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The tracheal relaxant activities and action mechanisms of
flavone derivatives, including 6-hydroxyflavone,
7-hydroxyflavone, chrysin, baicalein, luteolin,
5-methoxyflavone, 6-methoxyflavone, diosmetin, diosmin,
acacetin, tangeretin and luteolin-7-glucoside were analyzed to
understand their structure-activity relationships (SAR). The
above tweleve flavones concentration-dependently relaxed the
histamine (30 μM)-, carbachol (0.2 μM)-, and KCl (30 mM)-
induced precontractions of isolated guinea-pig trachea. Roughly,
according to their IC50 values, the order of their relaxant
potency was 6-hydroxyflavone, 7-hydroxyflavone, luteolin,
tangeretin > chrysin, 5-methoxyflavone, > baicalein, acacetin,
luteolin-7-glucoside > 6-methoxyflavone, diosmetin, diosmin. The
SAR was concluded as follows: (a) The substitution of sugar
group at position 7, such as luteolin to luteolin-7-glucoside
and diosmetin to diosmin, reduced their relaxant activities ;
(b) The substitution of OH group at position 6 or 3', such as
flavone to 6-hydroxyflavone or apigenin to luteolin,
respectively, increased their relaxant activity, but at position
7 , such as flavone to 7-hydroxyflavone, and even at both
positions 5 and 7, such as flavone to chrysin did not change its
relaxant activity. On the contrary, substitution of OH group at
positions 5, 6, and 7, such as flavone to baicalein, or at
position 6 of 5, 7-dihydroxyflavone compound, such as chrysin to
baicalein decreased their relaxant activities. (c) The
substitution of OCH3 group at position 5, such as flavone to
5-methoxyflavone, or further substitution at many other
positions, such as 5-methoxyflavone to tangeretin, did not
change its relaxant activity, whereas at position 6, such as
flavone to 6-methoxyflavone attenuate its relaxant activity. (d)
The substitution of OCH3 to OH group at position 6, such as
6-hydroxyflavone to 6-methoxyflavone or at position 4', such as
luteolin and apigenine to diosmetin and acacetin, respectively,
markedly decreased their relaxant activity. The preincubation of
the six more potent flavones, 6-hydroxyflavone,
7-hydroxyflavone, chrysin, luteolin, 5-methoxyflavone or
tangeretin among the above twelve compounds, non-competitively
inhibited contraction induced by cumulatively adding histamine,
carbachol or KCl in isolated guinea-pig trachea. In general,
their pD2' values were significantly less than their -logIC50
values. Therefore, their abilities of inhibition on calcium
release from intracellular calcium stores may be less potent
than those of suppression on calcium influx from extracellular
fluid. They also non-competitively inhibited contractions of the
trachealis induced by cumulatively adding calcium into high
potassium (60 mM)-Ca2+ free medium in the trachealis. After
maximal relaxation on histamine (30 μM)-induced precontraction
by nifedipine (10 μM), they caused further relaxation of the
trachealis. The result suggests that they may have other
relaxing mechanisms in addition to inhibiting voltage (VOC) and/
or receptor operated calcium channels (ROC) in the trachealis.
With exception of the following three flavones, their relaxant
responses were not affected by the removal of epithelial cells
or by the preincubation of propranolol (1 μM), glibenclamide
(10 μM), methylene blue (25 μM) or 2',5'-dideoxyadenosine (10
μM), suggesting their relaxing effects may not be related to
epithelium derived relaxing factor(s), activation of β-
adrenoreceptor, opening of ATP-sensitive potassium channels, or
activation of guanylate cyclase or adenylate cyclase. First,
2',5'-dideoxyadenosine (10 μM) parallelly rightward shifted the
log concentration-response curve of 6-hydroxyflavone, suggesting
that 6-hydroxyflavone may activate adenylate cyclase. Secondary,
methylene blue (25 μM) parallelly to the rightward shifted the
log concentration-response curve of luteolin, suggesting that
luteolin may activate guanylate cyclase. Third, glibenclamide
(10 μM) parallelly leftward shifted the log concentration-
response curve of 5-methoxyflavone with unknown mechanism.
6-hydroxyflavone (20 μM) and luteolin (20 μM) parallelly
leftward shifted the log concentration-response curve of
forskolin, and enhance the pD2 value of forskolin.
6-hydroxyflavone (10, 20 μM), luteolin (20 μM),
5-methoxyflavone (20 μM) and tangeretin (20 μM) also
parallelly leftward shifted the log concentration-response curve
of nitroprusside and enhanced the pD2 value of nitroprusside. It
seems that 6-hydroxyflavone, luteolin, 5-methoxyflavone, and
tangeretin may inhibit phosphodiesterase (PDE) activity. From
determination of PDE activity, we found that luteolin (100 and
300 μM) markedly inhibited cAMP-PDE and cGMP-PDE activity and
that the inhibition on cGMP-PDE activity was significantly
larger than on that of cAMP-PDE. Other flavone derivatives, such
as 6-hydroxyflavone, 7-hydroxyflavone, chrysin,
5-methoxyflavone, and tangeretin partially inhibited cAMP-PDE
and cGMP-PDE activities. Even at a high concentration such as
300 μM the inhibition was less than 70%. However, the
inhibition on cAMP-PDE activity by 6-hydroxyflavone (100 μM)
was significantly larger than that on cGMP-PDE, but the
inhibition on cGMP-PDE activity by tangeretin (300 μM) was
significantly larger than that on cAMP-PDE. These results
suggest that the relaxant mechanism of luteolin is mainly the
inhibition on PDE activity, especially on cGMP-PDE activity.
6-Hydroxyflavone may activate adenylate cyclase and slightly
inhibit PDE activity. 7-Hydroxyflavone, chrysin,
5-methoxyflavone and tangeretin only slightly inhibit the
activity of PDE. The above six flavones inhibit both calcium
influx and calcium release from calcium store. In addition to
luteolin, these six flavones may inhibit calcium influx more
markedly than that on calcium release.
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