ABSTRACT
ObjectiveBased on the inhibitory activity of phosphodiesterase （PDE）， a method for determining the anti-inflammatory activity of Qingjin Huatantang was established to supplement and improve the quality control system of this famous classical formula. MethodHigh performance liquid chromatography （HPLC） was used to determine the activity of PDE， and the dose-effect relationship of inhibiting PDE activity of Qingjin Huatantang was investigated. The mobile phase consisted of methanol-0.5% acetic acid aqueous solution （5∶95）， and the detection wavelength was 254 nm. By measuring the PDE inhibition rate of multiple batches of Qingjin Huatantang water extract lyophilized powder， biological activity was marked with the activity of the neutralizing enzyme in the international unit U. ResultWhen the concentration of reaction substrate （cyclic adenosine monophosphate） was 50 μmol·L-1 and the reaction time was 60 min， the enzymatic reaction was stable with 4 U·mL-1 of PDE. In this reaction system， when the concentration of Qingjin Huatantang water extract lyophilized powder was 0.11-3.0 g·L-1， the inhibitory effect of PDE showed a concentration-dependent relationship. It was determined that the concentration of Qingjin Huatantang water extract lyophilized powder to be tested was 1 g·L-1， which showed a significant and stable inhibitory effect on PDE， and the inhibitory rate was >45%， that is， 1 mg of Qingjin Huatantang water extract lyophilized powder could neutralize the activity of 1.8 U PDE at least. ConclusionThis study establishes a biological activity evaluation method of Qingjin Huatantang based on the inhibitory activity of PDE， and the anti-inflammatory activity of Qingjin Huatantang is characterized by international unit U of PDE activity， which can provide a new method for the determination of biological activity of traditional Chinese medicine compounds.
ABSTRACT
Qingjin Huatanpang， first contained in Yixue Tongzhi， was composed of eleven medicinal materials of Scutellariae Radix， Gardeniae Fructus， Fritillariae Thunbergii Bulbus， Mori Cortex， Trichosanthis Semen Tostum， Citri Exocarpium Rubrum， Platycodonis Radix， Ophiopogonis Radix （core removed）， Anemarrhenae Rhizoma， Poria and Glycyrrhizae Radix et Rhizoma. It is a classic prescription created by YE Wen-ling in Ming dynasty for treating pulmonary disease with phlegm-heat obstructing lung syndrome. With the significant functions of clearing heat and moistening lung， reducing phlegm and relieving cough， it has been included in the "Classic Catalogue of Ancient Classics （First Batch）". Modern pharmacological studies have shown that Qingjin Huatantang has multiple activities such as relieving cough and eliminating phlegm， anti-inflammatory， bronchodilation， and immunoregulatory， and now it is commonly used for treating infectious lung diseases， such as acute exacerbation of chronic obstructive pulmonary disease， community acquire pneumonia， bronchiectasis， acute and chronic bronchitis in a form of its modified prescription or its combined use with western medicine， consistent with the clinical application in ancient times. According to the literatures on the study of Qingjin Huatantang published in recent years， this paper summed up the historical evolution， compatibility analysis， chemistry constituents， quality control， advances in pharmacology research， and clinical uses， which can provide theoretical and experimental data reference for further research and development， and proposed to establish a biological activity assay for quality control based on the pharmacological effect such as immunoregulatory activity， which can improve its quality control method and provide a reference for other famous classical formulas.
ABSTRACT
Objective:To establish a method for evaluating the biological activity of water extract lyophilized powder of Qingjin Huatantang based on the phagocytic and secretory functions of macrophages， and to control the quality of this formula from the biological activity level. Method:The phagocytic and inflammation models of RAW264.7 macrophages were established， the inhibition rates of water extract lyophilized powder of Qingjin Huatantang on interleukin-6 （IL-6） secretion and phagocytic index of neutral red of RAW264.7 macrophages were chosen as indicators to investigate the biological activity of Qingjin Huatantang， and the biological limit was searched. Result:The optimal inoculation density of RAW264.7 macrophages was 3×10<sup>5</sup> pcs/mL， and the concentration of lipopolysaccharide （LPS） was 1 mg·L<sup>-1</sup> after treatment for 24 h. When the concentration was 500 mg·L<sup>-1</sup>， water extract lyophilized powder of Qingjin Huatantang had no toxicity and no obvious promotion effect on the proliferation of RAW264.7 macrophages， and at this concentration， the phagocytosis of RAW264.7 macrophages for neutral red was significantly promoted， the phagocytic index was >113%. In addition， the lyophilized powder had a significant and stable inhibitory effect on IL-6 secretion of RAW264.7 macrophages induced by LPS， the inhibitory rate was >45%. Conclusion:Combined with the anti-inflammatory and immunomodulatory effects of Qingjin Huatantang， this study establishes an <italic>in vitro </italic>biological limit method for evaluating the quality of water extract of Qingjin Huatantang based on the phagocytic and secretory functions of RAW264.7 macrophages， and 500 mg·L<sup>-1</sup> was confirmed as the limit concentration. Under the limit concentration， Qingjin Huatantang water extract can significantly promote the phagocytic index of macrophages or significantly inhibit the secretion of IL-6 of RAW264.7 macrophages induced by LPS， which can be judged as qualified.
ABSTRACT
Objective:To regulate autophagy protein p62 of airway epithelial cells in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) rats with Qingjin Huatantang, in order to explore its effect on interleukin (IL) -1β and tumor necrosis, tumor necrosis factor-α (TNF-α), leukotriene B4 (LTB4), and leukotriene C4 (LTC4). Method:Airway epithelial cells and AECOPD airway epithelial cells were cultured. Sixty SPF male SD rats were randomly divided into 6 groups, namely high, medium and low-dose Qingjin Huatantang groups, western medicine group, model group and normal group. Except for the normal group, the remaining groups were included into the AECOPD model by lipopolysaccharide (LPS) tracheal instillation method + fumigation method. After modeling, the dosage of the high-dose traditional Chinese medicine group was 30 g·kg-1·d-1, that of the middle-dose group was 15 g·kg-1·d-1, that of the low-dose group was 7.5 g·kg-1·d-1, the positive control group was given luo erythromycin (0.017 5 g·kg-1·d-1), the model group and the blank control group were orally given normal saline with the volume of 20 mL·kg-1·d-1. Serum was extracted two weeks after administration, and the cells were intervened with drug-containing serum. The content of interleukin IL-1β, TNF-α, and LTB4 in cell supernatants were detected by enzyme-linked immunosorbent assay (ELISA). And LTC4 content, p62 mRNA and protein expressions in lung airway epithelial cells were detected by quantitative real-time fluorescence polymerase chain reaction (Real-time PCR) and Western blot. Result:Compared with the normal group, ELISA results showed that IL-1β, TNF-α, LTB4, and LTC4 in the model group were significantly increased (P<0.01). Compared with the model group, IL-1β, TNF-α, LTB4, LTC4 in cell supernatants in each administration group were significantly reduced (P<0.01), mRNA and protein expressions in p62 showed that compared with the normal group, mRNA and protein expressions in p62 of model group significantly decreased (P<0.01). Compared with the model group, the mRNA and protein expressions of p62 in each administration group significantly increased to different degrees (P<0.01). The expression of autophagy in Qingjin Huatantang high-dose group and western medicine group was comparable. Conclusion:Qingjin Huatantang can reduce the inflammatory response in airway epithelial cells, which may be related to the regulation of autophagy protein p62.