| Flavonoid distributes widely in plant kingdom, such as vegetables, fruits, soy nuts and natural products, and has lots of physiological activity such as anti-inflammatory, antibacterial, anti-aging, antivirus, antitumor and the antioxidant properties. Although flavones have these benefits, rapid and extensive metabolism in gut and liver reduces their bioavailabilities (<10%), plasma concentrations, and other claimed biological activities. Sulfates and glucuronides are the principal products of flavone metabolism in vivo. Therefore, revealing its absorption and metabolism can provide helpful information and guidance for the drug development. The aim of this thesis is to deeply elucidate the metabolic mechanism of flavonoids which is useful for pre-clinical research.Metabolism of flavonoids in vivo is very complicated, and biotransformation by UDP - Glucuronosyl transferase (UGTs) and sulfotransferase (SULTs) is the main reason for its low bioavailability. In addition, there were literatures reported that 7-hydroxyflavone and fisetin once absorbed can be metabolized into glucose aldehyde acidification derivatives and sulfonic derivatives which could decrease their oral bioavailability. Nearly all of quercetins were metabolized into conjugate compounds such as sulfonic conjugate and glucose aldehyde acidification conjugate in plasma after oral administration. Besides,5-hydroxyflavone and BaiYangSu (5,7 -dihydroxyflavone) were metabolized into glucose aldehyde acidification conjugate after absorption. Some other reports suggested that the phaseⅡmetabolism of isoflavone compounds were dertermined by the position of hydroxyl and the isoforms of UGT enzymes. Wang reported the glucuronidation of 7-hydroxycoumarin in different species including human, dog, monkey, rat, mouse were different and had significant species-dependent differences. Studies before also showed that different genders can affect metabolic activities and selectivities of enzymes; Lin found that metabolism of clivorine in vitro also had significant species-dependent differences.In the present study, we measured the conjugating rates of flavones catalyzed by UDP-gluconosyltransferases (UGTs) or sulfotransferases (SULTs) alone or together using a FVB mouse liver S9 fraction. We analyzed the glucuronidation and sulfation of seven mono-hydroxyflavones (HFs),2'-,3'-,4'-,3-,5-,6-, and 7-HF, and five di-hydroxyflavones (diHFs),3,7-,4',7-,5,7-,6,7-, and 3,4'-diHF. Three enzymatic reaction systems were used:(A) sulfation only, (B) glucuronidation only, or (C) simultaneous sulfation and glucuronidation or SULT-UGT co-reaction. Also, we made a species and gender comparison of SULT metabolism by mouse, rat, dog and human liver S9 fractions.1. The UGT-mediated metabolism of flavonesUsing FVB mouse liver S9 microsomal fraction to study the regularity and mechanism of the UGT-mediated metabolism of flavonoids in which 12 kinds of flavones included. Results showed that every mono-hydroxyflavones had one glucuronidation metabolite and 3,4'-diHF,3,7 - diHF,6,7 - diHF were detected to have two glucuronidation metabolites except 4',7-diHF and 5,7-diHF had one glucuronidation metabolite.And the glucuronidation rate of 7-HF was still the fastest of all seven mono-hydroxyflavones at all three substrate concentrations, while 5-HF was glucuronidated the slowest. Also, the rank order of glucuronidation at 5μM and 10μM was:7-HF> 3-HF> 3'-HF> 4'-HF> 2'-HF> 6-HF> 5-HF (F5μΜ=469.683,P5μΜ<0.001, F10μΜ=361.015, P10μΜ<.001). At 40μM substrate concentration, the rank order in the middle changed except for 7-HF and 5-HF and the order changed for: 7HF> 3'HF>4'HF>3HF> 6HF>2'HF> 5HF (F40μΜ=437.787, P40μΜ<0.001). The results clearly indicated that the glucuronidation of seven mono-hydroxyflavones were structure-independent by FVB mouse liver S9 fraction. In addition, the glucuronidation rate at 7-OH was always faster than that at other -OH groups (3 position,5 position,6 position,4'position)for all five di-hydroxyflavones at all three substrate concentrations. The glucuronidation activity of-OH groups in diHFs were 7-OH> 3-OH> 6-OH/4'-OH(F5μM=2.769, P5μΜ=0.057, F10μΜ=3.126, P10μΜ=0.039, F40μΜ=4.442, P40μΜ=0.011). So the results aslo indicated that the glucuronidation of five di-hydroxyflavones were structure-independent by FVB mouse liver S9 fraction. The hydroxyl group at position seven is always metabolized fastest, probably because position seven is a structural feature important for active site recognition. The hydroxyl group at position five is always metabolized slowest, and in the glucuronidation of 5,7-dihydroxyflavone, none of metabolite was detected in position five probably duo to the formation of an intra-molecular hydrogen bond between 4-carbonyl and 5-OH group and block the glucuronidation of 5-OH group.Meanwhile, we explored the concentration-dependent glucuronidation of twelve hydroxyflavones in single UGT reaction system, and found that glucuronidation rates for five selected mono-hydroxflavones (7-HF,4'-HF,3'-HF,6-HF,5-HF) and all of five selected di-hydroxflavones were increased as substrate concentrations increased (P<0.05). But for 2'HF, the glucuronidation rate was the highest in the middle concentration (P<0.05).We refitted the 7-OH position glucuronidation for 7-HF and other 7-OH groups (such as 3,7-diHF,4',7-diHF,5,7-diHF and 6,7-diHF). The glucuronidation rates at 7-OH appeared to decrease when an extra -OH group was added to the 3'-OH,4'-OH, 5-OH and 6-OH while the degree of decreasing was 3'-OH,6-OH,4'-OH and 5-OH which the order was similar as in mono-hydroxyflavones that the glucuronidation of 3-HF was faster than that in 6-HF,4'-HF and 5-HF. In other words, when having a 7-OH replaced, the order of glucuronidation rate in seven position was 3,7-diHF> 6,7-diHF> 4',7-diHF and 5,7-diHF, but all of them were slower than that of 7-HF. This suggested that when adding a phenolic hydroxyl group in position 3,4',5 and 6 may change the density of parent nucleus in "A" ring, thus inhibiting the glucuronidation of 7-OH.2. The SULT-mediated metabolism of flavonesUsing FVB mouse liver S9 microsomal fraction to study the regularity and mechanism of the SULT-mediated metabolism of flavonoids which 12 kinds of flavones included. Results showed that every mono-hydroxyflavones had one sulfation metabolite except for 3-HF and all of the diHFs formed only one sulfation metabolite, each of which was determined to be 7-O-sulfate, except for 4',7-diHF which formed two mono-sulfates.And the sulfation rate of 7-HF was still the fastest of all seven mono-hydroxyflavones at all three substrate concentrations in FVB mouse liver S9 while 6-HF was sulfated the slowest and 3-HF was not sulfated. For example, the rank orders of sulfation at 5μM and 10μM was:7-HF> 4'-HF> 2'-HF> 5-HF.> 3'-HF> 6-HF> 3HF=0 (F5μM=285.476, P5μM<0.001, F10μM=237.350, P10μM <0.001). As for di-hydroxyflavones, the sulfation rates at 7-OH were always faster than that at other—OH groups (4' position,3 position,5 position,6 position). Besides the formation rate of 7-O-sulfate from 4',7-diHF was always higher than formation rate at the 4'-OH position. When at 5μM and 10μM substrate concentrations, the fastest formation rate was 7-O-sulfate of 6,7-diHF, followed by 7-O-sulfate of 4', 7-diHF; 4'-O-sulfate of 4',7-diHF; 7-O-sulfate of 3,7-diHF; 7-O-sulfate of 5, 7-diHF and 4'-O-sulfate of 3,4'-diHF (F5μM=66.164, P5μM<0.001, F10μM=23.895, P10μΜ<0.001, F40μΜ=3.941, P40μΜ=0.026). The sulfation rate of 7-O-sulfate was always higher than that at other—OH groups (4'position,3 position,5 position,6 position) followed by 4'-OH position. When a substitutional group of hydroxyl existing at the seventh position, we can't detect sulfation metabolite at the third, fifth, sixth positions. As for 3-OH, sulfation metabolite can't be detectable currently in 3-HF probably because of excessive space resistance in position three; As for 5-OH, probably duo to the formation of an intra-molecular hydrogen bond between 4-carbonyl and 5-OH group and block the sulfation of 5-OH group. But as for 6-OH, it may be that the position six had occurred the sulfation reaction but metabolic rate was too slow, or quantity of metabolites were so little that current detectable methods cannot detect. So the results aslo indicated that sulfation of six monohydroxyflavones (except for 3-HF) and five dihydroxyflavones were structure-independent difference by FVB mouse liver S9 fraction.As for concentration-dependent sulfation of twelve hydroxyflavones in single SULT reaction system. Results showed that sulfation rates of 5-HF were increased as substrate concentrations increased. But for 4'-HF,7-HF,6HF,2'HF,3'HF and all of the five selected di-hydroxflavones(except for position seven in 3,7-diHF), sulfation rates were decreased as substrate concentrations increased (P<0.05,One-way ANOVA)In the study of 3,7-diHF,4',7-diHF,5,7-diHF and 6,7-diHF, we found that the sulfation rate at 7-OH appeared to increase when an extra-OH group was added to the 4'-OH and 6-OH, while the sulfation rate at 7-OH appeared to decrease (P<0.05) when an extra -OH group was added to the 5-OH and 3-OH. We presume that the -OH group added to 4'-,6-,5-, and 3- positions could change the electron cloud density of the "A" ring of the main backbone to facilitate or inhibit sulfation at 7-OH, Interestingly, the sulfations at 7-OH were decreased or increased in the presence of hydroxyl groups (3-,5-,4'-,and 6-OH).3. Interaction of glucuronidation and sulfation for hydroxyflavonesWe had used FVB mouse liver S9 microsomal fraction to study the interaction of glucuronidation and sulfation of 12 kinds of hydroxyflavones. Results showed that, sulfation and glucuronidation of hydroxyflavones were structure-independent and concentration-independent by FVB mouse liver S9 fraction in UGT-SULT co-reaction which was similar as in single UGT reaction system and in single SULT reaction system. As for structure-independent experiment in co-reaction, the metabolic order changed because of existence of another metabolic reaction in mono-hydroxyflavones while the order didn't change in di-hydroxyflavones. As for concentration-independent experiment in co-reaction, glucuronidation rates were increased as substrate concentrations increased while sulfation rates were decreased as substrate concentrations increased, which the phenomena was similar with in single reaction,but only a few compounds were different such as 2'HF. The specific mechanism is unclear, further discussion should be needed.One of the most important discovery in this chapter was that:in FVB mouse liver S9 fraction, whether mono-hydroxyflavones or di-hydroxyflavones, sulfation rates of these flavones were enhanced significantly in SULT-UGT co-reaction system compared to that in single SULT system while the glucuronidation rates were nearly unchanged in SULT-UGT co-reaction system compared to that in single UGT system and the specific mechanism is unclear.4. Species-, and gender-dependent differences of sulfation for three mono-hydroxyflavonesSpecies-dependent sulfation rates of three mono-hydroxyflavones (7-HF,6-HF, and 4'-HF) in liver S9 fractions prepared from four different species (mouse, rat, dog, and human) were compared. Results suggested that statistically significant differences among species were present in three mono-hydroxyflavones (P<0.05). For 7-HF and 6-HF, the rank order of the sulfation rate was dog> rat> human> mouse. However, for 4'-HF, the trend was different, the rate of 4'-O-S formation was always the highest in dog liver S9 at three concentrations, followed by mouse, rat and human at 5μM while at 10μM and 40μM, the order was dog> rat> mouse> human.In addition, gender-dependent sulfation rates of three mono-hydroxyflavones (7-HF,6-HF and 4'-HF) in liver S9 fractions prepared from the same animal species but of different genders were compared. Results suggested that, in rat liver S9 fraction the sulfation rates of all three mono-hydroxyflavones at three concentrations were much higher in males than that in females while in mouse.liver S9 fraction, the sulfation rates of three mono-hydroxyflavones were much higher in females than that in males. But in dog liver S9 fraction, the sulfation rates of three HFs had no or there was a small difference between females and males (except in 7-HF and 6-HF at 40μM). As a result, gender-dependence exists in the sulfation metabolism of mono-hydroxyflavones.In summary, this thesis investigated the metabolic mechanism of glucuronidation and sulfation of flavones, elucidated the interaction between glucuronidation and sulfation for the first time. Besides, we revealed the species-, gender-dependent differences of sulfation metabolism for three mono-hydroxyflavones. All of this can provide guidance for pre-clinical studies of flavones. |
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