Proteinase activity and stability of natural bromelain preparations
Introduction
Bromelain is a mixture of proteolytic enzymes that is derived from the stem of the pineapple plant, Ananas comosus. We and others have previously shown that bromelain proteolytically removes certain cell surface molecules that affect lymphocyte migration and activation [1], [2], [3], [4]. In addition, bromelain treatment markedly affects the production of cytokines and inflammatory mediators by isolated leukocytes or colon epithelial cells in vitro [5], [6]. These effects require that the bromelain be proteolytically active [1], [5].
Bromelain or proteinase mixtures containing bromelain have been suggested or proven to have anti-inflammatory effects in several different animal models of inflammation and human inflammatory diseases. These include carrageenan-induced pleurisy in the rat [7], [8], [9], immunologically mediated arteriosclerosis in rat aortic allografts [10], the experimental allergic encephalomyelitis (EAE) murine model for the human autimmune disease multiple sclerosis [11], collagen- or adjuvant-induced arthritis [12], [13], IgE-mediated perennial allergic rhinitis [14], and some human rheumatologic diseases [15], [16], [17]. Oral bromelain was also anecdotally reported to induce clinical and endoscopic remission of ulcerative colitis in two patients whose disease was refractory to multi-agent conventional medical therapy [18]. Our studies thus far show that treatment with oral bromelain decreases the development of spontaneous colitis and also decreases the severity of established colitis in the IL-10 knockout model of murine inflammatory bowel disease (L.P. Hale et al., manuscript in preparation). Proteolytic activity is required for the anti-inflammatory effect of oral bromelain in this model.
Bromelain is only poorly absorbed when administered orally, generating plasma levels of less than 10 ng/ml in humans given 4 g/day [19]. We previously showed that, although 1 μg/ml is sufficient to remove at least 50% of bromelain-sensitive molecules from cells in the absence of plasma, concentrations of ≥500 μg/ml bromelain are required when cells are present in whole blood [2]. This is primarily due to inhibition of bromelain by the plasma proteinase inhibitor alpha-2-macroglobulin [2]. Taken together, these studies show that the concentration of bromelain achieved systemically following large oral doses is many orders of magnitude less that what is required to significantly affect cell surface bromelain-sensitive molecules. In contrast, we recently showed [20] that the concentration of proteolytically active bromelain in stool after oral administration was sufficient to remove bromelain-sensitive molecules from the surface of colon epithelial cells. Local proteolytic activity of bromelain has also been demonstrated in small intestine of pigs following oral administration [21], [22]. Thus we feel that anti-inflammatory effects of oral bromelain in IBD are more likely due to local proteolytic activity within the intestinal lumen or lamina propria, rather than systemic activity.
Bromelain contains several distinct cysteine proteinases that have similar but distinct amino acid sequences, as well as differences in proteolytic specificity and sensitivity to inactivation. However, it is not clear how the different proteinases within bromelain contribute to its anti-inflammatory activity in vivo. Studies to test the potential efficacy of bromelain in clinical trials in animals or humans have been limited by the lack of assays to control for differences in the composition and proteolytic activity of naturally derived bromelain preparations. Stem bromelain (EC 3.4.22.32, formerly EC 3.4.22.4) is the most abundant proteinase within bromelain preparations derived from pineapple stem. Other proteinases that are present at lesser amounts include fruit bromelain (the major proteinase present in pineapple fruit; EC3.4.22.33, formerly EC 3.4.22.4 and 3.4.22.5) and ananain (EC 3.4.22.31). Several model peptide substrates of the form B–(P3)–P2–P1-indicator (where B=blocking group such as Z=benzyloxycarbonyl or Bz=benzoyl, and P1, P2, and P3 represent specific amino acids) have been used to characterize the proteolytic activity of purified bromelain enzymes. Amidolytic cleavage of the substrate results in release of free indicator that can be detected either fluorescently or colorimetrically. Stem bromelain preferentially cleaves the Z-Arg-Arg model substrate, whereas fruit bromelain and ananain show minimal activity against this substrate. In contrast, fruit bromelain and ananain but not stem bromelain efficiently cleave the Bz-Phe-Val-Arg substrate [23], [24]. These enzymes also differ in their susceptibility to inactivation. Ananain is reported to be rapidly inactivated by the chicken egg white proteinase inhibitor cystatin and by the suicide substrate, E-64 (trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane), but these inhibitors either very slowly or only minimally inactivate stem and fruit bromelain [23].
Development of assays that can assess its proteinase composition and enzyme activity are needed to facilitate the standardization of bromelain as a therapeutic product. The purpose of this study was to develop criteria for standardizing the content and activity of bromelain preparations and to determine the stability of their proteolytic activity under a variety of physiologically and therapeutically relevant conditions.
Section snippets
Reagents
Bromelain (catalog #B-4882) obtained from Sigma-Aldrich (St. Louis, MO) was used as the standard for these studies. Nutraceutical preparations of bromelain were obtained from GNC (Pittsburgh, PA), Natural Organics Laboratories (Amityville, NY), and Country Life (Hauppauge, NY). Unless specified, all other reagents were obtained from Sigma-Aldrich or Invitrogen (Carlsbad, CA).
Purification of bromelain proteinases
Bromelains were obtained from pineapple fruit by grinding fresh pineapple fruit with a mortar and pestle then
Purification and characterization of bromelain proteinases
Three major peaks (the largest one a doublet) were identified within bromelain using a combination of protein assays and enzymatic activity against model substrates (Fig. 1A). Fractions were then combined as shown to create 3 pools that were further analyzed for proteolytic activity using model peptide substrates and a panel of biologically relevant cell surface molecules. Western blotting with a rabbit polyclonal antiserum raised against the bromelain mixture showed that the 3 pools gave
Discussion
The existing data suggest that bromelain may have potential for therapy of inflammatory diseases, including arthritis and inflammatory bowel disease. The biological effects of bromelain depend on its proteolytic activity (1, 5; L.P. Hale, unpublished). Thus it is critical to accurately assess the proteinase content, activity, and specificity of the bromelain that is used pre-clinical studies in animals and in human clinical trials. The studies reported here provide methods to assess the
Acknowledgements
The authors would like to thank Dr. Salvatore V. Pizzo (Duke University Medical Center, Durham, NC) and Dr. Tracey L. Mynott (Queensland Institute for Medical Research, Brisbane, Australia) for helpful suggestions and Dr. Pizzo for critical review of this manuscript. This work was supported by the Broad Medical Research Program of the Eli and Edythe L. Broad Foundation (IBD-0024R) and by the National Center for Complementary and Alternative Medicine, National Institutes of Health (1R21
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2022, Food Hydrocolloids for HealthCitation Excerpt :Alcalase can recognize a wide range of amino acids hence has a strong tendency to give a hydrolysate with many peptides of small size and a high protein hydrolysis degree in a wide range of protein substrates (Tacias-Pascacio et al., 2020). On the other hand, bromelain belongs to papain family of cysteine proteases with preference for peptide bond cleavage when arginine and lysine residues are located at P1 position (Hale et al., 2005). Because of generally lesser cleavage sites for bromelain, the resulting bromelain hydrolysate is larger with lower degree of hydrolysis.