# Recent Advances and Insights into Bromelain Processing, Pharmacokinetics and Therapeutic Uses
## 1 Introduction  
Bromelain is a mixture of proteolytic enzymes that is extracted primarily from pineapples (_Anantas comosus_) that is well known and used in several fields, especially in the nutraceuticalI and cosmeceutical sectors [1]. It was first identified by [PERSON] in 1891 in pineapple juice, and _[PERSON]_ discovered higher amounts of bromelain in the stem than the fruit in 1957 [2]. Although bromelain was initially defined as the mixture of enzymes produced in the fruit, today, this term includes any protease extracted from any member of the _Bromeliaceae_ family [3]. Pineapple is the most common plant from which bromelain is extracted. The proteases that constitute bromelain are cysteine endopeptidaseases, which catalyze the hydrolysis of the peptide bonds of non-terminal amino acids [4]. Bromelain usually includes glycosidases, phosphatases, ribonuclelease, cellulases, peroxididases and glycoproteins, which primarily cleave alanyl, glycosyl and leucyl peptide bonds. These enzymes are present in different ratios that depend on a number of factors, including the geographic location of the plant, the land and mode of cultivation, the extraction method,and the part of the plant used [5]. In fact, although bromelain is present in almost all of the aerial parts of the plant--the peels, leaves, stems and fruit--only the fruit and stems are rich in bromelain. In particular, the bromelain in pineapple stems (EC number 3.4.22.32) is the most abundant protease, with an isoelectric point (pI) of 9.5 and optimum pH range of 6-7, while the bromelain found in pineapple fruit (EC number 3.4.22.33) is present in lower amounts, with a pI of 4.6 and an optimum pH range of 3-8 [6; 7]. These significant differences in bromelain composition, probably due to the presence of different thiol-endopeptidase, may, in part, explain the great heterogeneity of clinical and preclinical results for potential effects on a wide category of conditions, including inflammatory diseases, cancer, immune dysfunction and others [8].  
The demand for bromelain is increasing quickly, and the reason for this great interest in the clinical field is related to its anti-inflammatory, antiedematous, fibrinolytic, anticancer, anticoagulative and antithrombotic properties that have been thoroughly described in the literature [3]. In addition, this enzymatic complex is used in other sectors, including cosmetics, breweries, flesh processing and tenderisation, and textile industries [7; 9; 10]. However, the isolation and purification of bromelain from pineapple (fruit, stem, core, leaves) is a challenge and constitutes 70-90% of the total production cost of the final extract [11]. To date, the commercial cost of bromelain extracts is high, with prices hovering around 2400 USD/kg [12]. The development of methods to achieve highly purified bromelain in fewer steps is therefore necessary to decrease overall production costs [3]. Despite the new feasible methods of protein purification (e.g., membrane filtration, reverse micellar systems, aqueous two-phase extraction, chromatographic techniques) and the new biotechnological processes developed to mitigate production costs, several limitations still create problems for the efficiency of product recovery from crude-plant extracts and the effectiveness of the obtained extract [12]. In fact, the enzyme complex tends to be irreversibly inactivated at high temperatures (e.g., during the pasteurization process), while the progressive concentration of bromelain in crude pineapple juice during the purification process can induce spontaneous enzymatic deactivation [5].  
This review will describe the state of the art in the main conventional and unconventional extraction and purification methods of bromelain and discuss the advantages and limitations of these strategies. Pharmacokine and pharmacodynamic profiles and the clinical applications that have arisen from randomized controlled clinical trials (RCTs) are also discussed. Finally, future perspectives for bromelain extracts will be presented.