A histomorphometric study on the hepatoprotective effects of a green rooibos extract in a diet-induced obese rat model

Highlights

• Aspalathin-rich green rooibos extract (GRE) possible anti-obesity agent.

• GRE significantly reduced liver and body mass.

• GRE reduced hepatic steatosis.

• GRE reduced inflammation, decreased inflammatory foci.

• GRE possible therapeutic for fatty liver disease and metabolic syndrome.

Abstract

Obesity, type two diabetes mellitus and insulin resistance are associated with increased oxidative stress and inflammation. Unfermented green rooibos is an aspalathin rich variant of traditional fermented rooibos (Aspalathus linearis) and has a high polyphenol content. The present study aimed to determine the histologically observable effects of a commercially produced, aspalathin-rich green rooibos extract, Afriplex GRT™ (GRE) in a diet-induced obese rat model. Male Wistar rats (N = 28) were randomly assigned to four study groups (n = 7): control (C), green rooibos (GRT), high-fat diet (HFD) and experimental (HFD-GRT) group. Body mass was determined prior to euthanasia and liver mass was determined after death. The left lateral lobe of the liver was processed to wax and stained using haematoxylin and eosin (H & E), Masson’s trichrome stain, Gordons and Sweet’s reticulin impregnation and periodic acid-Schiff stain. Frozen liver tissue sections were used for Oil red O staining. Morphometric quantification of steatosis, semiquantitative pathology grading and scoring were performed and verified by a veterinary histopathologist. A significant increase in body and liver mass was observed in the HFD groups while co-treatment with green rooibos significantly reduced both. The volume and area of steatosis were significantly increased in the HFD groups while the area of steatosis significantly reduced with green rooibos co-treatment. The percentage, location and type of steatosis as well as presence of inflammation and hepatocellular injury were reduced in the HFD group co-treated with GRE. These findings suggest that a GRE has potential as an anti-steatotic, anti-inflammatory and weight reducing agent in vivo.

Introduction

Polyphenol compounds such as those found in rooibos have been shown to have beneficial effects in obesity-associated metabolic disorders such as metabolic syndrome and type two diabetes mellitus. Polyphenols have anti-oxidant and anti-inflammatory properties (Herranz-López et al., 2012; Rodriguez-Ramiro et al., 2016), as well as glucose and lipid metabolizing properties (Hanhineva et al., 2010). In glucose metabolism, polyphenols like aspalathin can modulate glucose related receptors and downstream molecular pathway activation, as well as the absorption and release of glucose from the liver (Hanhineva et al., 2010). Polyphenols have been suggested as complementary therapeutic agents in addition to healthy lifestyle changes and increased exercise to combat metabolic disorders (Hanhineva et al., 2010). In addition, polyphenols are readily accessible in fruits, vegetables and teas at relatively low cost (Hu et al., 2017). A shrub indigenous to South Africa, Aspalathus linearis (Brum.f) R. Dahlgren, commonly known as rooibos, is especially rich in polyphenols, particularly aspalathin, a dihydrochalcone (von Gadow et al., 1997; Joubert and Schultz, 2012; McKay and Blumberg, 2007). Unfermented rooibos is particularly rich in aspalathin and therefore has increased anti-oxidant potential (Villaño et al., 2010; De Beer et al., 2017). Afriplex GRT™ (GRE) is a laboratory standardized good manufacturing practice (GMP) unfermented (green) rooibos extract which has a high aspalathin content (12.8%); (Beelders et al., 2012; Patel et al., 2016). Fermented rooibos processing involves shredding of leaves and stems, bruising the material with hammers and sun drying in heaps resulting in a lower aspalathin content (6% aspalathin) and anti-oxidant capacity (Joubert and De Beer, 2011). In contrast, unfermented rooibos processing uses vacuum drying technology, lower temperatures and pH levels which reduce oxidation and loss of aspalathin (De Beer et al., 2002; Joubert and De Beer, 2011). To date, anti-inflammatory (Schloms et al., 2012) and anti-diabetic (Muller et al., 2013; Mazibuko et al., 2013; Kamakura et al., 2015; Mazibuko-Mbeje et al., 2019) effects of green rooibos have been demonstrated. Promising in vivo studies have established that green rooibos reduces hyperglycaemia in male Wistar rats and in KK-ay diabetic mice (Muller et al., 2013; Kamakura et al., 2015) and recently in C3A liver cells (Mazibuko-Mbeje et al., 2019) suggesting potential anti-diabetic properties of green rooibos. However, the effects of green rooibos on actual tissues such as the liver, as determined by using histomorphometric analysis, is underreported.

Metabolic syndrome, often associated with visceral obesity, leads to an increase in the production of excessive reactive oxygen species (ROS) with a subsequent decrease in endogenous anti-oxidants such as glutathione peroxidase (GPx) and superoxide dismutase (SOD); (Roberts and Sindhu, 2009). In obesity, the influx of dietary carbohydrates and fat may lead to increased oxidative stress resulting in significant strain on glucose and lipid balance maintained by the liver (Hanhineva et al., 2010). The liver plays a major role in glucose homeostasis and storage (via glycogenesis) as well as glucose secretion (via glycogenolysis and gluconeogenesis); (Campbell, 2006; Hanhineva et al., 2010). Glucose balance is controlled by insulin, however, excess glucose (hyperglycaemia) and the development of peripheral insulin resistance leads to hyperinsulinemia. Therefore, when hepatic glucose balance is compromised, hepatic insulin resistance occurs which has been linked to fatty liver disease (Marchesni et al., 1999). During excessive intake of dietary fat, (systemic hyperglycaemia and insulin resistance) an excess of free fatty acids in the liver leads to increased triglyceride synthesis resulting in an increase in very-low-density lipoprotein secretion (further increasing the lipid burden in other organs) and hepatic steatosis (Sanders and Griffin, 2016). Hepatic insulin resistance, compromised glucose, and lipid metabolism results in increased triglyceride deposition (>5%) in the liver which is clinically defined as non-alcoholic fatty liver disease (NAFLD); (Schultz et al., 2013; Sears and Perry, 2015; Brunt, 2016). Fatty liver disease includes liver pathology ranging from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH), which may further progress to fibrosis, liver cirrhosis and hepatocellular carcinoma (Marchesini et al., 1999; Brunt, 2016; Kleiner et al., 2005; Santiago-Rolón et al., 2015).

Many studies have shown improved hepatic glucose and lipid metabolism with polyphenol supplementation, including green tea (Bose et al., 2008), piperine (Choi et al., 2013), fermented rooibos (Beltrán-Debón et al., 2011; Sanderson et al., 2014), quercetin (Jung et al., 2013), resveratrol, soy, citrus flavonoids and grape polyphenols (Hanhineva et al., 2010). However, studies on the histologically visible effects of an aspalathin-rich green rooibos extract on liver tissue are lacking. Wistar rats are often used in obesity studies, as they readily and variably gain (body) mass with a high-fat diet (HFD), similar to that seen in human obesity (Buettner et al., 2007; Jang, 2017). Therefore, the present histomorphometric study aimed to evaluate the ameliorative effects of an unfermented green rooibos extract (GRE) on liver tissue in a diet -induced obese rat model. Specifically, the ameliorative effects of GRE on liver steatosis, inflammation, fibrosis, glycogen storage and reticulin architectural changes are evaluated.