Feilong Chen

Inhibitory Effect of Methyleugenol on IgE-Mediated Allergic Inflammation in RBL-2H3 Cells

Allergic diseases, such as asthma and allergic rhinitis, are common. Therefore, the discovery of therapeutic drugs for these conditions is essential. Methyleugenol (ME) is a natural compound with antiallergic, antianaphylactic, antinociceptive, and anti-inflammatory effects. This study examined the antiallergic effect of ME on IgE-mediated inflammatory responses and its antiallergy mechanism in the mast cell line, RBL-2H3. We found that ME significantly inhibited the release of β-hexosaminidase, tumor necrosis factor- (TNF-) α, and interleukin- (IL-) 4, and was not cytotoxic at the tested concentrations (0–100 μM). Additionally, ME markedly reduced the production of the proinflammatory lipid mediators prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), leukotriene B4 (LTB4), and leukotriene C4 (LTC4). We further evaluated the effect of ME on the early stages of the FcεRI cascade. ME significantly inhibited Syk phosphorylation and expression but had no effect on Lyn. Furthermore, it suppressed ERK1/2, p38, and JNK phosphorylation, which is implicated in proinflammatory cytokine expression. ME also decreased cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO) phosphorylation and cyclooxygenase-2 (COX-2) expression. These results suggest that ME inhibits allergic response by suppressing the activation of Syk, ERK1/2, p38, JNK, cPLA2, and 5-LO. Furthermore, the strong inhibition of COX-2 expression may also contribute to the antiallergic action of ME. Our study provides further information about the biological functions of ME.

Concurrent quantification and comparative pharmacokinetic analysis of bioactive compounds in the Herba Ephedrae-Semen Armeniacae Amarum herb pair.

The Mahuang-Xingren herb-pair (MX), the combination of Herba Ephedrae (Mahuang in Chinese) and Semen Armeniacae Amarum (Xingren in Chinese), is a classical combination used in traditional Chinese Medicine to treat asthma and bronchitis. A simple and reliable ultra-performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously quantify and compare the pharmacokinetics of 5 ephedra alkaloids and epimers of amygdalin and prunasin in rat plasma after oral administration of Mahuang, Xingren, and MX aqueous extracts. Samples were pretreated by a single-step protein precipitation with acetonitrile, and diphenhydramine hydrochloride and puerarin were used as internal standards. Pharmacokinetic parameters were investigated using DAS 3.2.2 (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The validated method demonstrated adequate sensitivity, selectivity, and process efficiency for the bioanalysis of 8 compounds, including 3 pairs of epimers. MX administration improved the bioavailability of amygdalin and prunasin. Furthermore, MX facilitated intake of lower doses of ephedra alkaloids and increased elimination rates in comparison with Mahuang alone. These results illustrate the rationale behind the preferred use of the combination of Mahuang and Xingren. To our knowledge, this is the first report of stereo-selective metabolism of amygdalin. Further, the metabolic mechanism underlying this phenomenon merits future research attention.

Neuroprotective Effect of Gui Zhi (Ramulus Cinnamomi) on Ma Huang- (Herb Ephedra-) Induced Toxicity in Rats Treated with a Ma Huang-Gui Zhi Herb Pair

Herb Ephedra (Ma Huang in Chinese) and Ramulus Cinnamomi (Gui Zhi in Chinese) are traditional Chinese herbs, often used together to treat asthma, nose and lung congestion, and fever with anhidrosis. Due to the adverse effects of ephedrine, clinical use of Ma Huang is restricted. However, Gui Zhi extract has been reported to decrease spontaneous activity in rats and exert anti-inflammatory and neuroprotective effects. The present study explored the possible inhibitory effect of Gui Zhi on Ma Huang-induced neurotoxicity in rats when the two herbs were used in combination. All Ma Huang and Ma Huang-Gui Zhi herb pair extracts were prepared using methods of traditional Chinese medicine and were normalized based on the ephedrine content. Two-month-old male Sprague-Dawley rats (n = 6 rats/group) were administered Ma Huang or the Ma Huang-Gui Zhi herb pair extracts for 7 days (ephedrine = 48 mg/kg), and locomotor activity was measured. After 7 days, oxidative damage in the prefrontal cortex was measured. Gui Zhi decreased hyperactivity and sensitization produced by repeated Ma Huang administration and attenuated oxidative stress induced by Ma Huang. The results of this study demonstrate the neuroprotective potential of Gui Zhi in Ma Huang-induced hyperactivity and oxidative damage in the prefrontal cortex of rats when used in combination.

Comparative pharmacokinetics of eight major bioactive components in normal and bacterial diarrhea mini-pigs after oral administration of Gegen Qinlian Decoction

Healthy animals are most widely used in current pharmacokinetic(PK) studies. However, neglecting the effects of specific diseases on drug absorption results in the PK parameters of those experiments not accurately reflecting in vivo drug concentration changes during treatment. In this study, an E. coli infective diarrheal minipig model was applied to explore the pharmacokinetics of Gegen Qinlian decoction (GQD). A simple and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine the concentrations of the eight GQD components in minipig plasma after intragastric administration of GQD. The PK parameters of the main GQD components in normal and model minipigs after oral administration of GQD were compared. There were statistically significant differences (p<0.05) in the pharmacokinetic parameters of Puerarin, Wogonin and Daidzein involving the AUC0-t, Cmax, MRT(0-t), t1/2z between normal and model minipigs. Results showed that bacterial diarrhea had a great impact on the biological availability of the main ingredients in GQD. More importantly, the results obtained suggest that the bacterial diarrheal minipig model can be successfully applied in PK studies and may be used in other PK studies of drugs targeting intestinal disease.

Antipyretic and Anti-Asthmatic Activities of Traditional Chinese Herb-Pairs, Ephedra and Gypsum

ObjectiveMahuang-Shigao herb-pair is a famous formula composed of Ephedra and Gypsum. The herb-pair is frequently used for treating cold symptoms and bronchial asthma in the clinical practice of Chinese medicine (CM). In the present study, we evaluated evidence for the benefit of combined use of Ephedra and Gypsum by analyzing the antipyretic and anti-asthmatic activities of Ephedra-Gypsum.MethodsThe antipyretic effects of Ephedra-Gypsum were evaluated in yeast-induced hyperthermia test. Thirty male Wistar rats were randomly divided into 5 groups, including control group, standard aspirin group, and 3 Ephedra- Gypsum groups of different doses (6, 12, 24 g/kg). Ephedra-Gypsum extract and asprin were administered orally 6 h after the injection of yeast solution and body temperature was measured every 1 h for 8 h. The antiasthmatic effects of Ephedra-Gypsum were evaluated using an ovalbumin (OVA)-induced asthmatic rat model. Thirty-six male SD rats were randomly divided into 6 groups. Rats were alternately sensitized and OVA+Al(OH) challenged by exposure to mists of ovalbumin. Ephedra-Gypsum extracts (6, 12, 24 g/kg) or dexamethasone were administered 45 min prior to the allergen challenge for 8 days. Latent period and the weight of wet to dry ratio of lung were determined. In addition, the eosinophils in blood and white blood cell (WBC) were counted by an YZ-Hemavet Analyzer.ResultsThe Ephedra-Gypsum extracts at test dose (6, 12, 24 g/kg) significantly and dose-dependently attenuated yeast-induced fever in rats. The Ephedra-Gypsum extracts also prolonged the latent period, reduced OVA-induced increases in eosinophils and WBC, and decreased the wet and dry weight ratio of the lungs in the anti-asthmatic test.ConclusionsThese findings indicate that the Ephedra-Gypsum extract has antipyretic and anti-asthmatic properties. Hence, the results support additional scientific evidence in prescriptions.

Simultaneous determination of phosphate anion and calcium cation in Plastrum testudinis by HPLC-ELSD.

A simple and rapid high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method was developed for simultaneous determination of phosphate anion and calcium cation in Plastrum testudinis. The analyte was separated on a Prevail C18 reserved-phase column (250mm×4.6mm, 5μm) using isocratic elution at a flow rate of 0.5mL/min. The column temperature was 25°C. The drift tube temperature and the nitrogen gas flow-rate of ELSD were set at 115°C and 2.5L/min, respectively. This developed method was able to determine phosphate anion and calcium cation with good linearity (r(2)>0.9997), precision (RSD<1.6%) and recovery (99.52-101.94%). The newly established method was successfully applied to evaluate the contents of phosphate anion and calcium cation in six commercial samples.

Intestinal absorption differences of major bioactive compounds of Gegenqinlian Decoction between normal and bacterial diarrheal mini-pigs in vitro and in situ

Intestinal condition plays an important role in drug absorption and metabolism, thus the effects of varied gastrointestinal diseases such as infectious diarrhea on the intestinal function are crucial for drug absorption. However, due to the lack of suitable models, the differences of absorption and metabolism of drugs between the diarrheal and normal intestines are rarely reported. Thus, in this study, Escherichia coli diarrhea model was induced in mini-pigs and single-pass intestinal perfusion and intestinal mucosal enzyme metabolism experiments were conducted. A simple and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine the concentrations of 9 major components in Gegen Qinlian decoction (GQD). Samples were pretreated by protein precipitation with methanol and naringin and prednisolone were used as internal standards. The validated method demonstrated adequate sensitivity, selectivity, and process efficiency for the bioanalysis of 9 compounds. Results of intestinal perfusion showed that puerarin, daidzein, daidzin and baicalin and berberine were absorbed faster in diarrheal jejunum than in normal intestines (p < 0.05). However, puerarin, daidzin and liquiritin were metabolized more slowly in diarrheal intestine after incubation compared with the normal group (p < 0.05). The concentrations of daidzein in both perfusion and metabolism and wogonin in metabolism were significantly increased (p < 0.05). In conclusion, absorption and metabolism of GQD were significantly different between the diarrheal and normal intestines, which suggest that bacterial diarrheal mini-pigs model can be used in the intestinal absorption study and is worthy to be applied in the other intestinal absorption study of anti- diarrheal drugs.

Comparative Metabolism of the Eight Main Bioactive Ingredients of Gegen Qinlian Decoction by the Intestinal Flora of Diarrhoeal and Healthy Piglets

Diarrhoeal diseases alter the composition of intestinal flora, thereby affecting the efficacy of herbal medicinal formulations. Gegen Qinlian decoction (GQD), a Chinese traditional herbal formulation, is widely used to treat infectious diarrhoea. However, little is known about the microbial disposition of GQD in the diarrhoeal state. In this study, the comparative metabolism of components of GQD by diarrhoeal and normal intestinal flora was investigated in vitro. UPLC-MS/MS was performed for simultaneous analysis of eight ingredients of GQD in bacterial solution. The type, activities, and sources of microbial enzymes were also investigated. Microbial metabolism of daidzin, genistin and liquiritin (metabolized by β-glucosidase); baicalin, wogonoside and glycyrrhizin (metabolized by β-glucuronidase); and berberine and coptisine (metabolized via nitroreductase) was faster in the diarrhoeal group than in the normal group. Moreover, the activities of these enzymes in the diarrhoeal group were higher than those in the normal group. This difference might be associated with the increase in Escherichia spp. Thus, a change in the metabolism of components by diarrhoeal intestinal flora is associated with a preponderance of Escherichia spp., which might improve the efficacy of GQD. These findings have implications for understanding the action mechanism of GQD for diarrhoea treatment in terms of the microbial milieu.

Soybean Milk Inhibits Absorption and Intestinal Transmembrane Transport of Gegen in Rats

Puerariae Lobatae Radix, known as Gegen in Chinese, is widely used to treat cardiovascular diseases, diabetes, and many other chronic illnesses. Flavonoids are the main active components in Gegen and are found in high concentrations in soybeans. Few studies, however, have focused on the effects of flavonoid-rich food on the absorption of Gegen. Here, we report an in vivo pharmacokinetic study on rats to explore the effects of soybean milk on the absorption of Gegen and an in vitro Ussing chamber study of puerarin intestinal transmembrane absorption. Area under the plasma concentration-time curve (AUC0–t) and maximum plasma concentration (Cmax) values of puerarin in rats were significantly decreased after drinking soybean milk, when taking Gegen decoction or a Gegen patent medicine (P < 0.01). In the Ussing chamber experiment, cumulative transmission (Qtn) after 2 h and apparent permeability coefficient (Papp) were lower in the puerarin-daidzin and puerarin-soybean milk solution groups than in the puerarin group. Daidzin in soybean milk inhibited the transmembrane transport of puerarin, resulting in decreased bioavailability of puerarin in Gegen. The results of this study strongly suggest that Gegen should not be taken with flavonoid-rich food, particularly soybean products.

Comparative pharmacokinetics of three phenylpropanoids in rat plasma after oral administration of Ramulus Cinnamomi and Ramulus Cinnamomi-Ephedrae Herba herb-couple extract

ObjectiveTo investigate the pharmacokinetic characteristics of three phenylpropanoids (cinnamic acid, cinnamic alcohol and coumarin) in Ramulus Cinnamomi (GZ) and Ramulus Cinnamomi-Ephedrae Herba (MH) herb-couple (GZMH).MethodsTwelve male Sprague-Dawley rats were randomly and equally divided into the GZ and GZMH herb-couple groups. Blood samples were collected at 0, 0.08, 0.25, 0.5, 0.75, 1.5, 3, 4, 6, 8, 12, 24, 36 and 48 h after oral administration. The three phenylpropanoids in rat plasma were quantified using an ultra-performance liquid-chromatography with tandem mass spectrometry (UPLC-MS/MS) method for pharmacokinetic study.ResultsIn GZMH group, the area under the curve (AUC), mean retention time (MRT) of cinnamic acid and coumarin were increased significantly (P<0.01, respectively), and biological half-life (t1/2z) was obviously shorter (P<0.05) compared with the GZ group. There were no significant differences in the mean retention time from 0 to ∞ (MRT0-∞), the peak concentration (Cmax), the time to peak (Tmax) and t1/2z, except for AUC and MRT0-t (the mean retention time from 0 to t) of cinnamic alcohol in the GZMH group by comparison to the GZ group (P<0.01, respectively). The AUC, MRT (both P<0.01) and t1/2z (P<0.05) of coumarin were increased significantly, while Cmax, and Tmax were decreased slightly by comparison to the GZ group (P>0.05).ConclusionsThere were statistically significant differences in some pharmacokinetic parameters of the three compounds between GZ and GZMH groups, which meant that MH could affect the absorption and elimination of the three compounds.