Bronchodilatory effect of Zingiber officinale Roscoe (ginger) in guinea pigs

Purpose: To determine the bronchodilatory effect of ginger on histamine-induced bronchospasm in guinea pigs. Methods: Thirty-six guinea pigs weighing 400 700 g were randomly divided into six groups. Group 1 received distilled water, while group 2 was given formoterol (1.55 μg/kg) + budesonide (0.02 mg/kg). Guinea pigs in groups 3 and 4 were given ginger (350 and 700 mg/kg, respectively), while those in groups 5 and 6 received ginger (350 and 700 mg/kg, respectively) in addition to formoterol (1.55 μg/kg) and budesonide (0.02 mg/kg). Pre-convulsion time and percent protection in each group was calculated. Results: There was statistically significant improvement in pre-convulsion times, with values of 156.64 ± 32.93, 299.33 ± 44.20, and 235.99 ± 34.55 s for groups 2, 5 and 6, respectively (p < 0.01). Moreover, statistically significant protection values of 73 and 68 % were obtained for groups 5 and 6, respectively, relative to normal control (p < 0.01). Conclusion: These results indicate that ginger has promising potential for use as an add-on treatment for bronchospasm.


INTRODUCTION
Asthma is characterized by chronic airway inflammation, with symptoms such as wheezing, shortness of breath, chest tightness and cough, as well as variable expiratory airflow limitation [1]. Asthma causes limitations in daily activities, loss of school and work days, lung function impairment, and reduced quality of life. Moreover, it is a source of huge socioeconomic burden. Studies have shown that about 15 million disability-adjusted life years are lost annually due to asthma, which represents 1% of the total global disease burden [2]. Although bronchodilators and inhalation corticosteroids are currently used for asthma management, the mortality, morbidity and asthma-related costs are challenging [3].
Complementary and alternative medicine (CAM) is widely used for managing asthma symptoms, but the exact mechanism of action of these agents is unclear [4]. Herbal drugs constitute a major portion of officially-recognized alternate systems of medicine practised in India, and more than 70 % of the Indian population of 1.1 billion still use these non-allopathic systems of medicine due to their evidence-based safety [4].
Zingiber officinale Roscoe (ginger) is one of the common plants consumed worldwide as a spice and flavoring agent. In India, the average daily consumption of fresh ginger root is 8-10 g [5]. Ginger exerts pharmacological effects such as cardioprotective, anti-inflammatory, antimicrobial, anti-proliferative, neuroprotective, hepatoprotective and antioxidant properties [5]. Ginger is also a useful remedy for respiratory ailments like cough, cold and other respiratory problems [6]. Moreover, ginger has also been shown to possess bioactivity against respiratory ailments in various studies either by acting directly as a smooth muscle relaxant, or as an anti-inflammatory effect [7][8][9][10].
The present study was designed to evaluate the bronchodilatory effect of aqueous extract of rhizomes of Zingiber officinale Roscoe in a guinea pig model of bronchial hyperreactivity.

Reagents
Histamine was procured from Sigma Aldrich, USA (H7125-Lot = BCBD6387V The guinea pigs were handled with care as per internationally accepted guidelines and norms for handling and care of animals, as provided by CPCSEA, India [11,12]. Thirty-six healthy guinea pigs weighing between 400 and 700 g were used for the experiments. They were housed in standard transparent polypropylene cages with wheat husk bedding, and kept under controlled room temperature and humidity (26 ± 3 °C; 40 ± 5 %) in a 12-h light/12-h dark cycle, and were given standard laboratory diet and water ad libitum.
After an overnight fast, the animals were exposed to inhalation of 0.5 % histamine in a histamine chamber through a nebulizer [13]. The pre-convulsion time (PCT), i.e. the time from inhalation exposure to the start of dyspnoea leading to the appearance of convulsion, was recorded in seconds (T1). As soon as the asphyctic convulsion occurred, the animals were removed from the chamber and resuscitated with humidified oxygen given through a face mask. After 14 days of treatment with extract/standard drug, the animals were again exposed to 0.5 % histamine inhalation, and pre-convulsion time was recorded (T2).

Statistical analysis
The differences between PCTs on day 0 and day 14 were compared using paired t-test. The values of percentage protection in the normal control group and active control group were compared with corresponding values for the other groups using One-way ANOVA. All statistical analyses were performed using GraphPad Instat trial version 3.06 (GraphPad Software Inc, San Diago, USA). Values of p < 0.01 were considered significant.

RESULTS
The mean pre-drug and post-drug PCT values in different groups are depicted in Table 1. Distilled water produced no effect on the PCT in guinea pigs in group 1. There were significant increases in post-drug PCT values in group 2 (p < 0.01), Data are expressed as mean ± SEM) (n = 6); *p < 0.01 (intra-group comparison between days 0 and 14 PCT, using paired t-test), # p < 0.01 (inter-group comparison amongst all groups with respect to PCT values for day 0 and day 14, using one-way ANOVA) group 5 (p < 0.01) and group 6 (p < 0.01), relative to pre-drug values. The mean values of % protection obtained for different groups are shown in Figure 1. There were significant increases in groups 5 and 6, when compared to normal control group (p < 0.01). However, there were favorable increases in % protection in the low-dose and high-dose Zingiber officinale Roscoe group, relative to normal control group, although these increases were not statistically significant (p > 0.01). Although the values of percentage protection in the low-dose Zingiber officinale Roscoe + formoterol + budesonide group (73.27 ± 3.08 %), and high-dose Zingiber officinale Roscoe + formoterol + budesonide group (67.82 ± 4.80 %) were higher than that of the active control, i.e., formoterol + budesonide group alone (56.67 ± 6.83 %), the difference between them was not statistically significant (p > 0.01).

DISCUSSION
Herbal drugs are used for the treatment of various common ailments as a part of the traditional system of medicine, due to their potential efficacy. However, there is need to scientifically validate their pharmacological effects and their clinical efficacies. Newer drug targets need to be discovered rather than synthesizing more and more me-too drugs [16]. In the present study, an aqueous extract of Zingiber officinale Roscoe (ginger) was investigated with respect to its bronchodilatory effect in terms of pre-convulsion time and percentage protection in guinea pigs. In the normal control group as well as low-and highdose Zingiber officinale Roscoe groups, there were favorable, yet non-significant increases in pre-convulsion times between day 0 and day 14.
However, in the low-dose ginger groups, there were substantial increases in pre-convulsion time between day 0 and day 14, when compared with the normal control group. In formoterol and budesonide control groups, low-dose Zingiber officinale Roscoe + formoterol and budesonide control group, as well as high-dose Zingiber officinale Roscoe group, there were statistically significant increases in pre-convulsion time between day 0 and day 14. An increase in preconvulsion time roughly corresponds to summation of the effect of the active control group and that of the respective ginger group alone group. This indicates that the effect of zingiber officinale Roscoe was additive. There were significant increases in percentage protection in formoterol + budesonide group, lowdose Zingiber officinale Roscoe + formoterol and budesonide group, and in high-dose Zingiber officinale Roscoe + formoterol and budesonide group, when compared to the normal control group.
In low-and high-dose Zingiber officinale Roscoe groups, there were also favorable, yet nonsignificant increases in percentage protection, relative to the active control group. The findings in the present study suggest potential protective effect of 5 % gingerol-based aqueous extract of Zingiber officinale Roscoe against histamineinduced bronchospasm in guinea pigs. These findings also suggest that addition of formoterol and budesonide to Zingiber officinale Roscoe is therapeutically advantageous.
A study by Townsend et al has established independent as well as add-on effects of constituents of Zingiber officinale Roscoe i.e. 6gingerol, 8-gingerol, and 6-shagaol on isolated guinea pig and human tracheas challenged with bradykinin. They reported that 6-shagaol caused significant relaxation of isolated guinea pig and human tracheas, when compared with 8-gingerol and 6-gingerol [9]. In addition, they established that there were significant potentiation of isoproterenol-induced relaxation with any of the constituents of Zingiber officinale Roscoe, while 6-Shagaol was the most effective compound with the largest shift in EC50 [10]. It was also reported that 6-gingerol, 8-gingerol or 6-shagaol significantly inhibited PDE4D, and that in addition, 8-gingerol and 6-shagaol inhibited phospholipase C activity [10].
In addition, Ghayur et al. reported that modulation in inward calcium current might be a probable mechanism involved in ginger-induced airway smooth muscle relaxation [8]. This mechanism might be responsible for the effects of Zingiber officinale Roscoe observed in this study.
The results of the present study suggest that Zingiber officinale Roscoe provides better bronchodilator effect in the presence of formoterol and budesonide than when it is used alone. This may be due to the fact that 5 % gingerol-based aqueous extract of Zingiber officinale Roscoe was used in the present study, while previous studies which reported significant positive effects used either the bioactive constituents alone (i.e. 6-shagaol) or crude extract which may have contained bioactive components other than gingerol. Previous studies reported positive effects of only one bioactive constituent of Zingiber officinale Roscoe i.e. isoproterenol. In present study, formoterol and budesonide were used.

CONCLUSION
The results obtained in this study show that the aqueous extract of Zingiber officinale Roscoe exhibits strong bronchodilator effect against histamine-induced bronchospasm in guinea pigs. The bronchodilator activity may be directly due to smooth muscle relaxation, inhibition of PDE4D enzyme or anti-inflammatory effect. Thus, 5 % gingerol-based aqueous extract of Zingiber officinale Roscoe has potential for clinical application in asthma treatment.