Antitussive and expectorant properties of growing and fallen leaves of loquat (Eriobotrya japonica)

ABSTRACT

Folium Eriobotryae, the dried leaves of loquat (Eriobotrya japonica, (Thunb.) Lindl., Rosaceae), is a traditional Chinese medicine used to treat cough with phlegm in China. Fallen and growing loquat leaves were tested for their effect on coughing and expectoration in mice. HPLC-ELSD and HPLC-MS analyses of aqueous and ethanol extracts of fallen or growing leaves were used to identify the chemical components responsible for this effect. Both the aqueous and ethanol extracts of growing and fallen leaves of loquat contained antitussive and expectorant activities. Moreover, an aqueous extract of growing loquat leaves with a higher flavonoid content displayed a stronger expectorant activity while the ethanol extract of fallen loquat leaves that contained a higher content of triterpenoid acids induced a stronger antitussive activity.

Keywords: Folium Eriobotryae; Triterpenoid acids; Flavonoids; Antitussive; Expectorant; Anti-inflammatory

Introduction

Cough is the most common symptom in patients subjected to endogenous or exogenous irritants and it is an important symptom of various airway inflammatory diseases. It is also a major public health issue worldwide because it reduces comfort and causes sleep disturbances. Over recent decades, herbal medicines and active ingredients of natural products have garnered growing attention as potential therapeutic agents to prevent and treat coughing, due to their high efficacy and low risk of side effects (Zhou et al., 2013).

Loquat is a multipurpose plant cultivated as a kind of fruit tree and its dried leaves have been used as a traditional Chinese medicine to treat cough with phlegm for thousands of years. Recent pharmacological studies found that Folium Eriobotryae also possessed several other activities, such as anti-diabetic effects (Chen et al., 2008; Lü et al., 2009b), anti-tumor and anti-inflammatory effects (Huang et al., 2009), antioxidant effects (Huang et al., 2006), and a beneficial effect on non-alcoholic fatty liver disease (NAFLD) (Jian et al., 2017; Jian et al., 2018). Phytochemical studies indicated that flavonoids (Jung et al., 1999), triterpenoid acids (Chen et al., 2008) and sesquiterpene glycosides (Zhao et al., 2015) were the main chemical constituents in loquat leaves; however, its active compounds responsible for the antitussive activity have not be identified. In the present study, the aqueous and ethanol extract of growing leaves and fallen leaves of loquat were investigated for their content of secondary metabolites and their beneficial effects on coughing in mice. The results indicated an advantage in evaluation of the pharmacological effects and the potential effective substances of both growing and fallen leaves.

Materials and methods

Growing leaves (GL) and fallen leaves (FL) of loquat (Eriobotrya japonica, (Thunb.) Lindl., Rosaceae) were both manually collected in December 2015 in Suzhou, China (120.296235° S; 31.086002° W). The plant was identified by Professor Wei-Lin Li at the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing (China). A voucher specimen (No. 151228) was deposited in the herbarium of the Institute of Botany (Nanjing). The leaves were washed, dried at 25–30 °C before being cut into strips.

Pentoxyverine was purchased from Sinopharm Rongsheng Pharmaceutical Co. Ltd. (Henan, China). Acetonitrile and methanol (HPLC grade) were obtained from Tedia Co. Inc. (Fairfield, OH, USA). Ammonium hydroxide, sodium nitrite, sodium carboxyl methyl cellulose (CMC-Na), aluminum nitrate, sodium hydroxide, and Phenol Red were all purchased from Nanjing Chemical Reagent Co. Ltd. (Jiangsu, China), and other reagents of analytical grade were provided from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Chemical standards were purchased from the National Institutes for Food and Drug Control (Beijing, China).

Batches of air-dried GL (200 g) were extracted twice with 2000 ml of boiling water for 2 h, and the filtrate was concentrated in a vacuum to obtain the aqueous extract of GL (AGL). The filtered residue was subsequently extracted twice with 2000 ml of 80% ethanol at 80 °C for 2 h, and the filtrate was concentrated in a vacuum to obtain the ethanol extract of GL (EGL). The aqueous and ethanol extracts of FL (AFL and EFL) were obtained as for GL.

Total flavonoid content of AGL, EGL, AFL, and EFL was measured by the aluminum chloride colorimetric assay as reported previously from our laboratory (Lü et al., 2009b).

Aqueous solutions of AGL and AFL were prepared [AGL 17.35 mg (equal to 100 mg crude drug)/ml, AFL 11.85 mg (equal to 100 mg crude drug)/ml] and filtered through a 0.45-µm PVD filter for HPLC-MS analysis. Spectra were generated on an Agilent 6530 accurate-mass quadrupole time-of-flight system (Agilent, USA), with an ESI source operating in the positive ionization mode. Analyses were made using a MassHunter Qualitative Analysis software (B.05.00). Separation was carried out with an Agilent Zorbax SB-C₁₈ column (1.8 µm, 4.6 × 100 mm; Waldbronn, Germany) using a capillary voltage of +4.0 kV. Methanol (A) and 0.1% formic acid (B) were used as the mobile phase under gradient conditions (0–5 min, 15% A, 85% B; 5–60 min, 15–55% A, 85–45% B; 60–75 min, 55–100% A, 45–0% B).

Methanol solutions of EGL and EFL and aqueous solutions of AGL and AFL were prepared [EGL 9.35 mg (equal to 100 mg crude drug)/ml, EFL 9.65 mg (equal to 100 mg crude drug)/ml] and filtered through a 0.45-µm PVD filter for HPLC-ELSD analysis. HPLC-ELSD analysis was performed with an Ultimate 3000 HPLC system (Thermo Fisher, USA) coupled with an Alltech 3300 ELSD detector. An Acclaim C₁₈ column (4.6 × 250 mm, 5 µm) was used and the temperature was held at 30 °C. The mobile phases, acetonitrile (A) and 0.5% ammonium acetate (B) were used under gradient conditions as follows: 0–5 min, 50% A, 50% B; 5–23 min, 50–54% A, 50–46% B; 23–48 min, 54–90% A, 46–10% B; 48–50 min, 90–100% A, 10–0% B, with a flow rate of 1 ml/min. The temperature of the ELSD drift tube was 70 °C, with a flow rate of nitrogen of 1.5 l/min.

Six triterpenoid acids including euscaphic acid, tormentic acid, corosolic acid, maslinic acid, oleanolic acid, and ursolic acid were used as reference samples to quantify the contents in extracts.

The 6-week-old male ICR mice (18–22 g) were provided from the Comparative Medicine Center of Yangzhou University, China. All the experimental procedures and animal treatments were according to the Guide for the Care and Use of Laboratory Animals (No. 2008001680632).

In the evaluation of antitussive activity, the animals were acclimatized for one week before being randomly selected and placed in ten groups of ten biological replicates (n = 10). Then, they were treated orally for three days as follows: mice of control groups were fed with 0.5% CMC-Na/day, while low dose [2.5 g (crude drug)/kg (mouse body weight)/day] and high dose [5 g (crude drug)/kg (mouse body weight)/day] of AGL, AFL, EGL, and EFL were administrated to mice of the test groups. The positive group was treated with Pentoxyverine (17.5 mg/kg (mouse body weight)/day). Antitussive effects were examined by using a classical mouse cough model induced by ammonium hydroxide (Liu et al., 2015), the mice were each exposed to a 500 ml special glass chamber sprayed with 15% ammonium hydroxide (0.2 ml) as described in a previous study (Wang et al., 2012). The cough incubation period and the frequency of cough were recorded from each mouse during 3 min.

To evaluate expectorant activity, the mice animals were grouped and treated as described above, except that the positive group was treated with ammonium chloride (NH₄Cl, 250 mg/kg (mouse body weight)/day). After the third day of treatments, mice were administrated after a 12 h, overnight fast, then 30 min later, they were given Phenol Red (5%, w/v) by intraperitoneal injection. Fifteen min later, the mice were sacrificed by cervical dislocation without damaging the trachea, which was tied-off and then lavaged three times with 0.5 ml sodium bicarbonate (5%, w/v). The collected washing fluid was centrifuged and the absorbance at 546 nm measured at 37 °C. The concentration of Phenol Red was determined as described previously (Li et al., 2012).

The experimental results were expressed as mean ± standard deviation (mean ± SD). The statistical significance of differences between two groups was analyzed using One-way ANOVA analysis of variance followed by a Newman–Keuls post hoc test used to perform multiple comparisons (GraphPad Software, Inc., San Diego, CA, USA). Values of p < 0.05 were taken to indicate statistical significant pharmacological effects.

Results and discussion

The total flavonoid content of AGL, EGL, AFL, and EFL was 28.079 ± 0.606, 3.643 ± 0.253, 9.880 ± 0.522, and 2.143 ± 0.312 mg/g, respectively, indicated that the total flavonoid content in growing leaves was richer than that in fallen leaves, and that the total flavonoids content in aqueous extract was higher than that in the ethanol extract. To investigate further, both AGL and AFL were subjected to HPLC-MS analysis, and twelve compounds were assigned using a HPLC-MS method in both the AGL and AFL (Fig. 1) (Lü et al., 2009b). As shown in Fig. 1, the aqueous extracts of GL and FL presented qualitatively similar chromatograms, but with clear differences in peak heights. For twelve compounds were identified and assigned. Compounds 3–12 were flavonoids or their glycosides. Except for compounds 10 and 12, the peak areas of other compounds in AGL were higher than those of AFL, which was consistent with measurements of the total flavonoid content.

Fig. 1
Chromatograms of AGL and AFL by HPLC-MS analysis (chlorogenic acid 1, vomifoliol-9-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranside 2, quercetin-3-O-galactosyl-(1→6)-glucoside 3, quercetin-3-O-sophoroside 4, quercetin-3-O-rutinoside 5, kaempherol-3-O-sophoroside 6, kaempherol-3-O-rutinoside 7, hyperoside 8, quercetin-3-O-glucoside 9, kaempherol-3-O-galactoside 10, quercetin-3-O-rhamnoside 11, kaempherol-3-O-glucoside 12).

In the evaluation of expectorant activity, mice were injected intraperitoneally with Phenol Red, which was partially discharged within the trachea secretion. Expectorant drugs enhance secretion and dilute the phlegm in the respiratory tract, thus increasing the excretion of Phenol Red (Zhou et al., 2013). As shown in Fig. 2, relative to the control group, all treatments, with the exception of low dose of ethanol extracts of growing and fallen leaves (EGLL and EFLL) induced remarkable expectorant activity. However, aqueous extracts of growing and fallen leaves induced a higher effect than those of ethanol extracts, and the most effective was the high dose of aqueous extract of growing leaves (AGLH) (p < 0.001).

Fig. 2
Effect of different samples and ammonium chloride on the changes of concentration of phenol red in mice (positive means ammonium chloride, AGLL, AFLL, EGLL and EFLL mean the low dose of AGL, AFL, EGL and EFL, AGLH, AFLH, EGLH and EFLH mean the high dose of AGL, AFL, EGL and EFL; each value represents the mean ± SD (n = 10); ns = not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the control group).

Airway mucus hypersecretion, the major cause of coughing and phlegm, is closely associated with the occurrence and development of chronic airway inflammation, and affects the lung function (Zhang and Zhou, 2014). Previous studies have demonstrated that loquat infusion has significant anti-inflammatory activities in both cell and animal models (Zar et al., 2014). Some of the identified flavonoids in the aqueous extract of loquat leaves are known to have anti-inflammatory activity through STAT-1 and NF-κB inhibition (Hamalainen et al., 2007; Hoensch and Oertel, 2012). NF-κB is known to play critical roles in the expression of proinflammatory cytokines. In our study, AGL with higher contents of flavonoids showed better expectorant activities; therefore, flavonoids may be the main constituents responsible for reducing phlegm.

Ammonia-induced cough is a commonly used model for assessing the antitussive effects of medical, bioactive components. In this model, the cough incubation period and cough incidence are often used as the assessing indices. Compared to the control group, the aqueous and ethanol extracts of both growing and fallen leaves of loquat delayed the cough incubation period for ammonia-induced cough and decreased the cough frequency (Fig. 3). The antitussive effects of ethanol extracts were higher than the aqueous extracts. Among the treatments, the high dose of ethanol extract of fallen leaves (EFLH) provided the best activity to delay the cough incubation period for ammonia-induced cough (p < 0.001) and decrease the cough frequency (p < 0.001).

Fig. 3
Effect of different samples and pentoxyverine on the cough incubation period (A) and frequency of cough (B) induced by ammonia in mice (positive means pentoxyverine, AGLL, AFLL, EGLL and EFLL mean the low dose of AGL, AFL, EDL and EFL, AGLH, AFLH, EGLH and EFLH mean the high dose of AGL, AFL, EDL and EFL; each value represents the mean ± SD (n = 10); ns = not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the control group).

Our previous results (Chen et al., 2008; Lü et al., 2009a) showed that the ethanol fraction of loquat leaves was rich in triterpenoid acids. In this study, we found that the content of six triterpenoid acids in the ethanol extracts of growing leaves were higher than that in fallen loquat leaves (Table 1). Inflammation plays an important role in the severe cases of cough. A lower airway inflammation in eosinophilic bronchitis can relieve cough severity and sensitivity (Brightling et al., 2000). The anti-inflammatory effects of the identified triterpenoids in loquat may be related to the inhibition of MAPK signal transduction (Chang et al., 2011; Huang et al., 2009). In the present test, EFL with higher contents of triterpenoids showed better antitussive activity. These results validated the previous conclusion that triterpenoids may be the main constituents responsible for relieving coughing.

Table 1
The contents of triterpenoid acids in EGL and EFL (%).

Extracts	Euscaphic acid	Tormentic acid	Corosolic acid	Maslinic acid	Oleanolic acid	Ursolic acid
EGL	0.154 ± 0.006	0.246 ± 0.009	0.116 ± 0.004	0.341 ± 0.011	0.064 ± 0.002	0.750 ± 0.012
EFL	0.617 ± 0.013	1.077 ± 0.045	0.398 ± 0.014	0.453 ± 0.006	0.096 ± 0.005	1.158 ± 0.009

• Each triterpenoid acid content was expressed as triterpenoid acid/crude drug*100%.

Conclusions

Folium Eriobotryaea is traditionally used to treat cough and eliminate phlegm. In the present study, both growing leaves and fallen leaves achieved therapeutic effects to relieve cough and reduce airway mucus secretion. The pharmacological results also demonstrated that extracts of fallen leaves were more effective in relieving cough, which may be due to their higher content of triterpenoids, while extracts of growing leaves had a better antitussive activity, which may be related to the higher content of flavonoid compounds in growing leaves.

• Ethical disclosures
  Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).
  Confidentiality of data. The authors declare that they have followed the protocols of their work center on the publication of patient data.
  Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Acknowledgements

This study was supported by the Jiangsu Province Science and Technology Social Development Plan (No. BE2015690), the National Natural Science Foundation of China (Nos. 21102058, 81703224 and 81773885), the Natural Science Foundation of Jiangsu Province of China (No. BK20141387) and the Jiangsu Key Laboratory for the Research and Utilization of Plant Resources (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) (No. JSPKLB201502).

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Publication Dates

• Publication in this collection
  Mar-Apr 2018

History

• Received
  28 Aug 2017
• Accepted
  22 Feb 2018
• Published
  5 Apr 2018