Effects of ginger and its constituents on airway smooth muscle relaxation and calcium regulation

Abstract

The prevalence of asthma has increased in recent years, and is characterized by airway hyperresponsiveness and inflammation. Many patients report using alternative therapies to self-treat asthma symptoms as adjuncts to short-acting and long-acting β-agonists and inhaled corticosteroids (ICS). As many as 40% of patients with asthma use herbal therapies to manage asthma symptoms, often without proven efficacy or known mechanisms of action. Therefore, investigations of both the therapeutic and possible detrimental effects of isolated components of herbal treatments on the airway are important. We hypothesized that ginger and its active components induce bronchodilation by modulating intracellular calcium ([Ca(2+)](i)) in airway smooth muscle (ASM). In isolated human ASM, ginger caused significant and rapid relaxation. Four purified constituents of ginger were subsequently tested for ASM relaxant properties in both guinea pig and human tracheas: [6]-gingerol, [8]-gingerol, and [6]-shogaol induced rapid relaxation of precontracted ASM (100-300 μM), whereas [10]-gingerol failed to induce relaxation. In human ASM cells, exposure to [6]-gingerol, [8]-gingerol, and [6]-shogaol, but not [10]-gingerol (100 μM), blunted subsequent Ca(2+) responses to bradykinin (10 μM) and S-(-)-Bay K 8644 (10 μM). In A/J mice, the nebulization of [8]-gingerol (100 μM), 15 minutes before methacholine challenge, significantly attenuated airway resistance, compared with vehicle. Taken together, these novel data show that ginger and its isolated active components, [6]-gingerol, [8]-gingerol, and [6]-shogaol, relax ASM, and [8]-gingerol attenuates airway hyperresponsiveness, in part by altering [Ca(2+)](i) regulation. These purified compounds may provide a therapeutic option alone or in combination with accepted therapeutics, including β(2)-agonists, in airway diseases such as asthma.

Figure 1.

Ginger and its active constituents relax precontracted human airway smooth muscle. (A) Force studies in human airway smooth muscle show that food-grade ginger relaxes precontracted airways in a dose-dependent manner. Human tracheal strips were contracted to acetylcholine (ACh) at approximately half maximal concentration (∼ EC₅₀), and showed rapid and substantial relaxation upon increasing concentrations of food-grade ginger. In subsequent studies, only the 50 mg/ml concentration was used. (B) Food-grade ginger (50 mg/ml) induced approximately 25% relaxation. The purified active components of crude ginger, [6]-gingerol, [8]-gingerol, and [6]-shogaol, produced relaxation of 60–90% (100 μM). Interestingly, [10]-gingerol (100 μM) did not significantly induce relaxation compared with vehicle control (DMSO, 0.2%). *P < 0.05, compared with vehicle. ^$P < 0.05, compared with food-grade ginger (n = 5–9).

Figure 2.

The relaxant effects of gingerols and [6]-shogaol persist, irrespective of a specific contractile agonist. (A) [6]-gingerol, [8]-gingerol, and [6]-shogaol (300 μM) significantly relaxed both acetylcholine-induced (∼ EC₅₀) and (B) substance P–induced (1 μM) tracheal contractions in ex vivo guinea pig airway smooth muscle. Again, [10]-gingerol did not induce significant relaxation. (A) *P < 0.001, compared with vehicle (n = 8–11). (B) *P < 0.01, compared with vehicle (n = 5–8).

Figure 3.

Ginger constituents block acetylcholine-induced increases of intracellular calcium ([Ca²⁺]_i) in human airway smooth muscle cells. (A and B) In Fura-2–loaded human airway smooth muscle cells, pretreatment with 100 μM gingerols or [6]-shogaol, followed by subsequent exposure to 10 μM acetylcholine (ACh), revealed that [6]-gingerol, [8]-gingerol, and [6]-shogaol prevented increases in [Ca²⁺]_i. This result was not observed with [10]-gingerol. (A) Representative tracing. (B) Summary bar graph. *P < 0.05, compared with vehicle (0.2% DMSO; n = 8). s, seconds. CTL, control; F/F₀, change in fura-2 fluorescence ratio.

Figure 4.

Ginger constituents cause a transient rise in [Ca²⁺]_i. In Fura-2–loaded cells, the addition of [6]-gingerol, [8]-gingerol, or [6]-shogaol alone (100 μM) caused an initial transient increase in [Ca²⁺]_i. This response returned to baseline within 5 minutes. No change in [Ca²⁺]_i was observed with [10]-gingerol (100 μM). *P < 0.05, compared with vehicle (n = 9).

Figure 5.

Initial transient increases in [Ca²⁺]_i caused by ginger components are attributable to influx and store depletion. In airway smooth muscle cells, the initial [Ca²⁺]_i spike was attenuated by the L-type Ca²⁺ channel blocker, nifedipine (10 μM), the transient receptor potential vanilloid channel blocker, capsazepine (100 μM), the ryanodine receptor antagonist, ruthenium red (20 μM), and the removal of external Ca²⁺. It was not attenuated by the inositol triphosphate receptor antagonist, xestospongin C (XeC, 1 μM). *P < 0.05 (n = 7–8).

Figure 6.

[6]-shogaol decreases the synthesis of inositol phosphate. After a 15-minute preincubation with ginger constituents (100 μM) or vehicle (0.1% or 0.2% DMSO), 10 μM of bradykinin were added for an additional 30 minutes before the extraction and column separation of newly synthesized total [³H]-inositol phosphates. Only [6]-shogaol significantly attenuated the production of inositol phosphate. Data are expressed as a percentage of vehicle control values. *P < 0.05, compared with vehicle (n = 3–8).

Figure 7.

The nebulization of [8]-gingerol prevents hyperresponsiveness to methacholine. In male A/J mice, increasing concentrations of methacholine (MCh) result in increases in central airway resistance (Newtonian resistance, Rn) measured using a constant phase model on a Flexivent FX1 module. Increases in resistance at 37.5 and 50 mg/ml MCh were attenuated in animals that received a 10-second nebulization of 100 μM [8]-gingerol, 15 minutes before MCh challenge. *P < 0.05, compared with vehicle (n = 6–11).