Interaction Between Daidzein and Hesperetin on Antispasmodic Action in Isolated Sensitized and Non-sensitized Guinea-Pig Tracheas

In traditional Chinese medicine (TCM), a combination of kudzu and Chen-Pi is frequently prescribed for relieving colds, fever, bronchitis, and cough. It contains daidzein and hesperetin, selective inhibitors of family 3 (PDE3), and 4 (PDE4) of phosphodiesterases (PDEs), respectively. In passively sensitized human airways, allergen-induced contraction was reported to be inhibited only by the simultaneous inhibition of PDE3 and PDE4, but not by single inhibition of either isozyme. Therefore, we are interested in investigating the interaction between daidzein and hesperetin on their antispasmodic effects in the isolated sensitized and non-sensitized guinea-pig tracheas, to clarify the difference between these two tissues, because effects of TCM prescription on patients with or without allergic asthma are often different. Guinea-pigs were sensitized by subcutaneous injection of ovalbumin (OVA) into legs. After sensitization, the baseline and cumulative OVA-induced contractions of the sensitized trachea were isometrically recorded on a polygraph. In the same way, the histamine (30 μM)-induced tonic contraction of non-sensitized guinea-pig trachea was recorded. The isobole method was used to analyze the antagonism and synergism between daidzein and hesperetin. The isoboles showed antagonism between daidzein and hesperetin on baseline relaxant effect and OVA (100 μg/ml)-induced contraction in the sensitized guinea-pig trachea. In contrast, the isobole showed synergism between daidzein and hesperetin on the relaxant effect of histamine-induced tonic contraction in non-sensitized guinea-pig trachea. These results suggest that the combination of kudzu and Chen-Pi for relieving colds, fever, bronchitis and cough is effective in patients without, but might show little effect in patients with allergic asthma.


INTRODUCTION
is about 1% present in the tuber of kudzu (Pueraria pseudohirsuta Tang et Wang, Leguminosae), known as Radix Puerariae. In TCM, kudzu is used against colds, fever, influenza, diarrhea, urticaria, tonsillitis, headache, and spasm (Yen, 1970). The alcoholic extract of kudzu or daidzein has been proved to have antispasmodic action against FIGURE 1 | Relaxing effects of daidzein (daid), hesperetin (hesp), and combination of both on baseline (A,C) and on OVA (100 µg/ml)-induced contraction (B,D) in isolated sensitized guinea-pig trachealis. All data including combination of daidzein and hesperetin in isoboles (C,D) are the mean ± SEM. The number (n) of experiment was 6∼10. * P < 0.05, * * P < 0.01, * * * P < 0.001 when compared to the control (vehicle). I: interaction index.
acetylcholine-induced contraction in isolated mouse intestine. The antispasmodic action is non-competitive and papaverinelike (Yen, 1970). Thus it may be a result from their selective inhibition of family 3 (Ko et al., 2004) of PDEs which consists of 11 families reported up today, and by which adenosine (cAMP) or guanosine cGMP is hydrolyzed to form inactive AMP or GMP. After inhibition of PDEs, cAMP or cGMP level is enhanced and subsequently activates cAMP-or cGMP-dependent protein kinase which may phosphorylate and inhibit myosin light-chain kinase, thus inhibiting contractions (Westfall et al., 1998). Hesperetin (5,7,3trihydroxy-4 -methoxyflavanone) is about 1.5% present in the fruit peel of Citrus aurantium L. (Rutaceae), well-known as "Chen-Pi" in TCM, which is used as an expectorant (Yen, 1971). Men with higher hesperetin intake have lower mortality from lung cancer, and lower incidences of asthma (Knekt et al., 2002). Allergic asthma is a chronic respiratory disease characterized by airway hyperresponsiveness (AHR), mucus hypersecretion, bronchial inflammation, and elevated immunoglobulin (Ig) E levels. T helper type-2 (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 (Busse and Lemanske, 2001). Hesperetin was reported to selectively inhibit PDE4 activity (Ko et al., 2004), and to have a suppressive effect on OVA-induced AHR (Shih et al., 2012). In TCM, a combination of kudzu and Chen-Pi is frequently prescribed for relieving colds, fever, bronchitis, and cough (Yen, 1971).
PDE3 and PDE4 isozymes are cGMP inhibited and cAMP specific, respectively. They were identified in the guinea pig airway (Silver et al., 1988). In passively sensitized human airways, allergen-induced contraction was inhibited only by the simultaneous inhibition of PDE3 and PDE4, but not by a single inhibition of either isozyme (Schmidt et al., 2000). Therefore, we were interested in investigating the interaction between daidzein and hesperetin in combination on their antispasmodic effects in the isolated sensitized and non-sensitized guinea-pig tracheas, to clarify the difference between these two tissues, because an effect of prescriptions in TCM on patients with or without allergic asthma is often different. The number (n) of experiment was 6∼10. * P < 0.05, * * P < 0.01, * * * P < 0.001 when compared to the control (vehicle). I: interaction index.
All values including drug combination in isobole are given as the mean ± SEM (n), n is the number of experiment. The difference between two values was determined by Student's t-test. Differences with P < 0.05 were considered statistically significant.

DISCUSSION
In isolated sensitized guinea-pig trachea, nifedipine (1 µM) significantly relaxed the baseline about 40%, but did not affect cumulative OVA (0.01∼100 µg/ml)-induced contractions, which may involve the release of tachykinins from sensory nerve ending (Bertrand et al., 1993), suggesting that the baseline tension is maintained in a part by a Ca 2+ influx through L-type of voltage-dependent calcium channels from the extracellular fluid. The nifedipine-insensitive baseline tension was unrelated to prostaglandins as indomethacin (3 µM) present throughout the experiment. However, daidzein (10, 30 and 100 µM), hesperetin (100 µM), and combination (daidzein plus hesperetin 30 and 100 µM each, daidzein 20 µM plus hesperetin 50, 75, and 100 µM, and daidzein 30 µM plus hesperetin 30, 60, and 100 µM) significantly relaxed the baseline, suggesting that the nifedipine-insensitive baseline tension is related to PDE3 and PDE4 activities, but other isozymes may be involved (Silver et al., 1988). The inhibitory effects of daidzein (30 and 100 µM) on the OVA (100 µg/ml)-induced maximal contraction were obviously greater than those of hesperetin (30 and 100 µM), although their IC 50 values (28.6 and 28.2 µM, respectively) on partially purified PDE3 and PDE4 are similar (Ko et al., 2004). These results suggest that the PDE3 activity may decrease or/and PDE4 activity increase after sensitization. This results in that daidzein is more potent than hesperetin at the same concentration. Both isoboles showed antagonism between daidzein and hesperetin. Our present results revealed an antagonism between hesperetin and daidzein are consistent with the previous report (Clayton et al., 2004).
In non-sensitized guinea-pig trachea, the isobole showed relaxing synergism between daidzein and hesperetin on histamine-induced tonic contraction. Consistently, the bronchodilatory and anti-inflammatory effects of dual PDE3/4 inhibitors are more obvious when compared with those of either isozyme inhibition (Abbott-Banner and Page, 2014). These results point to differing effects in patients with or without allergic asthma. From TCM such examples are known, e.g., in clinic, Sun-Su-In, a preparation containing Panax ginseng, Lithospermi radix, Glycyrrhizae radix, kudzu, Chen-Pi, etc., is mainly used for relieving infection of upper respiratory tract, bronchitis, pneumonia, and emphysema in children or elderly patients without asthmatic history (Zuong, 1995).

CONCLUSION
These findings in sensitized and non-sensitized guinea-pig tracheas suggest that the combination of kudzu and Chen-Pi for relieving colds, fever, bronchitis and cough is effective in patients without, but might show little effect in patients with allergic asthma.

AUTHOR CONTRIBUTIONS
C-HS and W-CK conceived and designed the study. T-YC performed the experiments and analyzed the data. C-HS and W-CK wrote the manuscript. All the authors read and aproved the final manuscript.