Biochanin a, a phytoestrogenic isoflavone with selective inhibition of phosphodiesterase 4, suppresses ovalbumin-induced airway hyperresponsiveness

Abstract

The present study investigated the potential of biochanin A, a phytoestrogenic isoflavone of red clover (Triflolium pratense), for use in treating asthma or chronic obstructive pulmonary disease (COPD). Biochanin A (100 μmol/kg, orally (p.o.)) significantly attenuated airway resistance (R(L)), enhanced pause (P(enh)), and increased lung dynamic compliance (C(dyn)) values induced by methacholine (MCh) in sensitized and challenged mice. It also significantly suppressed an increase in the number of total inflammatory cells, neutrophils, and eosinophils, and levels of cytokines, including interleukin (IL)-2, IL-4, IL-5, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF) of the mice. However, it did not influence interferon (IFN)-γ levels. Biochanin A (100 μmol/kg, p.o.) also significantly suppressed the total and ovalbumin (OVA)-specific immunoglobulin E (IgE) levels in the serum and BALF, and enhanced the total IgG(2a) level in the serum of these mice. The PDE4(H)/PDE4(L) value of biochanin A was calculated as >35. Biochanin A did not influence xylazine/ketamine-induced anesthesia. Biochanin A (10~30 μM) significantly reduced cumulative OVA (10~100 μg/mL)-induced contractions in the isolated guinea pig trachealis, suggesting that it inhibits degranulation of mast cells. In conclusion, red clover containing biochanin A has the potential for treating allergic asthma and COPD.

Figure 1

Displacement of [³H]-rolipram by biochanin A (a) and rolipram (b) in high-affinity rolipram binding sites of guinea pig whole-brain particulates. Each value represents the mean ± SEM. The numbers of experiments for biochanin A and rolipram were 4 and 8, respectively.

Figure 2

Effects of biochanin A (30~100 μmol/kg, p.o.) on R _L (a) and C _dyn (b) in sensitized mice receiving aerosolized methacholine (MCh, 0.78~25 mg/mL) 2 days after primary allergen challenge. ^## P < .01, and ^### P < .001, compared to the nonchallenged group. ***P < .001, compared to the control (vehicle) group. The number of mice in each group was 10. PBS: phosphate-buffered saline.

Figure 3

Effects of biochanin A (30~100 μmol/kg, p.o.) on P _enh (a), inflammatory cells (b), and cytokines (c) in sensitized mice receiving aerosolized methacholine (MCh, 6.25~50 mg/mL) 2 days after primary allergen challenge. ^# P < .05, ^## P < .01, and ^### P < .001, compared to the nonchallenged group. *P < .05, **P < .01, and ***P < .001, compared to the control (vehicle) group. The number of mice in each group was 10. PBS, phosphate-buffered saline; Total, total cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils; IL, interleukin; TNF-α, tumor necrosis factor-α; TNF-γ, tumor necrosis factor-γ.

Figure 4

Effects of biochanin A (10~100 μmol/kg, p.o.) on the total IgG_2a (a) level in the serum, and total IgE (b, c) and OVA-specific IgE (d, e) levels in the serum (b, d), and BALF (c, e) of sensitized mice receiving aerosolized methacholine (MCh, 6.25~50 mg/mL) 2 days after primary allergen challenge. ^## P < .01, and ^### P < .001, compared to the nonchallenged group. *P < .05, and **P < .01, compared to the control (vehicle) group. Each value represents the mean ± SEM. The number of mice in each group was 10.

Figure 5

Effects of subcutaneously administered (a) biochanin A and Ro 20-1724 (b) on the duration of xylazine (10 mg/kg, i.p.)/ketamine (70 mg/kg, i.p.)-induced anesthesia in mice. Biochanin A or Ro 20-1724 was administered 15 min or 1 h prior to anesthesia, respectively. ***P < .001, compared to the vehicle (control). Each value represents the mean ± SEM. The numbers of mice used for biochanin A and Ro 20-1724 were 14 and 21, respectively.

Figure 6

Effects of biochanin A (a) and Ro 20-1724 (b) on cumulative ovalbumin (OVA)-induced contractions in isolated sensitized guinea pig trachealis. *P < .05, **P < .01, and ***P < .001, compared to the control (vehicle). Each value represents the mean ± SEM (n = 5 ~ 8).

Figure 7

Mechanisms involved in the action of Biochanin A. Biochanin A selectively inhibits phosphodiesterase (PDE)4 activity resulting in an increase in cAMP, activating cAMP dependent protein kinase (PKA) and increasing calcium extrusion from the intracellular space and uptake to sarcoplasmic reticula (SR). Biochanin A largely decreased the concentration of intracellular calcium ([Ca²⁺]_i) resulting in trachobronchial smooth muscle relaxation. The increase in cAMP also had anti-inflammatory and immunoregulatory effects. AC, adenylate cyclase; Th, T-helper cells; Ig, immunoglobulin; IL, interleukin; TNF-α, tumor necrosis factor-α. Up and down arrows indicate increases and decreases, respectively.