Chemoprevention Effect of mixed Medicinal Herb Hibiscus sabdariffa L.(contains Anthocyanins)and Nanoseleniumon DEN-induced Hepatocellular Carcinoma in Rats

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide; however it is rarely detected early. The burden of HCC has been increasing in Egypt with a doubling in the incidence rate in the past two decades. An active surveillance and secondary prevention programs for patients with chronic hepatitis are the most important steps to reduce the risk of HCC. Accordingly chemoprevention could play important role in management of hepatocellular carcinoma. Thus selective and efficient targeting of cancer cells is the foremost goal in the development of anti-cancer therapeutics. Hibiscussabdariffa (Roselle) is rich in anthocyanins and protocatechuic acid (PCA). Water extract of the plant is considered as a local soft drink material and medicinal herb. The H. sabdariffa extract consider being save that the LD50 of roselle calyx extract was found to be above 5000 mg kg-1.Anthocyanins are heterocyclic compound pigments thus have antioxidant activities because of their cloud of hydroxyls around. Selenium (Se) is a trace nutrient element, excess of this important element is toxic, and therefore nano-form of Se is used for element toxicity avoidance. N-diethylnitrosamine (DEN) is a potent hepatocarcinogenic agent present in tobacco smoke, water, cured and fried meals, cheddar cheese, agricultural chemicals, cosmetics and pharmaceutical products. The present study aimed at evaluating the efficacy of Hibiscus sabdariffa L. (Karkadeh) water extract(HEx), containing anthocyanins and nanoselenium (nSe)as chemopreventive substances against hepatocellular carcinoma induced by DEN in experimental animals. Assessment of chemopreventive efficacy of HExand nSe that conducted in this study included in vivo and in vitro tests. 1- Preparation of Drugs: a- H. sabdariffa extract (HEx): was prepared by aqueous extractionin hot water (80 – 90 ᵒC), then lyophilized to powder form using freeze dryer. About 250g/ Kg b.w. were prepared freshly and administrated in the animals by oral tube thrice/week for ~ 6months. b- Nanoselenium (nSe):was purchased as powder selenium, grinded in energy Ball Mill, photographed by TEM. The suspension is prepared by dissolving in 10% DMSO with gentle heat-stirring forming nSe homogenate of which 2mg/Kg b.w. is injected i.p. twice/week for~ 6 months. 2- Biochemical studies: A- In vitro tests: The in vitro of the HEx or/andnSeshowed50% inhibitory concentration (IC50)of 9.1, 11.6 and 13.6 µg/ml for HEx, nSe and (HEx + nSe),respectively against human cell lineHepG2. B- Theinvivoexperiments: The in vivo tests were conducted on 120 Swiss albino rats divided into 8 equal groups of two main groups according to the carcinogen DEN-induction. Four without tumor: G1; normal control, G2; HEx, G3; nSe and G4; (HEx+nSe) groups and the other four with tumor induction, G5; DEN-induced, G6; (DEN+HEx), G7; (DEN+nSe) and G8;(DEN+HEx+nSe). After the end of the experimental period, the animals were anesthetized then scarified; the blood (whole blood and plasma) and the liver (homogenate and lysate) were collected,in addition to kidneys, spleens and parts of intestinefor further investigation. The in vivo studies comprise assessment of oxidative stress, and antioxidant parameters, assessment of apoptosis, inflammation and tumor markers. Liver function, kidney function biomarkers were measured. The study comprises also testing of lipid profile and measurement of selenium accumulation in different organs using inductively coupled plasma-optical emission spectrometry (ICP-OES) in addition to histopathological examination. The findings of DEN-injection led to liver intoxication appear as significant elevation in lipid peroxidation level, which expressed as malondialdehyde (MDA). On other hand liver intoxication led to decrease in the level of antioxidant parameters, reduced glutathione as well as superoxide dismutase (SOD), and catalase (CAT) activities. The study recorded significant increase in inflammation marker, tumor necrosis factor alpha (TNF-α), tumor marker, alpha fetoprotein (AFP) and significant diminish in caspase-3. Albuminas well as the diagnostic enzymes of liver function aspartate aminotransferase (AST) alanine aminotransferase (ALT), Gamma Glutamate transferase (γ-GT) in plasma was recorded highly significant elevation. Kidney function was affected also by the carcinogen induction which appears as highly significant increase in plasma urea and creatinine concentration. These results ensure the increased oxidative stress in the liver due to DEN intoxication relative to the normal control groups of each parameter. More over the survival rate of the animals, 100% in HEx, nSe and (DEN+HEx) groups, 73.33% in DEN-group ensured the safety use of hibiscus extract, our findings in histopathological examinations emphasized these results and suggestions. ICP-OESfindings recorded that nSe is accumulated in high concentrations in spleen and then in intestine whereas in low concentrations in liver and very low concentration in kidney. The significantchanges of tumor bearing groups compared to the corresponding normal control of each parameter, markedly altered compared with corresponding DEN-induced group of each parameter after administration of the drugs particularly post-treatment of HEx (250 mg/Kg body weight thrice a week for ~ 6 months) or/and nSe (2 mg/Kg body weight twice a week for ~ 6 months), indicating recovery of natural antioxidant defense system and physiological function of affected tissues. It is suggested that H. sabdariffa extract reduce the risk of oxidative stress via decreasing production of free radicals and increasing the defense mechanism against reactive oxygen species (ROS), restoring antioxidant capacity within liver hepatic tissues due to its phenolic phytochemical compounds, anthocyanins that act against oxidative stress. Nanoselenium incorporated in formation and function of selenoproteins that enhance immunity and natural enzymatic defense system. The results emphasize that H. sabdariffa extract of anthocyanins contains and nanoselenium separately or somewhat, synergistically have potent attenuation effect against DEN-induced HCC. They exhibited chemopreventive and treatment effect against HCC in rats.