At Sublethal Doses of Gammacyhalothrin

1 Human and Animal Health Vol.59: e16150010, January-December 2016 BRAZILIAN ARCHIVES OF http://dx.doi.org/10.1590/1678-4324-2016150010 ISSN 1678-4324 Online Edition BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL Bromelain: Methods of Extraction, Purification and Therapeutic Applications Zoya Manzoor1, Ali Nawaz1*, Hamid Mukhtar1, Ikram Haq1. 1Institute of Industrial Biotechnology GC University Lahore, Pakistan. ABSTRACT Bromelain is a concoction of sulfhydryl proteolytic enzymes. Depending upon the site of extraction it can be regarded as either stem bromelain (SBM) (EC 3.4.22.32) or fruit bromelain (FBM) (EC 3.4.22.33). Bromelain remain enzymatic active over a broad spectrumand endure a range of pH (5.5 to 8.0) and temperature (35.5 to 71 ºC). It is one of the extensively investigated proteolytic enzyme owing to its astonishing applications in various industries. This necessitated employing a strategy that result in highest purified bromelain in less steps and lowest cost. Use of modernistic approach such as membrane filtration, reverse micellar systems, aqueous two phase extraction and chromatographic techniques have shown promise in this regard. Besides its industrial applications, bromelain has been widely utilized as a potential phytomedical compound. Some of its reported actions include inhibition of platelet aggregation, anti-edematous, anti-thrombotic, anti-inflammatory, modulation of cytokines and immunity, skin debridement and fibrinolytic activity. It also assist digestion, enhance absorption of other drugs and is a potential postoperatively agent that promote wound healing and reduce postsurgical discomfort and swelling. Key words: Phytomedical, Anti-edematous, Fibrinolytic, Anti-thrombotic. *Authors for correspondence: [email protected] Braz. Arch. Biol. Technol. v.59: e16150010, Jan/Dec 2016 2 Nawaz, A et al. INTRODUCTION PROPERTIES Bromelain is a chief protease enzymes found in Bromelain is actually a group of sulfhydryl pineapple plant (Ananascomosus) (Smith-Marshall proteolytic enzymes (Smith-Marshall and Golden and Golden 2012). It has been known chemically 2012) and encompasses variety of cysteine proteases since 1876 (Tochi et al. 2008) and was identified for (Tochi et al. 2008) when extracted from the stem and the first time by Marcano in 1891 (Upadhyay et al. fruit of pineapple plant (Neta et al. 2012; Pillai et al. 2010). The investigation and isolation of bromelain 2013). Stem bromelain and fruit bromelain are both has been started since 1894 (Neta et al. 2012). single-chain glycosylated enzymes but they possess Sulfhydryl proteolytic enzymes are the chief different characteristics (Barrett et al. 2004). SBM constituents of bromelain (Tochi et al.2008; Gautam has reduced proteolytic activity and exhibit low et al. 2010). Bromelain is abundant in stem and fruit specificity for peptide bonds then FBM. Researchers of pineapple plant and it can also be isolated in small have reported several distinct pH and temperature amount from pineapple waste such as core, leaves, optima for the activity of bromelain towards its peel etc. (Hossain et al. 2015). Bromelain present in substrates (De Lencastre et al. 2016). The reported fruit of pineapple has assigned the EC number EC optimum pH range for SBM by various researchers is 3.4.22.33 and is regarded as fruit bromelain (FBM). 6-7 and its optimum temperature range is 50-60 ºC Likewise bromelain present in the stem of pineapple (Harrachi et al. 1998; Gautam et al. 2010; Xue et al. is called stem bromelain (SBM) and its EC number is 2010). While the optimum pH range for FBM is 3-8 EC 3.4.22.32. Stem bromelain possess different and optimum temperature ranges from 37-70 ºC biochemical properties and composition as compared (Lopes et al. 2009; Jutamongkon and Charoenrein to fruit bromelain (Pavan et al. 2012) and contains a 2010; Silvestre et al. 2012). Likewise, the molecular variegated blend of different thiol-endopeptidases. weight range for SBM is 26-37kDa (Grzonka et al. Efforts are being made by researchers to achieve 2007; Kumar et al. 2011) and for FBM molecular highly purified bromelain in less steps and low cost. weight range is 24.5-32kDa (Grzonka et al. 2007; Modern strategies, such as membrane filtration Lopes et al. 2009; Gautam et al. 2010). When (Lopes et al. 2009), reverse micellar systems bromelain is kept at -20 ºC its stability remains intact (Upadhyay et al. 2010; Kumar et al. 2011), aqueous for a defined time period (Rowan et al. 1990). two phase extraction (Coelho et al. 2013; Novaeset Amongst other, cysteine is the most efficient al. 2013) and chromatographic techniques (Yin et al. compound for the activation of bromelain (Grzonka 2011) have shown promise in this regard. Pineapple et al. 2007). SBM is in fact a combination of several plant also contains minor quantities of other thiol endopeptidases (Pavan et al. 2012) and also proteinases like ananain and comosain (Nadzirah et contains compounds like peroxidases, acid al. 2013) but bromelain is regarded as a primary and phosphatase, several protease inhibitors extensively investigated component (Amini et al. glucosidases, cellulases, glycoproteins, 2016). The reason of being such valuable is due to its carbohydrates and organically intact Ca2+ (Maurer miraculous utilization as phytomedical compound 2001; Smith-Marshall and Golden 2012). (Larocca et al. 2010). Bromelain displays antiedematous, fibrinolytic, anticancer, anti- EXTRACTION AND PURIFICATION inflammatory, antibiotic, anticoagulative and METHODS antithrombotic functions (Kavitha et al. 2013). It is also a potential postoperatively agent that assists Besides its clinical applications bromelain has been healing and decrease post surgical discomfort and subjected to many other industries due to its swelling (Graf 2000). Besides clinical approach, enormous benefits. Researchers thereby are trying bromelain has also employed in various industries various conventional as well as latest purification including food industries (Maurer 2001), such as techniques to achieve bromelain in highest purified breweries (Soares et al. 2012) and flesh processing form at reduced cost (Arshad et al. 2014). As and tenderization, textile and cosmetic industries compared to fruit, bromelain concentration is high in (Babu et al. 2008; Ketnawal et al. 2009). With the stem and is thus a cheaply available source of advent of recombinant DNA technology, scientists bromelain (Tochi et al. 2008). Other parts of and researchers across the globe have been working pineapple are also investigated for the presence of on recombinant bromelain to achieve exaggerate and bromelain (Ketnawa et al. 2012) including peel, core novel applications in future (Arshad et al. 2014). Braz. Arch. Biol. Technol. v.59: e16150010, Jan/Dec 2016 Strategies to obtain bromelain for cure 3 and crown etc. Extraction of bromelain from these exposed to numerous purification stages to eradicate parts is attractive not only from environmental point impurities that may interfere with bromelain to of view but also economically (Novaes et al. 2013). hinder its application and reduce the specific activity Bromelain can be easily extracted from the juice of of the enzyme (Illanes 2008). Product purity is the pineapple by ultrafiltration (Larocca et al. 2010) but key factor which may constitute a large proportion of still FBM is not commercially available due to being the total enzyme production cost (Lightfoot 1990). different from SBM (Pavan et al. 2012). The Several conventional isolation and purification marketable bromelain is mostly extracted from the techniques are now obsoleted because of their low stem of pineapple through centrifugation, purification potential (Soares et al. 2012).So the ultrafiltration, lyophilization (Corzo et al. 2011) and extraction and purification strategy (Figure 1) two-step Fast Protein Liquid Chromatography designed should be selective for the desired product, (FPLC) (Harrach et al. 1998). Once extracted, the cheap, high yielding and speedy (Gupta et al. 2004). crude mixture containing required enzyme is then Figure 1 - Overview of extraction and purification of Bromelain. Due to increased interest of scientists toward 5) Different chromatographic techniques bromelain, several new purification techniques have (Devakate et al. 2009; Li et al. 2009; Paulo et al. been employed for its extraction and purification 2012). (Table no. 1). These include: 1) Aqueous two phase systems (Babu et al. Reverse micellar system 2008; Ferreira et al. 2011; Coelho et al. 2013; Spir et Micelle is an aggregate of molecules possessing both al. 2015). polar and non-polar regions. Reverse micelles are 2) Membrane processes (Doko et al. 2005; thermodynamically stable, minute surfactants that Lopes et al. 2009) hold water in their interior surrounded by organic 3) Precipitation (Doko et al. 2005; Devakate phase (Andray et al. 2001). Only protein of interest is et al. 2009; Soares et al. 2012). entrapped in micelle (Figure 2) whereas other 4) Reversed micellar systems (Hemavathi et impurities remain in organic phase (Lee and Chong al. 2007; Navapara et al. 2008; Kumar et al. 2011). 2011). Reverse micellar system is an encouraging strategy for downstream processing. It is ideal to Braz. Arch. Biol. Technol. v.59: e16150010, Jan/Dec 2016 4 Nawaz, A et al. extract biomolecules through diluted samples. with ultrafiltration was also studied which resulted in Reverse micellar system possess higher sample an activity recovery of 95.8 % and purification factor loading capacity, are specific

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