Cocoa bean (Theobroma cacao L.) phenolic extracts as PTP1B inhibitors, hepatic HepG2 and pancreatic β-TC3 cell cytoprotective agents and their influence on oxidative stress in rats
Introduction
One of the richest known natural sources of antioxidants is cocoa bean, which contains more phenolic compounds and has higher antioxidant activity than tea or red wine (Lee, Kim, Lee, & Lee, 2003). Cocoa beans are the seeds of the tropical cocoa tree Theobroma cacao L. and are one of the most valuable ingredients of noble confectionery and pharmaceutical or cosmetic industries (Ali et al., 2015, Farahany et al., 2008, Liendo et al., 1998). Among three main varieties of cocoa bean: Forastero, Criollo, and Trinitario, the first one is the most common and widely cultivated for mass production. Cocoa beans of Forastero variety are dark brown and their aroma is less pleasant and aromatic comparing to the Criollo variety. They have more robust, sour–bitter flavor with moderate acidity, are richer in valuable cocoa butter and have higher content of phenolic compounds (Motamayor et al., 2003, Oracz et al., 2015, Rusconi and Conti, 2010). Phenolic compounds of cocoa beans represent on average around 10% of the dry weight of the whole bean (Ali et al., 2015, Gallo et al., 2013). Most of them, due to the nature of their construction and the presence of hydroxyl groups, have a high biological in vitro and in vivo activity and play an important role in the prevention of many diseases. They exhibit a wide range of physiological properties, such as antioxidant, anti-free radical, anti-allergic, antibacterial, antiviral, anti-inflamatory, antiatherogenic, antithrombotic, antihypertensive, anticarcinogenic, cardioprotective and vasodilatory effects (Arlorio et al., 2005, Aron and Kennedy, 2008, Faisal et al., 2015, Field and Newton, 2013, Keen et al., 2002, Kirch and Ellinger, 2014, Kumar Pandurangan et al., 2015, Maskarinec, 2009, Quinones et al., 2011, Rein et al., 2000, Rodriguez-Ramiro et al., 2011, Sies et al., 2005, Zanotti et al., 2015). It is known that the consumption of modest amounts of cocoa products is associated with ~ 40% reduction in cardiometabolic risk, decrease of blood glucose levels, improvement of glucose tolerance in diabetic patients, improvement of sensitivity to insulin and modulation of oxidative stress markers in skeletal muscle (Ramirez-Sanchez et al., 2013). However, the antioxidant defense mechanism for the preventive activities of cocoa during obesity and diabetes is not fully understood (Ali et al., 2015, Arlorio et al., 2005, Helal et al., 2015, Oracz et al., 2015, Ramiro et al., 2005). More recently it has been shown, that some phenolic compounds possessing antidiabetic effect can inhibit activity of PTP1B protein and protect pancreatic beta cells against oxidative stress (Zakłos-Szyda, Majewska, Redzynia, & Koziołkiewicz, 2015). Due to the high concentration of phenolic compounds in cocoa bean this variety will constitute research material in these studies.
Cocoa bean is the main raw material used for the preparation of chocolate and cocoa powder. One of the most important processes of the cocoa processing is roasting. As a result of roasting among others Maillard reaction products (MRP) are formed in the beans. Maillard reactions play very important role among multiple conversions taking place during the thermal processing of cocoa beans. This process is very complex and in the advanced and final steps of the Maillard reactions it leads to formation of high molecular weight polymers or co-polymers with molecular masses up to 100 kDa that are named melanoidins (Oliviero, Capuano, Cammerer, & Fogliano, 2009). These compounds are responsible for the taste, color, aroma and texture of roasted food products such as cocoa beans, coffee and malt (Wang, Qian, & Yao, 2011). Melanoidins have been shown to possess the strong antioxidant activity, antimicrobial ability and antihypertensive properties (Liu, Kong, Han, Sun, & Li, 2014). MRP isolated from roasted coffee and biscuits protect human hepatoma HepG2 cells against oxidative stress (Goya et al., 2007, Martin et al., 2009, Wang et al., 2011).
The aim of the study was to determine the biological activity of two freeze-dried aqueous extracts obtained from cocoa bean of Forastero variety (CBEs), i.e. raw and roasted beans and fractions of the compounds isolated from these extracts as potential anti-obesity agents using the hepatoma HepG2 and pancreatic β-TC3 cell lines. In addition to the in vitro study, the above mentioned extracts were used as dietary supplements in laboratory rats fed high-fat diets for 4 weeks. In in vivo study it was hypothesized that: i) polyphenols along with other cocoa bean compounds possess antioxidant properties irrespective if they are extracted from raw or roasted beans, ii) the antioxidant activity should be ascribed to the main polyphenolic fraction, i.e. monomeric flavan-3-ols, and iii) the dietary cocoa extracts may mitigate negative consequences of high-fat diet consumption.
Section snippets
Chemicals and reagents
Standards of (+)-catechin (≥ 99%), (−)-epicatechin (≥ 98%), epigallocatechin (≥ 90%), procyanidin B2 (≥ 90%), procyanidin C1 (≥ 75%), quercetin (≥ 95%), quercetin-3-O-glucoside (≥ 98%), quercetin-3-O-galactoside (≥ 97%), quercetin-3-O-arabinoside (≥ 95%), acetonitrile of HPLC grade (≥ 99.9%), formic acid for LC–MS (~ 98%), DCFH-DA dye, DPPH and ABTS radicals were all obtained from Sigma-Aldrich (St. Louis, MO, USA) and dichloromethane of HPLC grade from CHEMPUR (Piekary Śląskie, Poland). All other
Characteristic of CBEs and their fractions
Freeze-dried CBE obtained from raw cocoa bean, CBE prepared from roasted cocoa bean and isolated from them fractions of following bioactive compound groups obtained by CPC: 1. monomeric flavan-3-ols, 2. procyanidins, 3. colored compounds from raw cocoa bean and 4. colored compounds from roasted cocoa bean were analyzed for the content of phenolic compounds and Maillard reaction products. The results of these determinations are summarized in Table 2.
Preserved by freeze drying CBEs before
Conclusions
In the present study extracts obtained from raw and roasted cocoa bean of Forastero variety (full, fractionated and purified) were screened as potential protein phosphatase PTP1B inhibitors and protective agents against oxidative stress in human hepatoma HepG2 and mouse insulinoma β-TC3 cells. Additionally their influence on oxidative stress and lipid metabolism in rats fed high-fat diet was studied. Cocoa bean polyphenolic content is already known but for the first time we have characterized
Acknowledgments
Authors are grateful for the financial support provided by Polish National Center for Research and Development (project No. WNP-POIG.02.01.00-10-171).
Authors also want to thank Barry Callebaut in Poland for giving cocoa beans for research.
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