The Activity-Integrated Method for Quality Assessment of Reduning Injection by On-Line DPPH-CE-DAD

et al. (2014) The Activity-Integrated Method for Quality Assessment of Reduning Injection by On-Line DPPH-CE- DAD. PLoS ONE 9(9): e106254. doi:10.1371/journal.pone.0106254 The Activity-Integrated Method for Quality Assessment of Reduning Injection by On-Line DPPH-CE-DAD Yan-xu Chang 0 Jiao Liu 0 Yang Bai 0 Jin Li 0 Er-wei Liu 0 Jun He 0 Xiu-cheng Jiao 0 Zhen-zhong Wang 0 Xiu-Mei Gao 0 Bo-li Zhang 0 Wei Xiao 0 Irina V. Lebedeva, Columbia University, United States of America 0 1 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine , Tianjin , China , 2 State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Kanion Pharmaceutical Co., Ltd , Lianyungang , China A sensitive on-line DPPH-CE-DAD method was developed and validated for both screening and determining the concentration of seven antioxidants of Reduning injection. The pH and concentrations of buffer solution, SDS, b-CD and organic modifier were studied for the detection of DPPH and seven antioxidants. By on-line mixing DPPH and sample solution, a DPPH-CE method for testing the antioxidant activity of the complex matrix was successfully established and used to screen the antioxidant components of Reduning injection. Then, antioxidant components including caffeic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid were quantified by the newly established CE-DAD method. Finally, the total antioxidant activity and the multiple active components were selected as markers to evaluate the quality of Reduning injection. The results demonstrated that the on-line DPPH-CE-DAD method was reagent-saving, rapid and feasible for on-line simultaneous determination of total pharmacological activity and contents of multi-components samples. It was also a powerful method for evaluating the quality control and mechanism of action of TCM injection. - Funding: This research was supported National Natural Science Foundation of China (81374050) and Program for Innovative Research Team in Universities of Tianjin (TD12-5033) and Tianjin Research Program of Application Foundation Advanced Technology (12JCQNJC08800) and Stated Key Development Program for Basic Research of China (No. 973:2012CB723504). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: There are no competing interests and financial disclosure that declare the affiliation(s) to Kanion Pharmaceutical Co., LTD, along with any other relevant declarations relating to employment, consultancy, patents, products in development or marketed products etc. This does not alter the authors adherence to PLOS ONE policies on sharing data and materials. . These authors contributed equally to this work. Traditional Chinese medicines (TCMs) have shown an increasing prospect in recent years as an alternative therapy. Considering that TCMs usually contain one or more substances, multiingredient methods for assessing the quality of TCMs are important in order to meet the clinical requirements of safety and efficacy. Some common methods of quality control of TCMs include High-Performance Liquid Chromatography (HPLC) [1 3], Ultra-High Performance Liquid Chromatography (UPLC) [4], Gas Chromatography-Mass Spectrometry (GC-MS) [5], HPLCMS [6] and UPLC-MS [7]. These methods are limited in the determination of the chemical contents of TCMs and they fail to reflect the comprehensive pharmacological effect. Taking these problems into account, it has become imperative to develop a new method of linking pharmacological effects and quality control closely. In light of these, the dual-standard quality assessment, which was defined as a method to evaluate the quality using the total activity and contents of multi-ingredients, was proposed to evaluate the quality of TCMs. Reduning injection made from extracts of Gardenia jasminoides Ellis, Artemisia annua L. and Lonicera japonica Thunb. is a widely used TCM preparation for the treatment of common cold, cough, acute upper respiratory infection and acute bronchitis in the clinic [8]. The prospective clinical trial demonstrated that Reduning injection could be safely used in the treatment of severe hand, foot, and mouth disease (HFMD) [9]. Clinical studies reported that Reduning injection was safe and effective in curing pneumonia [8]. Reactive oxygen species (ROS) are believed to be crucial in the induction of lung damage caused by pneumonia, while therapeutic agents that could effectively scavenge ROS may prevent or reduce the deleterious effects of pneumonia. Reduning injection could increase the activity of superoxide dismutase (SOD) in lungs of rats treated with lipopolysaccharide [8]. Thus, Reduning injection has antioxidant properties. From available literatures, the components which possess antioxidant properties in Reduning injection have not yet been reported. Therefore, it is considered paramount to investigate its antioxidant properties. Free radical species such as ABTS (2, 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) and DPPH (1, 1-diphenyl-2-picrylhydrazyl) were primarily selected to test antioxidant capabilities of ingredients or extracts of TCMs by spectrophotometry [10]. Recently, HPLC-DPPH [11] and TLC-DPPH [12] methods were reported to analyze the antioxidant activity of ingredients in extracts of TCMs. Although these analytical methods prevented interference from color pigments of analytes, they often involved usage of large amount of organic reagents which were considered harmful to environment. As a powerful analytical technique, capillary electrophoresis (CE) has gained much attention in separation science [13], since it owned the advantages such as simple preparation process, short analysis time, excellent efficiency and few organic reagents. Capillary electrophoresis with the Diode Array Detector (CE-DAD) method has been used to separate components and successfully applied to quality control of herbal medicines [1416]. In this study, an on-line DPPH-CE-DAD method was developed and validated for the determination of the antioxidant activity of Reduning injection and its quality control. To our knowledge, the on-line DPPH-CE-DAD method for the analysis of Reduning injection has not been reported yet in literature. In this study, CE was used to separate the chemical components of Reduning injection and then further used to develop the antioxidant activity-integrated method of this herbal preparation. The antioxidant activity-integrated method was used to evaluate the quality of different batches of the sample. The feasibility and precision of this on-line DPPH-CE-DAD method for quality control has been discussed in our report. The on-line DPPH-CE-DAD could not only determine total antioxidant activity of samples, but also screen active components rapidly from the comp (...truncated)