Anti-cancer effects of Ajwa dates (Phoenix dactylifera L.) in diethylnitrosamine induced hepatocellular carcinoma in Wistar rats

Hepatocellular carcinoma (HCC) accounts for major cancer-related deaths despite current advanced therapies. Treatment and prognosis of HCC is better in patients with preserved liver function. Many natural products including Ajwa Dates Price in Pakistan dates (Phoenix dactylifera L.), are claimed to have hepatoprotective and HCC inhibitory effects, but most lack scientific validation. To prove our hypothesis, we attempted to evaluate the HCC inhibitory effects, and other beneficial properties of the aqueous extract of ajwa dates (ADE) in a rat model of diethylnitrosamine (DEN) induced liver cancer.

DEN treatment Groups (B, C, D) showed histological features of HCC and in rats treated with ADE (Groups C, D) partial to complete reversal of normal liver architecture was observed. Antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR), glutatione peroxidase (GPx) and catalase (CAT) were increased, while the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels and lipid peroxidation were significantly decreased in Group C and Group D compared to Group B. Pro-inflammatory cytokines such as interleukin (IL)-1?, IL-1?,, GM-CSF) were increased in the serum of rats in Group B while the anti-tumor cytokines (IL-2, IL-12) were increased in ADE treated Groups (C, D). In addition, Alpha-Feto Protein (AFP) and IL-6 gene expression levels were upregulated in Group B, while they were significantly downregulated in ADE treated Groups (C, D).

ADE helped in the reversal of DEN damaged liver towards normal. Restoration of anti-oxidant enzymes, liver enzymes, cytokines balance and gene expression to normal levels following ADE treatment indicates that ADE improves liver function and inhibits HCC. ADE can, therefore, be used together with conventional therapeutics for HCC.

Hepatocellular carcinoma (HCC) is one of the leading cause of cancer-associated deaths, and globally it is three times more common in men than women . HCC is one of the leading cause of cancer related deaths in the Kingdom of Saudi Arabia (KSA). HCC is the 4th most common cancer in males and 8thcommon cancer in females accounting for 4.3% of the reported cancer cases in the year 2013 . A global estimation of 40,710 new cases and 28,920 deaths due to liver and intrahepatic bile duct cancers in 2017 highlights the need for improvement in current management strategies . Common risk factors for liver cancer include heavy alcohol consumption, hepatitis B virus (HBV), hepatitis C virus, obesity, tobacco smoking, diabetes and genetic factors. Partial hepatectomy, embolization and sorafenib (Nexavar®) offer partial to complete success depending upon the stage of the disease.

The incidence of high mortality and associated side effects following chemotherapy and/or radiotherapy increase the demand for alternative medicine for the cancer treatment. Not surprisingly, many potent anticancer compounds were isolated from the plants viz. doxorubicin, taxol, etoposide, cisplatin, vinblastine, vindesine, vincristine and topotecan . Given the current interests in alternative medicine, it is imperative that much-concerted effects are undertaken to identify novel compounds that will aid therapy and also help to avoid its unjustifiable use.

Date fruit (Phoenix dactylifera L.) is a native fruit of arid region, largely cultivated as an economical and food crop in the Middle East, Southern Europe, North Africa, South America, India, and Pakistan . Of the many varieties of date fruits, ajwa dates are unique for its medicinal properties and these are specifically grown in the city of Al-Madina Al-Munawwarah, KSA . Ajwa dates are a rich source of energy, and readily provide sugars, proteins, vitamins, high dietary fibers, minerals and fats . They contain various phytochemicals like sterols, polyphenols, flavonoids and glycosides . Ajwa dates have hypolipidemic, antioxidant, anti-inflammatory, cardioprotective, nephroprotective and hepatoprotective effects . In addition, the concentrated polyphenols and ex vivo digested extracts from ajwa dates inhibited growth and proliferation of colon cancer Caco-2 cell line . Also, (1 ? 3)-?-D-glucan, which is an essential component of dates had an inhibitory effect on sarcoma-180 in vivo in mice . Recently, we identified that methanolic extract of ajwa dates inhibited human breast adenocarcinoma (MCF-7) by causing cell cycle inhibition and induction of apoptosis . Interestingly, so far no studies have been done to identify the effects of ajwa or other dates on hepatic cancer.

Many in vitro and in vivo models of hepatic cancer have been utilized in the past to evaluate various drugs. In vivo hepatic cancer model development is done using various chemical methods, of which diethylnitrosamine (DEN) is a chemical carcinogen, commonly utilized to induce liver carcinogenesis by increasing oxidative stress and liver damage . Hence in the present study, we used the DEN-induced hepatic cancer model in Wistar rats and evaluated the cancer inhibition effects of the aqueous extract of ajwa dates (ADE). Histological, biochemical, antioxidant enzyme status, as well as the related cytokines and gene expression profiles was studied.

The animals used in this study was approved by the Institutional Ethics Committee, King Abdulaziz University (KAU), Saudi Arabia vide approval number HA-02-J-008. Thirty-two albino male rats (Wistar strain, 5-6 weeks old) that weighed between 100 g - 120 g were purchased through the animal holding unit (AHU), King Fahd Medical Research Center (KFMRC, KAU). All rats were housed in the AHU under standard animal housing conditions of 25 °C ± 3 °C, 55 ± 2% humidity, 12 hrs day/night cycle, and had access to standard rat pellet and water ad libitum.

Preparation of aqueous extract of ajwa date (ADE)

Ajwa dates used in the present study were sourced from Al Madina Dates Co. (Tomoor), Madinah Al Munawarah, Kingdom of Saudi Arabia. Ajwa dates sample was deposited with the Biology Department, Faculty of Science, KAU. Dr. Dhafer Ahmed Al-Zahrani, Plant Taxonomist confirmed the genus, species and origin of date fruit (Specimen voucher number: P. dactylifera L. #PD17569). Ajwa dates flesh was mixed with ultrapure distilled water at a ratio of 1:3. The date suspension was continuously agitated and incubated at 25 °C on a shaker (at 60 rpm) placed within the incubator (25 °C) for 48 hrs. The date-water extract was then filtered through muslin cloth and Whatman no. 1 filter paper. The filtrate was then centrifuged at 4500 rpm for 10 min. The supernatant was again filtered and the aqueous extract of ajwa dates (ADE) thus obtained was stored at ?80 °C until use.

Wistar male rats (n = 32) were acclimatized for 1 week prior to start of experiments. Rats were randomly divided into four experimental groups (n = 8). HCC induction was done using diethylnitrosamine (DEN). Briefly, DEN was dissolved in corn oil and two doses (180 mg/kg body weight (bw); at 15 days interval) were administered orally to all rats in Group B, C and D using intragastric gavage. Untreated control (Group A) was administered vehicle (corn oil) alone. Treatment with ADE was started on the next day following DEN treatment. All rats in Group C and Group D were treated with ADE 0.5 g/kg bw and 1.0 g/kg bw respectively, daily for 10 weeks; while the rats in Group A (untreated control) and Group B (DEN control) were administered vehicle alone. Following completion of treatment, the animals were sacrificed using an injection of chloral hydrate (400 mg/kg bw; i.p). Blood and liver tissues were collected from all the anim