FLAVONOID DERIVATIVE FOR TREATING DENTAL CARIES

Abstract

The invention relates to a flavonoid derivative containing a flavonoid that is coupled to an alkylene group via an ether bond, said alkylene group having an adhesion group for adhesion onto a dental surface. The flavonoid derivative has a long-term suppressing effect on the dental disease-causing formation of biofilm on the dental surfaces treated with the flavonoid derivative. The invention further relates to a kit for producing such a flavonoid derivative, and to the use of same.

##STR00001##

Claims

1. A flavonoid derivative A-Et-Q-Z comprising i) a spacer group Q, coupled to a flavonoid A via an ether bond Et, wherein the spacer group Q is an alkylene group, which may optionally be interrupted by an oxygen atom, a nitrogen atom and/or a polyoxyalkylene group, and ii) at least one adhesion group Z coupled to the spacer group Q, said Z group being selected from the group consisting of: —COOR.sup.2, —SO.sub.2OR.sup.2, —OPO(OR.sup.2).sub.2, —PO(OR.sup.2).sub.2, —CR.sup.1(PO(OR.sup.2).sub.2).sub.2, preferably —PO(OR.sup.2).sub.2, —CR.sup.1(PO(OR.sup.2).sub.2).sub.2, with R.sup.1=H, OH or alkyl, R.sup.2=H, alkyl or M, where M is a monovalent cation, preferably a monovalent metal cation, in particular a sodium ion; particularly preferably —CH(PO(OH).sub.2).sub.2.

2. The flavonoid derivative as claimed in claim 1, wherein the flavonoid A inhibits at least one glucosyltransferase from streptococcal lactic acid bacteria, and said streptococci are preferably selected from the Streptococcus mutans group and the Streptococcus downei group, in particular Streptococcus mutans and Streptococcus sobrinus, preferably Streptococcus mutans.

3. The flavonoid derivative as claimed in claim 1 or 2, wherein said flavonoid derivative is selected from the group according to the formulae I, II or III: ##STR00010## where i) R.sup.4=H, OH or an oxo group (═O); ii) R.sup.3, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.2I, R.sup.3I, R.sup.4I, R.sup.5I, R.sup.6I are each independently H, OH, OR.sup.1 where R.sup.1=methyl or acyl, rhamnose, glucose, oligoglucose, rutinose or Et-Q-Z where Et=O, i.e. O-Q-Z, and iii) at least one of the radicals R.sup.3, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.21, .sup.R3I, R.sup.4I, R.sup.5I, R.sup.6I, preferably R.sup.6, R.sup.7, R.sup.8 R.sup.3I, R.sup.4I or R.sup.5I, particularly preferably R.sup.7 or R.sup.4I, is Et-Q-Z where Et=O, i.e. O-Q-Z; ##STR00011## where i) R.sup.4=H, OH or an oxo group (═O); ii) R.sup.3, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.2I, R.sup.3I, R.sup.4I, R.sup.5I, R.sup.6I are each independently H, OH, OR.sup.1 where R.sup.1=methyl or acyl, rhamnose, glucose, oligoglucose, rutinose or Et-Q-Z where Et=O, i.e. O-Q-Z, and iii) at least one of the radicals R.sup.3, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.2I, R.sup.3I, R.sup.4I, R.sup.5I, R.sup.6I, preferably R.sup.6, R.sup.7, R.sup.8, R.sup.3I, R.sup.4I or R.sup.5I, particularly preferably R.sup.7 or R.sup.4I, is Et-Q-Z where Et=O, i.e. O-Q-Z; ##STR00012## where i) R.sup.4=H, OH or an oxo group (═O); ii) R.sup.2, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.2I, R.sup.3I, R.sup.4I, R.sup.5I, R.sup.6I are each independently H, OH, OR.sup.1 where R.sup.1=methyl or acyl, rhamnose, glucose, oligoglucose, rutinose or Et-Q-Z where Et=O, i.e. O-Q-Z, and iii) at least one of the radicals R.sup.2, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.2I, R.sup.3I, R.sup.4I, R.sup.5I, R.sup.6I, preferably R.sup.6, R.sup.7, R.sup.8 R.sup.3I, R.sup.4I or R.sup.5I, particularly preferably R.sup.7 or R.sup.4I, is Et-Q-Z where Et=O, i.e. O-Q-Z and wherein said flavonoid derivative is preferably a flavonoid derivative according to formula IV ##STR00013## and more preferably according to formula V ##STR00014##

4. The flavonoid derivative as claimed in claim 1 or 2, wherein the flavonoid A in the flavonoid derivative is derived from the group of flavones, isoflavones, flavonols, flavanols, flavanones and flavanonols, preferably comprising at least one hydroxyl group on the benzyl and/or phenyl ring, is preferably selected from the group consisting of apigenin, acacetin, baicalein, chrysin, luteolin, kaempferol, kaempferide, galangin, isorhamnetin, rhamnetin, myricetin, fisetin, pinobanksin, pinobanksin 3-acetate, pinocembrin, sakuranetin, isosakuranetin, quercetin, hesperetin, naringenin, trihydroxymethoxyflavanones, tetrahydroxyflavanones, ermanin, 7-hydroxyflavone, 7,8-dihydroxyflavone, daidzein, genistein, gengwanin, quercitrin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, 3,5,7-trihydroxy-4′-methoxyflavanol, 5,6,7-trihydroxy-3,4′-dimethoxyflavone, 3,7-dihydroxy-5-methoxyflavanone, 2,5-dihydroxy-7-methoxyflavanone, 8-methylkaempferol, is more preferably selected from the group consisting of apigenin, kaempferol, chrysin, daidzein, quercetin and epigallocatechin gallate, and is most preferably apigenin.

5. The flavonoid derivative as claimed in any of the preceding claims, wherein a) the adhesion group Z is covalently bonded to the spacer group Q via a C—C bond, a C—S bond, a C—P bond, a C—O—P bond or a C—C—P bond and b) said spacer Q preferably has features selected from the following group: i) the spacer Q is a linear or branched alkylene, which may have alicyclic units; ii) comprises 3 to 20, preferably 5 to 18, more preferably 7 to 16 carbon atoms, particularly preferably a linear alkylene group having 8 to 12 carbon atoms, preferably a decanylene group; iii) the spacer Q is a polyether group having 2 to 10 alkoxy units, preferably selected from ethylene glycol units [—O—CH.sub.2—CH.sub.2—] and propylene glycol units [—O—CH.sub.2—CH(CH.sub.3)—], preferably a polyether group having 4 ethylene glycol or propylene glycol units.

6. The flavonoid derivative as claimed in any of claims 1-4, wherein the adhesion group Z-COOR.sup.2 is coupled to the spacer group Q and said spacer Q comprises a linear alkylene group having 8 to 12 carbon atoms.

7. A process for producing a flavonoid derivative A-Et-Q-Z as claimed in any of the preceding claims comprising the following steps: a) ether synthesis of i) a flavonoid A, wherein said flavonoid A is selected from the group consisting of flavones, isoflavones, flavonols, flavanols, flavanones and flavanonols, and comprises at least one hydroxyl group (—OH) on the benzyl and/or on the phenyl ring, with ii) a compound comprising firstly a bromine atom (Br) covalently bonded to the spacer group Q and secondly a group XZ covalently bonded to the spacer group Q, wherein said group XZ is preferably an acid ester group, which in a further process step may be converted to the adhesion group Z, as shown below:
XZ-Q-Br+HO-A.fwdarw.XZ-Q-O-A b) conversion of the group XZ bonded to the spacer group Q, preferably by hydrolysis, to the adhesion group Z and obtaining the flavonoid derivative A-Et-Q-Z as shown below, where Et=O:
XZ-Q-O-A.fwdarw.Z-Q-O-A

8. The flavonoid derivative obtainable by a process as claimed in claim 7.

9. A carrier and/or dental preparation comprising at least one flavonoid derivative as claimed in any of claims 1-6 and 8, preferably two or more flavonoid derivatives different from one another as claimed in any of claims 1-6 and 8, wherein said carrier and/or dental preparation are preferably selected from the group consisting of: paste, gel, coating, foam, mouthwash, chewing gum and chewable tablet.

10. The flavonoid derivative as claimed in any of claims 1-5 and 7 or carrier and/or dental preparation as claimed in claim 9 comprising at least one further active ingredient, preferably selected from the group of flavonoids, particularly catechins.

11. The carrier and/or dental preparation as claimed in claim 9 or 10 comprising a fluoridating agent, preferably selected from the group of metal fluorides, amine fluorides and fluoride complexes, in particular sodium fluoride.

12. The flavonoid derivative as claimed in any of claims 1-6 and 8 or carrier and/or dental preparation as claimed in any of claims 9-11 for use as a medicament.

13. The flavonoid derivative as claimed in any of claims 1-6 and 8 or carrier and/or dental preparation as claimed in any of claims 9-11 for the prophylaxis and/or treatment of oral disease, preferably for the prophylaxis and/or treatment of dental caries.

14. The flavonoid derivative as claimed in any of claims 1-6 and 8 or carrier and/or dental preparation as claimed in any of claims 9-11 for preventing the formation of a new biofilm and/or accumulation of a biofilm and/or for treating a biofilm, preferably a biofilm formed by Streptococcus mutans, wherein said biofilm is further preferably on the tooth surface in the oral cavity of a patient.

15. A method, preferably in vitro method, for preventing the formation of a new biofilm and/or accumulation of a biofilm and/or for treating a biofilm, preferably a biofilm formed by Streptococcus mutans on a surface comprising hydroxyapatite, comprising the following steps: a) providing i) a flavonoid derivative as claimed in any of claims 1-6 and 8 or a carrier and/or a dental preparation as claimed in any of claims 9-11 and ii) a surface comprising hydroxyapatite with a biofilm optionally present on the surface; b) treating ii) with i).

16. The method as claimed in claim 15, wherein said method is carried out during professional tooth cleaning (PTC), preferably immediately after professional tooth cleaning, and further optionally including repeating step b) at an interval of at least 1 week, preferably at least 1 month, more preferably at least 3 months and no longer than 24 months, preferably no longer than 12 months, particularly preferably after 6 months.

Description

EXAMPLE 1

Synthesis of “ApiBisPhos EG”

[0068] (Mixture of 4″-(12,12-bisphosphono-3,6,9-trioxadodecanoxy)-7,5-dihydroxyflavone and 7-(12,12-bisphosphono-3,6,9-trioxadodecanoxy)-4″,5-dihydroxyflavone)

Tetraethylenglycol Dibromide (1)

[0069] 17.7 g (204 mmol) of lithium bromide were suspended in 80 ml of acetone under nitrogen. The suspension was cooled to 0-5° C. in an ice bath and 17.6 g (35 mmol) of tetraethylene glycol di(p-toluenesulfonate), dissolved in 30 ml of acetone, were added dropwise. After stirring at 0° C. for 30 min, the solution was stirred at RT for 48 hours. The white precipitate was then filtered off and washed with acetone. The acetone solution was concentrated. The residue was then taken up in 45 ml of toluene and shaken three times with water. The toluene phase was then concentrated using a rotary evaporator and dried under high vacuum. 9.5 g of a brown, clear liquid were obtained (yield: 85%).

[0070] .sup.1H-NMR (MeOD, 300 MHz, ppm): δ=3.51-3.62(m, 12H, CH.sub.2), 3.74(t, 4H, CH.sub.2CH.sub.2Br).

Tetraisopropyl 12-bromo-4,7,10-trioxadodecan-1,1-diylbisphosphonate (3)

[0071] 0.35 g of sodium hydride (60% in mineral oil, 9 mmol) were washed 3 times with n-heptane under nitrogen and then suspended in 5 ml of dry tetrahydrofuran (THF). The suspension was cooled to 0-5° C. in an ice bath and 2.5 g (7 mmol) of tetraisopropyl methylenebisphosphonate were added dropwise. 9.5 g (30 mmol) of the compound (1) were dissolved in 10 ml of dry THF and added to the reaction mixture. The solution was stirred at RT for 96 hours and then 20 ml of aqueous sodium hydrogen carbonate solution (0.1 mol/l) were added. The THF was then rotary evaporated and the aqueous phase was shaken twice with toluene. The combined organic phases were concentrated using a rotary evaporator. The crude product was purified by column chromatography (SiO.sub.2; ethyl acetate/n-heptane, 80:20, v/v) and dried under high vacuum. 1.9 g of a yellowish oil were obtained (yield: 45%).

[0072] .sup.1H-NMR (DMSO-d6, 300 MHz, ppm): δ=1.23-1.29(m, 24H, CH.sub.3), 1.85-2.05(m, 2H, CH.sub.2CH), 2.36(tt, 1H, CH), 3.41-3.61(m, 12H, CH.sub.2O+CH.sub.2Br), 3.74(t, 2H, CH.sub.2CH.sub.2Br), 4.53-4.72(m, 4H, CH(CH.sub.3).sub.2).

Mixture of 4′-(12,12-tetraisopropylbisphosphonato-3,6,9-trioxadodecanoxy)-7,5-dihydroxyflavone and 7-(12,12-tetraisopropylbisphosphonato-3,6,9-trioxadodecanoxy)-4″,5-dihydroxyflavone (5)

[0073] 0.5 g (1.8 mmol) of apigenin and 53 mg (0.9 mmol) of KOH were initially charged in 10 ml of DMSO. To this suspension was added 0.5 g (0.9 mmol) of tetraisopropyl 12-bromo-4,7,10-trioxadododecan-1,1-diyl bisphosphonate (3) and the mixture then stirred at 120° C. for 2 hours. After cooling to RT, the solution was precipitated with 40 ml of water and the precipitate was separated off by centrifugation. The precipitate was air dried for 72 hours. The crude product was then purified by column chromatography (1st chromatography n-heptane/ethanol, 60:40, v/v; 2nd chromatography SiO.sub.2, 1st eluent n-heptane/ethanol, 70:30, v/v, 2nd eluent, ethanol; 3rd chromatography SiO.sub.2, n-heptane/ethanol, 70:30, v/v), concentrated in a rotary evaporator and then dried under high vacuum. 0.15 g of a yellowish gel were obtained (yield: 22%).

[0074] .sup.1H-NMR (DMSO-d6, 300 MHz, ppm): δ=1.21-1.35(d, 24H, CH.sub.3), 1.85-2.05(m, 2H, CH.sub.2CH), 2.35(tt, 1H, CH), 3.41-3.63(m, 10H, CH.sub.2O) , 3.63-3.81 (m, 2H, CH.sub.2CH.sub.2OC.sub.4′/CH.sub.2CH.sub.2OC.sub.7) , 4.18-4.26 (m, 2H, CH.sub.2OC.sub.4′/CH.sub.2OC.sub.7), 4.53-4.70 (m, 4H, CH(CH.sub.3).sub.2), 6.19/6.38 (s, 2H, H-C.sub.6), 6.49/6.78 (s, 2H, H-C.sub.6), 6.83 (s, 2H, H-C.sub.3), 6.93/7.12(d, 2H, H-C.sub.3′, H-C.sub.5′), 7.95/8.02 (d, 2H, H-C.sub.2′, H-C.sub.6′), 12.93 (s, 1H, HO-C.sub.5).

Mixture of 4′-(12,12-bisphosphono-3,6,9-trioxadodecanoxy)-7,5-dihydroxyflavone and 7-(12,12-bisphosphono-3,6,9-trioxadodecanoxy)-4′,5-dihydroxyflavone (ApiBisPhosEG) (7)

[0075] 4 ml of 0.5N hydrochloric acid solution were added to 0.14 g of mixture (5). This mixture was then stirred in a microwave (microwave: Discover CEM, power: 150W) at 150° C. for 1 hour. The dilute hydrochloric acid was then rotary evaporated and the product was dried under high vacuum. 0.11 g of a yellow-brown gel were obtained (yield: 100%).

[0076] .sup.1H-NMR (DMSO-d6, 300 MHz, ppm): δ=1.87-2.20(m, 2H, CH.sub.2CH+CH), 3.44-3.63(m, 10H, CH.sub.2O+CH.sub.2CH.sub.2OC.sub.4′/CH.sub.2CH.sub.2OC.sub.7), 3.75-3.82(m, 2H, CH.sub.2CH.sub.2OC.sub.4′/CH.sub.2CH.sub.2OC.sub.7), 4.18-4.26 (m, 2H, CH.sub.2OC.sub.4′/CH.sub.2OC.sub.7), 6.21/6.38(s, 2H, H-C.sub.6), 6.49/6.78(s, 2H, H-C.sub.8), 6.83(s, 2H, H-C.sub.3), 6.95/7.12(d, 2H, H-C.sub.3′, H-C.sub.5′), 7.95/8.02(d, 2H, H-C.sub.2′, H-C.sub.6′).

EXAMPLE 2

Synthesis of “ApiBisPhos UD”

(Mixture of 4′-(11,11-bisphosphonoundecanoxy)-7,5-dihydroxyflavone and 7-(11,11-bisphosphonoundecanoxy)-4′, 5-dihydroxyflavone)

Tetraisopropyl 11-bromoundecan-1,1-diylbisphosphonate (2)

[0077] 1.4 g of sodium hydride (60% in mineral oil, 35 mmol) were washed 3 times with n-heptane under nitrogen and then suspended in 15 ml of dry tetrahydrofuran (THF). The suspension was cooled to 0-5° C. in an ice bath and 10.0 g (29 mmol) of tetraisopropyl methylenebisphosphonate were added dropwise. 39.2 g (130 mmol) of 1,10-dibromodecane were dissolved in 25 ml of dry THF and added to the reaction mixture. The solution was stirred at RT for 48 hours and then 30 ml of aqueous sodium hydrogen carbonate solution (0.1 mol/l) were added. The THF was then rotary evaporated and the aqueous phase was shaken twice with toluene. The combined organic phases were concentrated using a rotary evaporator. Using column filtration (SiO.sub.2, 1st eluent: heptane/ethyl acetate 1:1, v/v; 2nd eluent: ethanol), the excess dibromodecane was then removed. The crude product was purified by column chromatography (SiO.sub.2; ethyl acetate/ethanol, 90:10, v/v). 6.4 g of a yellowish oil were obtained (yield: 39%).

[0078] .sup.1H-NMR (DMSO-d6, 300 MHz, ppm): δ=1.12-1.42(m, 36H, CH.sub.2+CH.sub.3), 1.43-1.59(m, 2H, CH.sub.2), 1.60-1.89(m, 4H, CH.sub.2CH.sub.2Br+CH.sub.2CH), 2.16 (tt, 1H, CH), 3.51(t, 2H, CH.sub.2Br), 4.53-4.75(m, 4H, CH (CH.sub.3).sub.2).

Mixture of 4′-(11,11-tetraisopropylbisphosphonatoundecanoxy)-7,5-dihydroxyflavone and 7-(11,11-tetraisopropylbisphosphonatoundecanoxy)-4′,5-dihydroxyflavone (4)

[0079] 0.5 g (1.8 mmol) of apigenin and 52 mg (0.9 mmol) of potassium hydroxide (KOH) were initially charged in 10 ml of dimethyl sulfoxide (DMSO). To this suspension was added 0.5 g (0.9 mmol) of tetraisopropyl 11-bromoundecan-1,1-diylbisphosphonate (2) and the mixture then stirred at 120° C. for 2 hours. After cooling to RT, the solution was precipitated by adding 40 ml of water and the precipitate was separated off by centrifugation. The precipitate was dried under high vacuum. The crude product was then purified by column chromatography (1st chromatography SiO.sub.2; gradient elution ethyl acetate/ethanol; 2nd chromatography SiO.sub.2, ethyl acetate/ethanol, 95:5, v/v; 3rd chromatography SiO.sub.2, n-heptane/ethanol, 90:10, v/v), concentrated in a rotary evaporator and then dried under high vacuum. 0.16 g of a yellowish gel were obtained (yield: 24%).

[0080] .sup.1H-NMR (DMSO-d6, 300 MHz, ppm): δ=1.17-1.37(m, 34H, CH.sub.2+CH.sub.3), 1.37-1.58 (m, 4H, CH.sub.2), 1.59-1.84 (m, 4H, CH.sub.2CH.sub.2O+CH.sub.2CH), 2.16 (tt, 1H, CH), 4.08 (t, 2H, CH.sub.2O), 4.53-4.72 (m, 4H, CH(CH.sub.3).sub.2), 6.19/6.32(s, 2H, H-C.sub.6), 6.49/6.73 (s, 2H, H-C.sub.8), 6.78(s, 2H, H-C.sub.3), 6.89/7.06(d, 2H, H-C.sub.3′, H-C.sub.5′), 7.92/7.94 (d, 2H, H-C.sub.2′, H-C.sub.6′) .

Mixture of 4′-(11,11-bisphosphonoundecanoxy)-7,5-dihydroxyflavone and 7-(11,11-bisphosphonoundecanoxy)-4′, 5-dihydroxyflavone) (ApiBisPhos UD)(6)

[0081] 4 ml of 0.5N hydrochloric acid solution were added to 0.15 g of mixture (4). This mixture was then stirred in a microwave (microwave: Discover CEM, power: 150W) at 150° C. for 1 hour.

[0082] The dilute hydrochloric acid was then rotary evaporated and the product was dried under high vacuum. 0.12 g of a yellow-brown gel were obtained (yield: 100%).

[0083] .sup.1H-NMR (DMSO-d6, 300 MHz, ppm): δ=1.14-1.38(m, 10H, CH.sub.2), 1.37-1.58 (m, 4H, CH.sub.2), 1.62-1.84 (m, 4H, CH.sub.2CH.sub.2O+CH.sub.2CH), 1.86-2.08 (m, 1H, CH) 4.08 (t, 2H, CH.sub.2O), 6.21/6.34 (s, 2H, H-C.sub.6), 6.51/6.75 (s, 2H, H-C.sub.8), 6.82 (s, 2H, H-C.sub.3), 6.94/7.09(d, 2H, H-C.sub.3′, H-C.sub.5′), 7.95/8.00 (d, 2H, H-C.sub.2′, H-C.sub.6′).

[0084] The proportion of the compound 7-(11,11-bisphosphonoundecanoxy)-4′, 5-dihydroxyflavone in the isomeric mixture (6) was significantly greater than the proportion of the compound 4′-(11,11-bisphosphonoundecanoxy)-7,5-dihydroxyflavone. The synthetic route is designed in such a way that this compound is present in the isomeric mixture (6) in a clear excess (>70%).

[0085] Using the SeeSAR simulation software (BioSolveIT GmbH), the binding affinity of the compounds was simulated using the example of glucosyltransferase C (3AIE, 2.1A) from S. mutans. This showed an increased binding affinity of 7-(11,11-bisphosphonoundecanoxy)-4′, 5-dihydroxyflavone compared to 4′-(11,11-bisphosphonoundecanoxy)-7,5-dihydroxyflavone.

EXAMPLE 3

Inhibitory Effect of Apigenin (Prior Art) and the Inventive Flavonoid Derivative “ApiBisPhos UD” on the Glucosyltransferase Activity of S. mutans

[0086] It was investigated whether the “ApiBisPhos UD” synthesized according to Example 2 had an influence on the activity of the glucosyltransferases of S. mutans as is to be expected from the apigenin known from the prior art. For this purpose, the test substance was resuspended in a DMSO/ethanol mixture (20/80%) and then diluted in the reaction mixture to the concentration to be tested (final concentration DMSO/ethanol 2/8%). The glucosyltransferase GtfC (0.3 μg, nzytech) was incubated in 100 mM potassium phosphate buffer, pH 6.0 with 30 mM sucrose for one hour at 37° C. in the presence of the test substances. As a positive control, the enzyme activity was measured in the presence of DMSO/ethanol (2/8%). To demonstrate Gtf enzyme activity, the fructose eliminated during glucan synthesis was detected via a multi-stage enzyme reaction. For this purpose, the D-fructose and D-glucose assay kit from Megazyme (K-FRUGL) was used.

[0087] Principle of Detection:

[0088] D-glucose and D-fructose are phosphorylated in the presence of adenosine-5-triphosphate (ATP) to glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P) by the enzyme hexokinase (HK), producing adenosine 5′-diphosphate (ADP) (1), (2).

##STR00007##

[0089] In the presence of the enzyme glucose-6-phosphate dehydrogenase (G6P-DH), G-6-P is oxidized to gluconate-6-phosphate by nicotinamide adenine dinucleotide (NADP.sup.+). This produces reduced nicotinamide adenine dinucleotide phosphate (NADPH) (3).

##STR00008##

[0090] The amount of NADPH formed in this reaction corresponds stoichiometrically to the amount of D-glucose. The absorption of NADPH is measured at 340 nm.

[0091] After completion of the reaction (3), F-6-P is converted to G-6-P by phosphoglucose isomerase (PGI) (4).

##STR00009##

[0092] The G-6-P formed reacts in turn with NADP.sup.+ to form gluconate-6-phosphate and NADPH, which leads to a further increase in absorption and correlates stoichiometrically with the amount of D-fructose.

[0093] The results are shown in FIG. 2. FIG. 2 shows the activity of glucosyltransferase C of S. mutans. 9 independent reaction batches were measured in triplicate and the enzyme activity was normalized to the solvent control. Apigenin 6 mM could not be tested due to insufficient solubility.

[0094] As can be seen from FIG. 2, the activity of glucosyltransferase C of S. mutans was surprisingly significantly more strongly inhibited by the flavonoid derivative ApiBisPhos UD according to the invention compared to the flavonoid apigenin. The remaining enzyme activity when using ApiBisPhos UD is less than 30% at 1 mM and about 10% at 3 mM (hatched bars), several times less than the enzyme activity when using apigenin (black bars). Expressed another way: The activity of glucosyltransferase C is inhibited in the presence of 1 mM ApiBisPhosUD by 72±9%, at 3 mM by 90±4% and at 6 mM by 99±2%. In comparison, the enzyme activity was inhibited by only 38±10% at 1 mM apigenin and by only 53±6% at 3 mM apigenin.

EXAMPLE 4

Adhesion of the Inventive Flavonoid Derivative “ApiBisPhos UD” to Hydroxyapatite

[0095] Investigated in addition was the adhesion of the inventive flavonoid derivative “ApiBisPhos UD” to hydroxyapatite, a substance that largely determines the tooth surface and is suitable as an adhesive contact. For this purpose, 5 mg of hydroxyapatite (HAP) were moistened with saliva for 30 minutes and then incubated with ApiBisPhos UD (5, 10, 50, 100 mM in DMSO/ethanol mixture; 20/80%) for 30 minutes. The amount of unbound ApiBisPhos UD in the supernatant was then determined by thin-layer chromatography. The results of this experiment are presented in FIG. 3 and show that the flavonoid derivative “ApiBisPhos UD” according to the invention adheres excellently to the hydroxyapatite moistened with saliva with more than 96% of the applied ApiBisPhos UD substance binding.

EXAMPLE 5

Effect of “ApiBisPhos UD” and “ApiBisPhos EG” Bound to Hydroxyapatite on S. mutans Gtf70C

[0096] ApiBisPhos UD was bound to HAP as described in Example 4 and then incubated with GtfC as described in Example 3. The HAP was then centrifuged off and the GtfC activity in the supernatant was then determined as described in Example 3. A similar procedure was followed with the further flavonoid derivative “ApiBisPhos EG” according to the invention. The results are presented in FIG. 4 and show that both ApiBisPhos UD and ApiBisPhos EG bound to hydroxyapatite inhibit the GtfC enzyme activity of S. mutans to a considerable degree. In other words, the further example of a flavonoid derivative according to the invention (“ApiBisPhos EG”) confirms the successful functioning of the present invention as the basis for achieving the advantage according to the invention based on the adhesion of the flavonoid derivative according to the invention to the tooth surface comprising hydroxyapatite, so that the flavonoid derivative can prevent or to a great extent inhibit the formation of biofilm causing dental diseases on the tooth surfaces treated with the flavonoid derivative in the long term.

EXAMPLE 6

Effect of “ApiBisPhos UD” Bonded to Hydroxyapatite (HAP) on the Formation of S. mutans Biofilm

[0097] In this example, it was investigated whether “ApibisPhos UD” has a significant influence on S. mutans-mediated biofilm formation using hydroxyapatite discs coated with saliva under conditions simulating the oral cavity.

[0098] For this purpose, sterile hydroxyapatite disks (Clarkson Chromatography Products Inc., Ø5 mm×2 mm) were coated with saliva (Lee Biosolutions, human pooled donors) as substrate for 60 minutes and then incubated with ApiBisPhos UD (60 μl; 10 mM in DMSO/ethanol mixture, 20/80%) for 60 minutes. After washing twice with DPBS, these pretreated hydroxyapatite disks were coated with S. mutans preculture (1.5×10.sup.8 bacteria per ml in Balmelli broth) and cultured aerobically at 37° C. for 24 hours. The grown biofilm was fixed with 100% ethanol (30 min, 25° C.), stained with 0.06% (w/v) crystal violet (60 min, 25° C.) and the dye eluted with 30% (v/v) acetic acid quantified by measuring the absorbance at 570 nm. The measured values were set in relation to the solvent control (DMSO/ethanol mixture; 20/80%), i.e. without the use of ApibisPhos UD.

[0099] The results of 5 independent experiments carried out in triplicate show that the flavonoid derivative “ApiBisPhos UD” according to the invention inhibits the formation of biofilms on HAP surfaces by 26±9% under the test conditions described.

[0100] In other words, the further example of the flavonoid derivative according to the invention (“ApiBisPhos UD”) also confirms the successful functioning of the present invention.

[0101] This example also demonstrates the statistically significant flavonoid derivative-mediated inhibition of S. mutans-induced biolfilm formation on a hydroxyapatite disk simulating the tooth surface with the flavonoid derivative ApiBisPhos UD bonded thereto.