EP0349607A1

EP0349607A1 - Preparation and use of plants of improved biological value

Info

Publication number: EP0349607A1
Application number: EP88909818A
Authority: EP
Inventors: Olga Bogdanyne Forgacs; Lászl BOGDANY; Katalin Fabianne Pehar; Mikl S Fabian; Péter LONYAI; Zsuzsa Abraham; Tibor Keve; Andrea Kevene Szenttornyai; István KAPOVITS; János BOGNAR
Original assignee: BOGDANY LASZL
Current assignee: BOGDANY LASZL
Priority date: 1987-11-24
Filing date: 1988-11-04
Publication date: 1990-01-10
Also published as: JPH02503985A; CN1034020A; AU2622988A; WO1989004599A1; KR890701004A

Abstract

La présente invention se rapporte à un procédé de préparation de plantes ou de parties de plantes de valeur biologique accrue, procédé qui consiste à cultiver des graines, des tissus ou des cellules de plantes dans un milieu de culture complété par un ou plusieurs éléments à l'état de traces selon une concentration de 10-9 à 10-1 M. La plante, la partie ou l'extrait de plante ainsi obtenus présentant une teneur élevée en éléments à l'état de traces peuvent former une composition diététique, pharmaceutique ou cosmétique.The present invention relates to a method for preparing plants or parts of plants of increased biological value, which method consists in cultivating seeds, tissues or cells of plants in a culture medium supplemented by one or more elements containing state of traces according to a concentration of 10-9 to 10-1 M. The plant, part or plant extract thus obtained having a high content of elements in the state of traces can form a dietetic, pharmaceutical or cosmetic.

Description

PREPARATION AND USE OF PLANTS OF IMPROVED BIOLOGICAL VALUE

The invention relates to a cultivating process for preparatiπg plants of improved biological value, first of all of plant germs, as well as plant organs, tissues and cells and the use of the thus-prepared active ingredient as cosmetical, dietetic and pharmaceutical compositions.

All living organisms consist of the elements of the periodic system. Eleven elements of this system can be found in the living creatures in great amount, these are: carbon, hydrogen, oxygen, nitrogen, sulphur, calcium, phosphorus, potassium, sodium, chlorine, magnesium. Further 16 elements can be found in the organism of living creatures only in a small amount, these are the so-called trace elements, such as iron, zinc, copper, manganese, nickel, cobalt, molybdenum, selenium, chromium, iodine, fluorine, tin, silicon, vanadium, arsenic, lithium.

In addition to these elements the presence of boron is absolutely necessary for growing of the higher plants. Most of the above elements are between the atoms No. 23 and 34. In this region the gallium and germanium can also be found, the biological role of which has been unknown and is being clarified lately.

From the above it can be seen that the presence of elements, first of all of trace elements in a suitable amount is very important for life.

In spite of the different artificial manures and fertilizers the trace elements-content of soil is more and more decreasing nowadays owing to the a gr i cu l tur a l producing which has grown to huge dimensions. As a consequence troubles arise in the trace element supply of the soil - plant - human and animal food chain, which leads to an increased lack of trace elements. The most important point here is the trace element supply of the plant both for people and the animals for this is the first source where people and the animals can get trace elements from.

Four main factors influence the trace element content of plants: a) genus, species b) type of soil, where the plant is growing c) climatic and seasonal circumstances during growing d) stage of development of the plant. In the specification according to the invention thos possibilities have been optimalized and used that certain plant species are able in themselves or by a gradual accustoming to enrich certain trace elements in their tissue in biological active form and high concentration.

Astragalus racemosus and Neptunia amplexicaulis are able for example to accumulate selenium. Nyssa sylvatica gathers cobalt from the same soil a hundred times better than other plants do. Similar differences can be observed between leguminous plants, Gramineae and cereals growing on the same soil, to the advantage of leguminous plants in case of Ca, Mi, Fe, Cu and Zn. Gramineae and cereals can gather Mn, Mo and especially silicon much better. During development these elements can decrease and increase in the whole plant or may become stable.

In the next step the trace element amount already decreases in the human and animal organism too, which may cause different symptoms. These symptoms can be (detailed literature can be found: Trace elements in Human and Animal Nutrition, Eric. J. Underwood; Academic Preww New York, San Francisco, London, 1977) and the same time the biological advantages of the trace elements used according to the process of the invention can be summarized as follows:

The presence of iron (Fe²⁺) in the human organism is of especially great importance, its whole amount is 4 to 5 g. which can be found mainly in the haemoglobine and myoglobine, flavopr oteins contain less than 1 % of same.

Lack of iron may cause serious diseases in formation of blood and this is the most obvious sign of lack of iron, but there are other lack symptoms such as palpitation, sore tongue, stomatitis, dysphagia.

Copper is also important in blood formation, its whole amount runs to 80 mg. Several proteins of copper content are known such as erythrocuprine, cerebracuprine, hepatocuprine, cytocuprine. These enzymes have a significant role in the oxydating mechanisms and in the inhibition and formation of free radicals. It has effect on the hair growth, for the disulphide bounds will be formed only in its presence during the keratin synthesis, and as it is part of the polyphenyloxidase enzyme forming tyrosine into melanine it has a role in becoming gray.

The biological role of nickel proved to be significant first of all in case of myocardial infarct and burning lesions. Lack of nickel also decreases the working of liver hydrogenase.

Manganese can be found in the organism in a total amount of 12-20 mg., first of all in the mitocondriums. In lack of manganese the growth of bones decreases as the synthesis of condroitine sulphate and hexosamine decreases, ataxia arises, the reproduction functions sustain injuries, the lipid metabolism alters and the cholesterol synthesis decreases. The active part of several enzymes as hydrolases, quinases, decarboxylases, traπsferases etc. decrases.

Molybdenum can be found in every tissue in a low concentration. The flavoprotein and xantinoxidase enzyme are metalloenzyme containing molybdenum, its lack causes caries. Selenium can be found in the liver and kidney in the greatest amount. Its importance in the oxidative mechanism was recognized lately. It is ingredient of the glutation peroxidase which protects the organism from the unbeneficial peroxides:

Therefore selenium protects the cells directly from oxidation, the exudative diathesis can be prevented by adding selenium or vitamine E. The effect of selenium is similar to that of vitamine E known as aπtioxidant, they both protect the biological membraπs from the oxidative attack and prevent the exudative diathesis. The cytochromes containing selenium play presumably a role in the oxidation-reduction mechanisms of tricarboxylic acid cycle. Its lack causes disorders in growth, sexual troubles, pancreatic fibrosis hepatosis detetica, exudative diathesis.

The examinations of the previous years have shown the very important role of selenium in prevention of cancer (Mark F. Mc . Carty, Medical Hypothesis 14, 213- 225, 1984). According to the test results the addition of 600 meg/day of selenium protects people from several chemical inductors causing cancer and supports the working of the DNA repair. During the examinations it became obvious that the effectivity of organically bound selenium is the same in spite of the fact that its toxicity significantly decreased. This examination was carried out by using sαdiumselenit and yeast fungus enriched with selenium (Schwarz K., 60-day toxicity trial with selenium yeast II. Dec. 21, 1975 unpublished manuscript).

A significant amount of silicon can be found in the skin, this element is integral part of mucopolysaccharides, being essential structural components of the skin. According to the examinations of Schwarz (Proc. Nat Acad. Sci. USA 70 1608, 1973) not less than 330-554 ppm of bound silicon can be found in hyaluronic acid. At the same time it was also proved that the α chains of collagen contain at least 3-6 atom of silicon. Silicon is essential for bone formation and growth, it has effect directly for the amount of phosphorus.

70-80 % of iodine can be found in the thyroid gland, in lack of iodine the cellularic oxidation suffers injuries, respiration increases in the mitocondriums. It has an effect on the differentiating and growth of cells, inducing the RNA and protein synthesis by controlling the gen expression.

The fluorine-content of the organism is concentrated in the bones and teeth and inhibits the caries and osteoporosis. Chromium can be found in the organism only in a very small amount its role is, however, significant. Typical symptoms of chromium-deficiency are: backwardness of growth, disorders of the metabolism of gluco, lipid and protein. The role of tin is less known but it is absolutely necessary for growth.

Vanadium is present in a very low concentration. It has effect on the reproduction and the lipoid metabolism. It hinders the cholesterol synthesis and the phospholipoid and cholesterol level of plasm and reduces the cholesterol concentration of the aorta. It protects against caries.

In a small amount (30 ppb) arsenic could have an adavantageous effect to growth, in higher concentration is as selenium antagonist disadvantageous.

The physiological role of lithium in the living organism is unknown though it has been used for a long time for treating psychosis (manic-depression). Its activity for the herpes viruses was recognized lately.

The role of boron by mammalia is unknown but it is essential because of its role in the flavonoid synthesis with higher plants.

Germanium was known until the late years as being necessary in the organisms of mammalia. According to the newest search results it plays a significant role in the oxidative mechanisms, its role is similar to that of selenium.

It has a strong effect against tumours and is especially good for the strong metastatising and hard influencable Lewis-Lung carcinoma in mice (Kumano N., Ishikawa T., Koinumaru S., et al. Antitimour effect of organogermanium compound G-132 on the Lewis-Lung carcinoma /3LL/ in C57 PL/6/86/ mice Tohoku J. Exp. Med. 1985 146/1 94-104.).

Here we would especially remark that first of all the representative of the organic germanium compounds have been examined and found to be effective. Germanium presumably has its required effect by modifying the immune response (Sato I., Yan B.D., Nishimura I., Tanaka N. Inhibition of tumor growth and metastasis in association with modification of immune response by novel organic germanium compound 3. Biαl. Response Modif. 1985 4/2, 159-168).

Magnesium has a various role in the organism. It takes part in the working of several enzymes. The most significant is to ensure the activity of these taking part in the nucleinacid, protein synthesis. Calcium is present in the organism in a relative great amount, its role in the bone and tooth formation is significant.

The aluminum needs of the organism is less than 1 μg per day. It is the promoter of succiπe dehydrogenase enzyme.

The importance of cobalt in the blood formation is known, the cobalt and iron are transportated together. It is ingredient of vitamine B₁₂ promoting the transformation of methylmalonyl CoA into succinyl CoA. 5-Methyl- tetrahydrofolcyl:homocysteine methyltransferase is also an important enzyme catalysing the formation of methionine from homocysteine. Daily needs: 3 mg./kg.

One fifth of zinc can be found in the skin. Its importance is great in the liver where it is present in the metallothionine. A relative great amount is in the eyes (139-436 ppm), in the testis (120 ppm), in the prostate (859 ppm), in the nail (93-292 ppm) and in the hair (92-255 ppm). In blood it is found bound to α₂ macroglobuline and albumine. Stress decreases the Zn level. In lack of zinc the growth is backward as the activity of timidine chinase is decreasing and also the DNA synthesis and cell division are decreasing, alopecia, dermatitis, deformations on the skin, chronic skin ulceration arises. The hyaluroπic acid amount in the skin also decrases (Thomson, R.W. 3 . Nutr. 105, 154, 1975), the collagen synthesis is reduced (McClain BBA 304 , 457, 1973}. Zinc is therefore of great importance for it plays a part in the collagen, DNA, RNA and protein synthesis, with other words the presence of zinc is necessa for the progresses of gen activation. Further enzymes containing zinc are the pankreas carboxypeptiase. lactas dehydrogenase (LDH), malonic acid dehydrogenase (MDH), alcohol dehydrogenase (ADH), alkalic phosphatase. 15-25 mg. of zinc is necessary a day for people.

At last we mention the precious metals as gold, silver, platinum direct the trace element effects of which are unknown but they are used as medicines. In for of silver protein silver is used mainly for disinfection gold by rheumatic articular diseases and platina for treating cancer.

From the above it is clear that the maintenance of the suitable level and the supplying of trace element in the living and first of all in the human organism, are of primary importance.

The healing of several unknown diseases can be achieved many times by the addition of the suitable trace element into the organism. According to the invention trace elements are enriched in the organism of the suitable plant arz used as cosmetics, dietetic compositions or pharmaceutical compositions in a biologically valuable form, that is in a form being directly usable for the organism of ammalia. According to what kind of trace elements it contains the composition is suitable for the medical treatment and healing of the above biological disorders detailed by the trace elements.

It is known that administering the trace elements in the form of inorganic salt is not suitably effective for the human organism as it cannot use this form suitably (e.g. iron compositions). A more advantageous form is when the metal ion in question is bound to a carrier molecule which after getting into the human organism is absorbed and gets to the active centrum of the given enzyme; it activates the enzyme so that it can fulfil it function in the organism. In case to the contrary the microelement in question (metal ions) leaves the organism without being used.

According to the process of our invention microelement-carrier complexes are prepared by using different plants, medicinal plants, agricultural plants or feral plants or parts of plants which organic complexes completely fulfil the above requirements.

According to the invention seeds, tissues or cells of different plants are grown in a culture medium completed with trace elements in a concentration of 10 -9-10-1 M.

From the plants, plant tissues or cells of high trace element-content dietetic compositions, pharmaceutical compositions or cosmetics containing trace elements of high biological value are prepared by processing or after extraction.

During growing (germinating) the seeds according to the invention and the cultivation of the plant tissues and cells according to the invention a solid or a liquid culture medium is used.

The following culture media can preferably by used: White, Murashige-Skoog, Gambαrg (B5) and that of Nitsch- type, ("Tissue Culture Methods and Applicaitons", Edited by Perul F. Kruse, JR. and M.K. Patterson, J.R. Academic Press, New York and London, 1973; Murashige, T.: Nutrition of Plant Cells and Organs in vitro. 9 , 81-85, 1973; B.V. Conger et al: "Cloning Agricultural Plants via in vitro Techniques", CRC Press, 1981; P.H.Calcoot: "Continuous Cultures of Cells", CRC Press, 1981).

Any elements which can be found in a little amount in living organism and which have biological activ ity can be used as trace element. Preferred trace elements are selected from group consisting iron, zinc, copper, manganese, nickel, cobalt, molybdenum, selenium, chromium, iodine, fluorine, tin, silicon, vanadium, arsenic, boron, lithium, gold, silver, platinum, bromine, gallium, germanium, magnesium, calcium, and aluminum. This enumeration is not of restricting nature.

The trace elements are added into the culture medium in form of organic or inorganic compound. Ionic compounds can preferably be used such as the suitable halogenides in case of metallic trace elements such as tiπchloride, lithiumchloride or goldchloride, other mineral acidic salts, as nickelsulphate, chromosulphate or silver nitrate, furthermore compounds of covalent bond, e.g. arsene trioxide and organic compounds such as selenocysteine. For administering the trace elements water of natural origin, as sweet water, mineral water, thermal water, medicinal water or the mixture thereof can completely or preferably partly be used, which contain the required trace elements. The given water can be used as the solvent or diluent of the culture medium used. Waters of natural origin such as the water of Parad, Heviz and Margitsziget (Margaret Island, Budapest), Hungary and Postyeπ, Czechoslovakia, as well as the mineral water named Mohai Agnes can preferably be used. The great advantage of the waters of natural origin is that they show up an individual trace element- composition, which has been formed in itself in the nature during a long period and in addition to the. traceable trace elements of known biological activity (see the enumeration above) they contain further, unknown trace elements which cannot be aπalized yet according to 'the present state of prior art. By using the natural waters the advantageous trace element composition and the activity of the trace elements which are present in the nature but have not been tested yet, are utilized. One or more elements of the natural trace element composition can be enriched by an extra addition in the required manner.

During germination or cultivation the concentration range is registered, where the plants are growing well and their trace-element-content is higher than the natural value. The plants being able to develop also by a high trace-element-content are selected then cloned, subjected to further cultivation at the given high trace element concentration, than if required, they are furthered in form of a tissue- or a cell culture, preferably in a suspension culture. Continuous accustoming is used by certain plants storing hard the trace elements, that is the given trace element coπceπtraitoπ is not added at once into the culture medium but divided it is gradually increased for 1-28 days until the required level is achieved. Accordingto our experiments after 28 days the absorbtioπ of trace elements does not increase significantly.

The natural level of trace elements is increased by this process generally by 4-5 orders. This means that starting from the concentration of 10^-9 M of the natural level the concentration of 10^-1 M is neared depending on the given trace element and plant.

Germination is carried out in a culture medium completed with trace element pref-erably at a temperature of 5-35 °C for 1 to 3 weeks under aseptic or sterile con ditions. The cultivation of the plant tissues and cells is carried out preferably at a temperature of 10-30 °C for 7 to 28 days under sterile conditions by u sing lighting periods of 16 hours, if necessary. In principle any plant can be used according to our invention. First of all plants containing high amount of trace elements belonging to the legumes (Leguminosae- sal), papilionaceae (Fabaceae) and gramineae (Graminales) can preferably be used. The usable plants are for example as follows: bean (Phaseolus vulgaris), pea (Pisum sativum), lens (Lens culinaris), Astragalus racemosus, Neptunia amplexicaulis, white lupin (Lipinus albus), wheat (Triticum aestivum), barley (Hordeum nodosum), rye (Secale cereale), oat (Avena sativa), rice (Oryze sativa), mais (Zea mays), millet (Panicum miliaceum) rye grass (Lolium perenne) as well as garlic (Allium sativum), onion (Allium cepa), radish (Raphanus sativus), gipsy-wort (Lycopus exaltatus), borag (Borago officinalis), evening primrose (Oenothera biennis), syo bean (Glycine soja), cowslip (Primula veris), Lathyrus sativus, sorghum (Sorghum vulgare). The above enumeration is not of restricting nature.

From the above plants very hardy entities and tissue or cell cultures can be prepared by cloning, which grow well by a trace-element-content of high concentraiton they absorb the trace elements into their organism and build them in. After accumulation, drying and storing the substance of high biological value can be used as supplement, medicine and cosmetics. The plant material of improved biological value wit high trace-element-content obtained according to the process of the invention is processed for use. If desired germ is decomposed to its parts, i.e. roots, stem and leaves are separated from each other then dried, preground and micronised. The thus-obtained powder can directly be used. In case of a tissue- or cell culture, the culture is dried, the excess substance such as sugar and other culture medium residue is removed by washing with water then dried and processed in analogues way as germ.

Drying is preferably carried out by air current at a temperature up to 60 ºC. The pregrinding is carried out until a grain size below 0.1 mm, is achieved, while micronizing is carried out until 1 μ grain size is achieved.

The powder thus obtained is converted known per se into the usual compositions as tablets, capsules, soft gelatine capsules, dragees, pirules, granulates, aerosole, syrup, emulsion and suspension for using it as dietetic or pharmaceutical composition by means of intoxic pharmaceutically acceptable carriers or solvents.

The concentration of the plant substance of high trace-element-content is about 0.5 to 90 mass% relating to the whole mixture, i.e. it is a value being necessary for achieving the desired level of dose.

These compositions can be prepared for example by mixing a plant powder with a solvent and/or with a carrier optionally by using emulsifying and/or dispersing agents, whereas in case of using water as diluting agent, as auxiliary solvent optionally organic solvents can also be used.

The following auxiliary agents can be used: e.g. water, intoxic organic solvents, such as paraffines (e.g. oil frantions), plant oils (e.g. hazel-nut oil or sesame oil), alcohols (e.g. ethanol, glycerine), solid carriers, for example natural rock-dusts (e.g. caoline, clay, talc, chalk), artificial inorganic powders (e.g. silicic acid of high dispersion degreee or silicates), sugars (e.g. saccharose, lactose, glucose), emulsifying agents (e.g. poly/oxy-ethylene/-fatty acid ester, poly/oxy-ethylen/-fatty alcohol ether, alkyl-sulphonate, arylsulphonatte, dispersing agents (e.g. lignine, sulphite-sewage base, methyl-cellulose, starch and poly/vinyl-pyrrolidone/) and sliding materials (e.g. magnesium- stearate, talc, stearic acid and sodium-lauryl-sulphate)

The substances are administered in usually way, preferably orally. Of course, in case of oral administration the tablet may contain in addition to the above-mentioned carriers other materials, such as sodium-citrate, calcium-carbonate and dicalcium-phosphate together with other auxiliary agents, such as starch, preferably potato starch, gelatine and other similar materials. For forming tablets sliding materials, such as magnesium-stearate, sodium-lauryl-sulphate and talc can also be used.. In case of an aqueous suspension for oral administration the active ingredients can be mixed in addition to the above mentioned auxiliary agents with different flavouring and colouring substances.

For cosmetical purposes the plant powder of high trace-element-content is concerted into cosmetical compositions in a way known per se. The following compositions are preferably be used: body lotions, face creams, hydrating creams, regenerating creams, creams for treating wrinckles of the eye, different gels and jellies, mascs as well as shampoos, bath foams and dentifrices.

For preparing cosmetical compositions the liquid carriers, such as water, alcohol (e.g. ethanol and glycerine), paraffine oil, plant oils (such as sunflower oil), synthetic oils (e.g. silicon oil), glycerine-esters (such as glycerine-monostearate) and fatty alcohols (e.g. cetylalcohol), furthermore emulsifying agents, such as ionic or non-ionic emulsifying agents (e.g. poly/oxi-ethy lene/-fatty acid ester, poly/oxy-ethylene/-fatty alcohol ether, alkyl-sulphonate and aryl-sulphonate), gel forming agents (e.g. alginates, polyvinylcarboxylic acid) further more other additives, as rancidity-inhibiting agents (as butylhydroxy-toluol), conserving agents, essence of perfume, or flavouring agents and colouring ageπst can be used.

In addition to the plant powder the compositions according to the invention can contain other known active ingredients. As especially preferable effect can be achie ed by using germ oils of physiological activity. Cold- pressed plant oils, such as maisgerm oil, wheat germ oil punpkin-seed oil, oil of evening primrose, soy bean oil as well as the mixture thereof can preferably be used.

The invention is demonstrated more detailed by the following Examples.

Example 1

Beans (Phaseolus vulgaris) sterilized by a 3 % hydrogenperoxide are subjected to germination on a liquid containing 0.5 mM of CaSO₄, which liquid also contains cobalt chloride as trace element enrichment in a 10 -9-10-1 concentration line in increasing order. Germination is carried out with 50 seeds per concentration for 10-14 days at a temperature of 25 °C. The highest cobalt concentration is thereafter stated which the bean can bear by good growing. Leaves and roots of the here grown germ plants are furthered, cloned under steile conditions. The root and leaf-parts are grown on a modifie solid White cultur medium completed with the highest cobalt concentration already stated. The composition of the White culture medium is the following: 0.36 g./l. of magnesiumsulphate, 0.26 g./l. of calciumnitrate, 0.2 g./l. of sodiumsulphate, 0.08 g./l. of potassiumnitrate, 0.065 g./l. of potassiumchloride, 0.165 g./l. of sodium dihydrogenphosphate, 3 mg/l. of manganesesulphate, 0.5 mg./l. of zincsulphate, 0.5 mg./l. of boron acid, 0.025 mg./l. of copper sulphate, 1 mg./l. of iron citrate, 7.5 mg./l. of glycine, 1.15 mg./l. of nyacine, 0.25 mg./l. of thamine HCl, 0.25 mg./l. of Pyridoxine HCl, 0.25 ml. of calcium pantothenate, 2 mg./l of indolacetic acid, 20 g./l. of saccharose, 8 g./l. of agar-agar.

Growing is carried out at a temperature between 20-30 °C with redividing per 14-21 days, lighting was use periodically in every 16 hours.

From the leaf and root parts a suspension culture was prepared on the liquid Gamborg (B5) culture medium without agar-agar and they are grown here in a continuously working cultivating reactor.

After the suitable incubation time (14-21 days at 20-30 °C) plant parts containing great amount of cobalt are gathered by centrifuging or filtrating, decomposed, dried and prepared for use. Example 2

The process of Example 1 is followed with the difference that Gamborg (B5) solid culture medium is used in every case. Example 3

The plant seeds fertilized by using a 3 % hydrogen peroxide are subjected to germination in a plastic tray of 25 × 40 × 10 cm, where an acidproof steel net is strained at 8 cm. The plant seeds are placed depending on their size (50 to 250 pieces), and the trays are fille till line of the net with a sterile liquid culture medium

The liquid culture medium contains 0.5 mM of CaSO₄ and in addition until a concentration of 10^-9-10^-1 M one or more trace elements and/or up to 0-100 % mineral water, thermal water, medicinal water or the mixtures thereof. The end concentration of trace elements is achieved during 1-21 days by administration per 1 to 5 days. The seeds are lighted by neon lights during the whole period of germination, using lighting periods of 16 hours. Temperature: 5-35 °C.

After the germination is finished the plants are dried and milled to 1 um in a ball mill. The plant cell are so decomposed.

The grist thus obtained can directly be used for preparing the compositions. Example 4

The process according to Example 3 is followed. The germed plant is bean (Phaseolus vulgaris). Starting concentration of zinc sulphate as trace element is

1.8 x 10 -5 M, the end concentration is 1.8 x 10-2 M.

Example 5

The process according to Example 3 and 4 are follow with the difference that mineral water of Parad (Hungary) is added to the germinating solvent in an amount of 80 %.

Example 6

The process according to Example 3-4 are followed with the difference that medicinal water of Heviz (Hungar are used as germinating solvent in an amount of 100 % .

Example 7

The process according to Examples 3-4 are followed with the difference that thermal water of Margitsziget

(Margaret Island, Budapest, Hungary) is used as germinat- ing solvent in an amount of 90 %.

Example 8

The process according to Examples 3-4 are followed with the difference that an amount of 50 % mineral water of Ha duszoboszlό (Hungary) and 30 % mineral water named Mohai Agnes are used as germinating solvent. Number of Trace Starting End Examples Plant element concentration concentration 9 Bean (Phaseolus vulgaris) iron citrate 1. 7×10^-5 M 1. 7×10^-2 M copper sulphate 4 ×10^-7 M 4 ×10^-3 M zinc sulphate 1. 8×10^-5 M 1. 8×10^-2 M

10 Bean (Phaseolus vulgaris) nickel sulphate 2 ×10^-8 M 2 ×10^-3 M

11 Mais (Zea mays) manganese chloride 1 ×10^-9 M 6 .2×10^-3 M

12 Avena sativa sodium molybdenate 3 ×10^-8 M 3 ×10^-4 M

13 Rice (Oryza sativa) sodium molybdenate 3 ×10^-8 M 3 ×10^-3 M manganese chloride 1 ×10^-9 M 6 ×10^-3 M

14 Garlic (Alli'um sativum) sodium selenite 1 ×10^-9 M 1 ×10^-2 M

15 Garlic (Allium sativum) sodium selenite 1 ×10^-9 M 1 ×10^-2 M germanium chloride 1 ×10^-9 M 5 ×10^-2 M

16 Astragalus racemosus germanium chloride 1 ×10^-9 M 5 ×10^-2 M

17 Neptunia amplexicaulis germanium chlorid 1 ×10^-9 M 5 ×10^-2 M

18 Onion (Allium cepa) germanium chloride 1 ×10^-9 M 1 ×10^-5 M

19 Wtiont (Triticum aesticum) sodium silicate 1 ×10^-4 M 1 ×10^-1 M

20 Rye grass (Lolium perenue) sodium fluoride 1 ×10^-7 M 2. 8×10^-3 M

21 Gipsy wort (Lycopus exaltatus) germanium chloride 1 ×10^-9 M 1 ×10^-5 M

Number of Trace Starting End Examples Plant element concentreition concentraition

22 White lupin (Lupinus albus) germanium chloride 1 ×10^-9 M 1 ×10^-5 M

23 Lathyrus sativus germanium chloride 1 x10^-9 M 1 ×10^-5 M

24 Sorghum (Sorghum vulgare) sodium molybdenate 3 ×10^-8 M 3 ×10^-4 M

25 Barley (Hordeum nodosum) nickel sulphate 2 ×10^-6 M 2 ×10^-9 M

26 Rye (Secale cereale) chromosulphate 6. 5×10^-9 M 6. 5×10^-4 M

27 Radish (Raphanus sativus) platina chloride 1 ×10^-9 M 1 ×10 ^-4 M

28 Radish (Raphanus sativus) tin chloride 1 ×10^-7 M 9 ×10^-4 M

29 Barley (Hordeum nodosum) ammoniumvanadate 1 ×10^-9 M 1 ×10^-5 M

30 Borage (Borago officinalis) lithium chloride 5 ×10^-5 M 5 ×10^-2 M

31 Cowslip (Primula veris) lithium chloride 5 ×10^-5 M 5 ×10^-2 M

32 Evening primrose (Oenothera biennis) lithium chloride 5 ×10^-5 M 5 ×10^-2 M

33 Pea (Pisum sativum) gold chloride 3 ×10^-8 M 1 ×10^-4 M

34 Pea (Pisum sativum) silver nitrate 1 ×10-⁹ M 6 ×10^-5 M

35 Lens (Lens culinaris) gallium sulphate 2. ,3×10^-8 M 2, .3×10^-4 M

36 Pea (Pisum sativum) germanium chloride 1 ×10^-9 M 1 ×10^-5 M

37 Soy (Glycine soja) magnesium chloride 2 ×10^-5 M 2 ×10^-2 M calcium chloride 1 ×10^-3 M 1 ×10^-2 M

Number of Trace Starting End Examples Plant element concentration concentration

38 Bean (Phaseolus vulgaris) cobalt chloride × 6H₂O 4.2×10^-8 M 4.2×10^-3 M 39 Carrot (Daucus carota) sodium selenite 1 ×10^-9 M 1 ×10^-2 M 40 Carrot (Daucus carota) germanium chloride 1 ×10^-9 M 5 x10^-2 M

Example 41

The plant parts, leaf and root pieces, prepared for cloning, are grown on sterile, modified solit White culture medium. The composition of the White culture medium is: 0.2 g./l. of sodium sulphate, 0.08 g./l. of potassium nitrate, 0.065 g./l. of potassium chloride, 0.165 g./l. of sodium dihydrogeπ sulphate, 3 mg./l. of manganese sulphate, 0.5 mg./l. of zinc sulphate, 0.5 mg. of boron acid, 0.025 mg./l. of copper sulphate, 1 mg./l. of iron citrate, 7.5 mg./l. of glycine, 1.25 mg./l. of nyacine, 0.25 mg./l. of thiamin HCl, 0.25 mg./l. of py- ridoxine HCl, 0.25 ml. of calcium pantothenate, 2 mg./l. of iπdol-acetic acid, 20 g./l. of saccharose, 8 g./l. of agar-agar. Growing is carried out at 20-30 °C with redividing per 14-21 days, the plants are lighted periodically in every 16 hours. After the suitable amount of tissues is obtained a suspension culture is prepared from the leaf and root parts on the liquid culture medium Gamborg (B5) without agar-agar (Tissue culture Methods and Applications Edited by Perul F. Kruse, JR. and M.K. Patterson, J . R . Academic Press New York and London, 1973; Murashige, T: Nutrition of Plant Cells and Organs in vitro 9, 81-85, 1973; Conger, B.V. et: Cloning Agricultural Plants via in vitro Techniques CRC Press 1981, Calcott, PH: Continuous Cultures of Cells CRC Press 1931) and they are grown here in a continuously working cultivating reactor.

Incubation time is 7-28 days at 10-30 °C, the lighting periods are of 16 hours. The trace-element-content of the liquid culture medium is increased between the concentration 10^-9-10^-1 M by administration per 1-5 days until the end concentration is achieved and/or the culture medium is completed from 0 to 100 % with mineral water, thermal water, medi cinal water or with the mixtures thereof.

After the cultivation is finished the plant cells containing great amount of trace elements are gathered by centrifuging or filtration, they are decomposed, dried and prepared for 'use. Example 42

To 200 g. of lactose 20 g. of silicon acid of the trade-name Aerosil are added under stirring and 40 g. of plant active ingredient of Example 38 are added in form of flour with a 3 % humidity content, and it is homogenized in inert atmosphere.

After moistening the mixture is stirred in a malaxator for 30 minutes until a paste-like mass is obtained; granule is prepared from the obtained mass by a known method.

The granule is dried in fluid dryer in inert gas atmosphere. Drying is continued until the water content of -the sample decreases under the desired value measured by method of Karl-Fischer. After drying the granule is ground and filled into hard gelatine capsules of 250 mg. by known method. 1000 pieces of capsules are obtained soothing the symptoms of lack of cobalt. Example 43

40 g. of the plant active ingredient of Example 38 are homogenized with 220 g. of mais germ oil and in a cold pressed soft gelatine capsulating equipment it is filled into oval capsules of 250 mg. by known method. Example 44

40 g. of the plant active ingredient of Example 38 are homogenized with 140 g. of lactose, 20 g. of saccharose, 26 g. of potatoe starch, 4 g. of talc and 4 g. of magnesium stearate, then granulated and pressed into tablets of 250 mg. by known method. Example 45

The tablets prepared according to Example 44 are panned by a known method in a panning boiler and is coated with a yellow layer. Example 46

10 g. of the plant active ingredient prepared according to Examples 3-4 are suspended in 62.3 g. of water and heated to 70-75 °C and added to the fatty phase of 70 °C containing 7 g. cetyl alcohol, 10 g. of mais germ oil, 2 g. of vaseline, 5 g. of vaseline oil, 1 g. of white beeswax, 2.5 g. Tween 60.

The emulsion is stirred until cooling. At a temperature of 40 ºC the conserving agent (0.1 g.) and the flavour (0.1 g.) are added. A skin treating creame of high quality is obtained. Example 47

5 g. of cetyl alcohol, 10 g. of vaseline, 4 g. of vaseline oil and 2 g. of Tween 60 are melt together at 70-75 °C and the water phase being necessary to 100 mass%, prepared according to Examples 3-4 and contain ing 5.0 g. of plant active ingredient are added. The body lotion is stirred until cooling off, then preserving agent and favouring agent are added to the composition in 0.1 % and 0.15 %, resp. at 40 °C. The obtained composition is a body lotion with hydrating and skin regenerating activity of high quality. Example 48

5 g. of the plant active ingredient of Examples 3-4 are suspended in 100 g. of evening primrose oil which was preserved with 0.02 % of butyl hydroxytoluole. The composition is then filled into a 2 ml. tube-forming soft gelatine capsule. Ampouls are prepared by known method in a soft gelatine capsulating equipment. Example 49

5 g. of the active ingredient of Example 3-4 are suspended in water and added to 1 g. of Carbopol 940 which was swelled in 50 g. of water for 24 hours. Preserving agent content is 0.1 %. Thw two water-phases are mixed then stirred with 1.0 g. of triethanolamine until gel is formed.

An excellent hydrating, unwrinckling gel is obtained. Example 50

100 g. of polyvinylalcohol are swelled warm in 600 ml. of distilled water. The homogeneous solution is cooled to room temperature then 200 ml of 96 % alcohol and 10 g. of plant active ingredient prepared according to Examples 3-4 suspended in 190 g. of water are added. The obtained mask smoothens and hydrates the skin of face. Example 51

To 1. g. of plant active ingredient prepared according to Example 3-4 93 g. of Texapon N 25 then 6 ml. of a 10 mass% hypagine M are added. The obtained product is used as a hair tonic and shampoo.

Claims

1. Process for preparing plants or parts of plants of improved biological value which comprises growing plant seed, tissue or cell in a culture medium comp l eted with one or more trace elements in a concentration of 10^-9-10^-1 M.

2. A process according to claim 1 which comprises using one or more of the following elements as trace elements: iron, zinc, copper, manganese, nickel, cobalt, molybdenum, selenium, chromium, iodine, fluorine, tin, silicon, vanadium, arsenic, boron, lithium, gold, silver, platinum, bromine, gallium, germanium, magnesium, calcium and aluminum.

3. A process as claimed in claims 1 to 2 which comprises inserting the trace elements into the culture medium in form of organic or inorganic compound.

4. A process as claimed in claims 1 to 2 which comprises inserting the trace elements partly or complete ly into the culture medium in form of water of natural origin such as sweet water, mineral water, thermal water, medicinal water or the mixture thereof containing the desired trace elements.

5. A process as claimed in claims 1 to 4 which comprises cultivating the plant seed, tissue or cell for 1 to 28 days in a culture medium completed with one or more trace elements the concentration of which gradually increases.

6. A process as claimed in claims 1 to 5 which comprises using as plant bean (Phaseolus vulgaris), pea (Pisum sativum), lens (Lens culinaris), Astragalus racemosus, Neptunia amplexicaulis, white lupin (Lupinus albus), wheat (Triticum aestivum), barley (Hordeum nodosum), rye (Secale cereale), oat (Avena sativa), rice (Oryze sativa), mais (Zea mays), millet (Panicum miliaceum), rye grass (Lolium perenne), garlic (Allium sativum), onion (Allium cepa), radish (Raphanus sativus), gipsy-wort (Lycopus exaltatus), borge (Borago αfficinalis), evening primrose (Qenotherea biennis), soy bean (Glycine soja), cowslip (Primula veris), Lathyrus sativus, sorghum (Sorghum vulgare).

7. A dietetic composition which comprises containing a plant or part or extract of plant of high trace-element-content prepared according to claims 1 to 6.

8. Pharmaceutical composition which comprises containing a plant, part or extract of plant of high trace- element-content, prepared according to claims 1 to 6.

9. Cosmetical composition which comprises containing a plant, part or extract of plant of high trace-element- content prepared according to claims 1 to 6.

10. Use of the plant, and part or extract of plant of high trace-element-content prepared according to claims 1 to 6 in form of dietetic, pharmaceutical or cosmetical composition.