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Raphanus Sativus L. Var. Niger on Mice Fed with a Lithogenic Diet

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Raphanus Sativus L. Var. Niger on Mice Fed with a Lithogenic Diet Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2012, Article ID 161205, 8 pages doi:10.1155/2012/161205 Research Article Antilithiasic and Hypolipidaemic Effects of Raphanus sativus L. var. niger on Mice Fed with a Lithogenic Diet Ibrahim Guillermo Castro-Torres,1 Elia Brosla Naranjo-Rodrıguez,´ 2 Miguel Angel´ Domınguez-Ort´ ız,´ 3 Janeth Gallegos-Estudillo,3 and Margarita Virginia Saavedra-Velez´ 1 1 Facultad de Qu´ımica Farmac´eutica Biologica,´ Universidad Veracruzana, Xalapa, Veracruz, Mexico 2 Laboratorio de Neurofarmacolog´ıa, Departamento de Farmacia, Facultad de Qu´ımica, Universidad Nacional Autonoma´ de M´exico, M´exico, DF, Mexico 3 Laboratorio de Productos Naturales, Instituto de Ciencias Basicas,´ Universidad Veracruzana, Xalapa, Veracruz, Mexico Correspondence should be addressed to Elia Brosla Naranjo-Rodr´ıguez, [email protected] Received 22 June 2012; Revised 13 September 2012; Accepted 14 September 2012 Academic Editor: Ayman El-Kadi Copyright © 2012 Ibrahim Guillermo Castro-Torres et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In Mexico, Raphanus sativus L. var. niger (black radish) has uses for the treatment of gallstones and for decreasing lipids serum levels. We evaluate the effect of juice squeezed from black radish root in cholesterol gallstones and serum lipids of mice. The toxicity of juice was analyzed according to the OECD guidelines. We used female C57BL/6 mice fed with a lithogenic diet. We performed histopathological studies of gallbladder and liver, and measured concentrations of cholesterol, HDL cholesterol and triglycerides. The juice can be considered bioactive and non-toxic; the lithogenic diet significantly induced cholesterol gallstones; increased cholesterol and triglycerides levels, and decreased HDL levels; gallbladder wall thickness increased markedly, showing epithelial hyperplasia and increased liver weight. After treatment with juice for 6 days, cholesterol gallstones were eradicated significantly in the gallbladder of mice; cholesterol and triglycerides levels decreased too, and there was also an increase in levels of HDL (P<0.05). Gallbladder tissue continued to show epithelial hyperplasia and granulocyte infiltration; liver tissue showed vacuolar degeneration. The juice of black radish root has properties for treatment of cholesterol gallstones and for decreasing serum lipids levels; therefore, we confirm in a preclinical study the utility that people give it in traditional medicine. 1. Introduction and Niemann-Pick C1L1 protein, and these transporters are regulated in the liver by several transcription factors, Gallstones represent an important problem of public health; including nuclear receptors farnesoid X receptor and liver its prevalence is about 10% in middle-age persons and 20% Xreceptors[7–9]. Other important factors for gallstones in aged persons [1]. Cholesterol is the main component of formation are high levels of serum lipids (mainly cholesterol the majority of gallstones [2]. This disease is strongly asso- and triglycerides) [10]. New studies should focus on the ciated with atherosclerosis and metabolic syndrome [3], and more important proteins involved in biliary secretion and its pharmacological treatment is limited, being cholecystec- intestinal absorption of cholesterol, because those are key tomy, an invasive surgical treatment, the only treatment for sites of cholesterol transport, on which new drugs might symptomatic gallstones [4]. Cholesterol gallstones formation inhibit the formation of cholesterol gallstones; ezetimibe is a complex process mediated by genetic and environmental is a drug that showed potential benefit in cholesterol factors [5, 6]. Many proteins (ATP-binding cassette (ABC)) gallstone prevention, and ezetimibe inhibits the expression are implicated in its formation, mainly the biliary lipids of Niemann-Pick protein in the small intestine, thereby transporters ABCB4, ABCB11, ABCG5, ABCG8, ABCC7, decreasing the intestinal absorption of cholesterol [11]. 2 Journal of Biomedicine and Biotechnology There are pharmacological treatments, but there are (water), 175, 550, 1750, 2000, and 5000 mg/kg (0.1 mL/10 g also treatments based on traditional medicine; in Mexico, body weight). Juice of black radish was lyophilized and was knowledge about the medicinal properties of plants is the dissolved in purified water. The treatments were adminis- basis for their use as home remedies. Black radish (Raphanus tered intragastrically. Neither food nor water was given up sativus L. var niger) is a plant belonging to the Brassicaceae to 4 h after the treatment. Body weight, body weight change, family which contains a high concentration of glucosinolates signs of toxicity, behaviors, and mortality were observed [12]; experimental studies demonstrated that the aqueous during the first 4 hours. At the end of the experiment period, extract and juice of this root possess pharmacological the sacrifice of female mice was performed after anesthesia properties, mostly antioxidant, against urinary stones and for (sodium pentobarbital) in order to avoid animal pain or detoxifying enzyme activity [13–15]. In Mexican traditional stress. medicine, black radish root has ethnopharmacological uses for the treatment of pigment and cholesterol gallstones and 2.5. Experimental Animals and Treatments. Thirty-six adult for decreasing serum lipids levels; juice squeezed from the female C57BL/6 mice over 7–9 weeks of age and weighing 18– P< . root of black radish significantly decreases ( 0 05) serum 22 g were purchased in Harlan Laboratories of Mexico and lipids levels in C57BL/6 mice fed with a lithogenic diet [16]; were used in all experiments. Animal care and procedures however, the active metabolites of black radish responsible were conducted according to the guidelines approved by ff for its therapeutic e ects are unknown. The principal aim Norma Oficial Mexicana (NOM-062-ZOO-1999) and were ff of this work was to evaluate the e ects of the juice of black subjected to experimental protocols approved by the Pro- radish root in a biological model of cholesterol gallstones, grama Institucional para el Cuidado y Uso de Animales in order to establish the scientific basis that explains its de Laboratorio, Facultad de Qu´ımica, UNAM. All animals ethnopharmacological uses. were housed in plastic cages in a temperature-controlled room with 12 : 12-h light-dark cycles and provided ad libitum 2. Material and Methods access to food and water. Mice had free access to commercial rodent food (Purina Rat Chow). The animals were divided 2.1. Chemicals. Reagent grade cholesterol (C27H45OH) was into 6 groups (n = 6), and treatments were administered purchased in Hycel of Mexico; reagent grade cholic acid intragastrically. One group was fed a normal diet (ND); ≥98% (C24H40O5) purchased in Sigma-Aldrich; reagent five groups were also fed an experimental lithogenic diet grade β-cyclodextrin (C42H70O35)H2O 7.0 mol/mol pur- containing 2% cholesterol and 0.5% cholic acid (10 g chased in Sigma Aldrich; ursodeoxycholic acid (C24H40O4), cholesterol/kg and 5 g cholic acid/kg) [17]. The vehicle Ursofalk 250 mg; sodium pentobarbital, Pfizer. group received purified water 0.1 mL/10 g (VEH). After feeding with lithogenic diet for 34 days, four groups were 2.2. Plant Material. Fresh tubercles of black radish were treated against cholesterol gallstones for 6 days: one group collected in Veracruz State, Mexico, in 2011 and were used with ursodeoxycholic acid 0.5% in beta-cyclodextrin 2%, all experiments. The plant was classified taxonomically for 0.1 mL/10 g (UDCA) and the other groups with juice of black the Instituto de Investigaciones Biologicas´ in Universidad radish root (JBR) diluted one hundredfold (1 : 100), tenfold Veracruzana. The voucher specimen of the plant (CIB (1 : 10) in purified water (1 : 100) and juice concentrate. All 14655) is deposited at the herbarium for reference. The administrations were at doses of 0.1 mL/10 g of juice. During juice of black radish root was prepared in Laboratorio de treatment against cholesterol gallstones (6 days), the VEH + Neurofarmacolog´ıa in Facultad de Qu´ımica. A fresh tubercle LD group continued receiving lithogenic diet (40 days). (557.7 g) was divided in six parts (60 g), and the juice was squeezed with extraction equipment Moulinex; from each 2.6. Gallbladder and Gallstones Analysis. Gallstones were part approximately 10 mL of juice were obtained. usually visible through the gallbladder. To collect gallstones, the tip of the gallbladder was cut, the gallbladder was 2.3. HPLC Analysis. 500 mL of juice were lyophilized, and the squeezed gently with forceps to remove stones, and the inside dry material produced 4.5 g. The sample was treated using was washed with an isotonic saline solution to remove any a previously described method [14]; however, we did not gallstones adhering to the walls; the gallbladder mucosa quantify glucosinolates of black radish root. Measurements was observed also. This process was carried out under a were done on HPLC equipment (Agilent Technologies stereoscope and a dissecting microscope. The number of 1200 Series Binary SL), RP-C18 column (Zorbax Bonus cholesterol gallstones in mice was counted. 100 × 2.1 mm id, 3.5 μm). The mobile phase was 10 : 90 MeOH : H2O with a flow rate of 2.0 mL/min. 2.7. Measurements of Total Cholesterol, HDL Cholesterol
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