the invention. The flowers of this propagation material, sown in September-October and harvested in early June of the following year - when harvested when 30-70% of the tuberous flowers are open and dried immediately after harvest in a drying oven at 40 ° C for 72 hours - containing, for example, 150 mg% kamazulene, 300 mg% (-) -? -bisabolol and up to 50 mg% bisabolol based on the weight (dry weight) of the dried flowers.

Further examples of chamomile plants obtained by the process of the invention obtained by example are as follows:

1. Growth

Stem: Straight, slightly branched;

2. Foliage

Leaf: winged, 2-3 times;

Density: medium;

Winged leaf color: medium green;

Winged leaf subdivision (middle of stalk): medium to strongly divided;

3. Inflorescence

Flower head size: outer diameter about 30 mm, inner diameter about 15 mm;

Weight of a flower head in the dry state: about 45 mg;

Flower length: about 700 mm, however, it also depends on the climate of the place of production, sowing time, soil, fertilization, crop management and weather;

Flowering begins: from January 1, approximately 160 days (sowing in September, place of production in the Federal Republic of Freising, otherwise dependent on the factors listed above);

Virulence: mid-June (see above);

Essential oil content: about 1.0%, measured as a percentage of the stalk material of the stalkless inflorescence, and at least 15% of the azulene content of the essential oil;

Dried inflorescences, slight if harvesting is the last to disintegrate inflorescence ware tubular flowers: perform;

Pollen diameter: about 30 μτη;

Core length · about 1.25 mm;

Somatic cell chromosome number: 4 n-36;

4. Crop

Thousands of seeds: 0.06-0.13 g;

5. Germination capacity

The amount of germinating seeds: about 75%;

6. Purity

Purity of seed mass: 94-95%;

7. Other characteristics The drying ratio of fresh and dry flowers is 5.5-6: 1, the aroma has a characteristic aromatic smell, the aroma of the tea infusion is typical, aromatic.

In addition, the chamomile produced by the process of the present invention must have the following properties: Uniform growth height (uniformity) with narrow, low-layer inflorescence distribution, particularly suitable for machine harvesting, large flower heads, medium yield, coniferous branch (3-5) branching).

To win the item, proceed as follows:

The camomile obtained in the above example is sown in a conventional manner using a cultivation method. Once the plants have developed and the flower heads are in a vegetative state in which about 50% of the tuber flowers are open, we harvest these flower heads with a camomile tine machine whose tooth spacing is set so that only the flower heads cut off which flower heads have 50% of the tubular flowers open. The harvested crop is transported to a shaded place as quickly as possible and spread in a thin layer until further processing. Subsequently, in a sieve structure, the flower heads are freed from their stems and the flower heads are dried to a constant weight. The weight loss is about 80%. Drying is done in shade, in a well-ventilated area, in layers of about 10 cm thick on a drying grid.

After about 2-3 days, the drying process is completed. To remove the stems and flower debris, the chamomile is screened and then pressed into bales.

Analysis

Essential oil: 960 mg%

Camazulene: 162 mg% (-) -? -Bisabolol 330 mg%.

For example, when drying is carried out in a stationary belt dryer artificially heated to a temperature between 50 ° C and 70 ° C, drying is completed in about 4-5 hours. The sari

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HU 202034 Β to remove parts and flower debris, sift the cure and then squeeze it into bales.

For example, analysis of such a medicinal product gives the following values:

Essential oil: 870 mg%

Camazulene: 94 mg% (-) - α-bisabolol: 197 mg%.

Example 2 (The initial chamomile tetraploid)

As described by Dragon I. [Herba Hungar. 4 (1), 125-169, 1965.] A study of 10,000 plant species of tetraploid chamomile species reveals that there is a plant with a high content of 20 wt. It has a content of (-) - a-bisabolol by weight, but at the same time the amount of residual bisaboloids (mainly (-) - a-bisabolol oxide) is very small (less than 5% by weight). These individuals are selected and subjected to the following operations:

Step 1:

From the offspring of tetraploid chamomile plants selected as described above, individuals with a minimum content of 100 mg% or greater of kamazulene, a minimum content of 200 mg% or more of bisabolol and residual bisaboloids (mainly bisabololoxide) are selected. and are significantly below 50 mg%. All values refer to flower heads dried at about 40 ° C and harvested when 30-70% of all the tubers of a flower head were open.

Step 2:

The plants obtained after step 1 were removed from all flowered flower heads, and the plants were placed in a greenhouse and allowed to flower together under isolated conditions. The plants here are housed in 11 cm pots filled with garden soil. The plants are thus maintained at 18-24 ° C during the day and 12-14 ° C at night. The day length was at least 14 hours, which is achieved during the winter semester with additional lighting (200 watts / m ² ). Water supply was provided as needed.

From the total number of selected specimens, flower heads for flowering which have already bloomed and shortly before disintegration are harvested continuously for 4 weeks. Drying was carried out in a well-ventilated hall at 2030 ° C, and the seeds were sieved through a sieve (5 x 0.4 mm) and post-cleaned with a vertical cleaner.

The seed obtained after step 2 is the chamomile propagation seed produced by the process of the invention.

Flowers of this propagation material harvested from September to October, harvested at the beginning of June of the following year, harvested at a time when 30-70% of the tuberous flowers have been opened and immediately after harvesting in the oven at 40 'C Dried for 72 hours, containing at least 100 mg% kamazulene, at least 200 mg% (-) -? -Bisabolol and not more than 50 mg% bisaboloid, based on the dry weight of the flowers.

To win the item:

The chamomile obtained in this example is discarded in the usual manner. When the plants have developed and the flower heads are in a vegetative state in which about 50% of the tuber flowers are open, these flower heads are harvested using a camomile harvester. The spacing between the teeth of the camomile picking machine is set to pick only flower heads whose tubular flowers are 50% open. The harvested fruit is transported to a shaded place as quickly as possible and kept in a thin layer until further processing. The flower heads are then freed from their stems in a sieving structure and dried to constant weight. The weight loss is about 80%. Drying is carried out in the shade in a well-ventilated drying rack on which the chamomile is about 10 cm thick.

After about 2-3 days, drying is complete. To remove stems and flower debris, the chamomile curry is sieved and then pressed into bales.

Analysis

Essential oil: 940 mg%

Camazulene: 132 mg% (-) - α-Bisabolol: 243 mg%

For example, if the drying is carried out in a constant belt dryer artificially heated to 50-70 ° C, the drying is completed after about 4-5 hours. To remove the stems and flower debris, the herbal material is sieved and then tied into bales. Values obtained by analyzing such a commodity include, for example:

Essential oil: 775 mg%

Camazulene: 65 mg% (-) - .beta.-bisabol: 163 mg%.

Example 3 (The initial chamomile diploid)

The diploid type diploid, with little bisabolol but not bisabolol, described by Franz, Ch., J. Hölzl and A. Vöméi (Acta Horticulturae 73, 109-114, 1978), found that all up to 20% of the individuals have about 20% by weight of essential cazazulene and / or about 50% by weight of (-) -? - bisabolol in essential oil while the amount of remaining bisaboloids (mainly (-) -? - bisabolone oxide) is very low. These individuals are selected and tetraploidized as follows:

The seeds of the chamomile plants so selected are placed on filter paper impregnated with 0.05% colchicine solution in aqueous solution and allowed to swell at room temperature for 20 hours at 20 ° C. The seeds are then removed with filter paper, rinsed several times with water, and placed in crates in a greenhouse, discarded. The boxes have a 1: 1 mixture of peat and sand,

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HU 202034 Β the relative humidity between 18 'C and 20' C is about 60% and the day length is set to 14 hours with artificial lighting.

The germinated plants are observed until they bloom, and the result of polyploidization is determined by comparing pollen and seed size and by counting the chromosomes of the plants. (Fi progeny group - first seed progeny of colchicinated plants classified as tetraploid). The tetraploidization can be carried out as follows:

The 5-7 day well-developed camomile germs germinated on water-soaked filter paper are placed in a 0.05% colchicine solution with the germ leaves down for 4-6 hours at room temperature (20 ° C). Thus, the delicate germinal roots are spared. In these cases, the relative humidity of the ambient air must be close to 100% to eliminate drying damage. After treatment, the germs are rinsed several times with water and placed in crates. Further treatment is the same as for seeds.

Pollen size measurements can be performed with a Leitzbinocular microscope equipped with a micrometer eyepiece and micrometer holder.

Chromosome counting occurs at the root nodes. Fresh root tips of 1-2 cm in length from young plants or cuttings obtained in the greenhouse are collected and placed in a 5M solution of hydroxyquinoline for 5 hours and then in In HCl for 15 minutes. For the assay, approximately 1 mm of the root apex was stained with 2% orceinacetic acid and examined by microscopy in oil immersion to determine a 4-fold somatic cell chromosome pool for mitotic cells (4n-36).

Plants in which the pollen diameter is about 50% greater than the pollen diameter of the diploid starting material (about 30 pm instead of about 20 pm) and in which the somatic cell chromosome pool is doubled to 36 (corresponding number for the diploid starting material is 18) , they are tetraploids. We select these. Using the method described, about 0.1% to 0.5% of the seeds or germs are tetraploidized and turn into flowering healthy plants. For example, operations 1 and 2 are completed as in Example 2.

The camomile flowers thus obtained contain, for example, at least 100 mg% kamazulene, 200 mg% (-) - a-bisabolol and up to 50 mg% residual bisaboloid (when harvested at a time when 30-70% of the tuber flowers are open) and drying in an oven at 40 ° C for 72 hours).

We obtain the healing goods as follows:

The camomile obtained in this example is sown in the usual manner. Once the plants have developed and the flower heads are in a state of vegetation in which about 50% of the tuber flowers are open, these flower heads are harvested using a camomile tearing machine. The spacing between the teeth of the camomile tearing machine is adjusted to cut off only flower heads whose tubular flowers are already about 50% open. The crop is transported to a shaded place as quickly as possible and spread in a thin layer until further processing. The flower heads are then freed from their stems in a sieve structure and dried to constant weight. The weight loss is about 80%. Drying is done in the shade in a well ventilated area. The camomile is spread over a drying grid in a layer about 10 cm in the dryer.

After about 2-3 days, drying is complete. To remove stems and flower debris from the beets, sift the chamomile and the medicinal goods and then squeeze them in bales.

Analysis

Essential oil: 910 mg%

Camazulene: 117 mg% (-) -? -Bisabolol: 252 mg%.

Example 4 (The initial chamomile diploid)

The diploid type diploid species with little bisabolol but not bisabolol described by Franz, Ch. At 20%, the essential oil contains about 20% by weight of kamazulene and / or about 50% by weight of high (-) -? -Bisabolol content, while the amount of remaining bisaboloids (mainly (-) -? - bisabolone oxide) is very low. These individuals are selected and, as in Example 3, tetraploidized and the tetraploidized individuals selected.

About 0.5% to 5% of the seeds or germs are tetraploidized to develop flowering healthy plants. 1-6. operations are completed, for example, as in Example 1.

The camomile flowers thus obtained contain, for example, at least 150 mg% kamazulene, 300 mg% (-) -? -Bisabolol and up to 50 mg% residual bisaboloid (if harvested at a time when 30-70% of the tuber flowers are out). open and when oven-dried at 40 ° C for 72 hours). The other properties correspond to those of Example 1 obtained from chamomile.

To win the spa:

The camomile obtained in this example is discarded in the usual manner. When the plants have developed and the flower heads are in a vegetative state in which about 50% of the tuber flowers are open, these flower heads are harvested using a camomile tear machine. The distance between the chamfer's tearing teeth is set so that they only break off flower heads whose tubular flowers are already 50% open. The crop is transported to a shaded place as quickly as possible and spread here in a thin layer until further processing. The flower heads are then freed from their stems in a sieve structure and dried to constant weight. Weight loss is about 80%. The drying is carried out in the shade in a well-ventilated area with a layer thickness of about 10 cm on a drying grid.

After about 2-3 days, the drying process is completed. To remove the stems and the flower crumb, the chamomile mousse is sieved and then pressed into bales.

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Analysis

Essential oil: 1020 mg%

Kamazulen: 173 mg% (-) -? -Bisabolol: 418 mg%.

Example 5

Preparation of extract from chamomile goods.

The chamomile flowers used for the manufacture of the medicinal product are harvested as in Example 1 at a time when 30-70% of the tubular flowers are open and the harvested flower heads are dried at a temperature not higher than 50 ° C.

The thus obtained chamomile product containing 105 mg% of kamazulene and 212 mg% of (-) -? -Bisabolol is extracted with 2100 g of aqueous ethanol (40% by weight ethanol) in a tumbler mixer at 30 rpm for 3 hours. The drug substance is then squeezed and the extract filtered.

The content of the active ingredient in the extract is determined in a known manner.

Camazulene: 3.7 mg (-) -? - bisabolol: 10.2 mg%

Example 6

Preparation of extract or extract from fresh chamomile.

According to Example 1, from time-harvested camomile flowers in which 30-70% of the tubular flowers are open at the time (water content 74%), 758 g of fresh camomile flowers are mixed with 510 g of ethanol (84% by weight) in a trough mixer. While stirring, the agitator is rotated at 65 rpm and extracted from the mixture for 30 minutes. The drug substance is then extruded and the extract is filtered.

The amount of active ingredient in the extract is determined in a known manner.

Camazulene: 11.7 mg% (-) -? -Bisabolol: 16.3 mg%

Example 7

Production of essential chamomile oil

200 g of the dry camomile flower obtained in Example 1, dried at about 50 ° C with the exclusion of sunlight, are mixed with 3.6 l of water and 2 g of sodium ascorbate in a 5 liter sphere. The mixture was adjusted to pH 5.0 with 1N HCl. After the addition of pumice stone, the mixture was heated to phosphorus. Within about 3 hours, about 1.2 L of distillate is collected.

After the distillation is complete, the distillate is shaken three times with 100 ml each of petroleum ether and dried over anhydrous sodium sulfate. The dried solution is filtered and the solvent is then distilled off in a rotary evaporator.

The content of kamazulene and (-) -? -Bisabolol in the resulting essential oil is then determined.

The material obtained is 1.44 g of an essential oil and

Kamazulene: 3.6% (-) -? -Bisabolol: 10.2%.

The Appendix

Obtaining an essential oil

The starting material is a medicinal product prepared from the camomile of the present invention. Only flower heads in which 30-70%, preferably 40-60% of the tubular flowers are open, are used for the manufacture of a medicinal product. Drying in an oven at 40 ° C for 72 hours.

The essential oil of chamomile flowers is obtained by distillation of the two-hour water vapor of the medicinal product as follows.

A 2.0 g portion of the uncomminuted medicinal product is mixed with 250 ml of desalinated water in a 1 liter round-bottomed flask and subjected to a two hour reflux distillation in a Clevenger apparatus (water vapor distillation reflux apparatus to determine a small amount of essential oil). 1 ml of pentane is used as a sample for analysis. The backflow! speed 40 ± 4 drops per minute. After the distillation is complete, the essential oil dissolved in the pentane is drained as much as possible into a reagent bottle and any remaining essential oil adhering to the wall of the apparatus is rinsed with pentane. To remove any traces of water, a solution of Na ₂ SO 4, dried over a knife tip, is added to the solution and the solution is then filtered through a sintered glass filter. The sintered glass filter has a D 3 or D 4 porosity. After evaporation of the pentane in a water bath at 40 ° C and after drying in a desiccator, the amount of oil is determined gravimetrically.

The resulting oil (about 20 mg) is then assayed for the content of kamazulene and bisabolol.

Spectrophotometric determination of kamazulene Measurement solution: Dissolve all 2 g of the essential oil (about 20 mg) obtained from the drug in 25 ml of n-hexane or cyclohexane. Filter photometer (e.g. Eppendorf)

578 nm 1 cm

20.8 n-hexane or cyclohexane

Measuring Device:

Wavelength:

cuvette:

Specific extinction of kamazulene (1 g / 100 ml; lem):

Leveling fluid:

The amount of kamazulene found in mg / 100 g was 120 E578

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

Measuring instrument: Spectrophotometer (eg PM

Q II or PM Q III, ZEISS brake)

Wavelength: 605 nm

Cuvette: 1 cm

Specific extraction of kamazulene (1 g / 100 ml; 1 cm): 24.5

Equilibrating fluid: n-hexane or cyclohexane

The concentration of kamazulene in mg / 100 g was found to be 102 E05

Then determine the amount of bisabolol in the standard solution.

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HU 202034 Β

Gas chromatographic determination of bisabolol and residual bisaboloid

Gas chromatograph: Hewlett Packard Model 5750, Erba Fractovap 2350 or similar device 5 Detector: Flame ionization detector Cargo gas: Helium column: 3.175 mm; 200 cm; steel Column Packing: 3% nitrile silicon rubber "XE 60" silanized on diatomaceous earth 10 Chromosort WAW HP 125-150 μτη as carrier Temperature: Detector 320 'C Befecskendezőb Lokken: 220 'C 15 column: 85-220 'C The temperature programming: 4 'C / min Sample solution: Measuring for kamazulene- date related 20 Comparative About 15 mg standard solution: bisabolol is about Dissolved in 25 ml of cyclohexane injection From the sample solution and quantity: from reference solution 5-5 μΐ 25 evaluation: The evaluation is surface- is done Bisabolol 10 x test (Compare) CONTENTS: (Mg)

gyógyáru)

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

Appliance:

column:

Embankment:

Material Conveying Gas: Programming of Temperature: Injector / Detector Temperature Detector: Injection Quantity:

Packard Model 7721 Series 800 and Erba Fractovap Series 2350

Glass column, 3 m, diameter 2 mm and 2 m, diameter 2 mm

3% methylphenylsilicone rubber "OV 1" silanized on silica

Gaschrom Q '' 125-150 pm as carrier

N2, between 30 ml / min and 180 'C

2.5 (3) 'C / min

200 'C

Flame ionization detector About 2 µl of an essential oil diluted, for example, 1:50 are used

The evaluation is carried out partly without internal ethanol and partly with internal standard. They are suitable as an internal standard for lauric acid methyl ester or hexadecane for chamazulene-deficient and camouflage-free oils. For oils containing more than 5% kamazulene, this can be used as an internal standard, in which case the content is determined photometrically (at 578 nm).

Claims (4)

A process for producing new populations of tetraploid and bisabol soluble chamomile, belonging to the group of cultivars of true chamomile (Chamomilla recutita (L.) Rauscher, synonymous with Matricaria Chamomilla L., Asteraceae), which has a temperature of at least 40 ° C. 100 mg% kamazulene, at least 200 mg% (-) - a-bisabolol, and less than 50 mg% other bisaboloids, by selection for the active ingredient and optionally tetraploidization, characterized in that a diploid or tetraploid chamomile population, The diploid camomile of the species DEGUMILL - in its plant number range from 1000 to 10 000, is determined in each case in essential oil (-) - a-bisabolol and selected from chamomile plants whose flowers at 40 ° C are at least 100 mg% kamazulene, at least 50 -100 mg% (-) -? -Bisabolol and less than 50 mg% additional bisaboloids, these chamomile plants are further isolated from the initial population and A. At least 10 individuals are selected from these plants which exhibit a concurrent flowering time, a flower head size of 20 to 40 mm and a content of at least 100 mg% of kamazulene, at least 200 mg% of bisabolol and less than 50 mg% of additional bisaboloid active ingredient. , and optionally subsequently cloning the diploid or tetraploid plants so selected, which cloning or (a) by cuttings propagated by cutting back the plants, regrowing at 12 to 14 'C and rooting the resulting short shoots at high relative humidity and 12 to 18 ° C in peat-sand mixture, or b) propagating the meristem by treating divisible tissue portions, such as shoot tips or axillary buds, at a temperature between 15 ° C and 30 ° C, with the usual nutrients such that additional organs (buds, shoots, roots) are formed; optionally repeated several times in medium or in only one medium, and the plants thus obtained are further cultivated and propagated, as described above, at high relative humidity and 12-18 ° C, B, The plants obtained according to A are allowed to flower under separate conditions and continuously harvested for several weeks to obtain seed at flowering stage when 30-70% of all tubular flowers of a flower head are open, and the obtained flower heads are harvested. dried at ambient temperature between 30 ° C and 30 ° C, seed is obtained from the seeds thus obtained - under isolated conditions, at daytime temperatures between 18 ° C and 24 ° C and at night temperatures between 12 ° C and 14 ° C, optionally with additional lighting; they bloom at the same time, have evenly leafy branches, have flower heads of between 20 and 40 mm and contain at least 100 mg% of kamazulene, at least 200 mg% of (-) - a-bisabolol and less than 50 mg% of other bisaboloids ővé15 -15EN 202034 Β yielding seeds again and, if appropriate, growing plants at different sites from these seeds and selecting them based on the requirements described above, C. repeat steps B to 3 to 5 times, D. cultivating the seeds from the seeds obtained in accordance with C. under conditions of 18 to 24 'C at daytime and 12 to 14' C at night, and again flowering at the same time, with a smooth leaf branch and a flower head of 20 to 40 mm, and selecting at least 100 mg% of kamazulene, at least 200 mg% of bisabolol, and less than 50 mg% of other bisaboloid active ingredient, cloning the resulting plants as in A. and recovering seed from the resulting plants, and a diploid chamomile. - 0 to 25 'C with chemicals, 0 to 35' C with gamma rays, X-rays and UV rays, 33 to 50 'C High temperature between 5 ° C, low temperature 0 to 5 ° C, or by decapitation callus, or undergo anther culture and subsequent tetraploidization, and optionally Drug and / or propagation material is obtained from 10 camomile plants by harvesting the flowers at a stage of vegetation when 30-100% of the tubular flowers of a flower head are open and the flowers are dried at a temperature of 70 ° C, To obtain 15 propagation material, the resulting dry flower heads are crushed and the propagation material (seeds) screened.