Method of producing plants with an elevated content of flavonoids and phenolic compounds 5 The present invention relates to a method of increasing the content of flavonoids and phenolic constituents in plants, wherein a plant is generated, by methods of molecular genetics, in which the activity of the enzyme flavanone 3-hydroxylase is reduced. 10 Moreover, in the method according to the invention, the activity of the enzyme flavanone 3-hydroxylase is reduced fully or partially, permanently or transiently, in the entire plant or in parts of the plant by methods of molecular biology (for example 15 antisense constructs, cosuppression, the expression of specific antibodies or the expression of specific inhibitors). The invention furthermore relates to plants with an elevated content of flavonoids and phenolic constituents, whose enzymatic 20 activity of the enzyme flavanone 3-hydroxylase is reduced. Moreover, the invention relates to the use of plants, generated by the method according to the invention, or parts of these plants as foodstuffs, supplements or for producing curative 25 compositions, health-promoting compositions or tonics (juices, infusions, extracts, fermentation products) for humans and animals, and for the production of cosmetics. A variety of phenolic substances are found in plants, for example 30 caffeic acid, ferulic acid, chlorogenic acid, gallic acid, eugenol, lignans, coumarins, lignin, stilbenes (polydatin, resveratrol), flavonoids (flavones, catechines, flavanones, anthocyanidines, isoflavones), and polymethoxylated flavones. Accordingly, phenols are also generally a constituent of a number 35 of plant-derived foodstuffs and stimulants. Certain phenolic substances are of particular importance since, after having been taken up together with the food, they can exert an antioxidant effect in the human or animal metabolism (Baum, B. 0.; Perun, A. L. Antioxidant efficiency versus structure. Soc. Plast. Engrs 40 Trans 2: 250-257, (1962); Gardner, P.T.; McPhail, D.B.; Duthie, G.G. Electron spin resonance spectroscopic assessment of the antioxidant potential of teas in aqueous and organic media. J. Sci. Food Agric. 76: 257-262, (1997); Rice-Evans, C. A.; Miller, N. J.; Pananga, G. Structure-antioxidant activity relationship of 45 flavonoids and phenolic acids. Free Radic. Biol. Med. 20: 933-956, (1996); Salah, N.; Miller, N. J.; Paganga, G.; Tijburg, L.; Bolwell, G. P.; Rice-Evans, C. Polyphenolic flavonoids as 2 scavenger of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophys 322: 339-346, (1995); Stryer, L. Biochemistry S. Francisco: Freeman, (1975); Vieira, 0.; Escargueil-Blanc, I.; Meilhac, 0.; Basile, J. P.; Laranjinha, J.; 5 Almeida, L.; Salvayre, R.; Negre-Salvayre, A. Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL. Br J Pharmacol 123: 565-573, (1998)). Moreover, polyphenols also have a multiplicity of effects on the cell metabolism. Inter alia, they modulate signal 10 transduction enzymes such as protein kinase C, tyrosine protein kinase and phosphatidylinositol 3-kinase (Agullo, G.; Gamet-payrastre, L.; Manenti, S.; Viala, C.; Remesy, C.; Chap, H.; Payrastre, B. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with 15 tyrosine kinase and protein kinase C inhibition. Biochem Pharmacol 53: 1649-1657, (1997); Ferriola, P. C.; Cody, V.; Middleton, E. Protein kinase C inhibition by plant flavonoids. Kinetic mechanisms and structure activity relationship. Biochem Pharmacol 38: 1617-1624, (1989); Cushman, M.; Nagarathman, D.; 20 Burg, D. L.; Geahlen, R. L. Synthesis and protein-tyrosine kinase inhibitory activity of flavonoids analogues. J Meed Chem 34: 798-806, (1991); Hagiwara, M.; Inoue, S.; Tanaka, T.; Nunoki, K.; Ito, M.; Hidaka, H. Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine/threonin 25 protein kinases. Biochem Pharmacol 37: 2987-2992, (1988)) which down-regulates inducible NO synthase (Kobuchi, H.; Droy-Lefaix, M. T.; Christen, Y.; Packer, L. Ginkgo biloba extract (EGb761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7. Biochem Pharmacol 53: 897-903, (1997)) and 30 regulates the gene expression of, for example, interleucins and adhesion molecules (ICAM-1, VCAM-1) (Kobuchi, H.; Droy-Lefaix, M. T.; Christen, Y.; Packer, L. Ginkgo biloba extract (EGb761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7. Biochem Pharmacol 53:897-903, (1997); Wolle, 35 J.; Hill, R. R.; Ferguson, E.; Devall, L. J.; Trivedi, B. K.; Newton, R. S.; Saxena, U. Selective inhibition of Tumor necrosis Factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on transcriptional factor NF-kB. Atherioscler Thromb Vasc Biol 16: 1501-1508, (1996)). It 40 is now accepted that these effects are beneficial for precluding and preventing infarctions, cardiovascular diseases, diabetes, a variety of certain cancers, tumors and other chronic diseases (Bertuglia, S.; Malandrino, S.; Colantuoni, A. Effects of the natural flavonoid delphinidin on diabetic microangiopathy. 45 Arznei-Forsch/Drug Res 45: 481-485, (1995); Griffiths, K.; Adlercreutz, H.; Boyle, P.; Denis, L.; Nicholson, R.I.; Morton, M.S. Nutrition and Cancer Oxford: Isis Medical Media, (1996); 3 Hertog, M. G. L.; Fesrens, E. J. M.; Hollman, P. C. K.; Katan, M. B.; Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen elderly study. The Lancet 342: 1007-1011, (1993); Kapiotis, S.; Hermann, M.; Held, I.; 5 Seelos, C.; Ehringer, H.; Gmeiner, B. M. Genistein, the dietary-derived angiogenesis inhibitor, prevents LDL oxidation and protects endothelial cells from damage by atherogenic LDL. Arterioscler Thromb Vasc Biol 17: 2868-74, (1997); Stampfer, M. J.; Hennekens, C. H.; Manson, J. E.; Colditz, G. A.; Rosner, B.; 10 Willet, W. C. Vitamin E consumption and the risk of coronary disease in women. New Engl J Med 328: 1444-1449, (1993); Tijburg, L. B. M.; Mattern, T.; Folts, J. D.; Weisgerber, U. M.; Katan, M. B. Tea flavonoids and cardiovascular diseases: a review. Crit Rev Food Sci Nutr 37: 771-785, (1997); Kirk, E. A.; 15 Sutherland, P.; Wang, S. A.; Chait, A.; LeBoeuf, R. C. Dietary isoflavones reduce plasma cholesterol and atherosclerosis in C57BL/6 mice but not LDL receptor-deficient mice. J Nutr 128: 954-9, (1998) - references - ). A series of curative compositions, health-promoting compositions or tonics whose 20 effect is based on their content of phenolic substances is therefore already being obtained from suitable plants (Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A. Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression. Am J Pathol 25 147: 278-292, (1995); Lin, J. K.; Chen, Y. C.; Huang, Y. T.; Lin-Shiau, S. Y. Suppression of protein kinase C and nuclear oncogene expression as possible molecular mechanisms of cancer chemoprevention by apigenin and curcumin. J Cell Biochem Suppl 28-29: 39-48, 1997; Zi, X.; Mukhtar, H.; Agarval, R. Novel cancer 30 chemopreventive effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNF alpha. Biochem Biophys Res Comm 239: 334-339, 1997. Furthermore, it is known that certain plant-derived foodstuffs or stimulants prepared from them have a positive effect on various diseases. 35 Resveratrol, which is found in white wine, but in particular in red wine (in addition to other components), for example, acts against infarctions, cardiovascular diseases and cancer (Gehm, B.D.; McAndrews, J.M.; Chien, P.-Y.; Jameson, J.L. Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for 40 estrogen receptor. Proc Natl Acad Sci USA 94: 14138-14143, (1997); Jang, M.; Cai, L.; Udeani, G.O.; Slowing, K.V.; Thomas, C.F.; Beecher, C.W.W.; Fong, H.H.S; Farnsworth, N.R.; Kinghorn, A.D.; Mehtha, R.G.; Moon, R.C., Pezzuto, J.M. Cancer chemopreventive activity of resveratrol, a natural product 45 derived from grapes. Science 275: 218-220, (1997). A similar action is also found in substances such as catechin, epicatechin-3-gallate, epigallocatechin and -- 4 epigallocatechin-3-gallate, all of which are found in the leaves of tea (Camellia sinensis). Beverages made with, in particular, unfermented tea leaves (green tea) are health-promoting (Hu, G.; Han, C.; Chen, J. Inhibition of oncogene expression by green tea 5 and (-)-epigallocatechin gallate in mice. Nutr Cancer 24: 203-209; (1995); Scholz, E; Bertram, B. Camellia sinensis (L.) 0. Kuntze. Der Teestrauch [the tea shrub]. Z. Phytotherapie 17: 235-250, (1995); Yu, R.; Jiao, J. J.; Duh, J. L.; Gudehithlu, K.; Tan, T. H.; Kong, A. N. Activation of mitogen-activated protein 10 kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant responsive elements-mediated phase II enzyme gene expression. Carcinigenesis 18: 451-456, (1997); Jankun, J.; Selman, S.H.; Swiercz, R. Why drinking green tea could prevent cancer. Nature 387: 561, (1997). Moreover, 15 polymethoxylated flavones from citrus fruit also show a potential antitumor action (Chem, J.; Montanari, A.M.; Widmer, W.W. Two new polymethoxylierte flavone, a class of compounds with potential anticancer activity, isolated from cold pressed dancy tangerine peel oil solids. J Agric Food Chem 45: 364-368, (1997)). 20 It is an object of the present invention to find a simple, inexpensive method of increasing the content of flavonoids and phenolic constituents in crop plants. 25 We have found that this object is achieved, starting from physiological studies on growth regulators from the acylcyclohexanedione group, by genetic engineering methods which are now available, surprisingly, and with the aid of which plants can be generated which are characterized by an elevated content 30 of curative, health-promoting or tonifying constituents. Acylcyclohexanediones such as prohexadione-calcium and trinexapac-ethyl (earlier name: cimectacarb) are employed as bioregulators for inhibiting the longitudinal growth of plants. 35 The reason for their bioregulatory action is that they block the biosynthesis of gibberellins, which promote longitudinal growth. Owing to their structural relationship with 2-oxoglutaric acid, they inhibit certain dioxygenases which require 2-oxoglutaric acid as co-substrate (Rademacher, W, Biochemical effects of plant 40 growth retardants, in: Plant Biochemical Regulators, Gausman, HW (ed.), Marcel Dekker, Inc., New York, pp. 169-200 (1991)). It is known that such compounds also engage in the phenol metabolism and are therefore capable of causing, in a variety of plant species, the production of anthocyanins to be inhibited 45 (Rademacher, W et al., The mode of action of acylcyclohexanediones - a new type of growth retardant, in: Progress in Plant Growth Regulation, Karssen, CM, van Loon, LC, 6 luteoforol, luteoliflavan, apigeniflavan and tricetiflavan, and homogeneous and heterogeneous oligomers and polymers of the abovementioned structurally related substances are produced in higher quantities. 5 Elevated concentrations of the phenols hydroxycinnamic acid (p-coumaric acid, ferulic acid, sinapic acid), salicylic acid or umbelliferone, including the homogeneous and heterogeneous oligomers and polymers formed from them, are found in plants 10 after the enzyme activity of the enzyme flavanone 3-hydroxylase (F3H) has been reduced. Equally the concentration of the chalcones such as, for example, phloretin, and of the stilbenes, such as, for example, resveratrol, increases. 15 Since the enzyme activity of the enzyme flavanone 3-hydroxylase is reduced, the concentration of the glycosides of the flovonoids [sic], of the phenolic compounds, of the chalcones and of the stilbenes is also increased. 20 Starting from these findings and the hypotheses derived therefrom, genetically modified crop plants were generated in which the activities of F3H were reduced fully or partially, permanently or transiently, in the entire plant or in individual plant organs or plant tissues, by means of antisense constructs, 25 so that the content of curative, health-promoting or tonifying substituents was improved in terms of quantity and quality. The method according to the invention for increasing the content of flavonoids and phenolic compounds by expressing flavanone 30 3-hydroxylase in antisense orientation can be applied successfully to the following crop plants, but the method is not restricted to the plants mentioned: grapevines, cherries, tomatoes, plums, sloes, blueberries, strawberries, citrus fruit (such as oranges, grapefruit), pawpaw, red cabbage, broccoli, 35 Brussel sprouts, cacao, kale, carrots, parsley, celeriac/celery, onions, garlic, tea, coffee, hops, soya, oilseed rape, oats, wheat, rye, Aronia melanocarpa, Ginko [sic] biloba. Moreover, the invention relates to plants with an elevated 40 content of flavonoids and phenolic constituents, generated by the method according to the invention and with a reduced enzymatic activity of the enzyme flavanone 3-hydroxylase. As an alternative to generating plants whose flavanone 45 3-hydroxylase activity is reduced with the aid of antisense technology, it is also possible to use other methods of molecular genetics which are known from the literature, such as 7 cosuppression or the expression of specific antibodies, in order to achieve this effect. In addition, the invention relates to the use of plants, 5 generated by the method according to the invention, or of parts of these plants as foodstuffs, food supplements or for producing curative compositions, health-promoting compositions or tonics (juices, infusions, extracts, fermentation products) for humans and animals, and for the production of cosmetics. 10 Surprisingly, it has now been found that plants which have been generated in accordance with the invention, or parts of these plants or products produced from them (teas, extracts, fermentation products, juices and the like) have the following 15 effects: (1) the antioxidant capacity in vitro (Electron Spin Resonance (ESR), LDL oxidation, total antioxidant capacity, NO scavenging) is improved; 20 (2) a modulating effect on enzymes, especially signal transduction enzymes (protein kinase C, tyrosine protein kinase, phosphatidylinositol 3-kinase) is observed; 25 (3) a modulation of redox-sensitive transcriptional factors (NF-kB, AP-1) is induced in endothelial cells, lymphocytes and smooth muscle cells; (4) the regulation of gene expression of target genes involved in 30 the pathogenesis of inflammatory diseases (cytokines IL-1 and IL-8, macrophage chemoattractant protein 1 (MCP-1), adhesion factors ICAM-1 and VCAM-1) is modulated; (5) an antiaggregatory effect is induced; 35 (6) the cholesterol synthesis in hepatocytes is inhibited; (7) antiproliferative/antineoplastic effects exist. 40 Example 1 Cloning the gene of a flavanone 3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker. 45 Ripe tomato fruits of Lycopersicon esculentum Mill.cv. Moneymaker were washed, dried and, using a sterile blade, the pericarp was freed from seeds, central columnella and woody parts. The 8 pericarp (approx. 50 g) was frozen in liquid nitrogen. Then, the material was comminuted in a blender. In a pre-cooled mortar, the comminuted material was treated with 100 ml of homogenization medium and mixed. Then, the suspension was transferred into a 5 centrifuge flask by squeezing it through sterile gauze. Then, 1/10 volume 10% SDS was added and the material was mixed thoroughly. After 10 minutes on ice, one volume of phenol/chloroform was added, and the centrifuge flask was sealed and the contents mixed thoroughly. After centrifugation for 15 10 minutes at 4000 rpm, the supernatant was transferred into a fresh reaction vessel. This was followed by three more phenol/chloroform extractions and one chloroform extraction. Then, 1 volume of 3 M NaAC [sic] and 2.5 volumes of ethanol were added. The nucleic acids were precipitated overnight at -20 0 C. The 15 next morning, the nucleic acids were pelleted for 15 minutes in the refrigerated centrifuge (4*C) at 10,000 rpm. The supernatant was discarded, and the pellet was resuspended in 5-10 ml of cold 3 M NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. When completely dry, the pellet was 20 taken up in approx. 0.5 ml of sterile DEPC water, and the RNA concentration was determined photometrically. 20 Rg of total RNA were treated first with 3.3 R1 of 3M sodium acetate solution, 2 p1 of IM magnesium sulfate solution, and the 25 mixture was made up to a final volume of 100 Rl with DEPC water. A microliter of Rnase-free [sic] Dnase [sic] (Boehringer Mannheim) was added to this, and the mixture was incubated for 45 minutes at 370 degrees [sic]. After the enzyme had been removed by extraction by shaking with phenol/chloroform/isoamyl alcohol, the 30 RNA was precipitated with ethanol, and the pellet was taken up in 100 Rl DEPC water. 2.5 Rg of RNA from this solution were transcribed into cDNA using a cDNA kit (Gibco BRL). Using amino acid sequences which were derived from cDNA clones 35 encoding flavanone 3-hydroxylase, conserved regions in the primary sequence were identified (Britsch et al., Eur. J. Biochem. 217, 745-754 (1993), and these acted as the basis for designing degenerated PCR oligonucleotides. Using the peptide sequence SRWPDK (amino acid 147-152 in the Petunia hybrida 40 sequence FL3H PETHY), the 5' oligonucleotide was determined and had the following sequence: 5'-TCI (A/C) G (A/G) TGG CC(A/C/G) GA (C/T) AA (A/G) CC-3. 45 9 The sequence of the oligonucleotide deduced by using the peptide sequence DHQAVV (amino acid 276281 [sic] in the Petunia hybrida sequence FL3H PETHY) was as follows: 5'-CTT CAC ACA (C/G/T) GC (C/T) TG (A/G)TG (A/G)TC-3. 5 The PCR reaction was carried out using the tTth polymerase by Perkin-Elmer, following the manufacturer's instructions. The template used was 1/8 of the cDNA (corresponds to 0.3 Vg of RNA). The PCR program was: 10 30 cycles 94 degrees 4 sec 40 degrees 30 sec 72 degrees 2 min 15 72 degrees 10 min The fragment was cloned into Promega's vector pGEM-T following the manufacturer's instructions. 20 The correctness of the fragment was checked by sequencing. Using the restriction cleavage sites Ncol [sic] and Pstl [sic], which are present in the polylinker of the vector pGEM-T, the PCR fragment was isolated, and the overhangs were made blunt-ended using T4-polymerase. This fragment was cloned into an 25 Smal-(blunt-)cut [sic] vector pBinAR (Hbfgen and Willmitzer, Plant Sci. 66: 221 - 230 (1990)) (see Figure 2). This vector contains the CaMV (cauliflower mosaic virus) 35S promoter (Franck et al., Cell 21: 285 - 294 (1980)) and the termination signal from the octopine synthase gene (Gielen et al., EMBO J. 3: 835 30 846 (1984)). This vector mediates, in plants, a resistance to the antibiotic kanamycin. The resulting DNA constructs contained the PCR fragment in sense and antisense orientation. The antisense construct was employed for generating transgenic plants. 35 Figure 2: Fragment A (529 bp) contains the CaMV 35S promoter (nucleotides 6909 to 7437 of the cauliflower mosaic virus). Fragment B [lacuna] the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the termination signal of the octopine synthase gene. 40 Cloning a larger cDNA fragment of the flavanone 3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker using the 5'RACE system. 45 To exclude that the generation of plants with a reduced mRNA flow equilibrium quantity of F3H is not successful due to the small size of the F3H PCR fragment used in the antisense construct, a 10 second antisense construct using a larger F3H fragment should be generated. The 5'RACE method (system for rapid amplification of cDNA ends) 5 was employed for cloning a larger F3H fragment. Extension of the F3H PCR fragment by means of the 5'RACE method using the 5'RACE system for rapid amplification of cDNA ends, Version 2-0 by Life TechnologiesTM. 10 Total RNA was isolated from ripe tomato fruits of Lycopersicon esculentum Mill.cv. Moneymaker (see above). The cDNA first strand synthesis was carried out using the GSP-1 15 (gene-specific primer) 5'-TTCACCACTGCCTGGTGGTCC-3' following the manufacturer's instructions. Following Rnase [sic] digestion, the cDNA was purified using the GlassMAX spin system by Life TechnologiesTM following the manufacturer's instructions. 20 A cytosine homopolymer was added onto the 3' end of the purified simplex F3H cDNA using the terminal deoxynucleotydil [sic] transferase, following the manufacturer's instructions. The 5'-extended F3H cDNA was amplified using a second 25 gene-specific primer (GSP-2), which binds in the 3' region upstream of the GSP-1 recognition sequence and thus allowed a "nested" PCR to be performed. The 5' primer used was the "5'RACE abrided [sic] anchor primer", which was provided by the manufacturer and which is complementary to the homopolymeric dC 30 tail of the cDNA. The cDNA thus amplified, which was termed F 3 Hextended, was cloned into Promega's vector pGEM-T following the manufacturer's instructions. 35 The identity of the cDNA was confirmed by sequencing. The F 3 Hextended cDNA fragment was isolated using the restriction cleavage sites Ncol [sic] and Pstl [sic] which are present in the 40 polylinker of vector pGEM-T, and the overhangs were converted using T4-polymerase and smooth ends [sic]. This fragment was cloned into an Smal-(blunt-)cut [sic] vector pBinAR (Hdfgen and Willmitzer, 1990) (see Figure 3). This vector contains the CaMV (cauliflower mosaic virus) 35S promoter (Franck et al., 1980) and 45 the termination signal from the octopine synthase gene (Gielen et al., 1984). This vector mediates, in plants, a resistance to the antibiotic kanamycin. The resulting DNA constructs contained the 11 PCR fragment in sense and antisense orientation. The antisense construct was employed for generating transgenic plants. Figure 3: Fragment A (529 bp) contains the CaMV 35S promoter 5 (nucleotides 6909 to 7437 of the cauliflower mosaic virus). Fragment B (lacuna] the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the termination signal of the octopine synthase gene. 10 Example 2 Generation of transgenic Lycopersicon esculentum Mill.cv. Moneymaker which express a subfragment of flavanone 3-hydroxylase in antisense orientation. 15 The method used was that of Ling et al., Plant Cell Report 17, 843 - 847 (1998). Cultivation was carried out at approx. 22 0 C under a 16-hour-light/8-hour-dark regime. 20 Tomato seeds (Lycopersicon esculentum Mill. cv. Moneymaker) were incubated by incubation [sic] for 10 minutes in 4% strength sodium hypochlorite solution, and subsequently washed 3-4 times with sterile distilled water and placed on MS medium supplemented with 3% sucrose, pH 6.1, for germination. After a germination 25 time of 7-10 days, the cotyledons were ready for use in transformation. Day 1: Petri dishes containing "MSBN" medium were overlaid with 1.5 ml of an approximately 10-day-old tobacco suspension culture. 30 The plates were covered with film and incubated at room temperature until the next day. Day 2: Sterile filter paper was placed onto the plates overlaid with the tobacco suspension culture in such a way that no air 35 bubbles were formed. The cotyledons, which had been cut crosswise, were placed on the filter paper upside down. The Petri dishes were incubated for 3 days in a culture chamber. Day 5: The agrobacterial culture (LBA4404) was sedimented by 40 centrifugation for 10 minutes at approx. 3000 g and resuspended in MS medium so that the OD was 0.3. The cotyledon sections were placed into this suspension and incubated for 30 minutes at room temperature with gentle shaking. Then, the cotyledon sections were dried somewhat on sterile filter paper and returned to their 45 starting plates to continue cocultivation for 3 days in the culture chamber.
12 Day 8: The cocultured cotyledon sections were placed on MSZ2K50+B and incubated for the next 4 weeks in the culture chamber. They were then subcultured. 5 Shoots which formed were transferred to root induction medium After successful rooting, the plants were tested and transferred into the greenhouse. 10 Example 3 Inhibition of cholesterol biosynthesis in cultures of primary rat hepatocytes 15 Preparation of the stock solutions Between 10 and 20 mg of the lyophilisate of ripe tomatoes cv."Moneymaker" containing A) only the native flavanone 3-hydroxylase gene (control) and B) as described in Example 2 20 additionally a subfragment of the flavanone 3-hydroxylase in antisense orientation, was weighed exactly and treated with such an amount of DMSO that a stock solution of 10 mM total flavonoids resulted. Immediately before the tests were started, dilutions of these stock solutions in the culture medium were prepared. 25 Ten-fold dilution steps were carried out between 10-4 and 10- 8 M. Preparation of the hepatocyte cultures Primary hepatocytes were obtained from the livers of male 30 Spraque-Dawley [sic] rats (240-290 g) by means of collagenase perfusion (Gebhardt et al., Arzneimittel-Forschung/Drug Res. 41: 800-804 (1991) 1990). The hepatocytes were cultured in collagen-coated Petri dishes (6-well plates, Greiner, NUrtingen) at a cell density of 125,000 cells/cm 2 in Williams Medium E 35 supplemented with 10% calf serum. More details, in particular on the culture medium, are found in Gebhardt et al., Cell Biol. Toxicol. 6: 369 - 372 (1990) and Mewes et al., Cancer Res. 53: 5135 - 5142 (1993). After 2 hours, the cultures were transferred to serum-free medium supplemented with 0.1 RM insulin. After a 40 further 20 hours, they were employed in the tests. The test substances were tested in each case in three independent cultures from 2-3 rats. Incubation of the hepatocyte cultures with the test substances A 45 and B 13 To demonstrate that the cholesterol biosynthesis is affected by the test substances A and B, the hepatocyte cultures were kept for a total of 22 hours. Then, they were incubated with serum-free Williams Medium E supplemented with 14 C acetate (only 5 tracer quantities) for 2 hours together with the test substances at the concentrations indicated. In each test series, a control was included. The methodology is described in detail by Gebhardt (1991) and Gebhardt, Lipids 28: 613-619 (1993). The tracer quantities of 14 C acetate exchange rapidly with the intracellular 10 acetyl-CoA pool and therefore allow the incorporation of 1 4 C acetate into the sterol fraction, which consists of >90% of cholesterol, to be determined in a trouble-free manner (Gebhardt, 1993). 15 Analytical methods for influencing the cholesterol biosynthesis The incorporation of 14 C acetate into the sterol fraction (non-hydrolyzable lipids) was measured by the method of Gebhardt (1991). In the extraction used, in which Extrelut@ columns 20 (Merck, Darmstadt) are employed, over 95% of the 14 C acetate (and small amounts of other low-molecular-weight metabolites formed from it) are removed. This test allows comparisons to be made between the relative synthesis rate of cholesterol and precursor sterols under the effect of test substances (Gebhardt, 1993). 25 Visual and microbial quality checks of the hepatocyte cultures Before and after the test incubation, all the cultures used were checked visually under the microscope for contamination with 30 microorganisms and for the integrity of the cell monolayer. A recognizable change in cell morphology (in particular at the higher concentrations) was not observed in any of the samples. This largely excludes that the test results were affected by cytotoxic effects of the test substances. 35 The routine sterility tests on all cultures revealed no signs of contamination whatsoever with microorganisms. Results 40 Samples A) from the tomatoes which were not genetically modified (control) showed no effect whatsoever on cholesterol biosynthesis. In contrast, the cholesterol synthesis was inhibited significantly by samples B from the tomatoes which 45 contained a subfragment of flavanone 3-hydroxylase in antisense orientation.