Hesperidin-3′-O-Methylether Is More Potent than Hesperidin in Phosphodiesterase Inhibition and Suppression of Ovalbumin-Induced Airway Hyperresponsiveness

Hesperidin is present in the traditional Chinese medicine, “Chen Pi,” and recently was reported to have anti-inflammatory effects. Therefore, we were interested in comparing the effects of hesperidin and hesperidin-3′-O-methylether on phosphodiesterase inhibition and airway hyperresponsiveness (AHR) in a murine model of asthma. In the present results, hesperidin-3′-O-methylether, but not hesperidin, at 30 μmol/kg (p.o.) significantly attenuated the enhanced pause (P enh) value, suppressed the increases in numbers of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils, suppressed total and OVA-specific immunoglobulin (Ig)E levels in the serum and BALF, and enhanced the level of total IgG2a in the serum of sensitized and challenged mice, suggesting that hesperidin-3′-O-methylether is more potent than hesperidin in suppression of AHR and immunoregulation. The different potency between them may be due to their aglycons, because these two flavanone glycosides should be hydrolyzed by β-glucosidase after oral administration. Neither influenced xylazine/ketamine-induced anesthesia, suggesting that they may have few or no adverse effects, such as nausea, vomiting, and gastric hypersecretion. In conclusion, hesperidin-3′-O-methylether is more potent in phosphodiesterase inhibition and suppression of AHR and has higher therapeutic (PDE4H/PDE4L) ratio than hesperidin. Thus, hesperidin-3′-O-methylether may have more potential for use in treating allergic asthma and chronic obstructive pulmonary disease.


Introduction
Phosphodiesterases (PDEs) are classified according to their primary protein and complementary (c)DNA sequences, cofactors, substrate specificities, and pharmacological roles. It is now known that phosphodiesterases (PDEs) comprise at least 11 distinct enzyme families that hydrolyze adenosine 3 ,5 cyclic monophosphate (cAMP) and/or guanosine 3 ,5 cyclic monophosphate (cGMP) [1]. PDE1∼5 isozymes, which are calcium/calmodulin dependent (PDE1), cGMP stimulated (PDE2), cGMP inhibited (PDE3), cAMP specific (PDE4), and cGMP specific (PDE5), were found to be present in the canine trachea [2], guinea pig lungs [3], and human bronchi [4]. PDE3 and PDE4 were identified in the guinea pig airway [5], but other isozymes might also be present. PDE4 may adopt two different conformations which have high (PDE4 H ) and low (PDE4 L ) affinities for rolipram, respectively. It is believed that inhibition of PDE4 H is associated with adverse responses, such as nausea, vomiting, and gastric hypersecretion, while inhibition of PDE4 L is associated with anti-inflammatory and bronchodilating effects. Therefore the therapeutic ratio of selective PDE4 inhibitors for use in treating asthma and chronic obstructive pulmonary disease (COPD) is defined as the PDE4 H /PDE4 L 2 Evidence-Based Complementary and Alternative Medicine ratio [6,7]. Although both asthma and COPD are associated with an underlying chronic inflammation of the airways, there are important differences with regard to the inflammatory cells and mediators involved. The key inflammatory cells in COPD are macrophages, CD8+ T-lymphocytes, and neutrophils. In contrast, the key inflammatory cells in asthma are mast cells, eosinophils, and CD4+ T-lymphocytes. Both diseases are sensitive to steroids. However, COPD shows a limited response to inhaled corticosteroids as compared to the efficacy achieved in asthma. Other therapeutic drugs such as selective PDE4 or dual PDE3/4 inhibitors are recently developing. However, these developing inhibitors are also limited for the use of asthma and COPD in clinic because of their emetic side effect. This side effect can be easily assessed in nonvomiting species, such as rats or mice, in which selective PDE4 inhibitors reduce the duration of xylazine/ketamine-induced anesthesia [8,9].
Hesperetin, one of the most-common flavonoids in Citrus, was reported to selectively inhibit PDE4 activity [10]. Men with higher hesperetin intake have lower mortality from cerebrovascular disease and lung cancer, and lower incidences of asthma [11]. Hesperetin frequently occurs in nature as glycosides, such as hesperidin and neohesperidin. They are abundantly present in the fruit peel of Citrus aurantium L. (Rutaceae), a well-known traditional Chinese medicine called "Chen-Pi", which is used as an expectorant and stomach tonic, and contains vitamin P, a remedy for preventing capillary fragility and hypertension [12]. These glycosides are easily hydrolyzed by glycosidase to form hesperetin after ingestion. Hesperidin was recently reported to inhibit inflammatory cell infiltration and mucus hypersecretion in a murine model of asthma [13]. Therefore, we were interested in comparing the effects of hesperidin and hesperidin-3 -O-methylether, a more-liposoluble derivative of hesperidin, on PDE1∼5 inhibition and suppression on ovalbumin-induced airway hyperresponsiveness (AHR). To clarify their potentials for use in treating asthma and COPD, their PDE4 H /PDE4 L ratios were also investigated.

Supression of Inflammatory Cells in BALF.
The numbers of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils from the BALF of control sensitized and challenged mice significantly increased compared to those of nonchallenged mice ( Figure 5(b)). Hesperidin (100 μmol/kg, p.o.) significantly suppressed the increases in numbers of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils ( Figure 5(b)).

Effects on IgG 2a and IgE in the Serum and BALF.
Levels of total and ovalbumin-specific IgE in the BALF and serum of control sensitized and challenged mice were significantly enhanced compared to those of nonchallenged mice. Hesperidin (100 μmol/kg, p.o.) significantly suppressed these enhancements (Figures 6(a), 6(b), 6(c), and 6(d)). The total IgG 2a level in the serum of control sensitized and challenged mice was significantly reduced compared to that of nonchallenged mice. Hesperidin (100 μmol/kg, p.o.) significantly reversed this reduction (Figure 6(e)).

Discussion
Allergic asthma is a chronic respiratory disease characterized by AHR, mucus hypersecretion, bronchial inflammation, Total IgG 2a (ng/mL) Figure 6: Effects of hesperidin (a-e) and hesperidin-3 -O-methylether (f-j) on total IgE (a, c, f, and h) and ovalbumin-specific IgE (b, d, g, and i) levels in bronchial alveolar lavage fluid (a, b, f, and g) and serum (c, d, h, i), and total IgG 2a (e, j) levels in serum of sensitized mice which had received aerosolized methacholine (6.25∼50 mg/mL) 2 days after primary allergen challenge. # P < 0.05, ## P < 0.01, and ### P < 0.001, compared to the nonchallenged group. * P < 0.05 and * * P < 0.01, compared to the control (vehicle) group. Each value represents the mean ± SEM. The number of mice in each group was 10. and elevated IgE levels. Th2 cells, together with other inflammatory cells such as eosinophils, B cells, and mast cells are thought to play critical roles in the initiation, development, and chronicity of this disease [27]. One hypothesis emphasizes an imbalance in Th cell populations favoring expression of Th2 over Th1 cells. Cytokines released from Th2 cells are IL-4, IL-5, IL-6, IL-9, and IL-13, and those from Th1 cells are IL-2, IL-12, IFN-γ, and TNF-α [28,29]. In the present results, hesperidin (100 μmol/kg, p.o.) and hesperidin-3 -Omethylether (30∼100 μmol/kg, p.o.) significantly attenuated P enh values at 25 and 50 mg/mL methacholine (Figures 5(a) and 5(d)) suggesting that it significantly suppresses AHR. At the dose of 30 μmol/kg (p.o.), hesperidin-3 -Omethylether, but not hesperidin, significantly suppressed AHR, suggesting that hesperidin-3 -O-methylether is more potent than hesperidin in the suppression of AHR. Similarly, hesperidin-3 -O-methylether, but not hesperidin, at the dose of 30 μmol/kg (p.o.) significantly suppressed the numbers of all inflammatory cells examined, including total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils in the BALF of mice (Figures 5(b) and 5(e)). Hesperidin-3 -O-methylether even at 10 μmol/kg (p.o.) significantly suppressed the level of IL-4 which are released from Th2 cells, although hesperidin at this dose did not perform in this study. However, hesperidin was reported to insignificantly inhibit the level of IL-4 at a dose of 10 mg/kg (16.38 μmol/kg, p.o.) in a similar animal model [13]. Thus it also suggests that hesperidin-3 -O-methylether is more potent than hesperidin in the suppression of IL-4, although the levels of IL-5 were suppressed to the same extent by both. Hesperidin-3 -O-methylether 10 μmol/kg (p.o.) significantly suppressed the level of IL-2, which are released from Th1 cells, although hesperidin at this dose did not perform in this study. However, hesperidin-3 -O-methylether was obviously more potent than hesperidin in inhibition of TNF-α level, suggesting that the former is more potent than the latter in inhibition of Th1 cells. In contrast, the levels of IFNγ were enhanced by both hesperidin and hesperidin-3 -O-methylether at 100 μmol/kg (p.o.). These results suggest that hesperidin and hesperidin-3 -O-methylether suppress Th2 cells, and partly activate Th1 cells, which ameliorate this imbalance and produce anti-inflammatory effects. Th1 and Th2 cells have been implicated in autoimmune and atopic diseases, respectively [30]. Overall, orally administered hesperidin-3 -O-methylether was more potent than hesperidin to have anti-inflammatory effects in this in vivo study. The different potency between them may be due to their aglycons, because these two flavanone glycosides will be hydrolyzed by β-glucosidase after oral administration [31]. The aglycons of hesperidin-3 -O-methylether and hesperidin are hesperetin-3 -O-methylether and hesperetin, respectively. We have reported the IC 50 values of hesperetin-7,3 -O-dimethylether and hesperetin for PDE4 inhibition are 3.0 μM [32] and 28.2 μM [10], respectively, although that of hesperetin-3 -O-methylether remains unknown. Moreover, in the present results, the IC 50 values of hesperidin-3 -Omethylether for PDE1, 3, and 4 inhibition were 13.6, 13.2, and 13.9 μM, respectively. Thus, that of hesperetin-3 -Omethylether for PDE4 inhibition should be less than 13.9 μM, because its the bulky glycosyl residue may be as a steric hindrance for binding to this PDE conformation [33]. By this reason, hesperetin is more active for PDE4 inhibition than hesperidin which was demonstrated to be inactive for PDE1∼5 inhibitions in the present results. Hesperidin at 30 μmol/kg significantly suppressed levels of IL-2, IL-4, and IL-5 ( Figure 5(c)), and hesperidin-3 -O-methylether at 10 μmol/kg significantly suppressed levels of IL-2, IL-4, and TNF-α ( Figure 5(f)), although all types of inflammatory cells were unaffected by both at these doses (Figures 5(b) and 5(e)). These inconsistencies may be due to the accuracies of these two measurements, because that cytokines were measured using flow cytometric methods, whereas inflammatory cells were measured using a hemocytometer under light microscopy.

Evidence-Based Complementary and Alternative Medicine
IL-4 and IL-13 were shown to induce AHR in mouse asthma models [34,35]. IL-4 has three primary effects. First, IL-4 promotes B cell differentiation to plasma cells that secrete antigen-specific IgE antibodies. Second, IL-4 promotes mast cell proliferation. Third, increased IL-4 upregulates endothelial cell expression of adhesion molecules for eosinophils [36]. IL-5 mobilizes and activates eosinophils, leading to the release of a major basic protein, cysteinylleukotriene, and eosinophil peroxidase that contribute to tissue damage and AHR [35,37]. Phosphoinositide 3-kinase δ (p110δ) was shown to play a crucial role in the development, differentiation, and antigen receptor-induced proliferation of mature B cells [38,39], and inhibition of p110δ attenuates allergic airway inflammation and AHR in a murine asthma model [38,40]. In addition, IL-4 and IL-13 are important in directing B cell growth, differentiation, and secretion of IgE [41]. However, IFN-γ released from Th1 cells preferentially directs B cell switching of IgM to IgG 2a and IgG 3 in mice [42,43]. In the present results, hesperidin (100 μmol/kg, p.o.) and hesperidin-3 -O-methylether (30∼100 μmol/kg, p.o.) significantly suppressed total and OVA-specific IgE levels in the serum and BALF, and enhanced the level of total IgG 2a in the serum of sensitized and challenged mice, suggesting that both have immunoregulatory effects. At the dose of 30 μmol/kg (p.o.), hesperidin-3 -O-methylether, but not hesperidin, significantly suppressed total and OVA-specific IgE levels in the serum and BALF, and enhanced the level of total IgG 2a in the serum of sensitized and challenged mice, suggesting that hesperidin-3 -O-methylether is also more potent than hesperidin in these immunoregulatory effects. 8-Methoxymethyl-3-isobutyl-1-methylxanthine, a selective PDE1 inhibitor, was reported to block lipopolysaccharide (LPS)-mediated biosynthesis of IL-6, but not to influence the TNF-α level. Furthermore, inhibition of PDE3 by amrinone was reported to abolish the effect of LPS on IL-6, but attenuate TNF-α production. Reversible competitive inhibition of PDE4 by rolipram was reported to exhibit a potent inhibitory effect on IL-6 and a dual, biphasic (excitatory/inhibitory) effect on TNF-α secretion [44]. Selective inhibition of PDE1, 3, and 4 by these three compounds was also reported to exhibit a tendency to augment the translocation of NF-κB 1 (p50), RelA (p65), RelB (p68), and c-Rel (p75) and associate with upregulating NF-κB transcriptional activity [45]. These immunopharmacological effects may be found in the administration of hesperidin-3 -O-methylether with a similar extent for PDE1, 3, and 4 inhibition.
Selective PDE4 inhibitors specifically prevent the hydrolysis of cAMP, a 3 ,5 -cyclic nucleotide, and therefore have broad anti-inflammatory effects such as inhibition of cell trafficking and of cytokine and chemokine release from inflammatory cells. The increased cAMP levels induced by these selective PDE4 inhibitors subsequently activate cAMPdependent protein kinase which may phosphorylate and inhibit myosin light-chain kinase, thus inhibiting contractions [46]. The precise mechanism through which relaxation is produced by this second-messenger pathway is not known, but it may result from decreased intracellular Ca 2+ ([Ca 2+ ] i ). The decrease in [Ca 2+ ] i may be due to reduced influx of Ca 2+ , enhanced Ca 2+ uptake into the sarcoplasmic reticula, or enhanced Ca 2+ extrusion through cell membranes [46]. Thus hesperidin-3 -O-methylether may have bronchodilatory effects and may be useful in treating COPD.
In the present in vitro studies, the PDE4 H /PDE4 L ratios of hesperidin and hesperidin-3 -O-methylether were calculated to be 3 and 15.7, respectively. However, neither hesperidin nor hesperidin-3 -O-methylether administered (s.c.) influenced xylazine/ketamine-induced anesthesia. This may be due to the administration route, but administered (s.c.) hesperetin, an aglycon of hesperidin hydrolyzed after oral administration, was reported to not influence xylazine/ ketamine-induced anesthesia [47]. Nevertheless, Ro 20-1724, a selective PDE4 inhibitor, reversed the anesthesia. The reversing effect may occur through presynaptic α 2adrenoceptor inhibition [48], because MK-912, an α 2adrenoceptor antagonist, was reported to reverse xylazine/ ketamine-induced anesthesia in rats [8] and trigger vomiting in ferrets [48]. In contrast, clonidine, an α 2 -adrenoceptor agonist, prevented emesis induced by PDE4 inhibitors in ferrets [48]. The present results also suggest that hesperidin and hesperidin-3 -O-methylether may have few or no adverse effects, such as nausea, vomiting, and gastric hypersecretion. In addition, PDE4 subtypes (A∼D) may be considered for drug development of new PDE4 inhibitors. PDE4D inhibition in nontarget tissues promotes emesis, since PDE4D knock-out mice showed reduction of xylazine/ketamine-triggered anesthesia which is used as a surrogate marker for emesis in mice, a nonvomiting species [9]. In contrast to PDE4D, selective inhibition of PDE4A and/or PDE4B in proinflammatory and immune cells is believed to evoke the therapeutically desired effects of these drugs [49]. Thus, hesperidin-3 -O-methylether did not influence xylazine/ketamine-induced anesthesia may be due to its selectivity for PDE4A and/or PDE4B inhibition(s). However, whether hesperidin-3 -O-methylether selectively inhibits the PDE4 subtype needs to be further investigated.
In conclusion, hesperidin-3 -O-methylether may be more potent than hesperidin in anti-inflammatory and immunoregulatory effects, including suppression of AHR, and reduced expressions of inflammatory cells and cytokines in the murine model of allergic asthma. In addition, neither hesperidin nor hesperidin-3 -O-methylether influenced xylazine/ketamine-induced anesthesia, suggesting that they have few or no emetic effect. Thus, hesperidin-3 -O-methylether may have more potential than hesperidin for use in treating allergic asthma and COPD.