Identification of the Allergenic Ingredients in Reduning Injection by Ultrafiltration and High-Performance Liquid Chromatography

Hindawi Publishing Corporation Journal of Immunology Research Volume 2016, Article ID 4895672, 7 pages http://dx.doi.org/10.1155/2016/4895672 Research Article Identification of the Allergenic Ingredients in Reduning Injection by Ultrafiltration and High-Performance Liquid Chromatography Fang Wang,1 Cun-yu Li,1,2 Yun-feng Zheng,1 Hong-yang Li,1 Wei Xiao,3 and Guo-ping Peng1,2 1 College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu, China 3 Jiangsu Kanion Pharmaceutical Co., Ltd., China 2 Correspondence should be addressed to Guo-ping Peng; Received 10 November 2015; Accepted 8 February 2016 Academic Editor: Nejat K. Egilmez Copyright © 2016 Fang Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Reduning injection is a traditional Chinese medicine injection which has multiple functions such as clearing heat, dispelling wind, and detoxification. Although Reduning injection was widely utilized, reports of its allergenicity emerged one after another. However, there is little research on its allergenic substances. The aim of this study is to evaluate the sensitization of Reduning injection and explore the underlying cause of the anaphylactic reaction. The main ingredients in Reduning injection were analyzed before and after ultrafiltration. Ultrafiltrate Reduning injection, unfiltered Reduning injection, egg albumin, Tween-80, and nine effective components in Reduning injection were utilized to sensitize guinea pigs. The serum 5-hydroxytryptamine level was used to assess the sensitization effect of Reduning injection. We found a significant decrease in Tween-80 content comparing to other components in the injection after ultrafiltration. Unfiltered Reduning injection, Tween-80, chlorogenic acid, and cryptochlorogenin acid caused remarkable anaphylactoid reaction on guinea pigs while ultrafiltration Reduning resulted in a significantly lower degree of sensitization. Our results suggest that ultrafiltration could significantly reduce the sensitization of Reduning injection, which is likely due to the decrease of Tween-80. We also conjectured that the form of chlorogenic acid and cryptochlorogenin acid within the complex solution mixture may also affect the sensitizing effect. 1. Introduction Reduning injection is a traditional Chinese medicine (TCM) injection refined from three Chinese herbal medicines, namely, honeysuckle, gardenia, and abrotani herba, and formulated for injection [1, 2]. This injection has multiple functions such as clearing heat, dispelling wind, and detoxification [3]. It is clinically used in the treatment of hyperpyrexia, slight aversion to cold, head and body pain, cough, yellow sputum, and other symptoms caused by respiratory tract infection (with external wind heat syndrome) [4, 5]. From the time Reduning injection was listed as a clinical treatment, it has been widely utilized with good clinical efficacy. However, there are also a few reports of severe anaphylaxis during the clinical application [6]. It is known that Reduning injection mainly contains Tween-80, chlorogenic acid, geniposide, caffeic acid, cryptochlorogenin acid, isochlorogenic acid (A, B, and C), neochlorogenic acid, and secoxyloganin [7–10]. Previous studies have shown that some components in TCM injections might be the cause of the allergic reaction and anaphylactoid reaction [11–13]. However, the specific compound responsible for the allergic reaction is still not clear. Ultrafiltration is a separation technique in which a porous membrane is used as filtering medium and the molecules are separated according to their size [14]. After ultrafiltration, macromolecules such as pyrogen, microbes, proteins, pigments, resin, and tannin are excluded [15]. In this study, we used ultrafiltration to separate the compounds in Reduning injection to explore the cause of anaphylaxis. 2 Previous studies have identified some key cytokines are the mediator of antibody-mediated systemic anaphylaxis [16– 18] and our studies had shown that 5-hydroxytryptamine (5-HT) can be detected rapidly by high-performance liquid chromatography (HPLC) and can be used as an index for anaphylaxis [19]. In our study, guinea pig plasma 5-HT level was measured to determine the sensitization of Reduning injection before and after ultrafiltration. Furthermore, the components in the injection were also analyzed to determine the main allergens in Reduning injection. 2. Materials and Methods 2.1. Animals. A total of 140 male guinea pigs were purchased from Nanjing Qinglongshan Animal Breeding Center. All animals were housed at 22∘ C and 55% ±5% relative humidity. All experiments were carried out according to the guidelines of the Animal Care Committee of Nanjing University of Chinese Medicine. 2.2. Reagents and Materials. Egg albumin and 5-HT were purchased from Sigma (St. Louis, USA). Chloral hydrate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Heparin sodium injection was purchased from Jiangsu Wangbang Pharmaceutical Co., Ltd. (Jiangsu, China). Heptanesulfonate sodium was purchased from J&K Chemical Ltd. (Shanghai, China). Sodium dihydrogen phosphate was purchased from Shantou Xilong Chemical Industry Factory Co., Ltd. (Guangdong, China). Ethylene Diamine Tetraacetic Acid (EDTA) was purchased from Shanghai Fuqi Industrial & Trading Co., Ltd. (Shanghai, China). Sodium chloride injection was purchased from Anhui Fengyuan Pharmaceutical Co., Ltd. (Anhui, China). Reduning injection was purchased from Jiangsu Kangyuan Pharmaceutical Co., Ltd. (Jiangsu, China), the batch numbers are 120211, 120209, and 120703. Tween-80 was purchased from Sigma (St. Louis, USA), the batch number is 018K00941. Chlorogenic acid was purchased from China Institute of pharmaceutical and biological products (Beijing, China); the batch number is 0753-9909. Geniposide was purchased from National Institute for Food and Drug Control (Beijing, China); the batch number is 110885-200102. Neochlorogenic acid and cryptochlorogenin acid were purchased from Shanghai Yuanye Technology Co., Ltd. (Shanghai, China); the batch numbers are PA0819RA13 and ZF0226BA14. Isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and secoxyloganin reference substance were produced in-house and possessed a purity above 98% as tested by chromatography. The ultrafiltration column was purchased from Millipore Corporation (Massachusetts, USA). 2.3. Solutions Preparation. Egg albumin solution was dissolved in saline to a concentration of 5.0 mg/mL. Tween-80 was prepared to 1% v/v by dissolving 1 mL of Tween-80 in saline. Standard solutions of chlorogenic acid, neochlorogenic acid, cryptochlorogenin acid, geniposide, caffeic acid, isochlorogen (...truncated)