Anti-inflammatory activity of 2,5-dihydroxycyclohepta-2,4,6-trienone in rats

Article abstract of DOI:10.1007/s00044-009-9174-z

In this study, the anti-inflammatory effects of a tropolone derivative, 2,5-dihydroxycyclohepta-2,4,6-trienone (AD-4), were investigated. The anti-inflammatory potency of AD-4 was compared with that of indomethacin in carrageenan-induced inflammation models in rats. The effect on vascular permeability was also determined by hyaluronidase-induced capillary permeability. AD-4 decreased carrageenan-induced paw edema at doses of 3.62 × 10², 7.24 × 10², and 14.48 × 10 ² μmol/kg by 45% (p < 0.001), 79% (p < 0.001), and 83% (p < 0.001), respectively, compared with the value of 49% (p < 0.001) for indomethacin (69.8 μmol/kg). Additionally, AD-4 decreased hyaluronidase-induced capillary permeability significantly. In conclusion, AD-4 was determined to have anti-inflammatory effects with lower toxicity than indomethacin. Anti-inflammatory effect of AD-4 may be related to its effects on vascular permeability.

Full text of DOI:10.1007/s00044-009-9174-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:84–93
DOI 10.1007/s00044-009-9174-z
ORIGINAL RESEARCH
Anti-inflammatory activity of 2,5-dihydroxycyclohepta-
2,4,6-trienone in rats
Feride Koc Æ Elif Cadirci Æ Abdulmecit Albayrak Æ
Zekai Halici Æ Ahmet Hacimuftuoglu Æ Halis Suleyman
Received: 5 October 2008 / Accepted: 10 February 2009 / Published online: 17 March 2009
¨
Ó Birkhauser Boston 2009
Abstract In this study, the anti-inflammatory effects of a tropolone derivative,
2,5-dihydroxycyclohepta-2,4,6-trienone (AD-4), were investigated. The anti-
inflammatory potency of AD-4 was compared with that of indomethacin in carra-
geenan-induced inflammation models in rats. The effect on vascular permeability
was also determined by hyaluronidase-induced capillary permeability. AD-4
decreased carrageenan-induced paw edema at doses of 3.62 9 102, 7.24 9 102, and
14.48 9 102 lmol/kg by 45% (p \ 0.001), 79% (p \ 0.001), and 83% (p \ 0.001),
respectively, compared with the value of 49% (p \ 0.001) for indomethacin
(69.8 lmol/kg). Additionally, AD-4 decreased hyaluronidase-induced capillary
permeability significantly. In conclusion, AD-4 was determined to have anti-
inflammatory effects with lower toxicity than indomethacin. Anti-inflammatory
effect of AD-4 may be related to its effects on vascular permeability.
Keywords Hyaluronidase Á Tropolone Á Anti-inflammatory Á
Capillary permeability Á Toxicity
Introduction
2,5-Dihydroxycyclohepta-2,4,6-trienone (AD-4) (Dastan and Balci, 2006; Mori
et al., 1993) is a tropolone derivative (Fig. 1). Tropolones have an aromatic seven-
membered ring system (Ogata et al., 1999). Tropolones are isolated from plants and
F. Koc
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine,
Atatu¨rk University, Erzurum, Turkey
E. Cadirci Á A. Albayrak Á Z. Halici Á A. Hacimuftuoglu Á H. Suleyman (&)
Department of Pharmacology, Faculty of Medicine, Atatu¨rk University, Erzurum 25240,
Turkey
e-mail: suleyman@atauni.edu.tr

Med Chem Res (2010) 19:84–93
85
Fig. 1 Chemical structure of
AD-4
HO
O
OH
AD4
2,5-dihydroxycyclohepta-2,4,6-trienone
bacteria or obtained by synthesis (Arima et al., 2003; Budihas et al., 2005; Choi
et al., 2006; Kitamura et al., 1986; Korth et al., 1982; Morita et al., 2002, 2004a)
and these compounds have chelating capacity with metal ions such as Fe, Cu, and
Zn (Doulias et al., 2004; Miyamato et al., 1998). Tropolones exert various
biological activities due to their interesting ring structure and chelation properties
(Diouf et al., 2002; Doulias et al., 2004). These compounds have been used as
deodorants and natural food additives (food preservatives) (Ogata et al., 1999;
Nakano et al., 2005). Recently, the biological activities of many compounds derived
from tropone and tropolone, used as a starting material have been shown to include
antibacterial, antiviral, antifungal, insecticidal, acaricidal, antitumoral, antiallergic,
antithyroidal, and antioxidant activities (Bagli et al., 1979; Lee et al., 1979;
Miyamato et al., 1998; Morita et al., 2004b). In addition, it was reported that,
b-thujaplicin demonstrated an anti-inflammatory effect (Nakano et al., 2005).
Azulen derivatives, synthesized from tropolone, were shown to inhibit lipid
peroxidation (Rekka et al., 2002). The effects of tropolone derivatives on inhibition
of metaloprotease were also reported (Inamori et al., 1999). The role of
metaloprotease enzyme in inflammation is known. It is known that vascular
dilatation and increased permeability occur in inflammatory area. Role of
hyaluronidase enzyme is known in increased vascular permeability (Houck and
Chang, 1979). It has been determined that there is a parallelism between increased
hyaluronidase enzyme activity and severity of inflammation (Procide et al., 1971).
Carrageenan-induced inflammation model is commonly used to determine anti-
inflammatory effects of new compounds (Demirezer et al., 2006). Vascular
permeability increases in the inflammatory region that occurs after carrageenan
application (Nacife et al., 2004). So the aim of this study is to investigate the anti-
inflammatory effect of AD-4 on carrageenan-induced paw volume test and also
investigate the effect of AD-4 on increased vascular permeability with hyaluron-
idase test.
Materials and methods
Animals
In this study, male albino Wistar rats (n = 84, 220–230 g) and albino rabbits
(n = 24, 3.5–4.0 kg) were used. These animals were obtained from the Medical
Experimental Research Centre, Ataturk University. Prior to conducting the
experiments, rats and rabbits were housed and fed as separated groups under

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standard conditions at 22°C in the laboratory. Animal experiments were performed
in accordance with the national guidelines for the use and care of laboratory
animals, and were approved by the local animal care committee of Ataturk
University.
Chemicals
Carrageenan and trypan blue stain were obtained from Sigma Chemical Co. (St.
Louis, USA), thiopental sodium was from Abbott (Camopoverde di Aprilia (LT),
Italy), and indomethacin was from Deva (Istanbul, Turkey), hyaluronidase was from
Kiyevskoye predpriyatiye po proizvodstvu Bakteriynih preparatov (Kiev, Ukraine).
Hyaluronidase (hyaluronoglucosaminidase, EC 3.2.1.35) was obtained from bovine
testicles. Its specific activity was 0.64 U/mg.
Synthesis of 2,5-dihydroxycyclohepta-2,4,6-trienone (AD-4)
AD-4 was synthesized in the light of previous literature (Coskun et al., 2007; Dastan
and Balci, 2006; Mori et al., 1993). The synthesis steps are summarized in Fig. 2.
By base-catalyzed rearrangement of the tropone endoperoxide, AD-4 and additive
end products have been synthesized.
The effects of AD-4 on carrageenan-induced inflammatory paw edema in rats
In this series of experiments, anti-inflammatory effect of AD-4 on carrageenan-
induced paw edema was investigated in a total of 30 rats (Suleyman et al., 2004).
The rats were divided into five groups (n = 6 for each group) prior to initiating the
experiment. AD-4 was administrated by oral gavage to the first, second, and third
groups at doses of 3.62 9 102, 7.24 9 102, and 14.48 9 102 lmol/kg, respectively.
Indomethacin (69.8 lmol/kg) was given to the fourth group by the same method.
The anti-inflammatory effect potency of AD-4 was compared with that of
indomethacin. The control group received an equal volume of distilled water.
(The mentioned amounts of drugs were administered in 1 ml distilled water.) One
hour after administration of drugs, carrageenan solution (0.1 ml, 1% w/v in distilled
water) was injected subcutaneously into the plantar surface of the hind paw of all
rats. Before administration of carrageenan, the paw volumes of each animal were
measured with a plethysmometer up to the knee joint. Carrageenan-induced paw
edema was measured five times at 1-h intervals. The anti-inflammatory effects of
AD-4 and indomethacin were determined by comparing with the results of the
control.
Fig. 2 Synthesis of AD-4

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87
Hyaluronidase-induced capillary permeability test
In this series of experiments, the effects of AD-4 and indomethacin on
hyaluronidase-induced capillary vascular permeability were investigated (Suleyman
et al., 2007a). Twenty-four albino rabbits weighing 3.5–4.0 kg were used for the
experiment. Rabbits were divided into four equal groups and the bilateral abdominal
area of each animal was shaved. The first two groups received 3.62 9 102 and
7.24 9 102 lmol/kg AD-4 orally with the aid of gavage, while 27.9 lmol/kg
indomethacin was given to the third group by identical route. The last group
(control) received the same volume of distilled water.
Hyaluronidase (128 units) was dissolved in 1 ml isotonic NaCl solution and
0.8 ml trypan blue (0.75%) was added to 0.5 ml hyaluronidase solution. One hour
after oral drug administration, 0.1 ml of this mixture was injected subcutaneously
into the shaved area. The appearance of the blue area was measured in mm2, 5 and
30 min after injection. Smaller size of blue area indicates decreased hyaluronidase
enzyme activity and capillary vascular permeability.
Acute toxicity test
Six rats were allocated for each dose to test acute toxicity. AD-4 was administered
at 18.1 9 102, 36.2 9 102, 54.3 9 102, 57.9 9 102, 61.5 9 102, 65.2 9 102, and
68.78 9 102 lmol/kg and indomethacin was administered at 69.8 and 139.6 lmol/
kg to different groups of animals using an oral catheter. Survival of the animals was
monitored for 24 h. Acute toxicity was evaluated according to the number of deaths
during this period (Suleyman et al., 2007a).
Statistical analysis
All results were shown as means standard error (SE). One-way analysis of
variance with post hoc least significant difference (LSD) test was used to evaluate
the results; p \ 0.05 was accepted as the level of statistical significance.
Results
Carrageenan-induced inflammation test
As seen in Fig. 3, AD-4 decreased carrageenan-induced paw edema at doses of
3.62 9 102, 7.24 9 102, and 14.48 9 102 lmol/kg by 45% (p \ 0.001), 79%
(p \ 0.001), and 83% (p \ 0.001), respectively, at the third hour after carrageenan
injection. This value was 49% (p \ 0.001) for indomethacin. The anti-inflammatory
effect of 3.62 9 102, 7.24 9 102, and 14.48 9 102 lmol/kg AD-4 was 48%
(p \ 0.001), 41% (p \ 0.001), and 76% (p \ 0.001), respectively, at the first hour
after carrageenan injection, and was 52% (p \ 0.001), 72% (p \ 0.001), and 75%
(p \ 0.001) at the second hour after carrageenan injection. At the fourth hour, anti-
inflammatory effect of the same doses of AD-4 was 42% (p \ 0.001), 67%

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Fig. 3 Anti-inflammatory effects of AD-4 and indomethacin on carrageenan-induced paw edema in rats.
*Significant at p \ 0.05
(p \ 0.001), and 75% (p \ 0.001). Anti-inflammatory effect of indomethacin at the
first, second, and fourth hours was 50% (p \ 0.001), 51% (p \ 0.001), and 45%
(p \ 0.001).
Hyaluronidase test
Five and 30 min after subcutaneous injection of hyaluronidase enzyme, the areas of
subcutaneous spreading of trypan blue were 288.5 and 498.8 mm2, respectively, in
the control group. In the rabbits receiving indomethacin (27.9 lmol/kg) the
corresponding values were 227 (p \ 0.05) and 279.3 mm2 (p \ 0.01) while the
values in rabbits receiving AD-4 (3.62 9 102 and 7.24 9 102 lmol/kg) were 212.5
(p \ 0.05), 196 (p \ 0.05), 256 (p \ 0.01), and 267.5 mm2 (p \ 0.01) at 5 and
30 min, respectively (Fig. 4).
Acute toxicity
None of the rats receiving AD-4 orally at the doses of 18.1 9 102, 36.2 9 102, and
54.3 9 102 lmol/kg died during the 24-h time period. However, some of the animals
receiving AD-4 at the doses of 57.92 9 102, 61.54 9 102, and 65.16 9 102 lmol/kg
died. Sixteen percent of rats that received the 57.92 9 102 lmol/kg dose of AD-4
died. While death was observed in 33% of the 61.54 9 102 lmol/kg group, this rate
was 66% in the 65.16 9 102 lmol/kg group. All animals that received
68.78 9 102 lmol/kg AD-4 died. There was no observable functional difference in

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89
Fig. 4 Effects of AD-4 and indomethacin on hyaluronidase-induced capillary permeability in rabbits.
*Significant at p \ 0.05
Fig. 5 Acute toxic effects of AD-4 on rats. LD50 value for AD-4 determined as 64.44 9 102 lmol/kg
the 18.1 9 102 and 36.2 9 102 lmol/kg AD-4 groups. However, the rats in the
54.3 9 102 lmol/kgAD-4 group were more inactive when compared with thosein the
18.1 9 102 and 36.2 9 102 lmol/kg AD-4 groups. The rats in this group drank water
between times (did not eat). Also they were positioned close to one another. The rats
that received 57.92 9 102 lmol/kg AD-4 were much more inactive and silent. Their
drinking frequency was lower than the rats in the 54.30 9 102 lmol/kg group. The
posture of the rats in the 61.54 9 102 lmol/kg AD-4 group was worse when compared
with others. Twelve hours after drug administration death was observed among the rats
of the 65.16 9 102 and 68.78 9 102 lmol/kg AD-4 groups. In the group of rats that
received a dose of 69.8 lmol/kg of indomethacin, no deaths were observed, while all
of the rats receiving indomethacin at a dose of 139.6 lmol/kg died. It was determined

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that the 50% lethal dose (LD50) value of AD-4 is 64.44 9 102 lmol/kg (Fig. 5). These
results demonstrate that toxicity of indomethacin is higher than that of AD-4.
Discussion
In the present study, the effect of AD-4 on inflammation was investigated by
observation of carrageenan-induced paw edema. In addition, the effect of AD-4 on
capillary permeability was investigated using a hyaluronidase-induced test in
rabbits.
The results of our experiments showed that AD-4 significantly inhibited
carrageenan-induced paw edema at all doses used. The anti-inflammatory effect
of AD-4 increased in a dose-dependent manner. The anti-inflammatory effect of
AD-4 in all doses studied was accepted as statistically significant. Doses of
7.24 9 102 and 14.48 9 102 lmol/kg of AD-4 were more effective than the
69.8 lmol/kg dose of indomethacin. Doses of 7.24 9 102 and 14.48 9 102 lmol/
kg of AD-4 significantly inhibited carrageenan-induced paw edema in rats during
the first 3 h following administration, while the 3.62 9 102 lmol/kg dose of AD-4
was only found to be effective at the third hour of the test. The anti-inflammatory
effect of indomethacin was found to be effective after the second hour of
carrageenan inflammation. These results demonstrated that AD-4 inhibited both
early and late phases of carrageenan inflammation at doses of 7.24 9 102 and
14.48 9 102 lmol/kg. A carrageenan-induced inflammatory reaction is known to
exhibit an early and a late phase (Gupta et al., 2003). The early phase is associated
with histamine, serotonin, and bradykinin, while occurrence of the late phase
depends on prostaglandin release (Marzocco et al., 2004). All phases of
inflammation have been inhibited by AD-4 administration.
The anti-inflammatory effect of AD-4 (at doses of 7.24 9 102 and
14.48 9 102 lmol/kg) on carrageenan-induced inflammation in both phases of
the inflammatory reaction may result from its inhibitory effect on histamine,
serotonin, bradykinin, and prostaglandin release. In addition, the ineffectiveness of
AD-4 (at 3.62 9 102 lmol/kg dose) in the early phase may result from the lack of
inhibition of early-phase mediators. Indomethacin was used in the experiment at a
69.8 lmol/kg dose because this dose of indomethacin is more potent against
carrageenan-induced inflammation when compared with lower doses (Suleyman
et al., 2004). Indomethacin exhibits an anti-inflammatory effect by the inhibition of
histamine, serotonin, bradykinin, and prostaglandin synthesis like other nonsteroidal
anti-inflammatory drugs (Suleyman et al., 1999, 2007b).
The mechanism of the anti-inflammatory effect of AD-4 has not been reported in
the literature. However, tropolone derivatives have been reported to inhibit
lipoxygenase, which has a role in inflammation in vitro (Suzuki et al., 2000). The
results of our experiments and others published in the literature show that AD-4
possesses anti-inflammatory effect via inflammation mediators.
In a second series of experiments, effects of AD-4 and indomethacin on
hyaluronidase-induced capillary permeability were investigated in rabbits. AD-4
(3.62 9 102 lmol/kg) decreased the hyaluronidase-induced capillary vascular

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91
permeability as effectively as indomethacin (27.9 lmol/kg). In previous studies, this
dose of indomethacin demonstrated a significant reduction of hyaluronidase-induced
capillary permeability (Suleyman et al., 2001, 2003, 2007a). AD-4 and indometh-
acin significantly decreased the spread area of hyaluronidase at 5 and 30 min when
compared with the control. An increase in vascular dilatation and permeability
occurred in the inflamed area. The role of the hyaluronidase enzyme on vascular
permeability has been reported (Houck and Chang, 1979). The acute phase of
inflammatory response, which shows a classic inflammatory response including
calor, dolor, rubor, and tumor, is characterized by transient local vasodilatation and
increased capillary permeability (Maslinska and Gajewski, 1998). The activity of
the hyaluronidase enzyme increases during the inflammation and decreases as the
inflammation reduces (Procide et al., 1971; Houck and Chang, 1979). The role of
the hyaluronidase enzyme is also termed the ‘‘spreading factor,’’ as the increase of
vascular permeability is known (Houck and Chang, 1979). Capillary permeability
increases with the effect of inflammation, for many reasons. When inflammation
occurs, the activity of hyaluronidase increases and inflammation decreases in
parallel with the activity of the hyaluronidase enzyme (Procide et al., 1971).
In the literature, no information exists concerning the effects of tropolone
derivatives on vascular permeability. However, it was reported that hydroxytropol-
ones inhibited enzymes such as inositol monophosphate, alkaline phosphates (ALP),
and b-monooxygenase by competitive or uncompetitive antagonism (Piettre et al.,
1997). Arachidonic acid is also converted to metabolites, including hydroxyeico-
satetraenoic acid (HETE), epoxyeicosatrienoic acid (EETA), and 19,20-
hydroxyarachidonates, by monooxygenase enzymes. The metabolites (involving
the epoxy group) of arachidonic acid (11, 12-EETA) have a potent vasodilatory
effect (Kayaalp, 1998). Inhibition of the monooxygenase enzyme by tropolones may
result in reduction in the production of epoxy metabolites, which are potent
vasodilators, from arachidonic acid. The reduced production of epoxy metabolites
may participate in reduction of increased vascular permeability. Also it is important
to point out that AD-4 is an a,b-unsaturated carbonyl compound. So it can act as a
Michael’s acceptor. However in this study we performed only activity experiments
and did not pay attention to AD-4’s mechanistic properties. A mechanistic approach
requires a new investigation of its own.
In conclusion, the results of the present study demonstrated that AD-4 is an anti-
inflammatory agent, and exhibited lower toxicity than indomethacin. However,
detailed studies are needed to clarify the mechanisms of anti-inflammatory effects of
AD-4.
Acknowledgements We would like to express our thanks to Prof. Dr. Metin BALCI and Prof. Dr. Arif
DASTAN who synthesized AD-4 for their contribution to this work.
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