Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113075 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Characterization of bromelain indicates a molar excess of inhibitor vs. enzyme molecules, a Jacalin-like lectin and Maillard reaction products Peter Gross a , Holger Seelert a , Peter Meiser b , Rolf Müller c,∗ a Hochschule Kaiserslautern, Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany b URSAPHARM Arzneimittel GmbH Industriestrasse 35, 66129 Saarbrücken, Germany c Helmholtz-Institut für Pharmazeutische Forschung Saarland (HIPS), Campus E8.1, 66123 Saarbrücken, Germany a r t i c l e i n f o Article history: Received 27 June 2019 Received in revised form 11 December 2019 Accepted 21 December 2019 Available online 8 January 2020 Keywords: Bromelain Bromelain-inhibitor Anti-HIV Jacalin-like lectin Glycation Disulfide scrambling Maillard reaction a b s t r a c t The phytotherapeutic bromelain is a heterogeneous protein mixture, extracted from pineapple stem, with high proteolytic activity based on cysteine proteases. Its global protein chemical composition was analyzed qualitatively and quantitatively by SDS-PAGE and RP-HPLC. A SDS-PAGE method with elab- orate sample pretreatment was developed, to cope with the bromelain’s self-digestion properties and the hypothetical disulfide scrambling during electrophoresis. Both can produce misleading results, if not considered. RP-HPLC was applied for its high separation power for bromelain proteinaceous compounds. A peak identification and assignment to different protein classes in bromelain was done by enzyme kinet- ics and MS. The method was successfully applied for the quantitative determination of the molar ratio between inhibitor and enzyme and resulted to be approximately 3:2. Bromelain contains, from a molar point of view, inhibitor molecules as major component, which thus might be considered as a natural pharmaceutical excipient in Bromelain, because it protects the enzymes against autolysis. We described two methods to separate the inhibitor fraction from the enzyme fraction, RP-HPLC and size exclusion chromatography. A pineapple derived Jacalin-like-lectin, herein called ‘Anlec’, was identified and quan- tified by RP-HPLC-MS in bromelain and its content was determined to be 5%, related to all proteins in bromelain. Anlec binds specifically to mannose-containing glycans and is discussed in literature to pos- sess anti-HIV medical potential. Bromelain could therefore be a possible and economic source for the production of Anlec. An isolation strategy of Anlec from bromelain, in high purity, is shown in this work. The presented RP-HPLC results are comprehensive in chemical information, and the method is expedient to provide appropriate bromelain protein isolations but also to accomplish quality control, covering all relevant protein components. It is furthermore shown, that proteins in bromelain may react with reduc- ing sugars in a Maillard reaction to form glycated proteins. Maillard reaction products in bromelain are detected and characterized and could be responsible for the limited stability and storage times at room temperature of bromelain. Even the active center thiol group could be potentially glycated. © 2020 Elsevier B.V. All rights reserved. 1. Introduction The acetone precipitate from the juice of pineapple stem is called bromelain and since the 1950s it is available in large quanti- ties. Because of its antiedematous and anti-inflammatory activities bromelain has proven beneficial as phytotherapeutic in several therapeutic areas [1]. The underlying molecular mode of action is still in discussion and not well understood. A further chal- lenge for the traditional way of thinking is the oral application route, since proteins mainly get degraded by gastrointestinal pas- ∗ Corresponding author. E-mail address: rolf.mueller@helmholtz-hips.de (R. Müller). sage. The high proteolytic activity of bromelain is regarded as the basis for the mode of action. An inherent problem to assign a single structure-activity relationship is the heterogeneous nature of bromelain, because it is made from the juice of pineapple stem, without polishing steps to gather a pure product. Numerous approaches were applied to isolate and characterize components from bromelain. One of the first attempts was done by Murachi and Neurath in 1960 [2], who isolated a protease fraction from bromelain by ion-exchange chromatography. A series of studies were presented afterwards by Ota et al., starting with a publica- tion in the year 1964 [3]. They described a procedure to isolate the proteolytic active fractions of bromelain by a combination of size-exclusion (SEC) and ion-exchange chromatography. They were aware, that their isolated fractions are not single proteins: https://doi.org/10.1016/j.jpba.2019.113075 0731-7085/© 2020 Elsevier B.V. All rights reserved.