MXGST is mainly used by traditional Chinese physicians to treat cough from pneumonia. A recent report indicated that MXGST prolonged the coughing onset and decreased the coughing frequency of ammonia-induced cough in rats [
23]. Citric acid-induced cough is a very useful model for assessing the antitussive effects of medicinal plants and synthetic compounds. Hence, we first performed a citric acid-induced cough to evaluate the antitussive effects of MXGST extract in guinea pigs. The dosage (0.4 g/kg) of MXGST was chosen according to the conversion of the clinical daily therapeutic dosage [
5] to animal equivalent dosage by body surface area and other reports [
6,
7,
23]. The present data are similar to the effects of MXGST on ammonia-induced cough [
23], indicating that MXGST and the aminophylline positive control had antitussive effects on the citric acid-induced cough model. Furthermore, citric acid stimulated rapidly adapting, histaminergic and acetylcholinergic receptors that are located in the larynx and upper airways of the tracheobronchial tree by opening the pH gated ion channels to induce cough and tracheobronchial contraction [
2]. The inhalation of citric acid also causes bronchial hyperresponsiveness to acetylcholine and hypersensitivity to histamine-induced microvascular leakage, which can then induce tracheobronchial contraction and cough [
2]. As a result, the antitussive mechanism of MXGST extract on the bronchial tract was explored by acetylcholine/histamine-induced tracheobronchial contraction in guinea pigs. We also found that MXGST and positive control aminophylline relaxed the tracheobronchial contraction caused by acetylcholine/histamine. Another researcher indicated that MXGST relieved airway resistance increases by
Dermatophagoides Pteronyssinus in guinea pigs [
7]. Moreover, Ephedrae possessed the antitussive effects of citric acid-induced cough in guinea pigs [
24]. Additionally, ephedrine and methylephedrine (both active ingredients of Ephedrae) have antitussive and anti-asthmatic effects in animal models and humans. They can dilate bronchial smooth muscle via activating both sympathomimetic α- and β-adrenergic receptors [
25,
26]. Amygdalin, a major constituent of Semen Pruni Armeniacae, also possesses antitussive activities via inhibiting the central cough center when it is biotransformed into cyanide, an active ingredient [
25]. Licorice also decreased the cough response induced by ammonia or capsaicin [
27,
28]. Moreover, co-treatment with Ephedrae and Semen Pruni Armeniacae has better antitussive effects than Ephedrae or Semen Pruni Armeniacae alone [
9]. Thus, we suggested that MXGST extract, at a lower dosage than the reported dosage of each medicinal component, possessed antitussive effects. These effects might be due to the synergic effects of its medicinal components, especially of Ephedrae and Semen Pruni Armeniacae [
9]. The antitussive mechanism of MXGST might be related to dilating the bronchial smooth muscle by inhibiting the histaminergic and acetylcholinergic receptors in the bronchial tract, activating both sympathomimetic α- and β-adrenergic receptors from ephedra alkaloids and inhibiting the central cough center from amygdalin [
25,
26]. However, citric acid-induced cough and tracheobronchial constriction are partially mediated by the nonadrenergic–noncholinergic-nonhistaminergic nervous systems, which include tachykinins, leukotrienes and prostaglandin E
2 from mast cells [
29‐
33]. Therefore, whether the antitussive mechanism of MXGST might be related to stabilizing mast cells and inhibiting other inflammatory mediators and nervous systems must be investigated in the future.
Second, the anti-pyretic effects of MXGST extract on LPS-induced hyperthermia in rats were evaluated because traditional Chinese physicians mainly use MXGST to treat fever caused by pneumonia. This result is consistent with other report that MXGST possesses anti-pyretic effects on a typhoid/paratyphoid vaccine-induced fever model in rabbits [
6]. Some researchers indicated that Gypsum possesses anti-pyretic effects in Brewer’s yeast-induced hyperthermia via decreasing the hypothalamus prostaglandin E
2 levels [
34]. Glycyrrhetic acid, a major ingredient of licorice, possesses anti-pyretic effects in Brewer’s yeast-induced fever in rats via the pituitary adrenal axis [
35]. The herb pairs, Ephedrae and Gypsum, significantly attenuated Brewer’s yeast-induced fever in rats [
36]. Therefore, it can be suggested that MXGST possesses anti-pyretic effects via the synergic effects of its medicinal components, especially Gypsum and Ephedrae [
8,
36], and this effect might be mediated through the modulation of prostaglandin E
2 synthesis in the hypothalamus adrenal gland axis from glycyrrhizic acid and Gypsum [
34,
35].
Each MXGST component has been reported to have the various adverse effects, such as cardiovascular and CNS toxicology of Ephedra [
11,
12,
18,
19], edema and mineralocorticoid excess syndrome of licorice [
20], cyanide toxic reaction of Semen Pruni Armeniacae [
13,
14,
17], and allergic response of Gypsum [
37]. Therefore, we further evaluated the subacute toxicology of MXGST at 0.4, 1.0 or 2.0 g/kg after 28-day repeated oral administration in rats because MXGST possesses antitussive and anti-pyretic effects at doses of 0.4–1.0 g/kg. Rats treated with MXGST extract at 0.4, 1.0 or 2.0 g/kg body weight daily for 28 days survived throughout the period and did not show any changes in their general behavior or other physiological activities. We also found that oral administration of the MXGST extract at 5 times the effective dose for 28 days did not cause any toxicological responses or histopathological changes, and then MXGST which consists of Ephedrae, Semen Pruni Armeniacae, licorice and Gypsum did not show any toxicities such as cardiovascular and CNS toxicology from Ephedra [
11,
12,
18,
19], mineralocorticoid excess syndrome from licorice [
20], cyanide toxic reaction of Semen Pruni Armeniacae [
13,
14,
17], and allergic response of Gypsum [
37]. Recent reports indicated that Ephedrae decreased the acute toxicology of Semen Pruni Armeniacae, and licorice decreased acute toxicology of Ephedrae when these herb pairs are co-treated [
9,
38]. Therefore, we found that MXGST extract has a higher safe therapeutic index because the ephedrine and amygdalin levels in the MXGST extract for the antitussive and anti-pyretic tests are approximately 24–60 μg ephedrine/g body weight and 176–440 μg amygdalin/g body weight (thousandth of LD
50 dose of ephedrine and amygdalin).