WO1997048404A1

WO1997048404A1 - Water-soluble organ extracts with improved biochemical effectiveness, method for production and use thereof

Info

Publication number: WO1997048404A1
Application number: PCT/EP1997/003214
Authority: WO
Inventors: Randolph Riemschneider
Original assignee: Randolph Riemschneider
Priority date: 1996-06-19
Filing date: 1997-06-19
Publication date: 1997-12-24
Also published as: DE19624476A1; CH692408A5; DE29610769U1; DE19624476C2

Abstract

The invention concerns diluted organ extracts with improved biochemical effectiveness, which are taken from cell lines in the respective organs of animals, or parts of plants (generally designated as 'organs'), a method for production thereof and their use individually or in combination with other extracts in pharmaceutical, cosmetic and other compositions.

Description

Water-soluble organ extracts with improved biochemical efficiency, process for their preparation and their use

The invention relates to aqueous organ extracts with improved biochemical

Efficiency that have been obtained from cell lines of the corresponding organs of animals or parts of plants (hereinafter generally referred to as "organs"), processes for their preparation and their use individually or in the form of extract combinations in pharmaceutical, cosmetic and other compositions .

Organ extracts are of great importance as raw materials for pharmaceuticals and cosmetics. Fractionated spleen, blood, placenta and thymus extracts, for example, are used as active ingredients in pharmaceuticals and cosmetics ("Soaps - Oils - Fats - Waxes" 112, 215-218 (1986)), and in many cases they are supplemented by low molecular weight additives become.

Organ extracts are characterized by a large number of constituents or active ingredients which are used either as a complete extract or in the form of individual substances soluted therefrom. For example, over 100 components have been detected for placenta extracts (cf. "Cosmetics International" 6.1-8 (1978)). The synthesis has so far not been successful for some of the individual substances of the organ extracts, especially since the effort required is often too great.

Numerous organ extracts are available today in more or less natural form, for example placenta extracts, blood extracts, thyme extracts, collagen extracts, connective tissue extracts, amnion extracts, and the like, which are produced from fresh organ material . The fresh organ material, for example fresh placenta, liver, kidney, spleen, thymus, blood and the like, is washed, crushed (lastly in colloid mills), and extracted by treating the organ pulp obtained at -10 to

-20 ° C in organic solvents for 6 to 12 days. In the case of blood, hemolysis is carried out with 3 times the amount of water (containing nipagin) for a longer time, for example at -15 ° C. If a roe extract (caviar Extracts), the odorants must first be removed by steam distillation at a suitable pH. Other preparatory measures are again necessary for connective tissue extracts and for plant extracts (cf. also Examples 8, 9 and 10 below).

After the aqueous crude extract has been obtained by centrifugation or filtration or other suitable measures, it is processed further to pure protein-containing or protein-free organ extracts which are sterile filtered before freezing until further use, for example using a 0.2 μm millipore Filters. Such preparations usually still contain proteins or high-molecular protein breakdown products which can have pathogenic and immunogenic properties and, with regular or repeated administration - usually combined with long incubation times - can lead to serious diseases. In particular, those caused by unconventional viruses, i.e. Prions, caused clinical pictures, such as TSE (transmissible spongiform encephalopathies), in particular BSE (mad cow disease), and the Jacob-Creutzfeld syndrome. However, protein-containing placenta extracts are also known which can be carcinogenic when used on humans. [Lit. 1 ].

Animal organ extracts have fallen into disrepute worldwide, above all because of the problems associated with prions, in particular with BSE prions. Proof that organ material comes from herds that were BSE-free can hardly be provided when large amounts of organs of certain ages are used. In order to prove the BSE freedom of an end product experimentally in the Gold hamster model test, it takes at least 9 months - in many cases impractical. A BSE rapid test has so far been lacking.

Combinations of low molecular weight components with organ extracts obtained from natural, fresh organ material are known from DE-OS 24 05 983 and DE-OS 20 21 969, but these also have the disadvantages described above. In order to avoid such complications, EP-A-0 552 516 showed a way of producing water-soluble synthetic organ extracts, in which low-molecular components obtained synthetically, as they also occur in natural organ extracts, are combined with one another.

While the natural organ extracts have the disadvantages described above, the synthetic organ extracts have the disadvantage that for the most part they do not develop the full biochemical efficiency of the natural organ extracts because they lack certain components.

The object of the invention described in the present application is to provide organ extracts which at least achieve the biochemical activity profile of natural and synthetic organ extracts, but preferably significantly improve them, in particular free of undesired substances, especially immunogenic and pathogenic proteins and protein degradation processes Products such as those found in natural organ extracts and their undesirable effects.

Although the use of exclusively synthetic organ extracts, as described in EP-A-0 552 516, can avoid the disadvantages described above, their biochemical activity profile is not always satisfactory. An increase in the biochemical activity profile is now possible according to the invention by producing water-soluble organ extracts from cell lines of the corresponding organs, which may be combined with synthetically produced or natural organ extracts and which are free from allergological effects, such as, for example, with the aid of the patch Tests can be proven.

According to a first aspect, the invention relates to aqueous organ extracts with improved biochemical efficiency which are derived from cell lines of the corresponding organs of animals or parts of plants have been produced, as well as any combinations thereof and their use as active ingredients in pharmaceutical, cosmetic and other compositions.

According to a further aspect, the invention relates to a process for the production of aqueous organ extracts with an improved biochemical efficiency from cell lines of the corresponding organs of animals or parts of plants, which is characterized by the following stages:

a) activation of the selected animal or plant cells in a water bath of preferably 25 to 30 ° C., b) addition of a preheated culture medium prescribed for the respectively selected cells with mixing and subsequent incubation in a manner known per se, preferably at about 37 ° C. and preferably in an atmosphere containing about 5% CO ₂ , c) obtaining the cells thus formed as a starting material for large cultures on surfaces or in suspension by decanting the nutrient medium, washing the resulting cell lawn with a suitable buffer and taking up the cells in a complete medium while adjusting a dilution suitable for mass production, d) culturing the cells on surfaces, preferably in perfusion systems, on solid media and / or using the plastic film culture method (for animal Cells) or in suspension culture (for plant cells) on a large scale familiar way through

Inoculating the cultivation apparatus with a cell suspension in a sterile CO ₂ / air mixture, e) producing an extract by treating the suspended harvested cell material in an organic solvent and / or water, obtaining the aqueous phase, preferably by centrifugation, Removal of residual lipid components from the aqueous phase by repeated treatment with the organic solvent and ter formation of an aqueous crude extract, optionally in the form of a suspension, f) cleaning or treatment of the crude extract depending on the starting material and subsequent dialyzing, preferably using a Visking C 150 membrane, against an aqueous solution containing nipagin or another preservative for recovery an extract free of high molecular weight compounds as dialysate and g) sterile filtration of the extract, preferably using a 0.2 or 0.1 μm (Millipore ^ filter or a PALL candle after adjusting the extract to the desired pH, preferably 5.0 to 7.2.

The water-soluble organ extracts according to the invention can be combined both with synthetic organ extracts, as described in EP-A-0 552 516, and with natural organ extracts or both. Even in the case of these combinations, improved biochemical activity profiles are achieved which are better than those of the synthetic organ extracts alone and are free from the undesired side effects of the natural organ extracts. According to the invention it is in particular possible to produce virus-free and prion-free combinations of synthetic organ extracts and cell line extracts in a technically simple and particularly elegant manner, which is particularly important in connection with the very acute TSE problems today, especially BSE.

Preferred embodiments of the method according to the invention described above are characterized in that

an animal cell mass obtained in stage (d) is suspended in ethyl ether or chloroform and left to stand in the cold room at about -10 to about -20 ° C. for about 8 to about 15 days and then centrifuged to obtain the aqueous phase; the last traces of ether or chloroform are removed from the dialysate (external solution) in stage (f) by blowing through nitrogen at pH 7.2 to 7.5; and or

in stage (a) cell culture from commercially available cell lines including the non-commercially available special cell lines mentioned in the description, or be used in a mixture.

To produce the organ extracts according to the invention and to carry out the method according to the invention, at least one of the cell lines MDBK (bovine kidney cells DSM ACC 174 from the kidney of a normal adult bovine), P1-1A3 (thymic epithelial cells DSM ACC 280 from the thymic epithelium) are preferably used as animal cell lines of a 6 month old child), EBL (bovine lung cells DSM ACC 192 from the lungs of a 7 month old bovine fetus, frozen 110th passage), AM-C6SC8 (porcine kidney cells DSM ACC 152 from a subclone from the cell line Am II , which was obtained from a normal pig kidney), BeWo (female human placenta cells ATCC CCL 98), AV 3 (human amniotic cells ATCC CCL 21), WISH (human amniotic cells ATCC CCL 25), B 95.8 (Blood cells, Monkey, ATCC CRL 1612 ECACC 8501 1419, PK (15) (welding kidney cells) ATCC CCL 33) and FL (human amniotic cells ATCC CCL 62 IZSBS BSCL 46); 3A-subE (human placenta cells) ATCC CRL-1584; BVDV + EJG (bovine capillary cells) ATCC CRL-8659; P-17 (calf thymus epithelial cells) Brastone by Dr. FR Pesserl, Curitiba, Parana, Brazil; FB 5.Bm (bovine bone marrow) ATCC CRL-6043; R.Sp. (Bovine) ATCC CRL-6019; RPL 10 (bovine placenta cells from Prof. Dr. Mariano da Rocha Filho, UFSM, Faculdade de Medicina, Santa Maria, Rio Grande do Sul, Brazil, SPI.K (dolphin) ATCC CRL-6556; CF 37 Mg (Beagle -Dog, mammary gland) ATCC CRL 6230, SRC (rye cells from Stöhr, Acipenser huso from Dr. FR Pessserl, 80,000 Curitiba, Parana, Brazil), calf thymus cell line WANK and NM FARIA, 10,000 S. Paolo, SP , Brazil by Prof. Dr. Dr. R. Riemschneider, Institute for Central de Quimica da UFSM, S. Maria RS, Brazil); XTH-2 (cells from South African clawed frog Xenopus leavis DAUDIN, South Afπcan clawed toad) DSM ACC 190 and / or as plant cell lines at least one of the cell lines Acer pseudoplatanus, Phaseolus vulgaris (from Hypocotyl of Pole Bean), Nicotiana tabacum, Soianum tuberosum (tetraploid biloba strain), Ginkgo Pisum sativum (root), Centurus cyaπus, Daucus carota, Apium graveolens, Daucus catora, Apium graveolens, Arthrospira and / or Saccharomaces cerevisiae or other Actinomycetes used

The organ extracts according to the invention obtained from animal cell lines are combined individually or in a mixture, preferably with at least one synthetically produced extract or with at least one extract made from fresh organs, or with both, the ratio by weight between that obtained from animal cell lines , Steπi filtered organ extract and the synthetically produced organ extract and / or the extract made from fresh organs preferably (1 to 99) (99 to 1), in particular (1 to 50) (50 to 1), especially (1 to 20) (99 to 80). The same weight-to-weight ratios are preferably also used when combining one or more of the animal cell extracts described above with one or more of the extracts obtained from a plant cell

If vegetable cell lines are used, in stage (e) the cell material suspended in water or in an organic solvent, if the cell wall material is important, is preferably broken up in an ultrasound generator and the digested material is also removed after centrifugation and washing Water is digested and dialyzed by chemical and / or enzymatic partial hydrolysis

Further preferred embodiments of the method according to the invention result from claims 10 to 20 According to a further aspect, the invention relates to the use of the aqueous organ extracts described above, individually or in the form of mixtures or combinations thereof, as a replacement or supplement for placenta, thymus, blood, serum, spleen, liver, Collagen, connective tissue, amniotic fluid, jellyfish, roe and udder extracts or combinations thereof, as well as for the production of cosmetic formulations and for the manufacture of medical preparations for strengthening the immune system, for activating the cell metabolism or for the treatment of gastroenterological diseases, in particular ulcers, and for topical, subcutaneous, intravenous or intramuscular application for the purpose of external or internal wound healing, for the treatment of arthrosclerosis or radiation damage. The aqueous organ extracts and extract combinations according to the invention described above are also suitable for the preparation of preparations having an aphrodisiac effect. This applies in particular to the extracts obtained from plant cell lines of preferably Ginkgo biloba, Acer pseudoplatanus, Saccharomyces, Torula or cyanophytes and their combinations with extracts based on animal cell lines, which can be used as active ingredients in preparations with aphrodisiac activity.

In addition, the invention relates to the use of the extracts and extract mixtures according to the invention, including the crude extracts, crude extract mixtures and their precursors for stabilizing and preserving dyed fabrics, paints and varnishes, in particular those with water-based varnishes, and for producing culture solutions, nutrient solutions and dietary solutions .

The invention, in particular the individual stages of the method according to the invention, are explained in more detail below with reference to the accompanying drawings. Ad 1) Thawing frozen cell cultures from competent sources, from our own breeding or cell lines as they are commercially available (see, for example, [Ref. 13, 14, 15]), such as animal cell lines, such as

MDBK (bovine kidney cells DSM ACC 174 from a kidney of a normal adult bovine in a medium from 95% Dulbecco's MEM + 5% FCS (fetal calf serum) as described by Martin and Darby in "Proc Soc. Expr. Biol. Med." 98, 574 (1958), deposited with Dr. R. Riebe, BFA, Insel, Riems (Germany), P 1 -1 A3 (thymic epithelial cells DSM ACC 280 from the thymic epithelium of a 6-month-old child in a medium from 80 % RMPI 1640 + 20% FCS, as described by Fernandez in "Blood", 83, 3245-3254 (1994), deposited with E. Fernandez, Dr. Toribio, University of Madrid (Spain), EBL, bovine lung cells DSM ACC 192 the lungs of a 7 month old bovine fetus, frozen 110th passage, in a medium of 85% RPMI 1640 or MEM + 15% FCS, as described by Bechman and Schöss in "Deutsche Tierärztl. Wochenschrift." 95, 283 (1988) and ibid. , 98, 310 (1992), as well as described by Rutter and Luther in "Vet. Rec." 114, 393 (1984), deposited with Dr. V. Öppling, Paul Ehrlich-Institut Langen (Germany), AM-C6SC8, pig kidney cells DSM ACC 152 from a subclone of the cell line Am II, which was obtained from a normal pig kidney, in a medium from 80% medium 199 (Hanks salts) + 20% FCS ), as described by Limezewski in "Mycotoxin Res." 3, 69-76 (1967), deposited with Dr. Limezewski, Federal Institute for Milk Research (Kiel), BeWo, female human placenta cells ATCC CCL 98 in a medium from Waymouth's + Gey's BBS + 10% Newborn Calf Serum [Lit. 13],

AV 3, human amnion cells ATCC CCL 21 in a medium from M199 with Earle's BBS + 20% FCS [Lit. 13],

WISH, human amniotic cells ATCC CCL 25 in a medium from Eagle's MEM with Eagle's-BSB + 10% FCS *, and PK (15), pig kidney cells ATCC CCL 33 in a medium from Eagle's MEM with Eagle's BSS + 10% FCS + Na Pyruvate [Lit. 13], B95 8 (Blood cells, strain Marmaset, suspension culture ATCC CRL 1612 ECAGC 85011419 Medium RPMI 1640 + 10% FCS + L-Glutamiπ + Na pyruvate + non-essential amino acids + 2-mercaptoethanol, deposited with GB Ferrara, Servizia di Immunogenetica, Istituto Nazionale per la Ricerca, 16132 Genoa (Italy),

FL (human amnion cells ATCC CCL62 IZSBS BSCL46 in a medium Eagle's MEM with Earle's BSS + 10% FCS, described in "Proc Soc Exp Biol Mediane", 94, 532 (1957), deposited with M Ferrari, Istituto Zooprofilattico Spertmentale, 25100 Brescia, Italy), RPL 10 (bovine placenta cells, bos taurus, in a medium from Waymoth's + Gey's BBS + 8% Newborn Calf serum, origin Professor Dr Maπano da Rocha Filho, Reitor da UFSM _^ Faculdade de Mediciπa, Santa Maria Rio Gran¬ de do Sul, Brazil, and Dr G da Röche Filho da UFSM, Bioquimica, Istituto Central de Quimica, Santa Maria, RS, Brazil SRC, rye cells from the Stohr, Acipenser huso, Volga, in a medium of 85% Dulbecco's MEM + 10% Gay's BBS + 5% FCS, deposited with Brastone, Dr FR Pesseri, 82500 Curitiba, Parana, Brazil,

P-17, thymic epithelial cells from thymic epithelium of a 4-month-old calf in a medium of 80% RMPI 1640 + 20% FCS, deposited with Dr F R Pesserl, Brastone, 82500 Curitiba, Parana, Brazil,

CHSE-214 Fish Chinook Salmon Embryo NBL-4 (EB Tr) Bovme embryonic trachea [Lit 15],

3A-sub E (human placenta cells) ATCC-CRL-1584 in medium from Waymouth's + Gay's BBS + 15% Newborn Calf Serum, catalog "ATCC Cell hnes and Hybridomas", Rockville, MD 20852, USA, 1996

BVDV + EJG (cattle, bos taurus, capillary epithelium ATCC CRL-8659 in medium

Eagle's MEM + 20% FCS,

Catalog "ATCC Cell lines and Hybridomas", USA 1996 (see above)),

FB 3 Sp / Thy (beef, bos taurus, normal) ATCC-CRL 6039, catalog as above, B Sp (beef, bos taurus, normal) ATCC-CRL 6019, catalog as above,

CF 37 Mg (dog, Canis familiaπs, Beagle ATCC CRL 6230, Mammary gland, normal), catalog as above, PTI.Sp. (Chimpanzee, Pan troglodytes spleen) ATCC CRL 6315, catalog as above;

FB5.BM (cattle, bos taurus, bone marrow, normal) ATCC CRL 6043, catalog as above;

DBI. Tes (Delphin, Pacific common, Delphinus bairdi, Testes, normal) ATCC CRL 6258, catalog as above;

Spl.K (dolphin, stenella plagiodon, kidney, normal) ATCC CRL 6262, catalog as above; and L01.HT (sea turtle, Lepidochelys olivacea, heart) ATCC CRL 6556, catalog as above, or from suitable plant cell lines. e.g. can be grown in suspension cultures, such as

Phaseolus vulgaris (from Hypocotyl of Pole Bean) in a modified White's medium, bred according to L.G. Nickell, "Proc. U.S. Nat. Acad. Sei." 42, 848-850 (1956),

Nicotiana tabacum after D.K. Dougall, & K. Shimbayshi, "Plant Physiology" 33, 396-404 (1960), Soianum tuberosum (tetraploid strain) according to D.T.A. Lamport, "Adv. Bot. Research" 2, 168 (1965): Michigan State University Atomic Energy Commission Plant Research Laboratory,

Ginkgo biloba, Pisum sativum (root), Centaurus cyanus, Daucus carota, Apium graveolens, (as above), Saccharomyces cerevisiae Hansen from the Asmussen yeast factory, Elmshorn near Hamburg (baking yeasts, distillery yeasts, top-fermented brewer's yeasts), conventional brewing yeasts or yeasts such as Torula utilis, Saccharomyces cerevisiae var. ellipsoideus (wine yeasts), Saccharomyces carlsbergensis (bottom-fermented beer yeasts), Saccharomyces fragilis and Saccharomyces lactis (milk-fermenting yeasts), Candida utilis (formerly Torulopsis utilis) and Candida tropicalisfen (technical feed) and other, Nostoc commune VAUCHER and other cyanophytes such as Arthrospira, Nostoc mucosum etc. Rabenhorst, Algae Eur. No. 28 etc. Wittr. Nordst., Alg.exsicc. No.

497 etc. Occurrence: cosmopolitan, on the edge of salt marshes [Lit. 11];

Tree pollen cells: Acer negundo (Box Eider); Betual alba (White Birch; Olea europeae (Evergreen olive);

Ulmus pumila (Chinese Elm); Quercus rubra (Northern Red Oak).

Pollen (grass pollen): Cynodon dectylon (Bermuda);

Poa pratensis (Kentucky Blue); Seeale cerate (Cultivated Rye)

Become pollen: Artemisia absinthum (Wormwood); Iva xantifolia (Giant Poverty); Ambrosia elatior (Short Rogweed).

Rivularia bullata (poir), Berkeley, Glean, Brit. Alg. P. 1833.

Occurrence: cosmopolitan, in the ebb and flow zone; [Lit. 11],

Calothrix pulvinata Kg., Syth.Alg., P. 71, 1824. Occurrence on rocks, wood,

Algae (Europe, North America, Australia); [Lit. 11], and Romeria elegans Wollszynska, found in Koczw, in a pond near Lwöw.

Ad 2) Incubate the respective cell cultures according to the supplier's instructions.

Ad 3) Obtaining cells as the starting material for large cultures on surfaces or in suspension in a manner known per se, specifically animal cells on surfaces, plant cells in suspension.

Culturing the cells on surfaces e.g. in perfusion systems (according to Weiss and Schleicher [ref. 2]), on solid media and / or according to the plastic film culture method (according to House, Scherer and Maroudas [ref. 3]) on a large scale,

Extraction by treating the suspended harvested cell material at preferably -10 to -20 ° C in organic solvents for preferably 6 to 12 days. Ad 4) Preparation of an extract by obtaining the aqueous crude extract, preferably by centrifuging at, for example, 3000 rpm and

Further processing to pure, protein-containing or protein-free organ extracts, which are sterile filtered before being stored.

In the event that the selected cell line grows in suspension culture, the "Rotary Shaker Technique" e.g. started in the roller bottle apparatus (according to the attached Fig. 1) and then in suspension culture bottles e.g. 2 and 3, continued to work.

The suspension cultures on a very large scale are then in 50 L to 1500 L fermenters, e.g. according to Moore et al. [Lit. 5].

Pipe coil cultivators IGV from 50L up to have also proven their worth

2000L for biomass production of plant cells according to the attached FIG. 6.

The invention is explained in more detail below with reference to the accompanying drawings. Show it:

1 shows a Burler or roller bottle apparatus in which the cells grow on the inner surface of the Winchester or Baxter bottles, which are slowly rotated by rollers or wheels on which they lie; [Lit. 4]

Fig. 2 shows a suspension culture bottle, which is made from a feeding bottle. In order to be able to add culture medium and take samples in between, the tube for renewing the culture medium and the sample tube are squeezed off with a clamp. For "feeding", the clamp is removed from the tube for renewing the culture medium and culture medium is injected through the rubber cap. The clamp is removed from the sample tube for taking samples. There- the universal container is then replaced by a sterile one. The container should not be more than a quarter full. It should be noted that the "collar" around the magnetic rod serves to minimize the contact between the magnet and the bottom of the bottle hold,

a vessel used to grow cells in suspension with

Plastic-coated magnet is rotated by a magnetic stirrer located outside the vessel,

a tissue culture apparatus with a much enlarged surface according to Weiss and Schleicher [Ref. 2] (available in Abbot laboratons) in which the principle of a perfusion system with a solid matrix according to McCoy et al. [Ref. 6] is also used. The superimposed titanium plates are made by one Support frame held together within a glass culture vessel

Layers of titanium plates are held together with metal rods in a cylindrical culture vessel. The amount of cells inoculated is pumped into the vessel through the access tube. The medium is pumped inside the vessel by an air pump (when the gas has passed through the gas tube, the bubbles rise in the pump tube upwards, taking medium with them) After the inoculation, the cells circulate for some time in this way, then the pumps are stopped for a few hours so that the cells can settle out

Gas pumps can then be replaced. Medium can be added or removed through the filler tube. At the end of the cycle, all medium is removed and replaced by trypsin. The cell suspension is then removed through the filler tube

The device is inoculated by pumping in a cell suspension. The device is with a sterile carbon dioxide-air mixture with the help the air pump to distribute the cells evenly. The carbon dioxide air supply is stopped for 2 hours so that the cells settle, then it is continued. As soon as a sufficient cell density is reached, the culture medium is pumped out and replaced with a trypsin solution. After an incubation, the cell suspension can be pumped out of the machine. This type of apparatus has the advantage that it is suitable for a very simple, closed circuit system.

5 an apparatus according to House, Scherer and Maracoudas [Lit. 3] in which a spiral made of flexible plastic in the form of a polystyrene film is used to create a large surface. Culture medium and gas are introduced through the openings. The cells grow on the plastic spiral.

A spiral made of flexible plastic with a pitch of approximately 4 cm is produced with a suitable former and inserted vertically into a cylindrical container with an air pump that runs through the center of the spiral. To inoculate the apparatus, culture medium with the Zeil suspension is added. The cells are then allowed to settle and are distributed evenly over both surfaces of the film by rotating them horizontally at a speed of 2 rpm on a roller stand. As soon as the cells adhere completely (after 3 to 18 hours, depending on the cell type), the bottles are removed from the roller and placed upright for fumigation. To have an exact fumigation with

To ensure air containing carbon dioxide, it is necessary to add an anti-foaming agent (eg Midland silicone, Antifoam Emulsion RD). When growth is complete, the cells can be harvested using trypsin, as with a normal roller bottle or by scraping; Fig. 6 shows a pipe coil cultivator IGV for the production of plant cells in suspension cultures.

7a shows a device for measuring skin moisture (Corneometer CM 820 PC);

7b shows the sensor of the CM 820 PC corneometer with a flat capacitor as the end face, which is pressed onto the skin for measuring the skin moisture;

8 shows a graphical representation of the skin's moisture retention capacity (in h) after a single application of a skin cream without additives (control) or a skin cream with added amnion extract Mb or Ha according to Example 4 below;

9 shows a graphic representation of the change in the surface structure of human skin when viewed microscopically on the basis of the percentage change in the profile line areas (FA) and the profile line length (LL) as a function of the use of emulsion preparations containing placenta extract according to the following Example 5;

10 shows the DC finished plate moving image of the blood extract described in Example 7 below, after two-dimensional development, from which the different ones contained in the blood extract

Amino acids are evident

11 is a graphical representation of the fermentation increase of yeast by the yeast extract dialysate (according to Example 9) and 12 shows a graphic representation of the hydroxyl radical inhibitions by organ extracts as radical scavengers (scavengers on the viscosity of hydraulic acid) (according to example 10).

In all cases it is important that sterile conditions are strictly adhered to, both with regard to the apparatus and with regard to the culture media which are required in considerable quantities and have to be tested and sterilized beforehand.

With regard to cell proliferation, it can be expected on the basis of arithmetic considerations that the described large culture processes will deliver sufficient amounts of cell material, starting from a frozen cell culture, within a relatively short period of time. Slow-growing cells ver¬ multiply in a week to 10 times, in 6 weeks to 10 ⁶ fold. One cell can produce 10 ¹² cells (4 kg) within ¹² weeks and 10 ¹⁵ cells (4000 kg) within 15 weeks.

When developing and using suitable nutrient solutions, care must be taken to ensure that only components are used which later do not cause any pharmacological-toxicological and dermatologically undesirable effects in the desired organ extract. This applies above all in the case of medical use of the organ extracts produced according to the invention.

The water-soluble organ extracts produced according to the invention can be combined both with synthetic organ extracts as described in EP-A-0 552 516 and with natural organ extracts or both. Even in the case of these combinations, improved biochemical action profiles are achieved which are better than those of the synthetic organ extracts and are free from the undesirable side effects of the natural organ extracts. In particular, the invention allows technically simple and particularly elegant production of guaranteed virus-free and prion-free organ extracts, which are a combination of synthetic and cell line represent extracts, which is particularly important in connection with the TSE problems, especially BSE.

The weight-quantity ratios which are to be used when combining cell extracts from animal and / or plant cell lines with corresponding synthetic cell extracts can be varied within a wide range, e.g. between (1 to 50) :( 98 to 50), preferably (1 to 20) :( 99-80), in particular (1 to 10) :( 99 to 90). The same also applies to the extract combination, which is made from fresh organs and associated cell lines. Combinations of all three types of extract are also of interest, and in many areas from, for example (1 to 20) :( 1 to 10) :( rest of synthetic extract), preferably (1 to 10) :( 1 to 5): (Rest of synthetic extract), in particular (1 to 5) :( 1 to 5) :( rest of synthetic extract), for cell line extract to the corresponding natural organ extract to the corresponding synthetic organ extract. The stated proportions are intended to make the invention easier to understand, but the invention is not restricted to this.

As stated in EP-A-0 552 516, cited several times, by increasing the proportions of certain components, e.g. Glycine up to 4.5%, by increasing the proportions of sorbitol, mannitol up to 6% and by increasing the proportions of panthenol up to 35%, achieve special cosmetic effects that natural extracts are lacking (cf. Example 2, Table II , from EP-A-0 552 516). In Example 8 below, a corresponding measure is described for the Acer pseudoplatanus extract VI.

Table I below summarizes some of the animal cell lines used for extract production in the examples described below and which can be used according to the invention.

The preparation of a bovine kidney extract from the MDBK cell line is described in Example 1 below. The preparation of a combination of the extract obtained in Example 1 with an aqueous synthetic bovine kidney extract is described in Example 2.

The combination of the extract obtained in Example 1 with an extract made from fresh beef kidneys is described in Example 3.

Further extract preparations, in which, for example, plant cell lines are also used, are described in Examples 4 to 11.

table

1) Madin & Darby, "Proc. Soc. Exp. Biol. Med", 98, 574 (1958) Filing: Dr. R. Riebe, BFA, Insel, Riems, Germany 2) Fernandez, "Blood", 83, 3245-3254 (1994) Filing: E. Fernandez, Dr. Toribio, University of Madrid, Spain

3) Beckmann and Schöss, "Deutsche Tierärztliche Wochenschrift", 95, 283 (1988); ibid, 98, 310, Rutter and Luther, "Vet.Rec", 114, 393 (1984) Filing: Dr. V. Öppling, Paul Ehrlich Institute, Langen, Germany

4) Limezewski, "Mycotoxin Res.", 3, 69-76 (1987) Filing: Dr. Limezewski, Federal Institute for Milk Research, Kiel ⁵⁾ CRC - IST Ansaldo, Data Bank for Biochemical Research, Animal Cell Lines Catalog 1990 by Manniello, Parodi, Aresu, Romano, Milan 1990. ⁶⁾ Filing: GB Ferrara, Servizion di Immunogenetica, Istituto Na - zionale per la Ricerca, 16132 Genova, Italy.

7) ^" Proc. Soc. Exp. Biol. Medicine", 94, 532 (1957): Filing: M. Ferrari, Istituto Zooprofi lattico Sperimentale, 25100 Brescia, Italy.

The extracts are usually characterized by the following key figures: pH value, dry residue (5 h at 105 ° C), N content (according to Kjeldahl), amino acid determination: qualitatively using ninhydrin and using thin-layer chromatography (TLC), Detection of peptides: using a biuret reagent, freedom from protein (using sulfosalicylic acid), nucleic acid components: TLC, carbohydrate components: TLC, UV spectrum (1:50 or 1: 100), HPLC, sterility tests according to DAB 10.

In the case of medical use of extracts, testing for pyrogens in the rabbit or LAL test is required. Particular key figures that are used to characterize extracts are the hydroxyprol in¬ determination, for example, for connective tissue extracts and plant extracts, as can be obtained from cyanophytes, or as described for Colla 120 in Example 8 of this invention. Roe extracts can be characterized by appropriate fatty acid determinations. The protein-free yeast extract treated in Example 9 can be characterized by its high fermentation activity in the WARBURG experiment (cf. FIG. 11). The same applies to the increase in breathing. Dermatological tolerance and toxicology were tested in the usual way using the following methods: table

Current toxicity test after intravenous application to the mouse. Cumulative toxicity test after 7 days of oral application to the mouse.

Clinical-toxicological observations after intravenous administration in the mouse (screening by R.T. Turner):

2 g / kg, 10 g / kg, 20g / kg mouse.

Open epicutaneous sensitization test on guinea pigs over 3 weeks (according to Bühler).

Rabbit eye irritation test based on "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" by The Staff of Division of Pharmacology, FDA, assessment of eye lesions according to Draize, 1959. Test for primary skin irritation in rabbits based on to "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" by The Staff of Division of Pharmacology, FAD, Skin Toxicity according to Draize, 1959. Clinical-allergological examination in a patch test in humans. Arnes test: To test for mutagenic effects, the Arnes test is used without and with metabolic activation: mutagenicity test (screening).

Absence of viruses and prions: it can be guaranteed by using cell cultures from competent sources. The problems with fresh organ material mentioned in the introduction to the description above (p. 2) are completely eliminated according to the invention.

The lege artis organ extracts according to the invention produced according to GMP (good manufacturing practice) passed all the tests mentioned without any problems - as was also observed for the already known synthetic organ extracts and also for the organ extracts obtained from fresh organ material, proper production provided. Further information can be found in the examples below. The mode of action of all of the above extracts was compared and supported using the following tests (biochemical and biological tests):

Table III

Methods for testing the effects of organ extracts

No. Method Description

Measuring the increase in tissue breathing e.g. of liver homogenate of a rat or a guinea pig in the Warburg test for loading from p. 41 [Lit. 8] mood of the respiratory increase factor (the respiration of the liver homogenate is set to 1, 0): factor specification

Growth test to assess the increase in metabolic activity, for example by measuring the growth of a) guppy fish by measuring the length in p. 74/75 mm after 20 days [Tp 80-100], (control values in parentheses) b) Tadpoles of Xenopus laevis DAUDIN P.73-74 by length measurement, weight gain and advance of the metamorphosis (start and end when converting to frog) in days

Measurement of skin smoothing using the modified PADBERG method using 3% w / o emulsions after 14 days of use; from p. 62 [lit. 10]

Measurement of the photometer values in% of "treated" compared to the initial state (control values in brackets)

Profile line measurement after image analysis; From page 55, the profile line areas FA and the line length LL are assessed (see also FIG. 9).

Comparison of fibroblast growth using thymidine incorporation rate with and without formalin damage; Check from p. 71/72 for compensation of the formalin-damaged fibroblasts after 6 days 6 Measurement of water retention capacity using a corneometer from p. 49 onwards 7 Patch test on humans, ie allergological testing from p. 84 onwards

8 Measurement of the increase in skin tension in the wound p. 39/40 healing test after 21 days (Δ values)

Some of the results obtained are summarized in Tables IV and V, which relate to Examples 1 to 4 of the present application.

Table IV

Comparison of the effectiveness of those prepared according to Examples 1, 2 and 3

Beef kidney extracts la. Ib and le The serial numbers in the left column refer to the test methods listed in Table III under Nos. 1-8:

la: bovine kidney extract from the MDBK cell line

Ib: cell line extract la combined with a synthetic bovine kidney extract le: cell line extract la combined with a natural bovine kidney extract from fresh bovine kidneys

2i

No. (according to la Ib Ic table III)

1 factor 1, 9 2.4 1, 7 2a length in mm 22.0 (18.0) 23.2 (18.0) 21, 0 (18.0)

2b Metamorphosis shortened to 6 days 10 days 4 days

3 70 (85) 60 (85) 70 (85)

4 FA + 6 + 10 +4

LL -2.8 -2 -4

5 Thymidine incorporation rate after 6 days without HCHO 350 x 10 ³ 350 x 10 ³ 350 x 10 ³ with HCHO 10 x 10 ³ 10 x 10 ³ 10 x 10 ³ with HCHO + addition of la Ib Ic compensated: 280 x 10 ³ 330 x 10 ³ 250 x 10 ³

6 ++ +++ +

7 0 0 0

8 Δ value 290 320 230 *

see. also Table VIII on page 40

The table above shows that the optimum effectiveness is achieved by the combination of synthetic extract with the extract obtained from a cell line: data under Ib.

Table V

Comparison of the effectiveness of the Amnion extracts Ila and Mb prepared according to Example 4

The serial numbers in the left column refer to the test methods listed in Table III

lla: Amnion extract from the WISH cell line (human) Mb: cell line extract Ila combined with a synthetic human amniotic extract

No. (according to lla llb table I

1 2.0 2.3

2a 21.0 (17.0) 22.5 (17.0)

2b Metamorphosis shortened to 2 days 6 days

3 60 (80) 55 (80)

4 no measurements

5 without HCHO 360 x 10 ³ 360 x 10 ³ with HCHO 20 x 10 ³ 20 x 10 ³ with HCHO, compensated 280 x 10 ³ 340 x 10 ³ 6 ++ ++ 7 0 0 8 Δ value 260 300

The table above shows that optimum effectiveness is achieved when combining (llb) a synthetic extract with the extract (IIa) obtained from a cell line: data under llb.

While there was mainly talk above about water-soluble organ extracts from cell lines of corresponding animal organs, from fresh animal organs or extracts obtained by the synthetic route and a combination thereof - above all with regard to their cosmetic use as cosmetic additives and with regard to their medical use - shall be discussed in the following plant extracts, which can also be obtained from the corresponding plant cell lines (see also p. 7). As already mentioned, this is generally carried out in suspension cultures, which is to be shown using the example below of Acer pseudoplatanus cell cultures and yeasts (see also Examples 8, 9, 10 below). The further processing of the plant cell mass obtained on a large scale then depends very much on the desired plant extract itself. In the case of the extracts obtained from Acer pseudoplatanus, the cell wall material that contains glycoproteins in a protein-carbohydrate ratio of approx. 50:50 and whose protein components are characterized by a high content of hydroxyproline, tendons, glycine, etc. and is therefore of particular interest close to animal collagen (hydroxyproline is known to be the amino acid typical of collagens, which has many special features; hydroxyproline is not a building block of protein biosynthesis, only proline).

The production scheme for a "collagen-like plant extract" from Acer pseudoplatanus cultures is shown in Table VI. A description of the mode of operation can be found in Example 8.

Table VI

Scheme of obtaining a plant extract (Colla 120) from Acer pseudo-platanus cell cultures and use as a plant cosmetic additive in the sense of collagen replacement

1) Obtaining Acer pseudoplatanus plant material from suspension cultures:

Acer pseudoplatanus in cell culture suspension (50 ml) i propagation using the Rotary Shaker technique (500 ml)

I in suspension culture bottles (Bekkico Glass Inc.) (5-20 L) i in 50 L to 4000 L fermenters or in 2000 L coil cultivators 4 Harvesting Acer pseudoplatanus cell material as powder or suspension

2) Cell wall breaking: i suspended Acer pseudoplatanus cell material in kilowatts

Ultrasonic generator broken i centrifuging the digested material and washing with water i

3) Splitting and deproteinization: i by chemical and enzymatic partial hydrolysis i concentration in a vacuum on 10 - 15% extract i use as a cosmetic additive analogous to collagen in creams, lotions, masks and the like. A similar procedure can also be used if from Soianum tuberosum, namely a tetraploid strain in suspension cultures.

The molecular weight of the cosmetic additives obtained is less than 3000. Moisture-holding capacity and other cosmetic effects of these hydrolysates are likely to be related to the low-molecular peptides contained therein, for example to oligopeptides, in accordance with DE-OS 41 27 790, or derivatives of similar constitution.

The organ extracts according to the invention and / or their combinations can not only be used cosmetically and medicinally. There are further possible uses if one considers that the cellular metabolism activation which can be demonstrated as an increase in breathing (or by other systems) is simultaneous an activation of oxygen means and, in connection with this, also an influencing of radical-generating systems (radical scavenging effect). As a result, the influence of various types of organ extracts could also be expected in areas where stabilization and preservation is desired. Corresponding experiments with paint coats and dyed fabrics have shown positive effects:

Paint coats of color solutions, to which animal or vegetable extracts or extract mixtures according to the invention have been added, optionally concentrated to 10-15% solids and optionally with the addition of solubilizers or solvents, have been proven in long-term tests and also in model tests under UV radiation as more colourfast than the control tests without additives. Corresponding results have also been achieved with dyed fabrics and water-based car paints of various types: longer color durability and less sensitive to washing out. Particularly good effects have been achieved with the extracts mentioned in Example 11 below.

The radical scavenging effect of the plant and animal organ extracts according to the invention determined for biological systems should also play a role in the effects described above. It is important to refer here to the model experiments described below in Example 10 below about the radical scavenging effect of hydroxyl radicals on hyaluronic acid and its inhibition by the organ extracts (see FIG. 12).

The invention is explained in more detail by the following examples, but without being restricted thereto. example 1

Production of a bovine kidney extract from the MDBK cell line

1.1 Provision of the cell material 1.2 Extraction and processing for protein-free or protein-containing beef kidney extract

Ad 1.1

The MDBK cell line (DSM ACC 174) serves as the starting material for the production of a bovine kidney extract.

The cells come from a kidney of a normal adult bull from 1957. The cells are free of BVD. These are adherent epithelium-like cells.

The following is required as the culture medium: 95% Dulbecco's + 5% FCS.

The following applies to the subculture routine: optimal division rate 1: 4 to 1: 6 with Tryp¬ sin / EDTA. The cells grow together within 2 to 3 days. Sowing: (2 - 4) x 10 ⁵ cells / 80 cm ² ; Harvest (4-6) x 10 ⁶ cells / 80 cm ² ; 5% CO ₂ , 37 ° C.

Starting from the frozen cell culture of the MDBK line, this is first thawed in a water bath at 30 ° C and transferred to a 15 ml tube. 10 ml preheated culture medium are added, mixed with it, incubated (37 ° C., 5% CO ₂ ). After the formation of the cell lawn, work is continued in the following way while avoiding centrifugation in order to avoid cell damage: decanting the medium, washing the cell lawn once with a phosphate buffer, wetting the cell lawn with trypsin / EDTA (1 ml per 25 cm ² - Bottle) by swirling the cell culture bottle 3 to 4 times, aspirating trypsin, incubating at 37 ° C for 2 to 3 min; after detachment of the cell lawn (gentle shaking or tapping on the culture vessel), the cells are taken up in complete medium; the desired cell concentration of 4 × 10 ⁵ cells per 80 cm ² is produced by microscopic counting and appropriate dilution.

In this way, the starting material for a large culture of cells on surfaces is provided in a large number of bottles.

It is used with a solid matrix perfusion system according to McCoy, Wittle, Conway and Weiss [Lit. 6], Schleicher, White [Lit. 2] from the Abott laboratories.

The culture surfaces consisted of layers of titanium plates placed one above the other, which are held together by a support frame within the culture vessel (cf. FIG. 4). A simple air pump can be used.

The apparatus is inoculated with a cell suspension which is pumped in from the culture bottles. The apparatus is then charged - always under strictly sterile conditions - with a sterile CO ₂ / air mixture using the air pump in order to distribute the cells evenly. The CO ₂ -containing air supply is stopped for 2 hours so that the cells can settle, then the feeding is continued. As soon as a sufficient cell density is reached (see harvest), the culture medium is pumped out and replaced with a trypsin solution. After an incubation, the cell suspension can be pumped out of the apparatus. Up to 20 g of cell material are obtained with each cycle.

At the same time, the plastic film culture method was used on an industrial scale. In this way, it was not a problem to grow 10 kg of bovine kidney cells within a relatively short time, which were then treated further for extract extraction, with the same procedure as with fresh bovine kidney organ material after washing and crushing in colloid mills.

Ad 1.2

10 kg of thawed cell material according to section 1.1 are suspended in ethyl ether and left to stand in the cold room at -20 ° C. for about 10 days. After centrifuging in a suitable apparatus, for example an explosion-proof drum centrifuge, 4 l of crude extract are obtained, which are first filtered for further purification, thoroughly etherified at least twice and then dialyzed against a solution containing 0.3% nipagin.

Ether residues can be removed from the dialysate (external solution) by blowing through nitrogen at pH 7.2. The dialysate is a protein-free beef extract. There is a protein-containing bovine kidney extract in the dialysis tubes.

The yield of protein-free bovine kidney extract la was 25 L. Ultrafiltration of the extract la is not necessary since the starting material was BSE-free and contained no prions

The beef kidney extract la, sterile-filtered with a 0.2 μm (Millipore ' ^s ') filter, has the following characteristics after the pH adjustment:

pH 6.8

Dry residue 7 mg / ml

N content (Kjeldahl)> 0.03%

Amino acids (ninhydrin) positive peptides (biuret) positive

Proteins negative Metabolic activity:

Breathing increase factor according to Warburg (homogenate, rat) 1, 9

Heavy metals <10 ppm arsenic <2 ppm

Sterility sterile and free of hormones,

Prions and viruses

Example 2 Combination of an aqueous synthetic bovine kidney extract with a bovine kidney extract produced from the MDBK cell line according to Example 1

5 L of the sterile, protein-free bovine kidney extract la prepared according to Example 1 are combined with 19.8 L aqueous synthetic bovine kidney extract, produced according to EPA 92 250 349.5, made up to 6.8 L to 25 L after pH adjustment and with a 0.2 μm (Millipore ' ^a ') filter sterile filtered (combined bovine kidney extract Ib).

The preparation of 20 l of aqueous synthetic bovine kidney extract was carried out as follows:

The constituents given in the following table were dissolved under sterile conditions and with stirring in 8 L of double-distilled water, the pH of the solution being kept between 6.0 and 7.5 during production (partial solution I, the pH of which was Was set to 6.7).

Table VII

To prepare partial solution I, dissolve in 8 L of double-distilled water (the stated amounts are calculated for a final solution of 20 L):

L-glutamic acid 6.0 g

D-ornithine 1, 6

L-aspartic acid 3.0

L-tyrosine 0.6 L-hydroxyproline 4.0

Creatinine 0.2

L-isoleucine 0.02

L-leucine 1,0

Cysteine 0.04 glycine 12.0

L-serine 6.0

L-lysine 7.0

Urea 1, 6

L-histidine 1.2 g

L-Asparagine 1,0

L-valine 1.0

DL-threonine 0.8

L-phenylalanine 0.8 L-methionine 0.4

L-tryptophan 0.4β-alanine 0.7

L-Alanine 4.8

L-proline 4.8 L-arginine 7.0

Hippuric acid 0.02 Adenine 0.4 g guanine 0.2 hypoxanthine 0.4 uracil 0.4 uric acid 0.02 AMP 0.2 glucose 3.0

Adenosine 0.4 g cytosine 0.6

Inosine 2.0

Uridine 0.6

Orotic acid 0.02

Sorbitol 40.0

Cytidine 0.8 g

Guanosine 0.4

Thymine 0.8

Xanthine 0.2 Cycl.AMP 0.8

Mannitol 3.0

Succinic acid 30.0 g

Ethanol 40 ml

Malic acid 0.2 g

Citric acid 2.0 g

Benzyl alcohol 3 ml

Thiamine dichloride 0.04 g

Ca pantothenate 0.04

Pyridoxol HCl 0.08 36

Tocopherol succinate 0.08

Panthenol 20.0

Mg aspartate 0.4

Mn gluconate 0.2

N-methylglucamine 8.0 p-hydroxybenzoic acid methyl ester Na salt 14

Nicotinamide 0.2 myo-inositol 1.0

Biotin 0.2

MgSO ₄ 7H ₂ O 2.0

Co-gluconate 0.01

NaH ₂ PO ₄ H ₂ O 1.0

Na lactate 50% 20.0

In parallel, a partial solution II was prepared by dissolving a peptone amount of 80 g, obtained from soy, corn and partially dehydrated yeast in a weight ratio of 1: 1: 10, for deodorization and decolorization in 9 L of double-distilled water with vigorous stirring and overnight with 15 g fresh activated carbon has been treated.

The solution obtained in this way was combined with partial solution I after filtering with suction and sterile filtering through a 0.2 μm (Millipore ' ^fi ) filter. The synthetic peptide fragments glutathione (180 mg) and Gly-His-Lys (2.3 mg) and 0.2 mg H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His were then added to the mixture - Leu-Leu-Val-Tyr-Ser-OH + 0.08 mg H-Arg-Lys-Glu-Val-Tyr-OH + 5 ml of renin hydrolyzate added and after adjusting the pH to 20 L. filled, sterile filtered with a 0.2 μm filter and analyzed:

pH 6.7

Dry residue 1.6%

N content (Kjeldahl)> 0.05% 37

Warburg factor (increase in breathing) 2.0

Amino acids (Ninhydnπ) positive

Peptide (biuret) positive

Heavy metals <10 ppm

Arsenic <2 ppm

Sterility sterile

When preparing partial solution I, it should be noted that of the constituents mentioned in Table I, the amino acids L-Glu, L-Asp, L-Val, L-Tyr, L-Phe, L-Isoleu in 2N NaOH The carbonic acids citric acid, succinic acid and apple acid are then added. After the pH has been adjusted to 6.4, the other components are added. The constituents xanthm and guanine must be pre-dissolved in 3N NaOH or 3n HCl and be neutralized before adding

For a technical application of a synthetic bovine kidney extract, which only consists of partial solutions I and II, is preserved but not sterile, see Example 11 below

The same applies to the preserved, but not dialyzed, crude extract described in Example 3 below and made from fresh bovine kidneys

EXAMPLE 3 Combination of an Extract Made from Fresh Beef Kidneys with an Beef Kidney Extract Made from the MDBK Cell Line According to Example 1

5 L of the sterile-filtered bovine kidney extract la prepared according to Example 1 are combined with 19.8 L aqueous bovine kidney extract, which was prepared from fresh bovine kidneys as described below. After the pH value has been adjusted to 6.8, the pH is increased to 25 L filled up and filtered with a 0.2 μm filter, whereby the combined beef kidney extract Ic is obtained The bovine kidney extract from fresh, washed, shredded bovine greases from BSE-free herds was prepared as follows

The prepared and crushed (last in a colloid mill) bovine kidney are suspended in ethyl ether, and about 10 days at -20 ^C C in the cold room leave After centrifugation in a suitable apparatus, for example a (explosion-proof) basket centrifuge, be from 100 kg of bovine kidney About 25 L of aqueous crude extract obtained. For further purification, this is first filtered, intensively etherified at least twice and then dialyzed against a solution containing 0.3% nipagin

After standing for 4 days at 5 ° C., the protein-free dialysate on the outside is freed of any last traces of ether by passing nitrogen through at pH 7.2 and, after adjusting the pH to 6.8, is ultrafiltration (Filtron, Centrasette, type OMEGA, NMWL 8K)

This protein-free sterile dialysate is the bovine kidney extract, which is combined with the cell line extract la to form the combined bovine kidney extract Ic

The protein-containing bovine kidney extract contained in the dialysis tubes can be used for other purposes, for example to obtain bovine kidney peptides after partial hydrolysis, which are important for the production of synthetic extracts. The bovine kidney extract remaining in the dialysis tube, its dialysate and a combination Ic have been tested for free-radical effect, and positive results have been achieved in all cases (see Example 10 and Fig. 12 below)

The following key figures have been determined for the combined bovine kidney extract Ic 39

pH 6.8

Dry residue 8 mg / ml

N content (Kjeldahl)> 0.03%

Amino acids (ninhydrin) positive

Peptides (biuret) positive

Proteins free of proteins

Metabolic activity:

Breath enhancement factor

(Liver homogenate, rat) 1, 7

Sterility sterile

The protein-free dialysate used for the combination to produce the combined extract Ic had a Warburg factor of 1.55.

Extensive efficacy tests have been carried out with various types of bovine kidney extracts prepared according to Examples 1, 2 and 3, the results of which have been summarized in Table IV (according to the key in Table III).

Among other things, to demonstrate the effectiveness of the extract according to the invention, wound healing tests in rats according to DE-PS 37 11 054 and then skin tension measurements were carried out (cf. also EP-A-0 552 516, Table II). These results are listed in Table VIII:

Table VIII

Values in Table IV under No. 8.

The results of the skin tension measurements listed in Table VIII document the superior effectiveness of the preparations according to the invention under a) and b) and in all cases over the control groups.

The breathing increase factors stated in Table IV under No. 1 and all other respiratory increase factors specified in the application were obtained according to the following working procedure on "Efficacy test of test solutions for increased breath in rat liver homogenate (Warburg method)" [Lit. 8th]. Method and work plan

A. Solutions

1. Sörensen buffer, pH 7.4 from 80.4 ml of a solution of 11.87 g of Na ₂ HPO ₄ .2H ₂ O / l and 19.6 ml of a solution of 9.08 g of KH ₂ POVI.

After combining the stated amounts of solution, the pH is checked and, if necessary, corrected by adding one or the other solution.

2. Rat liver homogenate: the liver is removed from a 24-hour hunger rat after decapitation and bleeding and immediately cooled on ice. After washing and drying with cellulose, 6 g of chopped liver are topped up to 30 ml in a Potter-Elvehjem homogenizer with smooth-running Teflon pistol with cooled Sörensen buffer and homogenized for 2 to 3 minutes with ice-water cooling. The finished homogenate is allowed to stand under ice cooling for about 3 minutes in order to let the air swirled in during the homogenization soak.

3. KOH 6n

4. Test solutions, e.g. the bovine kidney extracts la, ib and ic see table IV and others.

5. Aqua dest. B. Execution of the experiment

Conical Warburg gofäßo with a cylindrical salt (ZE) and a side arm (SA) and a volume of approx. 13 ml are filled as follows:

Fill plan, the details are given in ml.

Glasses no. 1/2 3/4 5/6 7/8

Solution filling location

1 HR ^* ) 3.0 1, 5 3.0 1.5

5 SA 0.25 0.25 - -

4 SA - - 0.25 0.25

3 ZE 0.2 0.2 0.2 0.2

2 HR - 1, 5 - 1, 5

*) HR = main room

The solutions are in the. pipetted in the specified order. To increase the KOH surface, folded filter strips are inserted into the cylinder inserts. Immediately after the filling has been completed, the vessels are sealed with the appropriate hose valve plugs on the side arm attachment, the vessels are attached to the pressure gauges and secured with clips or rubber bands. With the taps open, the pressure gauges are suspended in the apparatus heated to 37 ° C. and shaken for 50 minutes in order to control the temperature of the reaction solution and to let the initially very rapid breathing subside. Then the right manometer legs are set to the mark "15" with the help of the pinch tap, the difference between the left legs is noted to "15", the manometer taps are closed and the solutions from the side arms are tilted into the main room (O-Zeit ). After every 20 minutes, the pouring device is switched off, the right pressure gauge legs are set to "15" (V = const.) And the difference between the left legs is noted at "15". The test runs up to a measuring time of 100 minutes. If the oxygen consumption in some glasses is so large that the manometer length is not sufficient, pressure compensation is created in good time by opening the manometer valve immediately after the reading, adjusting the right leg to "15", the difference of the left leg to " 15 "and immediately closes the pressure tap. After the last reading, the manometer cocks are opened and the volumes of the vessels and the associated manometers are noted.

evaluation

First, tubes 3/4 and 7/8 are compared with the corresponding thermo-barometers (TB; tubes 1/2 and 5/6) to compensate for fluctuations in air pressure and temperature during the test. The vessel constants for O _{2 of the} glasses 3/4 and 7/8 are then calculated using the following equation:

Vg = volume of the gas phase (volume of the vessel and the manometer up to the mark "15" minus V _f ). V, = volume of the total liquid contained in the vessel. T = test temperature in "K. α = Bunsian absorption coefficient (O ₂ 37 ° C - 0.0239).

P o = 1 atm, measured in mm Brodie solution (manometer filling) at 0.1 MPa (760 mm Hg) = 10,000 Brodie solution. The oxygen used can now be used directly according to the equation

X (µl O ₂ ) = h. k

are calculated because the carbonic acid produced by breathing has been completely absorbed by the KOH (h = manometric difference). If you divide the used μl 0 ₂ of glasses 7/8 by that of glasses 3/4, you get a factor that represents the measure of the increase in breathing. [Lit. 8y.

Example 4

Preparation of an aqueous amnion extract by combining an extract from the WISH cell line with a synthetic amnion extract analogous to EP-A 0 522 516

4.1 Provision of the cell material, starting from the Amnion cell line WISH (human) 4.2 Extraction of the Amnion cell material and processing to a protein-free Amnion extract IIIa

4.3 Preparation of a synthetic amnion extract according to EP-A-0 522 516

4.4 Combination of the two Amnion extracts to the extract llb 4.5 Test experiments with Ila and llb.

Ad 4.1

The cell line served as the starting material for the production of the amnion extract

WISH, ATCC CCL 25, IZSBS BS C192.

The culture medium required is: Eagle's MEM with Earle's BSS + 10% FCS (details can be found in "Exp. Cell. Res. ' ^* , 23, 14 (1961)). The procedure was as described in Example 1.1. With regard to the production of a large culture, the plastic film culture method was used on a large scale. The cell material obtained was deep-frozen until further processing.

Ad 4.2

1 kg of thawed cell material according to 4.1 is suspended in ethyl ether and left to stand in the cold room at -20 ° C for 12 days. The crude extract obtained by centrifugation is filtered before further processing, treated intensively several times with diethyl ether (here always called "ether") and / or chloroform and then dialyzed against a solution containing 0.3% nipagin. The protein-free dialysate is ultrafiltered (FILTRONP): yield 2.4 L.

The analytical data of the amnion extract Ila obtained are as follows:

pH 6.7

Dry residue 1.0%

N content (Kjeldahl) 0.02%

Amino acid positive

Proteins negative

Peptides positive

Breath increase factor 2.0

Sterility sterile

Ad 4.3

The synthetic amnion extract is produced in the following way:

The constituents listed in the following table are dissolved under sterile conditions and with stirring in 300 ml of double-distilled water to prepare partial solution I with a pH of 6.7. Table IX

To prepare partial solution I, the following components are dissolved in 300 ml of double-distilled water (the stated amounts are calculated for a final solution of 1000 ml).

L-glutamic acid 0.3 g

L-histidine 0.06 g L-ornithine 0.08 g

L-asparagine 0.05 g

L-aspartic acid 0.15 g

L-valine 0.05 g

L-tyrosine 0.03 g DL-threonine 0.04 g

L-hydroxyproline 0.2 g

L-phenylaianin 0.04 g

Creatinine 0.01 g

L-methionine 0.02 g L-isoleucine 0.001 g

L-tryptophan 0.02 g

L-leucine 0.05 g β-alanine 0.035 g

Cysteine 0.2 g L-alanine 0.24 g

Glycine 4.0 g

L-proline 0.24 g

L-serine 0.26 g

L-arginine 0.3 g L-lysine 0.3 g

Urea 0.8 g Adenine 0.02 g

Adenosine 0.02 g

Cytidine 0.04 g

Guanine 0.01 g

Cytosine 0.03 g

Guanosine 0.02 g

Hypoxanthine 0.02 g

Inosine 0.1 g

Thymine 0.04 g

Uracil 0.02 g

Uridine 0.03 g

Xanthine 0.01 g

Orotic acid 0.00 'g cyclic AMP 0.04 g

Adenosine monophosphate 0.01 g

Glucose 0.45 g

Sorbitol 4.0 g

Mannitol 0.5 g

Succinic acid 1.5! G

Malic acid 0.01 g

Citric acid 0.1 g

Ethanol 2.0 i ml glycerin 0.4 i nl

Thiamindichlorid 0.002: g Nicotinamide 0.01 g calcium pantothenate 0.002 9 myo-inositol 0.05 g Pyridoxol.HCI 0.6 r ^"ng Biotin 0.1 mg

Tocophenol succinate 0.002 g

Magnesium sulfate 0.05 g

Magnesium aspartate 0.05 g

Manganese gluconate 0.01 mg

NaH ₂ PO ₄ H ₂ O 0.05 g

N-methylglucamine 0.2 g

Sodium lactate, 50% solution 2.0 g

Preservatives p-Hydroxybenzoic acid methyl ester sodium salt 0.2 g

(Nipagin) dehydroacetic acid 3.5 g

To prepare partial solution II, 2 g Collagel Sol (Stoess company) and 2 g partially dehydrated yeast are dissolved in 300 ml bidistilled water with vigorous stirring and treated overnight with 2 g activated carbon. After use and sterile filtration, this batch (partial solution II) is combined with partial solution I and, before filling up to 750 ml with 0.02 mg tripeptides Gly-His-Lys, Gly-Pro-His and Gly-Ala-Lys im Ratio 1: 1: 1 and 0.4 g of glutathione.

Ad 4.4

250 ml of the sterile protein-free amnion extract Ila prepared according to section 4.2 are combined with 725 ml of aqueous synthetic amnion extract according to section 4.3 after adjusting the pH to 6.7 and made up to 1000 ml to form the combined extract 11b.

The analytical data of extract llb are as follows: pH 6.7

Dry residue 1, 72% N content (Kjeldahl)> 0.03

Breath increase factor 2.3

Amino acids positive

Peptides positive

Proteins negative

Sterility sterile

Ad 4.5

A comparison of the efficacy of the amnion extracts llb and lla can be found in Table V. The following test results are given to demonstrate the cosmetic effects in creams in which ILA and ILB have been incorporated:

4% of the amnion extract IIb produced was incorporated into a day cream of the following composition: It is a moisturizing cream of the oil-in-water type:

Cetyl alcohol 3% Myrj 51 3% Arlatone 983 2% Arlamol HD 2% Miglyol 812 6%

1, 2-propylene glycol 3% glycerin 0.5% nipasteril 30K 0.2% amnion extract llb 4% perfume oil 0.2% water ad 00% The oily phase including Miglyol is heated to 75 ° C and homogenized. At the same time, propylene glycol, glycerol and nipasteril 30 K are dissolved in water and heated to 75 ° C.

The emulsification is carried out by adding the water phase to the oil phase with vigorous stirring. The mixture is then kept at a temperature of 5 min with gentle stirring and then cooled to 35 ° C. Amnion extract and perfume oils are added with stirring. All operations must be carried out under strictly sterile conditions.

In a corresponding manner, creams are then produced using Amni-on extract Ila and a control cream without Amnion extract and compared with the Ilb cream in terms of moisture retention:

The Corneometer CM 820 PC was used to measure skin moisture

(Courage & Khazaka GmbH, Cologne) used according to Fig.7a. The device uses the relatively high dielectric constant of water. The sensor according to FIG. 7b has a flat capacitor as the end face.

If the measuring head is pressed onto the skin, the horny layer reaches the scattering area of the capacitor field. A certain capacitance of the capacitor is set in proportion to the water content.

The device was chosen because it delivers a reliable measured value in seconds. Due to the glass vaporization, the measuring head is so robust that it can be cleaned with alcohol. Influences due to contamination are almost impossible.

Carrying out the measurements

Λ. 10 female subjects aged 40 to 76 (X = 58.2; s - 13.0) were acclimatized for 30 minutes at 20 ° C and 65% relative humidity. B. To determine the initial values, 3 measurement results from different places per test field were averaged. The volar sides of the forearms served as test fields. C. After a single application of a consumer-friendly amount of product, the moisture was measured every 15 minutes. The time after which the moisture had dropped back to the initial value was noted. During the entire measurement period, the test subjects sat on special chairs in the climate laboratory. D. A 14-day application test followed. The products were issued with the instruction to use them appropriately for a period of 14 days in the morning and in the evening on the assigned areas.

E. After the end of the application phase, 12 hours after the last application, acclimatization was again carried out for 30 minutes and the moisture was measured.

Results:

a) Skin moisture measurements after a single application: the time in hours after application is shown after the initial moisture has been reached again: cf. Fig. 8 and Table X.

The difference between the control cream without additives and creams with 4% lla and 4% llb is highly significant in the paired t-test [Lit.9] and with the error probability p <0.01.

b) Application test over 14 days: Table XI: The second measurement was made 12 hours after the last application. Table X

Skin moisture measurements after a single application of creams containing amnion extract llb and lla

^* Time in hours after application of the test creams, if the

Initial moisture level is reached again.

Table XI

Skin moisture measurements after 14 days of application of creams containing amnion extract llb and lla

A strong positive effect could be demonstrated for the amnion extract llb in the melanin inhibitor test using Sarcoma B 16 cells. When using 5% llb, a "whitening effect" of +++ (whitening degree: white - greyish white) was found, corresponding to an authentic commercial preparation. [Lit. 19] Example 5

Production of an aqueous placenta extract IIIb by combining an extract from the placenta cell line JAR with a synthetic placenta extract according to EP-A-0 552 516.

5.1. Provision of the cell material starting from the placenta cell line JAR -

Placental cell line ATCC HTB 144. 5.2 Extraction of the placental cell material and processing to protein-free placental extract purple 5.3. Preparation of the synthetic placenta extract analogous to EP-A-0 552 516 5.4. Combination of the two placenta extracts from 5.2 and 5.3 to lllb 5.5 test results with purple, lllb and lllc (placenta extract from fresh placenta).

Ad 5.1.

The cell line JAR served as the starting material for the production of IIIb. Epithelium-like morphology.

Medium: RPMI 1640 + 10% FCS. Split: 1: 4-6. Bibliography: "In Vitro", 6, 398 (1971)

Filing: Paolo Paolucci, Lb. Immunologia-Citogenetica e Biologia Cellular, III Clinica Pediatrica Osp. S. Orsola-Malpighi, 40138 Bologna, Italy.

The procedure was as described in Example 1.1. With regard to large culture, the plastic film culture method was used. The cell mass obtained was deep-frozen until further processing.

Ad 5.2.

10 kg of thawed cell material from 5.1. are suspended in ethyl ether and left to stand in the cold room at -15 ° C for 2 weeks. The crude extract obtained by centrifugation is filtered before further processing, intensively etherified at least three times and then contained against a 0.3% nipagin. solution dialyzed. The protein-free dialysate is ultrafiltered (FILTRON ^® ) yield 35 L: purple. The analytical data of the placenta extract obtained are: pH 6.5, dry residue: 0.9%, N: 0.08%; Amino acids: positive (TLC: 18 amino acids); Proteins: negative; Peptides: positive, respiratory increase factor: 1, 8; Sterility: sterile.

Ad 5.3.

The synthetic placenta extract was prepared according to EP-A-0 552 516.

In the event that the synthetic placenta extract produced according to Example 1 of EP-A-0 552 516 is to contain alkaline phosphatase, the amount of 0.03 g of alkaline placenta phosphatase can be added per liter of final volume. In order to guarantee freedom from BSE in this case, too, the phosphatase must be isolated from placentas which come from herds in which no BSE has occurred (preferably from countries such as New Zealand or Brazil).

The specified amount of phosphatase brings the synthetic placenta extract to a King Armstrong value of more than 180 U.

A suitable regulation for the production of alkaline phosphatase from bovine placenta and the stabilization of the phosphatase by magnesium salts such as magnesium gluconate, magnesium acetate and the like have R. Riem-Schneider and V. Schulz in [Lit. 16] developed.

The importance of alkaline phosphatase for cosmetics is controversial. According to the relevant literature, the content of alkaline phosphatase was evaluated as a criterion for the lege artis production of placenta extracts, but not as a characteristic for the cosmetic properties of placenta extracts that are used as cosmetic additives [Lit. 17]. Ad 5.4.

30 L of the protein-free placenta extract purple prepared according to 5.2 are mixed with 90L aqueous synthetic placenta extract from 5.3. combined, after pH adjustment to 6.7 at 0.2 μm, filtered as extract IIIb.

Analytical data from IIIb: pH 6.7; Dry residue: 1%.

N: 0.1%; all other dates like purple.

Ad 5.5.

Test results of the image analysis with emulsion preparations which produced the placenta extracts purple, IIIb and IIIc (placenta extract from fresh placentas, analogously to Example 3, page 38, lines 5 to 20) in amounts of 1%, 1 , 5% and 2% included.

The image analysis allows an assessment of the influence of the surface structure of human skin by the emulsion preparations containing placenta extract.

35 subjects between the ages of 39 and 61 were pulled down for the test. They were instructed not to use any other cosmetics on the test areas (forearms) 4 days before the start of the test and for the duration of the test. For each subject, 12 test fields were marked on both forearms (6 per arm). The areas were assigned in such a way that the specimens were distributed equally frequently to 35 test subjects in the test fields (uniform permutation). In addition to the test emulsions, an empty field was also checked. After the blank values of all areas had been determined, the subjects themselves used the preparations over a period of 2 weeks, morning and evening. 4 hours after the last application, the image analysis was carried out.

Method: After the subjects had acclimated for 45 minutes at 22 ° C. and 65% relative atmospheric humidity, slides coated with a silicone material (layer thickness 500 μm) were pressed onto the test areas. The impression mass cured within 2 min. The same procedure was repeated after 2 weeks of use. The impressions obtained in this way were evaluated under the transmission light microscope using image analysis. The image drawn into the image computer via a TV camera is electronically reshaped so that the area of the profile lines of an image appearing dark in the microscope (corresponding to parameters FA = area area, area profile area, profile line area) and their length (as total length) are in this Have the image (parameter LL = line length) determined.

This image analytical method captures relatively macroscopic surface properties of the skin, namely the changes in the profile lines as they occur during drying and moistening processes. Depletion of the profile, ie a decrease in FA and LL indicate skin dehydration, while the opposite effect occurs with improved moisture penetration. The profile becomes richer in profile lines. 4 hours after the last application, an overlapping "smearing effect" can be excluded for the products, so that the actual skin profile was recorded and measured at the respective time. The percentage change in FA and LL is shown in FIG. 9 as a function of the concentration of the preparations. In order to ascertain sufficient data that can be statistically evaluated, 5 images were taken from each skin imprint (at different locations), so that sufficient FA and LL data per residual substance concentration were determined at the time "untreated" and also at the time "treated" and saved (more than 6000 data for 3 preparations). Since the data often do not meet the conditions of normal distribution, the untreated data per product were checked in pairs in a Wilcoxon test for significant differences. For the parameters FA and LL, mean values and standard deviations were determined for all 35 test subjects per product. The percentage change from "untreated" to "treated" was then calculated and the values corrected with the blank value were determined. The formulations with 1, 5% and 2% placenta extract IIIb show a clear improvement in the skin structure. The FA data are better differentiated: (Fig. 9) A placenta extract based on a bovine placenta cell line RPL 10 has also been produced in an analogous manner, which is grown and deposited with Prof. Dr. Mariano da Rocha Filho, Reitor da UFSM (Universidade de Santa Maria), Faculdade de Medicina, Santa Maria, Rio Grande do Sul, Brazil, and Dr. G. da Rocha Filho, UFSM, Bioquimica, Istituto Central de Quimica, Santa Maria RS, Brazil.

Example 6 Production of an aqueous thymus extract by combining an extract from cell line P1-1A3 (thymus epithelial cells DSM ACC 280) or a cell line from calf thymus (A. Wank) with a synthetic thymus extract analogous to EP-A-0 552 516

6.1. Provision of the cell material, starting from the thymus cell line P1-1A3 or from the thymus cell line Kalb WANK

6.2. Extraction of the thymus cell material and processing to a protein-free thymus extract IVa

6.3. Preparation of a synthetic thymus extract IVb according to EP-A-0 552 516

6.4. Combination of the two thymus extracts from 6.2 and 6.3 to extract IVc

6.5. Test experiments with the thymus extract IVc obtained under 6.4 and comparison with synthetic thymus extract IVb and with thymus extract IVd from fresh calf thymus glands.

Ad 6.1

The was used from the starting material for the production of the thymus extract IVa

Cell line P1-1A3, thymic epithelial cells DSM ACC 280.

The culture medium required is 80% RPMI + 20% FCS [details can be found in "Blood" 83, 3245-3254 (1994) "].

In October 1973, a 2-2.5-month-old calf was used to breed the calf thymus epithelial cells WANK in Brazil. Culture medium analogous to P1-1A3.

The procedure was as described in Example 1. With regard to large culture, the plastic film culture method was used on a large scale. The cell material obtained was frozen until further processing. Yield in 5 weeks: 25 kg mass.

In the case of working with the calf thymus cell line (bos taurus) WANK, the procedure was completely analogous, yield 30 kg. Origin of the named cell line: Hwa Sook Chang and A. Wank, Bethesda, Md. 20014, Clarandon, USA, and N.M. Faria, Pc. Carlos Gomez, 10000 S. Paolo, SP, Brazil. Prof. Dr. Dr. R. Riemschneider, Istituto Central de Quimica da UFSM, Santa Maria, RS, Brazil.

Ad 6.2 25 kg of thawed cell material from P1-1A3, prepared according to 6.1, were suspended in ethyl ether and left in a cold cell at -15 ° C for 10 days. The crude extract obtained by centrifugation is filtered before further processing, thoroughly etherified and then dialyzed against 0.3% nipagin (p-hydroxybenzoic acid methyl ester sodium salt) in water. The protein-free dialysate is ultrafiltered (FILTRON * '): yield 30 L.

The analytical data of the thymus extract IVa obtained are as follows: pH 6.8

Dry residue 2.8%

N (Kjeldahl) 0.6%

Amino acids (ninhydrin) positive and TLC

5 peptides (biuret) positive and TLC

Nucleic acid components positive TLC (9 components detected) metabolic activity

(Warburg factor)> 2.3

) Heavy metals <10 ppm

As <2 ppm

Sterility sterile

Shelf life> 2 years acute toxicity in mice (60 animals)

DL ₅₀ iv> 20 ml / kg

DL ₅₀ ip> 30 ml / kg

DL ₅₀ po> 25 ml / kg Chronic toxicity study in albino rats (40 days): no toxicity

) Toxicity in dogs (10 beagle

Dogs, 26 weeks): no toxicity

Ad 6.3

The synthetic thymus extract IVb is prepared as described below:

150 g of methyl p-hydroxybenzoate in 0.7 L of ethanol (94%) and 20 g of benzyl alcohol are dissolved or diluted to 20 L in double-distilled water. The following are added with stirring and under sterile conditions: Table XII

Glycine 80 g

L-alanine, L-serine, L-lysine HCI,

L-proline, L-arginine-HCl 200 g each

DL-threonine, L-histidine, L-asparagine 40 g each

L-methionine 2 g

Urea 200 g pre-dissolved in 2 N NaOH:

L-aspartic acid, L-leucine,

L-tyrosine-L-phenylalanine,

L-lysine 10 g each

L-glutamic acid, L-valine 60 g each

Citric acid 200 g

Malic acid 10 g

Succinic acid 250 g pH adjusted to 6.8

Sodium dihydrogen phosphate 20 g

Sodium lactate, 50% 700 g

Glycerin 200 g

deodorized peptone solution made from

1000 g peptone, which was dissolved in water and treated with 500 g activated carbon for 24 h, after filtration approx. 25 L solution

Glucose 400 g

Inosine 10 g

Glutathione 25 g

Adenine, adenosine, guanosine, thymine, cytidine, cytosine, uridine, uracil, cyclic AMP, GMP, ADP, Thiamine dichloride 0.5 g each

Zinc acetate, magnesium acetate, cobalt gluconate, manganese gluconate 0.1 g each

Chondriosome extract from yeast (GfN, Wald-Michelbach) 100 ml

The following peptide solutions were added to this basic solution, which has a volume of approx. 45 L:

Oligopeptide solution with 0.05 g Gly-Pro-His and Gly-His-Lys each

Thymus factor fragment:

Ala-Lys-Ser-Glu-Gly-Gly-Ser-Asn 0.02 mg

The pH of the solution obtained was then adjusted to 6.8 and the batch was made up to the final volume of 100 L with distilled water. The pH of the solution was then adjusted again to pH 6.8 and sterile filtered through 0.2 μm Millipore "'or a corresponding PALL candle.

The thymus extract IVb obtained had the following key figures:

Appearance: clear solution

Color colorless to pale yellow pH 6.8

Dry residue 5.0%

N 0.7%

Amino acids positive

Peptides positive

Metabolic activity

Breath increase factor> 2.3

Sterility sterile Ad 6.4

25 L of the sterile, protein-free thymus extract IVa produced according to Section 6.2 are combined with 100 L of aqueous synthetic thymus extract IVb and, after adjusting the pH to 6.8, are sterile filtered at 0.2 μm.

The analytical data of extract IVc are: pH 6.8

Dry residue 4.4%

N 0.6% respiratory increase factor> 2.3

Amino acids positive (ninhydrin) and TLC;

Peptides positive (biuret)

TLC nucleic acid components (more than 8 components)

Heavy metals <10 ppm sterility sterile

Pyrogenicity pyrogen free (LAL, rabbit)

Shelf life 3 years

Ad 6.5 Test trials with thymus extract IVc from 6.4 and comparison with synthetic thymus extract IVb from 6.3 and comparison with thymus extract IVa from 6.2 and comparison with natural thymus extract from thymus glands IVd

The cosmetic effectiveness of the thymus extracts to be tested was determined using the Padberg method. The following were tested: a W / Ö cream with 2% thymus extract IVc, a W / Ö cream with synthetic thymus extract IVb, a cream with thymus extract IVa and a cream with natural thymus extract IVd and, as a control , a cream without added thymus extract. The Padberg test was carried out as follows: Mark the test site on the skin

Pretreatment of the skin area with a 1% solution of a nonionic surfactant to impair the skin ("subjectively rough skin")

Wash the test site with 37 ° C warm water. Wipe the test site

Acclimatize the subject at 20 ° C and a relative humidity of 60% for a period of 45 minutes. Apply 20 ml of a stain solution containing 20% 0.5% methylene blue and 80% 1% nonionic surfactant

The stain solution acts on the test site for 30 seconds

Dry the dye for 1 minute

Removal of excess dye with a 0.2% solution of the nonionic surfactant used, eluting twice, in each case for 60 seconds, with 2 ml portions of a sodium laurate solution (2.3% sodium laurate, 48.7% pure water, 49% isopropyl alcohol) Determination of the absorption of the eluate at 660 nm with a.

spectrometer

The test treatment was carried out by applying a sample twice a day for 20 days. It was ensured that the test subjects both 3 days before the test and during the. Test period had not used any other ^' cosmetic. On the 21st day after the start of the treatment, the above-mentioned process steps including the elution were carried out 4 hours before the final evaluation. Table XIII below shows the respective ratio of the absorption of

Methylene blue before and after application for each person for W / Ö-

Creams listed in%, with all 4 thymus extract-containing creams with the

Control cream were compared.

The amount of methylene blue absorbed is proportional to the roughness of the skin and the percentages listed in the table were determined according to the following equation:

Absorption after application skin roughness% = x 100,

Absorption of untreated skin assuming that a skin treated with a cream only absorbs a small amount of methylene blue. The mean values for 22 test persons showed that all 4 thymus extracts showed good roughness values compared to the control cream (column V), the formulation IVa (column I) of a thymus extract from thymus cell lines according to the invention being one had optimal value. The synthetic thymus extract (IVb) (column II) can be improved in its cosmetic effectiveness by combination with the cell line extract.

Table XIII: Padberg data

Column I: W / Ö cream with 2% thymus extract IVa, made from thymus cell line P1-1A3 Column II: W / Ö cream with 2% synthetic thymus extract IVb

Column III: W / Ö cream with 2% thymus extract IVc, produced by combining extracts IVa and IVb. Column IV: W / Ö cream with 2% thymus extract IVd, made from fresh beef thymus glands. Column V: W / Ö cream without the addition of a thymus extract (control cream)

For the detection of the radical scavenging effect of thymus extract IVc cf. 12 and the experimental description in Example 10 below. Example 7

Production of an aqueous blood extract by combining a synthetic blood serum extract with the extract of the cell line B95 8

7 1 Preparation of a synthetic blood serum extract

7 2 Production of a blood extract from cell line B95 8

ATCC CRL 1612 ECACC 8501 1419 to cell mass

7 3 Extraction of the cell material obtained under 7 2 to the blood extract

7 4 Combination of the extracts obtained under 7 1 and 7 3 to the blood extract V

7 5 Trials with blood extract V

Ad 7 1

Preparation of a synthetic calf blood serum extract (100 L batch)

40 g of methyl p-hydroxybenzoate sodium salt are dissolved in 40 L of double-distilled water (pyrogen-free), then the following components are then added with stirring and gassing with sterile nitrogen

Table XIV

Adenine 0.3 g

Adenosine 8.0 g

Uracil 0.3 g

Uπdin 0.2 g cycl AMP 0.4 g cycl GMP 0.2 g

ADP 0.1 g

GDP 0.1 g

Cytosm 0.3 g

Cytidine 0.1 g

Thymine 0.5 g Guanine 0.2 g

Guanosine 40.0 g

Hypoxanthine 0.2 g

Inosine 0.2 g

Glycine 12.0 g

L-alanine 60 g ß-alanine 14 g

L-lysine-HCI 30 g

L-arginine-HCI 24 g

L-serine 30 g

L-proline 20 g

L-cysteine 0.7 g

Peptone 100.0 g α-aminobutyric acid 28 g

L-Asparagine 10 g

L-iso-leucine 14 g

L-hydroxyproline 10 g

L-methionine 18 g

D, L-threonine 10 g

Glucose 100 g

Fructose diphosphate 2 g

Glucose-6-phosphate 2 g

Sorbitol 100 g

Mannitol 100 g

Urea 30 g

uric acid

(pre-dissolved in hot 2N NaOH) 11 g pre-dissolved in 2N NaOH the following amino acids:

L-glutamic acid 60 g

L-Aspartic acid 1, 6 'g

L-tyrosine 12, C »g

L-phenylalanine 14, C> g L-leucine 30.0 g

L-valine 30.0 g

Hippuric acid 2.6 g p-aminobenzoic acid 3 g

Creatinine 1.0 g pre-dissolved (extra): + 50 g L-tryptophan in 2N NaOH then addition of acids

Citric acid 140 g

4-hydroxybenzoic acid 15 g iso-citric acid 4 g

Malic acid 40 g α-ketoglutaric acid 10 g

Ethanol (94%) 400 ml

NaCI + 500 g Na ₃ PO ₄ .12H ₂ O 1900 g

Na ascorbate 30 g

Sodium phosphate NaH ₂ PO ₄ 10 g

Chondriosome extract (from yeast) 100 g

Succinic acid disodium salt.6H ₂ θ 280 g

Sodium lactate (50%) 1000 g

Trace elements are introduced by later filling up with pyrogen-free tap water, which has previously been filtered through 0.2 μm Millipore ' ^h . Serum albumin fragments Arg-Gly and Arg-Gly-Val-Phe-Arg each 0.4 mg, glutathione 1, 2 g.

The extract solution obtained is adjusted to a pH of 7.1 and made up to the final volume of 100 L with water. After a further pH check, the product is filled under N ₂ and pyrogen-free: 0.1 μm PALL candle.

Analysis of the synthetic blood serum extract showed: pH 7.1 N: 1.15 mg / ml; Dry residue: 4.3%; Breath increase factor>2.2; Peptide detection: positive, sterility sterile, pyrogen free Ad 7.2.

Production of a blood extract from cell line B95.8 in suspension culture. The starting point is the cell line B95.8, obtained from Italy via G.B. Ferrari and Luigi Chieco-Bianchi, Istituto di Oncologia, Università degli Studi, 16132 Padova, Italy.

B 95.8 - ATCC CRL 1612 ECACC 8501 1419: Blood, Species: Strain Marmoset, Monkey. Medium: RPMI 1640 + 10% FCS + L-glutamine + Na pyruvate + non-essential amino acids + 2-mercaptoethanol. Split 1: 5. Literature: "Proc. Natl. Acad. Sei. USA", 7_0, 190-194 (1973).

Starting from the frozen cell culture of line B95.8, this is first thawed in a water bath at 30 ° C and transferred to a 15 ml tube. 10 ml of preheated culture medium (see above) are added and mixed with it. Centrifuge at 200 xg for 5 min. Resuspension of the cells in the defined medium and determination of the number of cells. Wash the cells and resuspension in freshly prepared medium and incubate as prescribed: 37 ° C and 5% CO ₂ . The further propagation then takes place according to the rotary shaker technique up to a volume of 500 ml, later in suspension bottles from Bekkico Glass Ind. Up to 25 L, then in fermenters or pipe coil cultivators up to 1000 L.

The harvested cell mass is kept briefly at -10 ° C. until extraction. Yield 15 kg in about 4 weeks.

Ad 7.3.

Extraction of the under 7.2. obtained cell material to the blood extract.

15 kg of thawed cell material are left in 30 L pyrogen-free water, which contains 0.2% p-hydroxybenzoic acid methyl ester, for 3 weeks at -15 ° C, after thawing, filtered, repeatedly etherified, dialyzed and ultrafiltered: Filtron ^® (Centrasette, Type OMEGA, NMWL 8k). After adjusting the pH to 7.1, the mixture was concentrated in vacuo at 20 ° C. to a dry residue of 4.0-4.2%. Yield: 15 kg.

The data of the extract filtered at 0.1 μm pyrogen-free are: pH 7.1; N 1.17%; Dry residue 4.0%; Sterility: sterile; Pyrogenicity: pyrogen free. Toxicity: all tests in Table II negative.

Ad 7.4

Combination of the extracts obtained under 7.1 and 7.3 to form blood extract V.

15 kg of the blood extract from cell line B95.8 produced according to 7.3 are combined with 100 L of the synthetic blood serum extract from 7.1 and, after adjusting the pH to 7.1, are filled sterile and pyrogen-free with a 0.1 μm Pall candle.

V's analytical indicators are:

pH 7.1; N: 1.15%; Dry residue: 4.2%;

AT factor (Warburg)> 2.3; Peptide detection: positive: amino acids: ninhydrin positive and TLC; (see Fig. 10). Heavy metals: <10 ppm; As: <2 ppm; Sterility: germ-free Pyrogenicity: pyrogen-free (rabbit test, LAL test)

Procedure for performing two-dimensional thin-layer chromatography for amino acids (TLC):

Principle: two-dimensional TLC on silica gel plates from Merck Solvent: a) n-propanol: ammonia, 25% = 7: 3 (v / v) b) n-butanol: acetic acid: water = 4: 1: 1 (v / v) Method Each 2x5 μl test extract, eg BV, is applied with intermediate drying to a DC finished plate (silica gel 60, F ₂₅₄ , 20 x 20 cm) and air-dried. The two plates are developed in one chamber

The first solvent is n-propanol / 25% ammonia solution (7 3 vol / vol), drying height 17 cm overnight under the hood

The plate is turned 90 °, n-butanol / glacial acetic acid / water (4 1 1 vol / vol) is used as the second solvent, drying height 17 cm overnight under the hood

Spraying with Ninhydπn (visualization) Each plate is sprayed with 0.1% ninhydnn spray solution. The sprayed plates are first pre-dried for 30 min at room temperature and then the color development is accelerated for 10 min at 60 ° C labeled

0.1% solution of ninhydrin (Merck Art 6758 or the following solution) 0.1% solution of ninhydrin in a solvent consisting of 97% by volume n-butanol and 3% by volume glacial acetic acid

Ad 7 5

Test experiments with the aqueous blood extract V (from 7 4)

7 5 1 Influence of V on the growth of damaged filbroblasts

The study of the influence of blood extract V on the growth of damaged fibroblasts by the method of W Fraefel, research laboratory of Solco - Basel, showed

The thymidine incorporation rate was measured over a period of 6 days in culture groups with a defined number of cells Control group I: undamaged fibroblasts and epithelial cells, group II: fibroblasts and epithelial cells damaged with CHOH (reversibly damaged), group with CHOH damaged fibroblasts and epithelial cells treated with the addition of synthetic serum extract,

Group IV: damaged with CHOH and by adding V.

Results

Table XV

Group Thvmidine installation rates 1 x 10 ³ / min

Tao O day 2 day 4 day 6

I control 0 35 200 450

II 0 3 30 30

III 0 25 90 350

IV 0 30 120 410

The values given represent mean values from 10 experiments each. They make it clear that the fibroblast culture damaged by CHOH could be adapted with the additives in group III and IV in terms of their DNA synthesis rate to the culture group I serving as a control.

7 5 2 Influence of V on the growth and metamorphosis of tadpoles (Xenopus laevis DAUDIN)

The growth of tadpoles was observed from day 1 (spawn deposited by the frog pair) to day 67 and measured from day 3 after the free-floating tadpoles were obtained. Water temperature 24 ° C. On day 7, the tadpoles were divided into 5 groups, to carry out a test with blood extract V and 3 controls. Each test group comprised approximately 100 tadpoles

The amount of water was increased to 2.5 L by day 21, to 5 L by day 35 and to 7.5 L per group from day 36 to 67 aeration by means of commercially available porous aquarium stones. Water change of the pools every 7 days of food from day 7 nettle powder, previously liquifry

Add V per week in 3 portions, amount 0.2% length measurements not noted here

Start of metamorphosis at V on day 51, in the controls only from day 56 table XVII

The frogs of Xenopus laevis are camivores and get fresh liver as feed

Table XVI Metamorphoses and Frogs, d 51-67

First number: number of frogs Second number: number of metamorphoses

(The metamorphosis has ended when the tail has been completely or almost completely absorbed) 1, 2: controls

3, 4: blood extract V

7.5.3 Report on the influence of blood extract V on the growth of guppy fish from day 75 to 100.

All of the guppy fish used were the same age.

115 guppies were tested in 5 groups of approx. 22 to 24 each in 5 L aquariums,

Water temperature 24.0-24.6 ° C.

The samples to be tested were added every other day.

Amount per week is given in the table.

The results of the length growth x in mm after 100 days (start of experiment on day 75) are summarized in Table XVII. Table XVII

Mean values d 100

7.5.4. For the radical scavenging effect of blood extract V cf. 12 and for the description of the methodology, see Example 10 below.

Example 8

Production of a plant extract VI from the Acer pseudoplatanus cell line (Colla 120) and use as a cosmetic additive in the sense of a collagen replacement (see also Table VI above)

The starting material is an Acer pseudoplatanus cell culture (origin: Botanical Institute of the UFSM, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil. The Acer pseudoplatanus cell culture was derived from callus tissue, which was originally derived from explants secondary cambial tissue of the Acer pseudoplatanus tree).

Cultures of Acer pseudoplatanus were grown in suspension as described by Stoddart, Barrett and Northcode in "Biochemical Journal", 102, 194 (1967). The tissue in suspension was harvested by centrifugation and washed with water. The culture medium was a modified White's medium, which contained the following components in g / L

Ca (NO ₃ ) ₂ 0.2 g

MgSO ₄ 7H ₂ O 0.36 g

KCI 0.055 g

KNO ₃ 0.08 g

Na ₂ SO ₄ 0.2 g

MnSO ₄ 4H ₂ O 0.009

ZnSO ₄ 7H ₂ O 0.003

H ₃ BO ₃ 0.003

KJ 0.0015

NaH ₂ PO ₄ H ₂ O 0.0165

Fe ₂ (SO ₄ ) ₃ 6H ₂ O 0.0025

Thiamin.HCI 0.0001,

Ca pantothenate 0.0001

Glycine 0.003

Cysteine HCI 0.001 g

Sucrose 20.0 g with coconut milk 20% (deproteinized [15 min 15 at] and sterilized)

For further extract production, it is advisable to refer to the scheme in Table VI above

The cells are first propagated using the rotary shaker technique, then in a number of Bekkino glass suspension culture bottles of 10 L, then on a large scale in fermenters (1000 - 4000 L) or pipe coil cultivators (1000 - 2000 L).

The harvested cell material is dried and suspended in batches of 10 kg of water and processed in a kilowatt ultrasound generator which delivers a strong oscillation The centrifuge residue obtained contains the desired cell wall material, which serves as the basis for the following enzymatic and chemical hydrolysis processes.

After 4 days of treatment with 10% cellulase at 35 ° C., the mixture is centrifuged: residue I is stored at 4 ° C. for further treatment. The supernatant solution I is brought to a concentration of 2 N with sulfuric acid and autoclaved for 4 days at 15 at for partial hydrolysis. The separation leads to residue II (stored like I at 4 ° C.) and solution II, which is brought to pH 5.3. Working temperatures at 4 to 10 ° C

The residues I and II are partially hydrolyzed in an autoclave in 3N HCl and then combined with the hydrolyzate II (from I) and dialyzed.

Concentration of the dialysate in vacuo at 20 ° C. to about 10% dry residue, adjustment to pH 5.3. Sterile filtration through a 0.2 μm Millipore ' ^a filter.

The resulting protein-free cosmetic additive VI (Colla 120) has the following analytical key figures:

pH: 5.3, dry residue: 10.5%; N: 0.95%; Amino acids: 4.5% (after hydrolysis); Hydroxyproline: 0.3%; Carbohydrates: 5.2% (after hydrolysis); Metabolic activity: 1, 8 (Warburg factor): Sterility: germ-free; Preservative: 0.3% Phenonip; Proteins: protein free.

If necessary, the dry residue can be increased to 25% by adding 5% mannitol and 10% panthenol, which improves the cosmetic effects of the skin's moisture retention capacity and the radical scavenging effect of extract VI (cf. also Fig. 12). The acer pseudoplatanus extract VI with 10.5% dry residue, hereinafter called "Colla 120", was used in a number of cosmetic formulations and compared with a collagen preparation.

1) Skin moisture measurements after a single application and after 14 days of application of cream containing VI (corneometer)

2) Skin roughness measurements using the modified PADBERG method

3) Influence of an oil-water emulsion containing VI on the skin temperature of test subjects with dry skin 4) Skin conditioning by formulations containing VI 5) Patch test for testing for allergic effects.

Ad P

Colla 120 (VI) was incorporated 5% into the following recipe: Phase I melting to 70 ° C: C: 10% Cetiol V, 10% Lanette N; 5% miglyol

812, 2% Adeps lanae; 0.3% Pheπonip.

Warm phase II to approx. 70 ° C, containing: 0.3% Phenonip;

5% Karion fl., Karion F; 62.3% water,

Stir phase II into phase I, continue stirring until the mixture has cooled to 32 ° C., then stir in Colla 120 5% (VI) and perfume oils and homogenize.

Analogously produce a cream with 5% collagen (1%), as well as a control cream without the addition of an additive.

The measurements, carried out analogously to Example 4.5, gave the following for the moisture retention capacity: after a single application, skin moisture retention in tested hours

Base (control cream) 4.5 base + 5% VI Colla 120 6.5 base + 5% collagen 6.3

after 14 days of application: improvement in skin moisture in% tested

Basis (control cream) 8.4 Basis + Colla 120 VI 5% 22 Basis + Collagen 5% 19

Ad 2)

Description of the Padberg method

Padberg method:

In the Padberg test, the roughness of a skin is assessed based on the amount of methylene blue that is absorbed by the skin depending on the skin conditions. Studies with "subjectively rough skin" or with "skin damaged by tenside" showed that the amount of methylene blue absorbed is proportional to the roughness of the skin. In contrast, good skin or well-cared for skin only absorbs a small amount of methylene blue.

Ten test subjects between the ages of 40 and 66 years were used for the test. The test subjects volunteered for this test. You were instructed not to use other cosmetics on the test areas (forearms) three days before the start of the test and for the entire duration of the test. For each subject, six test fields were marked on both inner sides of the forearm (three per arm). The areas were like this assigned that the products were equally distributed among the ten test subjects across the six test fields (uniform permutation). In addition to the five emulsions, an empty field was also checked. After the blank values of all areas were determined using both methods, the test subjects applied the products themselves at home in the morning and evening for a period of 14 days. After that, 4 hours after the last application it was checked again for Padberg.

The following steps are used to carry out the Padberg test: (1) Marking the test fields

(2) Pretreatment of the test fields with a 1% solution of a nonionic surfactant in water

(3) Rinse with tap water at 35 ° C

(4) Dab the test fields (5) after acclimatizing the test persons for 30 minutes at 22 ° C and 60% relative humidity (6) Apply 20 μl of color solution (the color solution consists of 0.5% methylene blue (20%) and 80 % of a 1% solution of a non-ionic surfactant) and 30 s (7) Allow the paint to dry for 30 s

(8) Remove the excess color with 2 ml of a 0.25% nonionic surfactant solution

(9) Extraction with 2 x 2 ml of a mixture of:

2.0% sodium lauryl sulfate 48.0% water

50.0% isopropyl alcohol after 60 s each

(10) Measure the eluate in the spectrophotometer at 660 nm against air

(11) Result = skin absorbance (initial value) (12) The products were then issued with the instruction to apply them to the designated areas for 14 days in the morning and in the evening. It was previously ensured that no other cosmetic products were used on the test areas 3 days before the start of the test and during the entire test period.

(13) Repeat steps (1) through (9) on day 15. The last application before the test was made 4 hours earlier.

Result = absorbance after application

(14) _Absorbance after application _x ^ _{∞ m RaU} higkeιt <%> Extinction skin baseline

Discussion of the Padberg method:

The Padberg method provides data on the adsorption of methylene blue on the skin. Depending on the skin condition, the dye has more or fewer binding sites on the skin. Years of experience have again and again confirmed that subjectively "rough" skin or, for example, skin damaged by detergents has more binding sites for methylene blue, depending on the degree of damage. Conversely, a skin improved with creams shows a decrease in the binding sites and in proportion to the duration of use. The differentiation for different products is very good and reliable; here too, correlation is always achieved with the subjective assessment. For more details, see [Lit. 10].

Results of the Padberg test:

The photometer values, expressed as a percentage of the value for treated skin and based on the initial state, are summarized in Table XVIII below. The calculated mean values are also given in the table.

The results show that smoothing of the skin can be achieved with the test emulsions. Table XVIII

Roughness test using the Padberg method with water / oil emulsions with and without the addition of tripeptides (application time 14 days)

Age of the water-oil emulsions of the invention

Subjects Placebo Colla 120 (VI,

46 81, 0 50.1 60.0

41 91, 0 64.4 64.9

4,477,800.00 76.5 40.0

50 76.6 33.0 41.2

51 75.3 56.8 70.0

40 95.0 37.5 40.5

65 89.0 72.5 68.0 6 666 9 977., 88 57.8 55.0

62 92.6 70.0 60.0

45 76.9 60.7 62.7

Average: 85.6 58.2 56A

Ad 3)

Influence of a Colla 120 extract (VI) (produced according to Example 8) - oil / water emulsion on the skin temperature of test subjects

The influence of the emulsion on the skin temperature was tested on 30 test subjects between the ages of 40 and 60 with dry skin. The results are shown in the following table Tab. XIX.

Table XIX

Temperature (° C) oil-water emulsion temperature (° C) before application after application ΔT

30.6 - 33.0 Colla 120 (VI) 32.7 - 32.9 0.2 30.5 - 34.0 control emulsion 30.5 - 33.6 3.1 Without Colla 120 (VI) there was no noticeable stabilization (small ΔT value) of the skin temperature, hence a high ΔT value when using the control emulsion.

Ad 4)

A hand cream containing lanolin was formulated according to the following instructions, in which Colla 120 (VI) was incorporated. The final composition of the cream formulation was as follows:

soluble lanolin derivative 1.0% by weight acetylated lanolin alcohols 5.0% by weight liquid multisterol extract 5.0% by weight

Vaseline 5.0% by weight

Polyvinyl alcohol gel 0.75% by weight 10% NaOH 2.25% by weight

Colla 120 (VI) 6.0% by weight of ethoxylated fatty amines 3.75% by weight

Perfume oil 0.3% by weight

Water, double dist. ad 100.00% by weight

To make this hand cream, the fat phase components and petroleum jelly were heated to approx. 75 ° C. At the same time, the PVA gel was dispersed in hot water at approx. 80 ° C and gradually dissolved. The ethoxylated fatty amine (with about 25 EO units) and, if appropriate, an emulsifier were added to the latter dispersion, after which the previously heated fat phase was emulsified therein. After stirring for 5 minutes at the mixture temperature, the mixture was cooled to 60 ° C., then 10% sodium hydroxide solution was added and the mixture was cooled further to below 40 ° C. 6% Colla 120 (VI) and the perfume were then added with slow stirring. The approach was well homogenized. The hand cream was made up stably and showed an improved skin conditioning, compared to an approach without Colla 120 (VI). A baby shampoo was made from the following composition:

Sulfosuccinic acid half-ester sodium salt, diluted (Steinapol SBFA 30 and

Steinapol SBZ) 700 g

Alkyl ether sulfate 10 g

Colla 120 (VI) 50 g

Gum arabic 5 g surface fat 10 g

Water 275 g

Potassium sorbate 1.5 g

Manganese acetate 1.0 g

The pH of the mixture was adjusted to a final value of 6.5.

A cream base was compounded as follows:

Vaseline 40% by weight

Cetanol 18% by weight sorbitan sesquioleate 5% by weight

Polyoxyethylene lauryl ether 0.5% by weight p-hydroxyalkyl benzoate 0.2% by weight

Colla 120 (VI) 5.0% by weight

Water ad 100.00% by weight

Ad 5)

Clinical allergological examination of Colla 120 extract VI in a patch test on

People

Summary: Colla 120 has been tested in a patch test for allergic potential. Local tolerance was assessed in a preclinical examination clarified. This test showed no concerns about the use of the product in a human clinical trial (see also Table II above).

The clinical examination in the patch test did not raise any concerns for the question of the use of Colla 120 in the face of humans from an aliergological point of view, since no reaction occurred in the 60 tested subjects.

Test method: This was carried out under the conditions of an epicutaneous lobule test. A small amount of test substance is applied to hygroscopic test platelets and attached to the back skin of the patient by means of adhesive plaster, which was tested in the clinic's aliergological laboratory because of the suspicion of contact allergy. After 24 hours, the platelets loaded with the test substance were removed and the local skin reaction at the contact point of the test platelets was read half an hour after the test plasters had been removed. Further readings were made after 48 h and 72 h: Table XIX

Evaluation: 0 = negative, (-) = questionable positive, + = weak positive, ++ = positive,

+++ = very positive. The test is carried out under an extreme load which is not given under normal conditions.

Table XX

Results: Colla 120 was tolerated without reaction by all 60 patients in the patch test when used undiluted.

As can be seen from the test protocol, 20 of the 60 people tested proved to be contact allergic to various allergens of everyday life and working life.

Example 9

Production of a protein-free yeast extract VII from partially dehydrated cells from Saccharomyces cerevisiae Hansen.

10 kg of an approx. 20% suspension of freshly grown yeast Saccha¬ romyces cerevisiae in water (so-called yeast milk) are mixed with 200 g of metabolically active extract VII (as a starter) and with 150 g of a mixture of 114 g of NaCl, 20 g MgSO ₄ .7H ₂ O, 1 g of activator and 15 g of cane sugar, with gentle stirring in a dough machine, rapidly heated to 65 ° C. and cooled to 15 ° C. after about 100 minutes: Vll crude product. In the cold, dialysis is carried out against a 0.2% strength p-hydroxybenzoate, sodium salt solution. The amount of water is chosen so that the solids content of the dialysate is 4%.

After deodorization with activated carbon and sterile filtration of the dialysate, a protein-free yeast extract VII is obtained, which has the following analytical parameters (a) and properties (b):

a) Key figures: pH 6.5; N: 0.4%; Dry residue: 4.2%; Amino acids: positive, TLC: peptides: positive (biuret);

Nucleic acid components: TLC; Protein detection: negative. Heavy metals: <10 ppm: As <2 ppm; Sterility: sterile. b) Properties:

Table XXI

WARBURG test

Breathing increase factor (rat liver homogenate)

(Pp. 41-44) 4.07

Fermentation increase factor: Fig. 11 3.9

Swimming test mouse:

Survival time to drown increased from 10 to 13 min

Padberg test

(Pp. 62-65) 60 (83) ^*

Growth tests:

Guppy fish in mm length after 100 days if over

25 days extract 24 (17) ^*

Tadpoles (Xenopus laevis D.) brought forward the beginning of the metamorphosis: days versus controls 16 days

Corneometer test (method p. 50-52)

Improvement in skin moisture retention after 14 days of use 22% (8%) *

Radical scavenger test 76%

(Fig. 12 + p.98-100)

^* Controls without the addition of extract VII in brackets

The cell mass including protein remaining in the dialysis tube also shows a high growth effect in tadpoles, fish and farm animals and is suitable as an environmentally compatible feed additive, as is the entire non-dialyzed product from Example 9: Vll crude product. Determination of the fermentation increase factor of the above yeast extract dialvsate VII:

The fermentation increase factor is measured in a volume determined in a Warburg vessel, which is charged with a solution consisting of 2.5 g glucose, 0.4 g casein peptone, 50 mg MgSO ₄ , 7 H ₂ O and 50 mg baker's yeast and 50 ml tap water . The vessel is then filled with a certain amount of the test preparation according to a filling plan. The amount of CO _{2 released} at 30 ° C. is determined as a function of time, converted to normal conditions and devised by the standardized amount of CO ₂ , which is produced in the control test under otherwise identical conditions, but without test preparation.

The measurable fermentation activation is remarkable for the preparations according to the invention: factor 3.8.

11 shows the course of the CO ₂ formation in the Warburg test as a function of time, two groups of comparative tests being included, ie the preparation-free test solution which characterizes the course of the conventional fermentation and the yeast-free test solution with the preparation that has no significant influence on the fermentation.

The composition of the solutions was as follows:

Solution I: 50 ml basic medium + 50 mg baker's yeast

Solution II: 10 ml solution I + 50 μl extract VII

Solution III: 10 ml solution I + 50 μl extract VII

Solution IV: 10 ml base medium + 50 μl extract VII

Solution V: 10 ml basic medium + 50 μl extract VII Example 10

Combination of a metabolically active protein-free animal extract with a metabolically active protein-free vegetable extract, namely a combination of blood extract V (example 7) with yeast extract VII (example 9) with the formation of extract VIII

Blood extract V and yeast extract VII are mixed in a ratio of 3:10 and sterile filtered after adjustment to pH 6.7 (0.1 μm filter). The analytical parameters and properties are as follows: Extract VIII

pH 6.7

N 0.34%

Dry residue 4.2%

Breath increase factor (Warburg) 4.07

Amino acids positive

Peptides positive

Nucleic acid components positive

Sterility sterile

Pyrogenicity is pyrogen free

Advancement of Xenopus laevis metamorphosis 16 days

For compensation of KCN-damaged cell respiration from liver homogenate, see description from p. 91

Padberg test 59 (80) comeometer test:

Skin moisture retention 24% improvement The test described below shows that the extract VIII examined here can partially compensate for cell respiration of rat liver homogenate damaged by KCN:

In the Warburg model, the influence of extract VIII on a liver homogenate poisoned with KCN was tested:

1.1 Liver homogenate with the breathing factor F = 1.00 is characterized by a KCN content of 3 nM (final concentration in the approach)

Breathability reduced to F = 0.75

1.2 By adding the breath-increasing

Extract VIII with the value F = 4.07 can partially cause cellular respiration

KCN poisoned liver homogenate increased from F = 0.75 to F = 3.22

Carrying out the experiment: (see also breath increase test on pages 41-44

Solutions ibid.)

Cone-shaped Warburg vessels with a cylinder insert (ZE) and a side arm (SA) and a volume of approx. 13 ml are filled according to the filling plan (details in ml): Table XXII.

Table XXII

KCN concentration 3.0 nM in the mixture (preparation)

The solutions are pipetted in the order given. Filter strips folded into the cylinder inserts are used to increase the KOH surface. Immediately after the filling has ended, the vessels are sealed with the appropriate hose valve plugs on the side arm attachment, the vessels are fastened to the pressure gauges and secured with clips or package rubbers. With the taps open, the pressure gauges are suspended in the apparatus heated to 37 ° C. and shaken for 60 minutes in order to control the temperature of the reaction solution and to let the initially very rapid breathing abate somewhat. Then the right pressure gauge legs are set to the mark "15" with the help of the pinch tap, the difference between the left legs is marked to "15", the pressure gauge taps are closed and the solutions from the side arms are transferred to the main rooms by tilting (zero time). After every 20 minutes the shaking device is switched off, the right manometer leg set to "15" (V = const.) And the difference between the left leg to "15" (pressure difference h at constant volume and temperature) noted.

The test runs up to a measuring time of 100 minutes. After the last reading, the manometer taps are opened and the volumes of the vessels and the associated manometers are noted.

The h values in mm measured for the pressure differences as a result of the oxygen consumption are summarized in the following test protocols a-d.

evaluation

First, the vessels are adjusted to the thermobarometers (vessel 1/2) for 3 to 10 minutes in order to compensate for air pressure and temperature fluctuations during the test. The vessel constants for O ₂ of glasses 3 to 10 are then calculated using the following equation:

V = volume of the cas phase (volume of the vessel and the manometer up to the mark "15" minus V _f ). V _f = volume of the total liquid contained in the vessel. T = test temperature in ° K. ^Ck - = Bunsche absorption coefficient

(θLθ_ 37 ° C = 0.0239). P_ = 1 atm. measured in mm Brodie solution (manometer filling) 760 mm Hg = 10,000 mm Brodie solution. The oxygen used can now be used directly according to the equation

X (μl 0 ₂ ) = h - K can be calculated because the carbonic acid produced by breathing has been completely absorbed by the KOH (h = manometric difference). Test protocol a

Increased breathing of the liver homogenate without additives (AT factor 1, 0)

Test protocol b

Increased breathing of the liver homogenate (AT factor 1.0) by adding extract VIII

Test protocol c

Inhibition of the breathing of liver homogenate (AT factor 1.0) by addition of 3 nM KCN

Test protocol d

Increased breathing of KCN-inhibited liver homogenate breathing after the addition of extract VIII

Extract VIII also had an aphrodisiac effect, as can be seen from the following experiments

Extract VIII was concentrated to obtain the solid and administered to men who were not particularly keen on mating for 14 days in amounts of 0.15 g per kg of extract VIII (solid). The rats showed increased ejaculation from day 10 compared to the control animals. This effect lasted for about 3 weeks and could be confirmed

In another experiment with 5000 mice, the potency of male mice was inhibited by adding torula yeast to the drinking water, which has been known for a long time from the literature [Lit 12]. This potency inhibition could be eliminated by administering extract VIII, so that the number of newborn mouse again rose to a normal level. These experiments were carried out with 1000 male and 4000 female mice

The following shows that extract VIII also has a remarkable free radical scavenging effect

Detection method

The system "hyaluronic acid, xanthine, xanthine oxidase" (hereinafter referred to as "HXX system") can be used to determine the radical scavenging effect in vitro

principle

It is known that hyaluronic acid in phosphate buffer 7.4 is rapidly broken down by hydroxyl radicals which are released by the action of the enzyme xanthine oxidase on xanthine. The depolymerization of hyaluronic acid can be measured by measuring the decrease in viscosity. Addition of radical scavengers inhibits hyaluronic acid -Degradation and leads to an inhibition of the decrease in viscosity The decrease in viscosity of the hyaluronic acid solution is a measure of the effect of free hydroxyl radicals This results from the attached FIG. 12, in which the depolansation of the hyaluronic acid, determined by measuring the decrease in viscosity of the HXX system, is shown graphically with various additives

In the figure 12 mean

1 HXX system without enzyme

2 HXX system with enzyme

3 System HXX with extract VIII (example 10) 4 System HXX with yeast extract VII (example 9)

5 System HXX with thymus extract IVc (example 6)

6 System HXX with blood extract V (example 7)

7 System HXX with Colla 120 VI with additives (example 8)

8 System HXX with extract Ic and protein-containing beef kidney extract (preserved)

Experimental approaches

Hyaluronic acid solution

Final concentrations of the aqueous test solutions 0.425% hyaluronic acid, 66.0 mM phosphate buffer 7.4, xanthine 0.6 mM, xanthine oxidase 0.10 U / ml (cf. also: Lengenfelder, E., Fink, R., "The Oxidative Hyaluronic Acid Degradation, Mechanism and Consequences", Springer-Verlag, Berlin 1986).

The test solutions are incubated for 40 min at 37 ° C, cooled to 20 ° C in a water bath for 10 min; after a further 35 minutes, the viscosity measurements are carried out at 20.00 ° C. using a precision viscometer. Results in Fig. 12, for VIII cf. Nos. 1, 2 and 3.

Example 11 Use of animal and / or vegetable organ extracts with breath-increasing, i.e. Oxygen activating effect and with radical trapping effect in technology

In the above description it has already been pointed out that the cell metabolism activity, which is important for cosmetic and medical applications and which, among other things, results in increased energy production, led to the expectation of further possible uses, specifically because of the oxygen activation and metabolism activation that go hand in hand with it Radical scavenger effect.

The organ extracts produced in the above examples for biological applications have mostly been thoroughly cleaned, for example also deodorized and sterile filtered. In some cases, freedom from pyrogens was even sought. Such intensive treatment or cleaning of the extracts does not appear to be necessary in the case of technical applications, for example as stabilizers for color solutions, in particular water-based paints (as are preferred today in the automotive industry), or colored systems. Positive results were obtained both with crude organ extracts and with protein-containing and protein-free extracts. Instead of sterile filtration, good preservation with phenonip or similar preservatives and the addition of suitable solvents and solubilizers such as propylene glycols were sufficient. Paint coatings on walls could also be made more permanent are, as corresponding long-term series tests over 1 to 3 years have shown.

The following organ extracts from the examples and analogue preparations were used:

a) The synthetic crude kidney extract described in Example 2, produced from partial solution I and partial solution II, but without the addition of peptide components, without sterile filtration, preserved with 0.8% Phenonip and optionally other preservatives.

In the case of color solutions, the addition of solubilizers has proven to be advantageous. We also worked with quick-drying alkyd resin paint on various materials.

b) The protein-free and protein-containing bovine kidney extracts from fresh bovine kidneys and cell lines described in Example 3.

c) The synthetic thymus extract IVb described in Example 6, namely before sterile filtration and without peptide additives, preserved. Also a combination of IVa and IVc, treated analogously to IVb.

d) The serum blood extract described in Example 7 synthetically alone and also in combination with blood extract from fresh calf blood according to DE-OS 2 405 983, page 3, but without evaporating to dryness in a vacuum rotary evaporator.

e) The vegetable extract Colla 120 VI described in Example 8 and its raw form.

f) The extracts VII and VIII described in Examples 9 and 10 as well as the VIII crude product as obtained before dialysis (Example 9, first paragraph). In this context, reference should also be made to the experiments described in Example 10 for the detection of the radical trapping effect of VIII and other organ extracts produced (cf. also FIG. 12).

Literature distortion fLit.1

[1] Riemschneider R. and Source G., "Carcinogenity of Placenta Extracts" in "Fragrance Journal", 57, 4, 115.122 (1982); 57, 6, 105-1 12 (1982).

[2] Weiss and Schleicher J.B, "A multisurface tissue propagator for mass-scale growth of cell monolayer culture" in "Biotechnol. Bioeng." 10, 601 (1968) and ibid, 10, 617 (1968). [3] House Shearer M. & Maroudas N.G, "Method for bulk Culture of animal Cells on Plastic film" in "Exp. Cell. Research", 71, 293 (1972).

[4] Paul J. "Zeil and tissue cultures", W. de Gruyter, Berlin, 1980.

[5] Moore, George E., Hasenpusch, Peter, Gerner, Robert E. & Burns, A. Alex, "A pilot plant for mammalian cell culture" in "Biotechnol. Bio-engng." 10, 625 (1968).

[6] McCoy, TA, Whittle, W. & Conway, E, "A glass helix perfusion chamber for massive growth of cells in vitro" in "Proc. Soc. Exp. Biol. (NY)", 109, 235 (1962 ).

[7] Ubertini, B., Nardelli, L, Santero, G. & Panona. G., "Process report: Large-scale production of foot and mouse decease virus" in "J. Biochem. Microbiol. Technol. Eng. ", 2, 327 (1960).

[8] Riemschneider, R., and Hennig, K., "Detection of metabolic activations by organ extracts" in "RAK (fragrances, flavors, cosmetics)", 1977, issues 9 and 10, 12 pages.

[9] Sachs, L, "Angew. Statistics", 6th edition Springer 242 (1944).

[10] Padberg, G., "J. Soc. Cosmetic Chemists", 20, 719-728 (1969).

[11] Seitler, L., Cyanophytaceae, cryptogamous flora after L. Rabenhorst, Vol. 14, VI + 1196, Academ. Publishing company, Leipzig 1932

[12] Riemschneider R. Series of publications: Messages from the Physio-Chemical Institute of the University of Berlin, 1946.

[13] CRT-IST Ansaldo, Data Bank for Chemical Research, Animal Cell Lines Catalog 1990 by Maniello, Parodi, Aresu and Romano, Milan 1990 [14] DSM Catalog of Human and Animal Cell Lines 5th Edition 1995, Drexler, Dirks, MacLeod, Quentmeier, Steube, Braunschweig

[15] ICN "Cell Biology Catalog" 1995/96, ICN Biomedicals GmbH, 53340 Meckenheim

[16] Reference Guide to ATCC Cell Lines and Hybridomas, 8th edition

1994, Rockville, MD 20852, USA, in conjunction with 1996 Ordering Catalog ATCC Cell Lines and Hybridomas

[17] Riemschneider R., Schulz V. "Alkaline phosphatase from bovine placentas and their stabilization by divalent metal ions and tripeptides" in "Mitt. D. Physiolog.-Chem. Insituts der Universität Berlin", R 33 b, January 1950

[18] Riemschneider R. "Studies on placenta extracts", publication from the Institute for Biochemistry at the Free University of Berlin in January 1962, lecture in Toky on January 22, 1962 in the PULA colloquium (in Japanese)

[19] Schachtschnabel D., Fischer R.-D., Zilliken F., "Studies on

Control of Melanin Synthesis in Cell Cultures ", in" Z. for Physiological Chemistry ", 351, 1402-1410 (1970)

List of reference numerals in the accompanying FIGS. 1-12

Fig. 1

Number 1 1 = frame

Number 12 = wheels or roller

Number 13 = engine

Number 14 = drive belt or chain

Fig. 2

Number 21 = gas

Number 22 = feeding bottle

Number 23 = culture medium

Number 24 = magnetic stirrer

Number 25 = stir bar

Number 26 = rubber cap

Number 27 = hose for renewing the medium

Number 28 = sample tube

Number 29 = universal container

Fig. 5

Number 51 = sample tube

Number 52 = gas outlet pipe

Number 53 = clamping rod screw

Number 54 = cover plate

Number 55 = end plate

Number 56 = support rod

Number 57 = clamp rod Number 58 = grooves

Number 59 = end plate

Number 60 = base plate

Number 61 = support rod screw

Number 62 = air pump

Number 63 = elastic rubber buffer

Number 64 = glass plates

Number 65 = gas spray pipe

Number 66 = cylindrical vessel

Number 67 = sealing ring

Number 68 = gas mixture supply

Claims

Patent claims

1. Process for the production of organ extracts with improved bio-chemical efficiency from cell lines of the corresponding organs of animals or parts of plants, g e k e n n e e i c h n e t by the following stages:

a) activation of the selected animal or plant cells in a water bath of preferably 25 to 30 ° C., b) addition of a preheated culture medium prescribed for the respectively selected cells with mixing and subsequent incubation in a manner known per se, preferably at about 37 ° C. and preferably in an atmosphere containing about 5% CO ₂ , c) obtaining the cells formed as a starting material for large cultures on surfaces or in suspension by decanting the

Nutrient medium, washing the resulting cell lawn with a suitable buffer and taking up the cells in a complete medium while adjusting a dilution suitable for mass production, d) culturing the cells on surfaces, preferably in perfusion systems, on solid media and / or using the plastic film culture method (for animal cells) or in suspension culture (for plant cells) on a large scale in a manner known per se by inoculating the cultivation apparatus with a cell suspension in a sterile CO ₂ / air mixture, e) production of an extract by treating the suspended harvested cell material in an organic solvent and / or water, obtaining the aqueous phase, preferably by centrifugation, removing residual lipid fractions from the aqueous phase by treating the organic solvent several times with formation an aqueous crude extract, if appropriate in the form of a suspension, f) cleaning or treatment of the crude extract depending on the starting material and subsequent dialyzing, preferably using an Visking C 150 membrane, against an aqueous solution containing nipagin or another preservative to obtain an extract free of high molecular weight compounds as dialysate and g) sterile filtration of the extract, preferably using a 0.2 .mu.m (Millipore ^®) filter or a PALL-candle the setting of the expression rate tract to the desired pH, preferably from 5.0 to 7.2.

2. The method according to claim 1, characterized in that an animal cell mass obtained in stage (d) is suspended in ethyl ether or chloroform and left for about 8 to about 15 days at about -10 to about -20 ° C in the cold room and then centrifuged to recover the aqueous phase.

3. The method according to claim 1 or 2, characterized in that the last traces of ether or chloroform are removed from the dialysate (external solution) in step (f) by blowing through nitrogen at pH 7.2 to 7.5.

4. The method according to at least one of claims 1 to 3, characterized ge indicates that in step (a) cell cultures from commercially available cell lines including the non-commercially available special cell lines mentioned in the description are used individually or in a mixture.

5. The method according to at least one of claims 1 to 4, characterized ge indicates that

as animal cell lines at least one of the cell lines MDBK (bovine kidney cells DSM ACC 174 from the kidney of a normal adult bovine), P1 -1A3 (thymic epithelial cells DSM ACC 280 from the thymic epithelium of a 6 month old child), EBL (bovine lung cells DSM ACC 192 from the lungs of a 7 month old bovine fetus, frozen 110th passage), AM-C6SC8 (porcine kidney cells DSM ACC 152 from a subclone of the cell line Am II, which was obtained from a normal pig kidney), BeWo (female human placenta cells ATCC CCL 98), AV 3 (human amniotic cells ATCC CCL 21), WISH (human amnion cells ATCC CCL 25), B 95.8 (blood cells, monkey, ATCC CRL 1612 ECACC 8501 1419, PK (15) (pig kidney cells) ATCC CCL 33) and FL (human amnion cells ATCC CCL 62 IZSBS BSCL 46); 3A-subE (human placenta cells) ATCC CRL-1584; BVDV + EJG (bovine capillary cells) ATCC CRL-8659; P-17 (calf thymus epithelial cells) Brastone by Dr. FR Pesserl, Curitiba, Parana, Brazil; FB 5.Bm (bovine bone marrow) ATCC CRL-6043; R.Sp. (Bovine) ATCC CRL-6019; RPL 10 (bovine placenta cells from Prof. Dr. Mariano da Rocha Filho, UFSM, Faculdade de Medicina, Santa Maria, Rio Grande do Sul, Brazil, SPI.K (dolphin) ATCC CRL-6556; CF 37 Mg (Beagle -Dog, mammary gland) ATCC CRL 6230, SRC (rye cells from Stöhr, Acipenser huso from Dr. FR Pessserl, 80,000 Curitiba, Parana, Brazil), calf thymus cell line WANK and NM FARIA, 10,000 S. Paolo, SP , Brazil by Prof. Dr. Dr. R. Riemschneider, Institute for Central de Quimica da UFSM, S. Maria RS, Brazil); XTH-2 (cells from the South African clawed frog Xenos leavis DAUDIN, South African clawed toad) DSM ACC 190 and / or as plant cell lines at least one of the cell lines Acer pseudoplatanus L, Phaseolus vulgaris (from Hypocotyl of Pole Bean), Nicotiana tabacum, Soianum tuberosum ( tetraploid strain), Ginkgo biloba, Pisum sativum (root), Centurus cyanus, Daucus carota, Apium graveolens, Daucus catora, Apium graveolens, Arthrospira and / or Saccharomyces cerevisiae or other Actinomycetes.

6. The method according to at least one of claims 1 to 5, characterized ge indicates that mixtures of extracts obtained from several different animal cell lines, sterile-filtered extracts or mixtures of sterile-filtered extracts obtained from several different plant cell lines and optionally combined with one another be and optionally further combined with at least one synthetically produced extract or with at least one extract made from fresh organs or both

7. The method according to claim 6, characterized in that the extracted from animal cell lines, sterile-filtered extract with at least one synthetically produced or with at least one extract made from fresh organs or with both in a weight ratio (1 to 99) (99 to 1), preferably from (1 to 50) (50 to 1), in particular from (1 to 20) (99 to 80), is combined

8. The method according to claim 6 or 7, characterized in that an extract obtained according to claim 6 or 7 with at least one extract obtained from a plant cell line in a weight ratio of (1 to 99) (99 to 1), preferably of (1 to 50) (50 to 1), in particular from (1-20) (80-99)

9. The method according to at least one of claims 1 to 8, characterized ge indicates that when using plant cell lines in stage (e), if it depends on the Zellwandmateπal, the cell material suspended in water or in the organic solvent, preferably in broken by an ultrasound generator and the digested material after centrifuging and washing with water is digested and dialyzed by chemical and / or enzymatic partial hydrolysis

10 The method according to at least one of claims 1 to 9, characterized ge indicates that a synthetic blood serum extract is combined with a blood extract made from the cell line B95 8 or FB5 Bm or from a calf blood cell in suspension culture to produce a blood extract has been

11. The method according to at least one of claims 1 to 10, characterized ge indicates that a bovine placenta extract is produced from a suitable cell line of bos taurus in combination with synthetic bovine placenta extract for incorporation in cosmetic preparations.

12. The method according to claim 11, characterized in that a Rinder¬ placenta extract from a suitable cell line of bos taurus is produced in combination with a synthetic bovine placenta extract, the 0.02 to 0.06 g of BSE-free alkaline bovine placenta phosphatase per liter of final volume have been added for incorporation into cosmetic preparations.

13. The method according to at least one of claims 1 to 10, characterized ge indicates that a pyrogen-free calf blood extract from a suitable cell line from the calf is combined with a synthetic calf serum extract and / or with a calf serum extract which ge from natural calf blood has been won.

14. The method according to at least one of claims 1 to 7, characterized ge indicates that a calf thymus extract from a suitable cell line from the calf is combined with a synthetic calf thymus extract and / or with a calf thymus extract obtained from natural calf thymus has been.

15. The method according to at least one of claims 1 to 7, characterized in that a spleen extract from a suitable cell line of beef is combined with a synthetic beef spleen extract and / or with a beef spleen extract which is obtained from natural beef spleen has been.

16. The method according to at least one of claims 1 to 7, characterized in that an amnion extract from a suitable animal cell line is combined with a synthetic and / or natural amnion extract.

17 The method according to at least one of claims 1 to 9, characterized ge indicates that a plant extract containing hydroxyproline is produced from suitable plant cell lines and used as a collagen replacement

18 The method according to at least one of claims 1 to 17, characterized ge indicates that, as in the production of cell line extracts, work is also carried out in the production of synthetic or natural organ extracts without preservatives for the production of preservative-free end products or combinations thereof

19 The method according to at least one of claims 1 to 17, characterized ge indicates that when using the cell line Saccharomyces cerevisiae in suspension (step (e)) this before dialysis (step (f)) by simultaneous application of osmotic and thermal effects (preferably at 50 to 65 ° C) and partially deproteinized

20. The method according to at least one of claims 1 to 19, characterized in that a metabolically active yeast extract is combined with at least one of the calf blood extracts mentioned in claim 13

21 Use of the extracts and extract mixtures according to at least one of claims 1 to 20 as a replacement or supplement for placenta, thymus, blood, serum, spleen, liver, collagen, connective tissue, amniotic fluid, jellyfish , Roe, caviar, yeast and udder extracts or combinations thereof

22 Use of the extracts and extract mixtures according to at least one of claims 1 to 20 for the production of a cosmetic formulation

23 Use of the extracts and extract mixtures according to at least one of claims 1 to 20, preferably according to claims 19 and 20, for the preparation of a preparation with a radical scavenger effect, in particular for cosmetic and / or technical purposes

24 Use of the amnion extract according to claim 16, optionally in combination with other organ extracts, for the production of a cosmetic formulation with properties which inhibit melanin formation (whitening effect)

25 Use of the extracts and extract mixtures according to at least one of claims 1 to 20 for the manufacture of a medical preparation for topical, subcutaneous, intravenous or intramuscular application for the purpose of external or internal wound healing, for the treatment of atherosclerosis or radiation damage

26 Use of the extracts and extract mixtures according to at least one of claims 1 to 20 for the manufacture of a medical preparation for strengthening the immune system, for activating the cell metabolism or for treating gastroenterological diseases, in particular UIcera

27 Use of the extracts and extract mixtures according to at least one of claims 1 to 20 for the manufacture of a preparation having an aphrodisiac effect

28 Use of the extracts obtained from plant cell lines of preferably Ginkgo biloba, Saccharomyces, Torula or Cyanophyten as well as their combinations with extracts based on animal cell lines for the production of a preparation with aphrodisiac effect

29 Use of the extracts and extract mixtures according to at least one of claims 1 to 20 and the crude extracts and precursors then contained for the stabilization and preservation of dyed fabrics and paints, in particular lacquers.

30. Use of the extracts and extract mixtures according to at least one of claims 1 to 20 for the production of culture solutions, nutrient solutions and dietary solutions.

31. Organ extracts with improved biochemical efficiency, as can be obtained from cell lines of the corresponding organs of animals or parts of plants by the process according to at least one of claims 1 to 4.

32. Organ extracts and organ extract mixtures with improved biochemical efficiency, as can be obtained by the process according to at least one of claims 5 to 20.

33. Organ extracts and organ extract mixtures with improved biochemical efficiency, as can be obtained by the process according to at least one of claims 1 to 20, for the production of medical, cosmetic and dietary preparations and for stabilizing and preserving colored tissues and paints, in particular Paintwork with water-based paints.