Effect of in vitro and in vivo aerosolized treatment with geniposide on tracheal permeability in ovalbumin-induced guinea pigs

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Effect of in vitro and in vivo aerosolized treatment with geniposide on

tracheal permeability in ovalbumin-induced guinea pigs

Jiahorng Liaw*, Yen-Chin Chao

Department of Pharmaceutics, School of Pharmacy, Taipei Medical University, 250 Wu Hsing Street, Taipei 110, Taiwan Received 10 May 2001; received in revised form 30 October 2001; accepted 2 November 2001

Abstract

The primary objective of this study was to investigate the effect of geniposide, a potent anti-inflammatory, on ovalbumin-antigen-induced tracheal permeability and transepithelial electrical resistance in guinea pigs. Two weeks after sensitization with ovalbumin (100 mg/ml), the permeability of guinea-pig tracheas was evaluated by flux measurements using the transcellular tracer, [14_{C]estradiol, and the paracellular}

tracer, [14C]mannitol. The effect of extracellular Ca2 + with geniposide was also studied, using deletion of Ca2 + in the donor chamber. The in vivo treatment effect of aerosolized geniposide on tracheal permeability in the ovalbumin-sensitized guinea pigs was also evaluated. The results indicate that tight junction permeability of ovalbumin-sensitized trachea was significantly dose dependent and decreased by geniposide (1 – 10 mM), as evidenced by substantial recovery of transepithelial electrical resistance and decreased transepithelial permeability of [14C]mannitol at (1.32 ± 0.12) 10 5cm/s. The effect of combination of the removal of extracellular Ca2 +with geniposide had no effect on tight junction permeability of ovalbumin-sensitized trachea and revealed that transepithelial electrical resistance and junction permeability did not recover. In addition, the cAMP levels and phosphodiesterase activity were not significantly influenced in ovalbumin-sensitized tracheal tissues after geniposide treatment. Inhaled geniposide (50 mM, 30 min after ovalbumin sensitization) significantly restored junction permeability induced by ovalbumin (100 mg/ml, 2 min). Junction permeability did not recover on pretreatment with geniposide (50 mM for 30 min over 16 days consecutive before ovalbumin sensitization) after exposure of conscious guinea pigs to aerosol ovalbumin. In conclusion, geniposide has inhibitory effects on ovalbumin-induced junction permeability and recovery of transepithelial electrical resistance in guinea pig trachea, showing its potential as anti-asthma therapy.D 2001 Elsevier Science B.V. All rights reserved.

Keywords: Permeability; Tight junction; Geniposide; Aerosol; Ovalbumin

1. Introduction

It is known that the increase in permeability of respira-tory epithelium after exposure to antigens is due to dam-aged epithelial junctions (Hulbert et al., 1981). In addition, others (Boucher et al., 1980; Goto et al., 2000) have demonstrated that this altered permeability is associated with structural damage to the healing strands of tight junctions and adherent junctions. We also observed that tight junctions in sensitized guinea pigs were opened by ovalbumin (Liaw et al., 1999). The altered state of perme-ability associated with mucosal injury must eventually return to normal or these junctions of tracheal epithelium must be closed to prevent further exposure to the antigen (Mashito et al., 1999; Ohbayashi et al., 1999; Pons et al.,

2000). Thus, a potential for closing tight junctions and repairing membrane permeability may be of interest for the treatment of asthma.

Geniposide is one of the major iridoid glucosides in the fruit of Gardenia jasminoides Ellis, which has been reported to possess anti-inflammatory activity (inhibition of 5-lip-oxygenase) (Nishizawa et al., 1988), activity against tumor-promoting12-O-tetradecanoyl-phorbol-13-acetate (activa-tion of protein kinase C) (Ueda and Iwahashi, 1991; Lee et al., 1995), and which have been used for treatment of a liver disorder (inhibition of P450-3A monooxygenase) (Ka-ng et al., 1997). Some authors (Kaji et al., 1990; Hua(Ka-ng et al., 1998; Li et al., 2000) also found that geniposide could promote collagen synthesis in false-aged rats, stimulate the proliferation of endothelial cells but, interestingly, also act as a cross-linking agent with low cytotoxicity and biocom-patibility.

Additional studies on the in vivo pharmacological profile of geniposide demonstrated that it not only could counter

*

Corresponding author. Tel.: +886-2377-9873; fax: +886-2377-9873. E-mail address: jhorng@tmu.edu.tw (J. Liaw).

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resistance to Ca2 +-induced mitochondrial membrane per-meability but also could inhibit the rapid reduction of mitochondrial membrane potential (Yamamoto et al., 2000). Furthermore, Yamazaki et al. (1996) reported that geniposide could enhance responses of cells to carbachol and KCl-induced depolarization in terms of cytoplasmic free-Ca2 + concentration. They indicated that geniposide could induce cell differentiation through activation of components of the intracellular signal transduction pathway (cAMP and protein kinase). In addition, geniposide also inhibits the spontaneous pilocarpine-induced contraction of rat stomach and cathartic action (Endo and Taguchi, 1973). However, the effectiveness of geniposide on the recovery of junction permeability of the asthmatic trachea is still unknown. Therefore, the purpose of this project was to evaluate the repair of junction permeability of asthmatic trachea by geniposide, using radioactive markers in an in vitro guinea pig model of acute antigen ovalbumin-enhanced permeabil-ity. Secondly, the effects of geniposide on low extracellular

Ca2 + concentration, cAMP levels, and phosphodiesterase

activity in ovalbumin-induced tracheal permeability were also studied. Finally, the influence of aerosolized geniposide on the antigen-induced tracheal junction permeability was studied in the in vivo guinea pig.

2. Materials and methods 2.1. Animals and materials

Male Dunkin – Hartley guinea pigs that weighed 250 – 350 g from the Animal Center of National Taiwan University were used. Radioactive [14C]mannitol and [14C]estradiol were obtained from NEN Life Science Products (DuPont Chemical, Boston, MA, USA). Geniposide was obtained from the fruit of G. jasminoides Ellis with boiling water under reflux for 6 h. Geniposide was isolated following passage through charcoal and silica columns in methanol,

with a 1:5 MeOH – CHCl3solvent system. The identity of

geniposide was confirmed, based on measuring the melting

point, and using FAB-Mass, infrared spectrometry, 1

H-nuclear magnetic resonance (1H-NMR), and 13C-NMR as

well as commercial standards. The yield and purity of geniposide were 4.7% and 99%, respectively, as determined by high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC). Ovalbumin was obtained from Sigma (St. Louis, MO, USA). All chemical reagents were of analytical grade and were used as received.

2.2. Immunization

Procedures for sensitization of guinea pigs followed the methods from our previous study (Liaw et al., 1999). Male Dunkin – Hartley guinea pigs were assigned at random to four groups. One group was first sensitized by the inhalation of aerosolized antigen (10 mg/ml of ovalbumin) for 30 min

daily for 8 days, using a DeVilbiss nebulizer (25 15-cm chamber, Pulmo-Amide 5610D, Somerset, PA, USA) with a

particle size range of 0.5 – 5 mm. The second challenge

sensitization was performed on the 16th day after the first, using 100 mg/ml ovalbumin aerosol for 2 min. The second group, as a control group, was not sensitized with ovalbu-min, and thus, served as the non-sensitized group. The other two groups were used for the in vivo aerosolized geniposide study in Section 2.5.

2.3. In vitro perfusion studies

The guinea pigs were killed with an overdose i.p. injection of 3% sodium pentobarbital. The tracheas were immediately removed, mounted on acrylic rings (0.24 cm2), and placed in Ussing chambers as in our previous study (Liaw et al., 1999). Briefly, both surfaces of the trachea were bathed in glutathione bicarbonated Ringer (GBR) solution. All experiments were performed at 37C with a mixture of 95% O2and 5% CO2and at a pH adjusted to 7.4 with NaOH

or HCl. The permeating [14C]radioactive compound was

sampled from the receiver compartment at fixed intervals and replaced with an equal volume of previously warmed

Table 1

Effect of geniposide on the in vitro ovalbumin-sensitized tracheal permeability of guinea pigs

Treatment Apparent permeability coefficients ( 10 5cm/s) [14_C]estradiola _[14_C]mannitol Control 2.23 ± 0.27 (n = 5) 1.49 ± 0.11 (n = 6) Control + 5 mM geniposide ndb 1.46 ± 0.27 (n = 5) Control + 10 mM geniposide 2.46 ± 0.39 (n = 5) 1.46 ± 0.12 (n = 7) Control Ca2 + nd 2.21 ± 0.22 (n = 7)c Control + 10 mM geniposide Ca2 + nd 1.91 ± 0.21 (n = 8)c Sensitized 1.96 ± 0.27 (n = 5) 1.92 ± 0.12 (n = 9)c Sensitized + 1 mM geniposide nd 2.26 ± 0.08 (n = 6) Sensitized + 5 mM geniposide nd 1.53 ± 0.21 (n = 6) Sensitized + 10 mM geniposide 2.13 ± 0.49 (n = 6) 1.32 ± 0.12 (n = 8)d Sensitized Ca2 + _nd _{1.31 ± 0.09 (n = 7)}d Sensitized + 10 mM geniposide Ca2 + nd 1.63 ± 0.06 (n = 8)e Sensitized + 1 mM nifedipine nd 1.20 ± 0.07 (n = 4)d a

There was no statistically significant difference between the four estradiol treatment groups ( p> 0.1).

b

nd: not determined.

c

Denotes a statistical significance at p 0.01, as compared to the control mannitol group.

d

Denotes a statistically significant decrease at p 0.0005, as compared to the sensitized mannitol group.

e _{There was a statistically significant difference between treated and}

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GBR solution. The radioactive compounds were counted as the total number of disintegrations per minute (dpm) with a Beckman 6500 liquid scintillation counter.

2.4. Transepithelial electrical resistance measurement Electrodes were prepared from silver wire, and resistance was measured using an Ag – AgCl four-electrode system (Liaw et al., 1999). Variable current pulses were given, and a digital multimeter measured the corresponding potential differences. Resistance was calculated from the slope of the applied current and potential difference plot.

2.5. In vivo aerosolized geniposide study

For the in vivo aerosolized geniposide study, a separate group of conscious male Dunkin – Hartley guinea pigs was treated with 50 mM of geniposide using a DeVilbiss nebulizer for 30 min after 2 min of second challenge sensitization. To evaluate the prevention ability of genipo-side in ovalbumin-sensitized guinea pigs, the second group inhaled 50 mM geniposide for 30 min daily over 16 days, using a DeVilbiss nebulizer before the second challenge sensitization. These guinea pigs were then killed with an overdose injection of 3% sodium pentobarbital after 2 min of second challenge sensitization for further evaluation in an in vitro perfusion study.

2.6. Assay of adenosine 3,5-cyclic monophosphate (cAMP) and inhibition of c-AMP phosphodiesterase by geniposide

c-AMP was assayed with a c-AMP assay kit from NEN Life Science Products (Boston, MA, USA) which measured phosphodiesterase activity. The experimental procedures followed those of our previous studies (Liaw et al., 1999).

2.7. Apparent permeability coefficient calculation and data analysis

In vitro apparent permeability coefficients ( P) were calculated from the equation

P¼ V =A 1=C dC=dt

¼ ðFraction of dose transportedÞ =dt V =A;

where the fraction of the dose transported through the trachea can be calculated after correction for sampling and solution replacement at each time point. These values were then plotted versus time. Therefore, the apparent permeability coefficient, P (cm/s), could be calculated from the slope, using the receiver compartment volume, V (7 ml), and sur-face area of the tissue A (0.24 cm2). Data from all experi-ments were pooled to determine the mean and standard error of the mean (S.E.M.). Analysis of variance (ANOVA), with Dunnett’s multiple comparison tests at 95% confidence levels, was used to determine the significance of differences between each group of experiments.

3. Results

Paracellular permeability coefficients (mannitol) of the control and ovalbumin-sensitized guinea pig tracheas are shown in Table 1. The [14C]mannitol permeability of the trachea in the sensitized group was significantly greater than that of the control group ([1.92 ± 0.12] vs. [1.49 ± 0.11] 10 5cm/s). Table 1 also shows the effects of three different concentrations (1, 5, and 10 mM) of geniposide on the permeability of mannitol in ovalbumin-sensitized tracheal tissues; the apparent permeability coefficients of mannitol

Fig. 1. Typical resistance – time profiles of ovalbumin-sensitized guinea-pig trachea, using three concentrations of geniposide (G). Error bars represent 1 S.E.M.; n = 6 – 8. * indicates a significant difference at P < 0.01 from the ovalbumin-sensitized group.

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were (2.26 ± 0.08), (1.53 ± 0.21), and (1.32 ± 0.12) 10 5 cm/s. Among these treatments, only 10 mM geniposide could decrease permeation of mannitol, and there was no significant difference from control tissues. In addition, in control tissues, there was no significant difference between effects for the two concentrations of geniposide. The apparent permeability coefficient of estradiol, an intracellular marker, also showed no significant differences between treatments ( p > 0.05). Fig. 1 shows the electrical resistance profile of guinea-pig tracheas sensitized with aerosolized ovalbumin and treated with three concentrations of geniposide. The range of transepithelial electrical resistance of the control and 10 mM geniposide-treated tracheal tissues fell in the range of 300 – 425 V cm2 over a 5-h period. On the other hand, in the 1 and 5 mM geniposide groups, resistance values decreased to around 98 – 280 V cm2, indicating that these two concentrations could not repair tracheal junctional integrity after ovalbumin challenge.

To understand the relation between geniposide and the effect of extracellular Ca2 + on tracheal permeability, Ca2 + was removed from the medium in the donor chamber during a 5-h experiment. Control and ovalbumin-sensitized tracheal permeability were affected by the removal of Ca2 + from the donor chamber and mannitol junction permeability was (2.21 ± 0.22) and (1.31 ± 0.09) 10 5 cm/s, respectively. After combination removal of Ca2 + from the extracellular bathing medium with the 10 mM geniposide-treated sensi-tized group, mannitol junction permeability increased from (1.31 ± 0.09) to (1.63 ± 0.06) 10 5cm/s, and a significant difference was found compared with that of the sensitized trachea group (1.92 ± 0.12 10 5 cm/s). However, there was no protection of junction permeability (1.91 ± 0.21 10 5cm/s) in the control group with 10 mM geniposide in a

low-Ca2 + extracellular medium. Furthermore, incubation

with 1 mM nifedipine restored junction permeability of

mannitol (1.20 ± 0.07 10 5 cm/s) to the same extent as 10 mM of geniposide did. A typical resistance – time profile of guinea pig trachea without Ca2 + is illustrated in Fig. 2. The resistance of samples without Ca2 + in the donor chamber decreased to around 90 – 200 V cm2. For the 10 mM genipo-side, 1 mM nifedipine, and control groups, the resistance values were all above 300 V cm2. Using the trichloroacetic acid extraction method, the cAMP content in ovalbumin-sensitized tracheal tissues was found to be 127.5 ± 2.5 to 122.8 ± 2.4 pmol/ml ( p > 0.05, n = 6) after 10 mM geniposide treatment. In addition, the inhibition of phosphodiesterase activity by 10 and 100mg/ml geniposide was evaluated, and the effects on this enzyme activity were found to be only 19.62% and 10.02%, respectively. Taken together, these results suggest that the extracellular calcium concentration in the environment of the tissues influences the effect of geniposide on sensitized trachea more than the cAMP second messenger pathway.

Inhalation of 50 mM geniposide 30 min after ovalbu-min challenge significantly decreased the apparent

per-Fig. 2. Typical resistance – time profiles of ovalbumin-sensitized guinea-pig trachea with 10 mM geniposide (G) (!), 10 mM geniposide without Ca2 +in the donor solution (5), and the control group with 10 mM geniposide without Ca2 +

in the donor solution (&), 1 mM nifedipine (

5

). Error bars represent 1 S.E.M.; n = 6 – 8.

Table 2

Effect of aerosolized geniposide on the in vivo ovalbumin-sensitized tracheal permeability of guinea pigs

Treatment Apparent permeability coefficients [14_{C]mannitol ( 10} 5_cm/s) Sensitized 1.92 ± 0.12 (n = 9) Aerosolized geniposide 50 mM + sensitizeda 1.98 ± 0.06 (n = 5) Sensitized + aerosolized geniposide 50 mMb 0.75 ± 0.19 (n = 6)c a

Pre-treatment with 50 mM geniposide for 30 min over 16 consecutive days before ovalbumin sensitization.

b

Inhalation of 50 mM geniposide 30 min after ovalbumin sensitization.

c

Denotes a statistically significant decrease at p 0.0005, as compared to the sensitized mannitol group.

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meability coefficients of mannitol from (1.92 ± 0.12) to (0.75 ± 0.19) 10 5 _{cm/s (Table 2). However, there was} no influence of pretreatment with 50 mM geniposide for 30 min for 16 consecutive days before ovalbumin

sensiti-zation at (1.98 ± 0.06) 10 5 cm/s. Fig. 3 shows the

electrical resistance property of guinea-pig tracheas with aerosolized 50 mM geniposide after ovalbumin sensitiza-tion. Transepithelial electrical resistance with aerosolized

geniposide increased to around 389 – 460 V cm2 which

differs significantly from that of ovalbumin-sensitized trachea of guinea pigs.

4. Discussion

Ranga et al. (1983) have reported that after antigen (ovalbumin) challenge, there was a significant increase in the plasma levels of horseradish peroxidase, dextran, man-nitol, and its correlates of enhanced transepithelial perme-ability of tight junctions. In our studies, after aerosol ovalbumin administration in guinea pig, tracheal permeabil-ity mainly consistently influenced a paracellular (tight junction) pathway up to (1.96 ± 0.27) 10 5cm/s as well as resulting in decreased transepithelial electrical resistance. Yamamoto et al. (2000) found that oral delivery of geniposide in mice could counter the Ca2 +-induced mito-chondrial membrane permeability and inhibits the rapid reduction of membrane potential. In the present study, we have extended these observations to show that geniposide reduces tight junction permeability in guinea-pig trachea. These results are consistent with those of previous studies that showed a reduction in antigen-induced microvascular leakage in guinea-pig trachea by baicalin and recovery of junction permeability in vitro (Liaw et al., 1999). Bhat et al. (1993) reported that high extracellular Ca2 + levels and low intracellular Ca2 + levels are required for normal epithelium.

In addition, decreasing extracellular Ca2 + may induce

intracellular Ca2 + release from internal stores (mitochondria and endoplasmic reticulum) followed by opening of tight junctions of the trachea. This is consistent with our perme-ability and transepithelial electrical resistance results show-ing that both in control trachea with low extracellular Ca2 + medium and in ovalbumin sensitized.

Yamazaki et al. (1996) reported that geniposide could

enhance the increase of cytoplasmic Ca2 + concentration

induced by high K+ and the carbachol-induced depolariza-tion with cytoplasmic Ca2 +-free condition and indicated that it could activate the intracellular signal pathway (protein kinases). Others (Anderson et al., 1993; Sandoval et al., 2001) suggested that the compound could affect tight junc-tion permeability and may exert its acjunc-tion through mecha-nism involving Ca2 +, cytoskeleton, cadherin, and junction proteins (ZO-1,-2, cingulin, and Rab13, etc.). The inferred blocking properties of Ca2 + influx channels or inhibition of Ca2 + release from intracellular stores, or promotion of Ca2 + sequestration by intracellular stores we showed in our geniposide studies in guinea pig trachea, had also been demonstrated by Ivorra et al. (1992). Similar results were

obtained with 1 mM nifedipine (a Ca2 + channel blocker)

treatment in ovalbumin-sensitized trachea, namely decreased mannitol permeability of trachea. This was found with other calcium antagonists (diltiazem) that attenuate antigen-induced bronchoconstriction in various animal species (Fanta et al., 1982; Malo et al., 1983) and show some activity in asthmatic patients (Barnes, 1985). These characteristics make this iridoid attractive for exploration of its anti-inflam-matory activity in vivo. Taken together, our results suggest that treatment of ovalbumin-sensitized tracheal tissues with 10 mM geniposide involves Ca2 + and plays a dominant role in repairing the junction structure.

When the concentration of geniposide was increased to over 10 mM, tracheal permeability increased to (1.93 ±

Fig. 3. Typical resistance – time profiles of ovalbumin-sensitized guinea-pig trachea with aerosolized 50 mM geniposide (G) (!). Preventive ability of aerosolized 50 mM geniposide for 30 min, 16 days on ovalbumin-sensitized guinea pigs (5). Error bars represent 1 S.E.M.; n = 5 – 9.

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0.77) and (2.19 ± 0.69) 10 5 _{cm/s for 15 and 20 mM} geniposide, respectively. Furthermore, geniposide was reported to be able to stimulate biliary excretion, to prevent hepatic damage induced by aflatoxin B1, and to have anti-inflammatory functions; however, hepatotoxicity was seen when it was administered at a high dose (Yamano et al., 1990). This is consistent with our finding that geniposide can be toxic to the structure of trachea tissues in high con-centrations. However, mechanisms of damage to asthmatic trachea by higher doses of geniposide will need further investigation.

Inhalation of aerosolized geniposide (50 mM, 30 min after antigen exposure) by conscious guinea pigs prevented the junction permeability that appeared and restored the tissue integrity, affected in sensitized animals immediately after their exposure to the antigen aerosol. Lower doses of genipo-side did not appear effective (data not shown). The dose of inhaled geniposide (50 mM) necessary to significantly restore the damage to the ovalbumin-sensitized trachea was higher than the corresponding inhaled or intratracheal doses of

rolipram (10 – 500mM) which markedly inhibited

broncho-constriction by an antigen in actively sensitized guinea pigs (Cortijo et al., 1993). Therefore, the preventive effect of 16 consecutive days-inhaled geniposide on antigen-induced acute asthma may be related to its low ability to inhibit phosphodiesterase IV activity.

However, the possible contribution of a variety of intra-cellular second messenger mechanisms to the inhibitory effect of geniposide should also be considered. Ullrich et al. (1993) indicated that inhibitors of dibutyl c-AMP hydro-lytic and phosphodiesterase activities could increase the concentration of c-AMP in epithelial cells and preserve the structure of tight junctions. Although geniposide did not significantly increase the cAMP content in trachea, and the inhibition of phosphodiesterase activity only reached

19.62% with 10 mg/ml geniposide, it could augment the

cAMP accumulation produced by the adenylyl cyclase activator. Pons et al. (2000) reported that glaucine did not increase cAMP, but could augment the cAMP accumulation produced by forskolin. On the other hand, cyclic nucleo-tides, such as cGMP, coupled to the nitric oxide system, are also of outstanding interest in this respect (Bredenbroker et al., 2001). Therefore, the contribution of these various actions to the inhibitory effect of geniposide cannot be ruled out by the present experiments.

In summary and conclusion, inhaled geniposide effec-tively reduced antigen-induced junction permeability, caus-ing the recovery of transepithelial resistance in actively sensitized guinea pigs. The main mechanism of these inhibitory effects of geniposide is likely to be its known property of blocking calcium channels but the contribution of other pharmacological properties cannot be excluded. Geniposide has been used for years as an anti-inflammatory, but its potential use as an anti-asthmatic is uncertain since other more potent and selective agents appear advantageous for treating asthma.

Acknowledgements

The authors thank Dr. K.H. Hsieh for the technical help provided in the ovalbumin sensitization of the guinea pigs. Also the authors thank Drs. L.L. Yang and K.Y. Yen for their hospitality during my stay in the Graduate Institute of Pharmacognosy. This work was supported by a grant from the National Science Council, Republic of China (NSC84-2331-B038-030-M03).

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