Red Clover (Triflolium pratense)

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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 (RL), enhanced pause (Penh), and increased lung dynamic compliance (Cdyn) 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 IgG2a level in the serum of these mice. The PDE4H/PDE4L 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.

Introduction

Red clover (Triflolium pratense L., Fabaceae) contains formononetin (4′-O-methyldaidzein), biochanin A (4′-O-methylgenistein), daidzein, and genistein in relative proportions of 5.46%, 1.97%, 0.43%, and 0.11% [1]. These phytoestrogenic isoflavones are also present in soy; however, the contents of formononetin and biochanin A are substantially higher in red clover [2]. Since the 19th century, red clover tea or tincture has been used in North America as an antispasmodic for whooping cough, measles, bronchitis, laryngitis, and tuberculosis [3]. According to an early edition of the National Formulary, red clover has also been used as a treatment for asthma. However, the efficacy of most of the above-mentioned traditional red clover treatments has not been tested in randomized, placebo-controlled clinical studies [2]. Recently, botanical dietary supplements containing red clover have received a great deal of attention for treating the symptoms of menopause, the maintenance/improvement of bone, and cardiovascular health. It has also been reported to have a benign effect on breasts and the endometrium [2]. In addition, estrogen receptor-targeted therapeutics have proven successful in treating breast cancer and metabolic disorders, since phytoestrogens were reported as an estrogen-related receptor α agonist [4]. The cancer-protective effects of flavonoids are attributed to a wide variety of mechanisms, including modulating enzyme activity resulting in a decrease in the carcinogenicity of xenobiotics. A number of naturally occurring flavonoids have been shown to modulate the cytochrome P450 (CYP) enzyme system, including the induction of specific CYP isozymes, and the activation or inhibition of these enzymes. Isoflavones inhibit the activity of aromatase (CYP19), thus decreasing estrogen biosynthesis and producing antiestrogenic effects, important in the treatment of breast and prostate cancer [5]. In 2004, we reported that biochanin A selectively inhibited phosphodiesterase (PDE)4 activity with an IC50 value of 8.5 μM, although PDE1 (IC50, 29.1 μM) and PDE2 (IC50, 27.9 μM) activities were also inhibited by the compound. However, biochanin A did not inhibit (IC50 > 100 μM) PDE3 or PDE5 activities [6]. Whether formononetin inhibits PDE4 activity remains unknown. In our previous report, genistein and daidzein selectively inhibited PDE2 and PDE3 activities, respectively [6].

PDEs are classified according to primary protein and complementary (c)DNA sequences, cofactors, substrate specificities, and pharmacological roles. It is now known that PDEs comprise at least 11 distinct enzyme families hydrolyzing cAMP and/or cGMP [7]. 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 [8], guinea pig lung [9], and human bronchi [10]. PDE3 and PDE4 were identified in the guinea pig airway [11], but other isozymes might also be present. Rolipram, a prototype PDE4 selective inhibitor, has both a high (PDE4H) and low (PDE4L) affinity for PDE4. In general, it is believed that the inhibition of PDE4H is associated with adverse responses, such as nausea, vomiting, and gastric hypersecretion, and that the inhibition of PDE4L 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 PDE4H/PDE4L ratio [12]. In this study, biochanin A showed a higher PDE4H/PDE4L ratio (>35) than the selective PDE4 inhibitor AWD 12-281 (11) [13]. The aim of the present study was to determine the potential of biochanin A, contained in red clover, in treating asthma or COPD.

Summary

Since the 19th century, red clover tea or tincture has been used in North America as an:

  • anti-spasmodic
  • whooping cough
  • measles
  • bronchitis
  • laryngitis
  • tuberculosis
  • asthma
  • COPD

However, the efficacy of most of the above-mentioned traditional red clover treatments has not been tested in randomized, placebo-controlled clinical studies

Recently, botanical dietary supplements containing red clover have received a great deal of attention for treating the symptoms of:

  • menopause
  • maintenance/ improvement of bone health
  • cardiovascular health
  • reported to have benign effects on breast and endometrium
  • treatment for breast cancer
  • treatment for metabolic disorders
  • cancer protective
  • treatment for prostate cancer

Supporting Data

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