GB2142535A - Preparation of a pharmaceutical camomile product - Google Patents

Abstract

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

Description

SPECIFICATION Preparation of a pharmaceutical camomile product This invention relates to a process for the preparation of a pharmaceutical camomile product.

Preparations of the flower heads of the true camomile (Chamomilla recuita (L.) Rauschert, Synonymous with Matricaria Chamomilla L.) are widely used therapeutically for their antiphlogistic and spasmolytic action and constitute an important item of medicinal plant products. In these preparation, the active ingredients (-)-ol-bis-abolol and chamazulene have particular therapeutic importance. A good pharmaceu tical camomile product should therefor contain as much of these two substances as possible.

Avariety of camomile having both a high (-)-oi-bis-aboloi content and a high chamazulene content is known under the name of DEGUMILL (this is a variety of camomile mentioned in the Patent Claim of German Patent Specification 2,402,802; DDR variety protective right Degumill; Italian Patent 1,035,096). The chamazulene and bisabolol content of this camomile variety DEGUMILL however, is only constant if all cross-breeding or cross-pollination with other varieties of camomile containing considerably less bisabolol and chamazulene or with the ubiquitos wild camomile, which is also low in active ingredient content, is prevented. The risk of cross-pollination almost always exists since the wild camomile occurs virtually everywhere.

Various varieties of tetraploid camomiles are also known (for example, BODEGOLD, DDR; POHORELICKY, CSSR; ZLOTY LAN, Poland; BK-2, Hungary). These tetraploid camomile varieties have a chamazulene content not substantially below that of the new camomile variety, Manzana, but the important active ingredient, (-)-a-bisabolol is only present in extremely small quantities in these known tetraploid camomiles while the other bisaboloids (bisabolol oxides A and B and bisabolone oxide) predominate and together make up more than half of the ethereal oil content in known tetraploid camomile varieties.

A process for producing a new tetraploid camomile variety having advantageous properties and named Manzana has now been found.

The present invention provides a process for the preparation of a pharmaceutical camomile product which comprises harvesting and drying flowers of the tetraploid camomile variety Manzana.

The newtetraploid variety of camomile named Manzana obtained according to the invention is surprisingly distinguished from the known camomile variety DEGUMILL as well as from other known tetraploid varieties of camomile by numerous new and advantageous properties.

In contrast to DEGUMILL, it is not susceptible to cross-pollination with the naturally occurring camomile population (wild camomile). In addition, it contains a considerably higher level of the important active ingredient (-)-a-bisabolol than the DEGUMILL variety. Compared with the known tetraploid varieties of camomile, the Manzana variety according to this process surprisingly differs by having an incomparably higher content in the important active ingredient (-)-a-bisabolol as well as having a very low content of other bisaboloids.

Furthermore, the variety Manzana obtained according to the process suprisingly differs from the known tetraploid camomile varieties by the following properties: improved germination of the seeds, less growth of useless foliage (therefore higher yield of flowers); lower water content of the flowers (hence higher yield of dried flowers and shorter drying time); better suitability for mechanical harvesting since the flowers are largely localised in one plane and most of the flowers bloom at the same time (so that more uniform harvesting dates are possible); better stability of the pharmaceutical product (less tendency to decomposition and crumbling); exceptionally aromatic and typical odour of camomile.

Furthermore, in contrast to the previously known tetraploid varieties of camomile, the camomile variety Manzana according to this process is homogeneous in its content of chamazulene and (-)-a-bisabolol and the characteristics of chamazulene - and (-) -a-bisabolol -bearing are unvarying (i.e. homozygous). By contrast, when individual plants of the known tetraploid camomile varieties are tested, it is found that they belong to different types with regard to their contents, which means that every radom sample of a camomile drug prepared from them produces a result which is inhomogeneous both qualitatively and quantitatively.

It is an object of the present invention to prepare a new tetraploid variety of camomile having improved properties, in particular an increased (-)-os-bisabolol content.

The new camomile variety according to this process is produced by tetraploidisation of the camomile variety DEGUMILL (gene mutation) followed by further stages of selection and propagation (breeding of mother pedigree for cross-fertilizers which are capable of vegetative propagation). The variety Manzana is obtained from 34 stocks or lines which have been cultivated separately.

Tetraploidisation may be carried out in known manner, for example by treating parts of plants (seeds, roots, tips of shoots, seedlings or axial buds,) or tissues of the camomile plant DEGUMILL with chemicals, X-rays, gamma rays or UV rays. It may also be achieved by the decapitation-callus method, by anther culture or by the application or high or low temperatues to the camomile plant DEGUMILL or to parts of the plants or plant tissue of DEGUMILL. Reference may be had for this purpose for example, to the book by Werner Gottschalk, "Die Bedeutung der Polyploidie fur die Evolution der Pflanzen", Gustav Fischer Verlag, Stuttgart, 1976, in particular pages 13 to 22.

Tetraploidisation by means of chemicals. The following chemicals, for example, may be used for tetraploidisation: Colchicine, acenaphthene, alkaloids such as atropine, veratrine, nicotine, saguinarine, derivatives of benzene, diphenyl and phenanthrene, naphthalene and naphthalene derivatives, diphenylamine, tribromoaniline, paradichlorobenzene, methylnapthoquinone, methylnaphthohydroquinone, salicylic acid and related substances, hexachlorohexane, pervitine (hydrochloride), alkyl-alkali metal carbamates such as isopropyl sodium carbonate, phenyl urethane, salts or cacodylic acid (for example, the sodium salt), glycosides of convallaria such as convallarin, convallatoxin and convallamarin, heteroauxin, germisan (phenylmercuric pyrocatechol), organic mercury compounds such as ethyl mercuric phosphate, ethyl mercuric chloride, phenyl mercuric hydroxide, phenyl mercuric dinaphthylmethane disulphonate, chloroform, nitrous oxide (N20) and mixtures of these substances. Oil cake, compost and cow dung may also be used.

Treatment with the substances mentioned above may be carried out, for example, at temperatures from 0 to 35"C, preferably from 12 to 30"C, in particular from 15 to 25"C.

In practice, the treatment may be applied, for example, to seeds, shoot tips, roots (in particular root tips or roots of seedlings), ovaries, cut surfaces of leaves or stalks, cell suspensions of meristem tissue or callus cultures, or the treatment may consist of injection into the basal region of the stalk or the region of axil buds.

The chemicals are generally applied in the form of aqueous, weak alcoholic (alcohol content generally below 5%) or weak acid solutions. The pH of the weak acid solutions may be, for example, from 5.5 to 6.5, and low organic aliphatic acids such as acetic acid, for example, may be used for acidification. If alcoholic solutions are used, these may also be slightly acid. The concentration of chemicals in these solutions may amount to, for example, 0.01 to 0.5%, preferably 0.02 to 0.2%, in particular 0.05 to 0.1%. Gaseous substances may be used as such, optionally under pressure (for example 1 to 10 bar). The treatment time may be, for example 1 to 36 hours, preferably 2 to 12 hours, in particular 4to 6 hours.

The most effective concentrations and duration of action should preferably be ascertained by preliminary experiments.

One particularly suitable method, for example is the treatment with colchicine at temperatures of from 0 to 35"C, preferably from 12 to 30or, in particular from 15 to 25on. This may be carried out, for example, by leaving seeds of the camomile variety DEGUMILL to swell in a 0.01 to 0.2% solution, in particular a 0.02 to 0.1%, preferably a 0.05% solution of colchicine or by immersing germinated, to 7-day old, well developed seedings of the camomile variety DEGUMILL (with the cotyledons facing downwards) in a 0.01 to 0.2% solution, in particular a 0.02 to 0.1%, preferably a 0.05% solution of colchicine. When the latter method is used, the ambient atmosphere should have a relative humidity of almost 100%.The duration of the colchicine treatment may be, for example, 3 to 36 hours, in particular 4 to 10 hours. When seedlings are used, a treatment time of up to 10 hours is generally sufficient. When seeds are used, the treatment time may extend to 36 hours.

After the treatment with the chemicals, the swelled seeds, seedlings or other parts of the plant are rinsed several times with water. The swelled seeds, for example, may then be sown out. Plants in which the roots or other parts have been treated or treated seedlings, for example, are pricked out into plant boxes. The plants are raised from the treated seeds or seedlings (for example, in a greenhouse: temperature from 18 to 25"C by day and 10 to 16"C by night) and those plants in which the pollen is about 1 times larger than in the starting material or in which the somatic cells have a chromosome count of 36 are selected.If other parts of plants (above or below ground) are subjected to the chemical treatment, only those shoots, roots or fiowers/seeds which have developed from the treated parts are subsequently investigated for tetraploidisation. If, for example, the treatment was carried out on a shoot or on an axil, only the new growth arising from this shoot or axil and the flowers or seeds developed from this growth are examined for the chromosome number.

Measurement of the pollen size and counting of the chromosome number may be carried out, for example, as indicated in Example 1.

Tetraploidisation by irradiation.

This treatment is carried out, for example, on seeds or root tips at temperatures of from 0 to 35"C, preferably from 10 to 30"C, in particular from 15 to 25"C. Total irradiation: 5 to 50 Krad. Gamma rays or X-rays are preferably used, UV rays used may have a wavelength for example, of from 400 to 30 nm, preferably 350 nm. The irradiated plants or plant parts are then treated in the same way as after a chemical treatment.

Application of high or low temperatures The high temperatures used may be, for example, from 33 to 500C, preferably from 42 to 45"C. The following, for example, may be exposed to these temperatures: Swelled seeds, seedlings, tips of new growths and meristem tissue. Treatment time: for example, 1 to 48 hours, preferably 12 to 24 hours.

The following temperatures, for example, may be used for low temperature treatment: 0 to 5"C, preferably 0.5 to 4 C, in particular 2 C. The following, for example, may be exposed to these temperatures: Swelled seeds, seedlings, shoots and meristem tissue. Treatment time: for example, 1 to 100 days, preferably 20 to 40 days. Subsequent treatment of plants and parts of plants which have been treated as described above is the same as that carried out after treatment with chemicals.

The decapitation callus method (decapitation = heading or rubbing out tips or back cutting). Decapitation is carried out, for example, on the stalk of young plants preferably on the growing cone at the tip after the formation of 4 to 6 leaves or on the stalks of leaves or sideshoots. The buds or shoots developing from the wounded tissue (callus tissue) are cut off, made to root, and grown on in pots, and the tetraploidised plants are then selected in the same manner as after chemical treatment.

Anther culture (production of plants with a single set of chromosomes from the pollen sacks or anthers, followed by spontaneous or artificial tetraploidisation).

Closed florets whose anthers (pollen sacks) are at the stage of the first pollen mitosis are taken from plants in flower. The anthers are taken from the buds of the flowers by a micromanipulator and transferred to petri dishes containing, for example, a Nitsch and Nitsch nutrient medium (Table 1). The petri dishes are then kept in a culture chamber for 16 days at 28"C by day and 20"C by night. After about 4 weeks, the anthers begin to break open and the plants to grow out. These plants are haploid if the parent is diploid and dihaploid if the parent is tetraploid. After development of the roots, these young plants may be potted out, for example, in garden soil and raised to develop flowers in the greenhouse.These (di)haploid plants are sterile but can be converted into polyploids by treating the shoot tips, roots or stalks with chemicals, e.g. colchicine, so that homozygous plants are obtained which can then be propagated from seeds. For further procedures, see the passage dealing with the treatment with chemicals (for example, colchicine treatment).

TABLE 1 Culture medium of Nitsch and Nitsch (Science, 1969) mg/l KNO3 950 NH4NO3 720 MgSO4.7H2 185 CaCI2 166 KH2PO4 68 MnSO4.4H2 25 H3B03 10 ZnSO4.7 H20 10 Na2MoO4.2 H2 0.25 CuSO4.5H2 0.025 5 ml of a solution of 7.45 g of the disodium salt of ethylene diaminotetracetic acid and 5.57 g of FeSO4. 7 H20 made up to 1000 ml myo-inositol 100 Glycine 2 Nicotinic acid 5 Pyridoxine-HCI 0.5 Thiamine-HCI 0.5 Folic acid 0.5 Biotin 0.05 Sucrose 20 g Ready-made nutrient soil (DIFCO Bacto Agar) 89 Indole acetic acid 0.1 pH of the medium adjusted to 5.5 From the tetraploid camomile plants selected for their size of pollen and/or chromosome count, which may be obtained by the methods described above, there is made a further selection of plants which satisfy the following conditions:: a) approximately simultaneous flowering b! uniform branching from the base and narrow flowering zone of about 5 cm c) large flower heads with an external diameter of 30 mm (25 to 35 mm) d) minimum chamazulene content of 150 mg% and minimum bisabolol content of 300 mg%, with the other bisaboloids (in particular biasbolol oxides) amounting to less than 50 mg% (based on the dry flowers, see Example).

The plants which have thus been selected are now propagated vegetatively from cuttings (cloned) and selected over 3 to 5 generations, the selection always being made according to the above criteria a) - d). This means that plants which have been selected on the basis of criteria a) - d) are cloned, seeds are obtained from the cloned plants, plants raised from the said seed are selected on the basis of criteria a) - d), and seeds are obtained from the plants last selected. The sequence of sowing - selection according to a) - d) - collection of seeds may be repeated 2 to 4 times. This sequence is then followed by another sequence of sowing out selection according to a) - d) - cloning - collection of seeds.

The last lot of seeds obtained is then the propagation material of the camomile variety of Manzana obtained according to the process.

The propagation of cuttings is carried out as foilows: To propagate cuttings, the initial plants (clone mother plants) must develop short roots free from flowering buds under short day conditions. This is carried out during the winter half year in the greenhouse without additional lighting or in air-conditioned chambers at a day length of 6 to 10 hours, preferably 8 hours, and temperatures of from 10 to 15"C, preferably 1 2"C.

Cuttings of leaves, shoots and in particular short shoots (side shoots) are suitable for cloning or propagation.

Their root development takes place at 12 to 18"C, preferably 15"C and day length of from 12 to 16 hours, preferably 14 hours, in a high tension atmosphere (above 100% relative humidity). The substrate used may be, for example, a 1:1 mixture of peat and sand but pure quartz sand, rock wool cubes for cuttings, peat cubes for cuttings or the like are also suitable.

The following soils may be used for sowing: Garden soil; medium heavy loam soil with humus; sandy soil with loam or humus. The seeds may be planted in the greenhouse or out in the open. The temperatures for germination and growth of the plants may be, for example, from 12 to 24do, in particular from 18 to 20"C.

Sowing out in open ground is preferably carried out in the autumn (September/October) or spring (March/April). These periods apply to all the possible regions (for example, northern hemisphere, moderate to sub-tropical climatic regions).

The new camomile variety Manzana according to this process belongs to cultivated species of the true camomile having the botanical name Matricaria Chamomilla L. (synonymous with Chamomilla recutita (L.), Rauschert) and is defined by the active ingredient particulars given above. The values given there for the active ingredient contents refer to that stage of flower development at which 30 to 70%, in particular 40 to 60% of all florets of a flower head are open (i.e. the flowers used for determining the active content were picked at this time and then dried for 72 hours in a drying cupboard at 400C).

Should the harvesting of the camomile flowers occur at a time when the stage of flower development is further advanced, that is, when, for example, 100% of all the florets of a flower head are open i.e. full bloom stage, and/or should the harvested flowers be dried at a temperature above 40"C, then the content of the active materials, (-)-a-bisabolol and chamazulene, is lower.If the flowers are harvested in a stage of development where from 30 to 100% of the florets of a flower head are open and are dried at a temperature of up to 60"C (preferably with the exclusion of sunlight, and under drying conditions as are, for example, described in the following pages), the resulting camomile pharmaceutical product contains at least 120 mg % of chamazulene and at least 200 mg % of (-)-a-bisabolol while the content of other bisaboloids remains less than 50 mg %.

By the term "other bisaboloids" in the more definition are meant in particular the following: (-)-a-bisabolol oxides A and B; (-)-a-bisabolol oxide A; other constituents of the ethereal oil of the camomile variety according to this process are ene-ine-dicyclo-ether, farnesol, spathulenol and small concentrations of various volatile terpene hydrocarbons. The flowers of the Manzana variety according to this process dried in the drying cupboard at 40"C (harvested at the stage of flower development when from 30 to 70% of the flower head are open) contain, for example, from 150 to 200 mg% of chamazulene, from 300 to 450 mg% of (-)-o-bisabolol and only a small quantity, i.e. from 5 to 50 mg% of other bisaboloids, based on the dry substance (i.e. based on the absolute dry weight of the flowers).This absolute dry weight is determined by drying a separate sample of camomile flowers of the Manzana variety of this process in a drying cupboard at 105 C until constant weight is reached (72 to 96 hours). The active ingredient content of flowers dried at 35 to 50"C (pharmaceutical product), for example, is then converted to the amount in the dry mass of flowers determined at 1050C.

In its phenotype, the variety Manzana according to the process is similar to the previously known tetraploid camomile varieties (for example, Bodegold, Zloty Lan, BK-2, Pohorelicky Velkokvety) but it differs from these in particularly by the fact that in the variety Manzana according to this process, (-)-o-bisabolol is the main component of the ethereal oil of the flowers, and the chamazulene content is higher and the content in other bisaboloides (bisabolol oxide A and B, bisabolone oxide) is considerably lower. Other constituents of the ethereal oil in the Manzana variety include farnesol, spatulenol and ene-ine-dicycloether.

The camomile variety according to the process may be planted successfully in all soils except soils containing more than 20% of organic substance (humus substances and soil organisms). No special agrotechnical processes or methods of cultivation are required, only a long day with over 13 hours maximum day length, which means that the temperature zones and sub-tropical zones are particularly suitable for cultivation.

The camomile variety Manzana according to the process has a medium late harvesting time, uniform growth height with narrow flowing zone and large flower head and is therefore particularly suitable for mechanical harvesting. To this is added the advantage that plants of the Manzana variety which have been sown at the same time generally all finish flowering at virtually the same time, whereby harvesting is greatly simplified and facilitated. The camomile variety Manzana according to the process provides a high yield.

Possible uses are, for example: Preparation of a pharmaceutical camomile product, camomile oil, camomile extracts and natural (-)-a-bisabolol.

A pharmaceutical preparation prepared from the camomile variety Manzana obtained according to the process contains the maximum amount of the active ingredients, chamazulene and bisabolol, if the flower heads of the camomile are harvested at the stage of growth when, for example, from 30 to 70%, preferably from 40 to 60%, ie. generally 50% of the florets of a flower head are open and drying is carried out at an air temperature of at the most 50"C, for example from 35 to 50"C. Drying may be carried out either with artificial supply of air or by drying in the shade or optionally also in the sun, but care must be taken to ensure that the supply of heat does not exceed the amount required for complete drying. It is therefore advantageous to check when constant weight has been reached by carrying out control weighings.Drying may take place spontaneously or be carried out artificially (for example with artificially heated air). The yield of active ingredients is highest when drying takes place spontaneously with the exclusion of sunlight. The drying process should take place as soon as possible or immediately after harvesting. Drying may be carried out in thin layers, for example layers from 5 to 20 cm, preferably 10 cm in thickness.

If the harvesting of the camomile flowers takes place at a time when the flower development has progressed further, ie, when, for example 100 % or up to 100 % of all of the florets of a flower head are open (for example, stage of full bloom) and/or if the drying of the harvested flowers takes place at higher temperatures than 40 C, the content of active agents (-)-ex-bisabolol and chamazulene is lower.When the harvesting thus takes place at a stage of growth when 30 to 100 per cent of the florets of a flower head are open and drying is effected at a temperature of up to 600C (preferably under exclusion of sunlight, for example, under the drying conditions described a few pages further on) the camomile drug thus obtained contains at least 120 mg per cent of chamazulene and at least 200 mg per cent of (-)-o-bisabolol, whilst the content of the other bisaboloids remains under 50 mg per cent as before.

The term "other remaining bisaboloids" as used herein means in particular bisabolol A, bisabolol oxides A and B and bisabolol oxide.

Determination of the chamazulene content is carried out spectophotometrically by a method similar to that conventionally used for camomile extracts. Determination of the (-)-ci-bisabolol content and of the other ingredients of the oil of camomile is carried out by the usual gas chromatographic method used for this purpose. A detailed description of the analytical methods of determination is given below.

The active ingredient chamazulene is not present as such in the camomile flowers but in the form of the sesquiterpene lactone, matricine. Matricine has a pharmacological action similar to that of chamazulene.

This precursor matricine immediately gives rise to chamazulene, for example when heated (for example by steam distillation or preparation not to give the matricine content of camomile but the analytically measureable chamazulene content obtained therefrom.

This invention is illustrated by the following examples: Example 1 Seeds of the camomile variety DEGUMILL were placed on filter paper which had been impregnated with a 0.05% aqueous colchicine solution, and the seeds were left to swell at room temperature (20"C) for 6 hours.

They were then removed from the filter paper, washed several times with water and planted out in seed boxes (greenhouse). Earth: Peat-sand mixture 1: 1, temperature: 18 to 20"C, relative humidity about 60%, length of day under artificial light: 14 hours.

The germinating plants were kept under observation until they flowered, and successful polyploidisation was ascertained by carrying out comparison measurements of the pollen size and seed size and by chromosome counting of the plants (F1 generation = first offspring raised from seeds of colchicine-treated plants which had been confirmed to be tetraploid).

Tetraploidisation may be carried out as follows: 5-to 7-day old, well-developed camomile seedlings of the DEGUMILL variety which had germinated on filter paper impregnated with water were placed in a 0.05% colchicine solution with their cotyledons downwards for 4 to 6 hours at room temperature (20 C). The delicate radicals were protected; to avoid damaging them by dryness, the ambient atmosphere must have a relative humidity of almost 100%. After the treatment, the seedlings were rinsed several times with water and pricked out into plant boxes. Subsequent treatment was carried out as for the seeds.

Measurements of the pollen size are carried out with a Leitz binocular research microscope using a micrometer measuring eyepiece and micrometer specimen slides.

Chromosome counting was carried out on the root tips. Fresh root ends 1 to 2 cm in length are collected from young plants raised in the greenhouse, and the root tips are placed for 5 hours in a 0.002-molar hydroxyquinoline solution and then for 15 minutes in 1 N HCI. For the investigation, about 1 mm pieces of root tips are dyed with 2% orecin acetic acid and examined microscopically in an oil immersion. The 4-fold chromosome set of the somatic cells can then be determined on the cells which are in the process of mitosis (4n = 36).

Those plants in which the pollen diameter is about 50% greater than that of the diploid starting material (about 30 instead of about 20 'ism) and in which the chromosome set of the somatic cells has been doubled to 36 (the corresponding number in the diploid starting material is 18) are tetraploid. These plants were selected. About 0.1 to 0.5% of the seeds or seedlings were tetraploidised by the above method and developed intact plants capable of flowering.This procedure is followed by the following steps: Step 1: Individuals which satisfied the following conditions were selected from the tetraploid camomile plants which have previously been selected (as described above): a) approximately simultaneous flowering, b) uniform branching from the base and a narrow flowering zone of about 5 cm, c) large flower heads with an external diameter of about 30 mm, d) minimum chamazulene content of 150 mg% or more and minimum bisabolol content of 300 mg% or more, with the other bisaboloids (in particular bisabolol oxides) amounting to considerably less than 50 mg% (all the values are based on the flower heads which have been dried at 40"C after being harvested at the stage where only about 50% (40 to 60%) of the florets of a flower head were open).

These plants were then cloned. For this purpose, the plants (clone mother plants) were first cut back to a length of shoot of about 15 cm and made to develop new growth (short side shoots) under a day length of 8 to 10 hours at 12 to 1 40C. The short shoots were cut and placed in a peat-sand mixture. At about 100% relative humidity, 1 5"C ambient temperature and a day length of 14 hours, rooting of the cuttings took 7 to 14 days.

Instead of conventional cloning as described above (propagation of cuttings), in vitro propagation of parts of plant tissue capable of dividing may be employed (so-called meristem propagation. Various parts of the plant are suitable for establishing a camomile culture, particularly the tips of shoots or the axil buds.

After the plants have been rinsed with H202 growing tips of buds from the leaf axil are removed and transferred to test tubes containing a nutrient medium, for example the medium of Murashige and Skoog (Physiol.Plant. 15,473-497, 1962). The test tubes are then placed in an air conditioned chamber with a day length of 12to 18 hours, preferably 16 hours (obtained by fluorescent lighting tubes) under a light intensity of 500 to 10,000 lux, preferably 1000 to 3000 lux, at a temperature of 15 to 30"C, preferably 22 to 27"C.

As soon as the explantates show good growth, they are transferred to a nutrient medium of the kind mentioned above but with a higher concentration of cytokinine (30 mg/l of N6 -isopentenyladenine) and little or no auxine (0 - 0.3 mg/i of indole acetic acid). The axils stretch out and adventitious organs and an increased number of axil buds are formed. These may then be removed in the manner described above and cultivated.

The explantates intended for plant propagation in relatively large numbers (the 3rd passage = 3rd propagation generation) are transferred, after growth and formation of the leaves on the first mentioned nutrient medium, to a nutrient substrate containing 10 mg/l of indole acetic acid or 3-indole butyric acid or 0.1 to 0.3 mg/l of cr-naphthylacetic acid. The plants take root and may be planted out after about 4 weeks into pots filled with sterilized garden soil (steam treated at 120"C for 12 hours) and then further cultivated in the greenhouse (under the usual conditions for conventional propagation of cuttings).

Nutrient medium of Murashige and Skoog mg/l mg/l NH4NO3 400 Indole acetic acid 2.0 Ca(NO3)2.4H2O 144 Furfuryl adenine 0.1 KNO3 80 Thiamine 0.1 KH2PO4 12.5 Nicotinicacid 0.5 MgSO4.7H2O 72 Pyridoxine 0.5 KCI 65 Glycine 2.0 NaFe-EDTA 25 myo-inositol 100 H3B03 1.6 Hydrolysed case in 1000 MnSO4.4H20 6.5 Sucrose 2% ZnSO4.7H2O 2.7 purified KI 0.75 Agar powder 1% Step 2: The plants obtained after step 1 finished flowering at the same time in the greenhouse under isolated conditions. The plants stood in 11 cm pots filled with garden soil and were exposed to a temperature of 18 to 24"C by day and 12 to 14"C by night. The day length was at least 14 hours, obtained during the winter half year by additional lighting (200 Watt/m2). Water was supplied as and when required.

From all the selected individuals, the flower heads which had finished blooming and were about to die were continuously removed for their seeds over a period of 4 weeks. After the picked flowers have been dried at room temperature, the seeds were obtained from them by blowing off the lighter parts of the flower.

Step 3: A random sample was removed from the seeds obtained by step 2, and about 2000 offspring were raised from these seeds (ecological conditions as in Step 2) and then selected according to the crieteria a) to d) of Step 1. The selected individuals were then treated as in Step 2.

Step 4: A portion of the seeds obtained according to Step 3 was planted out in the autumn in two different regions.

Region A) 450m NN,48.50N/11.500, Total annual rainfall 750 mm, moist-moderate climate, January -10 to 0 C, July lotto 20"C Region B) 200m NN,42'N/1'0, Total annual rainfall 400 mm Mediterranean Climate, January 0 to 10 C July 20 to 30"C.

NN = northern mean sea level = sea level "N = northern latitude (n degrees) "0 = eastern longitude (n degrees) The seeds were planted out at the end of September to the beginning of October in both regions.

The products of the field experiments obtained were examined and assessed (graded) according to homogeneity of growth, size of flowers and harvesting time, and random samples of the flowers were also taken to examine the active ingredient contents. A selection was made from the flowers in the fields to pick out those individuals which conformed to the parameters mentioned in Step 1. Seeds are again obtained from these plants as described in the last paragraph of Step 2.

Step 5: Steps 3 and 4 in the sequence and manner described were carried out on the seeds obtained by Step 4.

Step 6: About 1500 individuals were raised from the seeds obtained by step 5 (ecological conditions as in Step 2) and a selection was made on the same principle as indicated in Step 1.34 Individuals were selected from these selected plants and cloned according to Step 1. 10 plants from each clone were planted out in the open (Region A, see Step 4) in random distribution in an isolated spot, spaced apart by 40 x 30 cm. The ground was loessial loam, pH 7.0; the seeds were planted at the beginning of June and the first harvest of seeds was obtained in the middle of July. The plants were then cut back and again bloomed to deliver a second harvest of seeds at the middle to end of August.

The recovery of seeds from this material was carried out as in Step 2.

The seeds obtained from Step 6 constitute the propagation material of the camomile variety Manzana according to this process. Flowers of plants obtained from this propagation material (sown out in September/October, harvest early June the following year) picked at a stage when about 50% (40 to 60%) of the florets of a flower head are open and then immediately dried in a drying cupboard for 72 hours at 40"C contain, for example, based on the weight of dried flowers (dry substance): 150 mg% of chamazulene, 300 mg% of (-)-a-bisabolol and at the most 50 mg% of other bisaboloids.

Additional particulars given by way of example concerning the plants of the camomile variety obtained by the process according to the Example: 1. Growth Uniform growth height (balanced growth) with narrow flowering zone, therefore particularly suitable for mechanical harvesting, large flower heads, medium high yield; Form: branched from the base (3-to 5-fold); Stalks: upright, only slightly branched; 2. Foliage Leaf: pinnate, to 3-fold; Thickness: medium; Pinnate leaf, colour medium green; Pinnate leaf (middle of stalk); pinatifid splitting: medium to strong pinnate formation; 3.Inflorescence Flower heads: external diameter about 30 mm, internal diameter about 15 mm; Weight of individual flower head (dry): about 45 mg; Flowering shoot, length (mm): about 700, but depending upon region (climate), planting time, soil, soil fertilization, plant treatment and weather conditions; Beginning of flowering time (number of days from 1st January): About 160th day (seeds sown in September, Region Freising, Federal Republic of Germany, otherwise dependent upon the factors indicated above).

Blooms: middle of June (see above); Flowers without stalk, ethereal oil content (% of dry substance): about 1.0%; azulene content of ethereal oil; at least 15%; Decomposition of dried inflorescences (=pharmaceutical floral product): slight if harvested before last florets open; Pollen diameter: about 30 CLm; Length of seed: about 1.25 mm; Chromosome number of somatic cells: 4 n = 36; 4. Fruit Thousand grain mass (TKM) of seeds: 0.06 to 0.13 g; 5. Capacity for germination (KF) 75.75%; 6. Purity 94-95%; 7. Other features Dry ratio of fresh : dry (blooms) = 5.5 1 to 6:1, characteristic aromatic odour of pharmaceutical product, fine, aromatic, typical flavour of tea infusion.

Recovery ethereal oil The starting material is a pharmaceutical product of the camomile variety Manzana. To prepare the product, only those flower heads are used in which 30 to 70%, in particular 40 to 60% of the florets are open. Drying takes place in a drying cupboard at 40"C for 72 hours.

The ethereal oil of the camomile flowers is obtained by 2 hours' steam distillation of the pharmaceutical product as described below.

250 ml of salt-free water are added to 2.0 g of size reduced pharmaceutical product in a one litre round bottomed flask and reflux distillation is carried out for 2 hours in a Clevenger apparatus (steam distillation reflux apparatus for gravimetric determination of small quantities of ethereal oils). 1 ml of analytical grade pentane is introduced into the receiver. The reflux velocity is 40 +4 drops per minute. After termination of the distillation, the ethereal oil dissolved in pentane is transferred to test tubes, as far as possible without water, and any residues of ethereal oil adhering to the apparatus are washed into the test tubes with pentane. To remove any residues of water, a spatula tip of dried Na2SO4 is added to the solution and the solution is then suction filtered into round edged flasks through a glass suction filter of porosity D 3 or D 4.

After evaporation of the pentane at 40"C in a water bath and drying in a desiccator, the quantity of oil is determined gravimetrically.

The oil thus obtained (about 20 mg) is then examined to determine the chamazulene and bisabolol content.

Spectrophotometric determination of chamazulene Solution to be measured: The entire quantity of ethereal oil (about 20 mg) (obtained as described above) from 2 g of the pharmaceutical product is dissolved in 25 ml of n-hexane or cyclohexane.

Measuring apparatus: Filter photometer (for example, Eppendorf).

Wavelength: 578 nm Cuvette: 1 cm Specific extinction of chamazulene(1 g/100 ml; 1 cm): 20.8 Compensating liquid: n-hexane orcylohexane Content of chamazulene found in mg/100 g: 120.E578 If no filter photometer is available, the determination may also be carried out with a spectrophotometer.

Measuring apparatus: Spectrophotometer (for example, PM O II or PM Q lil "ZEISS" Wavelength: 605 nm Cuvette: 1 cm Specific extinction of chamazulene (1 9/100 ml; 1 cm); 24.5 Compensating liquid: n-hexane or cyclohexane Chamazulene content found in mg/100 g: 102.E605 The bisabolol is then determined in the solution used for measurement.

Gas chromatographic determination of bisabolol and the other bisaboloids Gas chromatograph: Hewlett Packard model 5750, Erba Fractovap 2350 or similar apparatus Detector: Flame ionisation detector Carrier gas: helium Column: 1/8 inch; 200 cm; steel Column filling: 3% nitrile silicone rubber "XE 60" Silanised on Kieselguhr "Chromosorb WAW HP" 125 to 150,LLm as carrier material Temperature: Detector: 320"C Injection block: 220"C Column: 85-220"C Temperature programme: 4 /minute Sample solution: The solution used for determining the chamazulene is used Comparison solution:About 15 mg of standard bisabolol are dissolved in cyclohexane to a volume of 25 ml Injection quantity: 5 CLI each of sample solution and comparison solution Assessment: The assessment is carried out by a comparison of the peak surface areas Bisabolol content in mg/100 g 10 x weighing (comparison) of pharmaceutical product: [mg] x surface area (sample) surface area (comparison) Determination of the other bisaboloids may also be carried out by gas chromatography, for example under the following conditions: Apparatus: Packard, Model 7721, series 800 or Erba Fractovap series 2350 Columns:Glass columns, 3m/2mm in diameter; 2m/2mm in diameter Filling: 3% methyl phenyl silicone rubber "OV 1" silanised on Kieselguhr "Gaschrom Q" 125 to 150 Fm as carrier material Carrier Gas: 30 ml/minute N2 Temperature programme: 80 to 1800C, 2.5 (3) C/minute Injector/Detector temperature 200"C Detector: Flame ionisation detector Injection quantity: about 2 pl of the ethereal oil diluted about 1:50.

The assessment is carried out partly with and partly without internal standard. The internal standard used may be lauric acid methyl ester or hexadecane for oils containing little or no chamazulene. For oils having a chamazulene content above 5%, the latter is to be preferred as internal standard, the content being determined photometrically (at 578 nm).

Claims (11)

1. A process for the preparation of a pharmaceutical camomile product which comprises harvesting and drying flowers of the tetraploid camomile variety Manzana.

2. A process as claimed in claim 1 wherein the dried flowers contain at least 150 mg % of chamazulene and at least 300 mg per cent of (-)-o-bisabolol, remaining bisaboloids amounting to less than 50 mg %.

3. A process as claimed in claim 1 or 2 wherein the flowers of the camomile are harvested at a stage of growth when only 30 to 70 % of the tubular florets of the flower head are open, and the flowers are then dried at an air temperature not higher than 50"C.

4. A process as claimed in claim 1, wherein the dried flowers contain at least 120 mg/% of chamazulene and at least 200 mg/% (-)-a-bisabolol and the content of remaining bisaboloids is less than 50 mg/% if the flowers are dried at an air temperature of from 40 to 600C and/or the flowers are harvested in the development stage where from 70 to 100% of the florets of a flower head are open.

5. A pharmaceutical camomile product including flowers of the tetraploid camomile variety Manzana which have been harvested and dried.

6. A pharmaceutical camomile product as claimed in claim 4 wherein the dried flowers contain at least 150 mg% of chamazulene, at least 300 mg% of (-)-a-bisabolol and less than 50 mg% of other bisaboloids.

7. Use of the camomile flowers of the tetraploid camomile variety Manzana for the preparation of a pharmaceutical camomile product.

8. A process for producing a new, tetraploid camomile variety (named Manzana) of the cultivated species, genuine camomile (Chamomilla recutita (L.) Rauschert, synonymous with Matricaria chamomilla L., Asteraceae), in which the flowers, dried at 40"C, contain, based on the dry substance, at least 150 mg% of chamazulene, at least 300 mg% of (-)-o-bisabolol and less than 50 mg % of other bisaboloids, said process comprising tetraploidising the diploid camomile variety DEGUMILL and subjecting the selected tetraploid plants to further stages of selection and propagation.

9. A process according to claim 8, wherein tetraploidisation is carried out by means of chemicals at temperatures from 0 to 35"C, by means of gamma rays, X-rays or UV-radiation at temperatures from 0 to 35"C, by means of high temperatures of 33 to 50"C, by means of low temperatures of from 0 to 5"C, by means of the decapitation-callus method or by anther culture.

10. A process according to claim 8 or 9 wherein following tetraploidisation, a) a selection is made of the tetraploid plants according to their active ingredient content (at least 150 mg% chamazulene, at least 300 mg% of bisabolol and less than 50 mg% of other bisaboloids, all based on the dry substance), simultaneity of flowering period, uniform branching from the base and a flower head size of from 25 to 35 mm, the plants thus selected are cloned, and seeds are obtained from the plants resulting from cloning, b) offspring are raised from the resulting seeds, a selection is then carried out in accordance with a) and seeds are again obtained from these selected plants, c) the measures according to b) are repeated 2 to 4times d) offspring are raised from the seeds obtained according to c), these offspring are selected in accordance with a), the plants thus selected are cloned, and seeds are obtained from the plants resulting from cloning.

11. Camomile or camomile propagation material obtained according to any of claims 8 to 10.