Raphanus Sativus L. Var Niger As a Source of Phytochemicals for the Prevention of Cholesterol Gallstones

PHYTOTHERAPY RESEARCH Phytother. Res. (2013) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.4964

Raphanus sativus L. var niger as a source of Phytochemicals for the Prevention of Cholesterol Gallstones

Ibrahim Guillermo Castro-Torres,1* Minarda De la O-Arciniega,2 Janeth Gallegos-Estudillo,3 Elia Brosla Naranjo-Rodríguez4 and Miguel Ángel Domínguez-Ortíz3 1Instituto de Química, Universidad Nacional Autónoma de México, México, D.F., México, 2Área Académica de Farmacia, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Pachuca de Soto, Hidalgo, México 3Laboratorio de Productos Naturales, Instituto de Ciencias Básicas, Universidad Veracruzana, XalapadeEnríquez,Veracruz,México 4Laboratorio de Neurofarmacología, Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, México, D.F., México,

Raphanus sativus L. var niger (black radish) is a plant of the cruciferous family with important ethnobotanical uses for the treatment of gallstones in Mexican traditional medicine. It has been established that the juice of black radish decreases cholesterol levels in plasma and dissolves gallstones in mice. Glucosinolates, the main secondary metabolites of black radish, can hydrolyze into its respective isothiocyanates and have already demonstrated antioxidant properties as well as their ability to diminish hepatic cholesterol levels; such therapeutic effects can prevent the formation of cholesterol gallstones. This disease is considered a current problem of public health. In the present review, we analyze and discuss the therapeutic effects of the main glucosinolates of black radish, as well as the effects that this plant has on cholesterol gallstones disease. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: Raphanus sativus L. var niger; gallstones; glucosinolates; cholesterol.

levels, important effects that not only serve for treating the INTRODUCTION disease, but also to prevent it (Castro-Torres et al., 2012). The secondary metabolites of the black radish are mainly Cholesterol gallstones are a highly prevalent affection glucosinolates, of which glucoraphasatin represents more in Western countries such as the United States; in Latin than 65% of them (Hanlon et al., 2007); this component America, it is prevalent in countries such as Chile, and its hydrolysis product, called raphasatin, have Mexico and Argentina (Portincasa et al., 2006). As this demonstrated to possess antioxidant properties in disease produces a high number of medical consultations different biological models and that these effects can and hospital admissions, it is thought to be a public prevent the appearance of oxygen reactive species, health problem; its pharmacological treatment is very which can appear in conditions that are also suitable for limited (Di Ciaula et al., 2010), and the invasive prophylactic the appearance of gallstones, due to the high concentration therapy for symptomatic gallstones is cholecystectomy, of hepatic cholesterol. Oxygen reactive species damage the or the removal of the gallbladder (Portincasa et al., gallbladder epithelium and, due to the constant secretion 2012). Because of this, research focuses on the search of biliary cholesterol, produce a pathophysiological for preventive treatments; the possible preventive effects imbalance which ends with the formation of a gallstone of numerous proteins that participate in the transport (Koppisetti et al., 2008); therefore, glucoraphasatin and and metabolism of biliary lipids have been investigated, raphasatin could be two natural agents that prevent both such as ATP or ABC-binding cassette: ABCB11, ABCB4, the formation of oxygen reactive species and cholesterol ABCG5 and ABCG8 (Castro-Torres, 2012; Stokes gallstones. Another glucosinolate present in black radish and Lammert, 2011). Treatments based on traditional is glucoraphanin, which yields sulforaphane as a product medicine are highly relevant, and researching them is of its immediate hydrolysis; in addition to the antioxidant a complex matter. In Mexican traditional medicine, the properties of these metabolites, they also diminish hepatic black radish has been used since ancient times for treating cholesterol levels by inhibiting the expression of enzymes gallstones (Castro-Torres and Naranjo-Rodríguez, 2012), and transcription factors associated with cholesterol stimulating the biliary function and to treat some digestive metabolism (Rodríguez-Cantú et al., 2011). Black radish problems caused by the high intake of fats (Lugasi et al., contains important metabolites for the prevention of 2005). It is now known that black radish juice can be used gallstones; in this review, we discuss the reports that for treating gallstones in mice fed with a lithogenic diet support this therapeutic mechanism. (rich in cholesterol and cholic acid) and that it has the property of diminishing serum triglycerides and cholesterol Raphanus sativus L. var niger * Correspondence to: Ibrahim Guillermo Castro-Torres, Prof. Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, C.P. 04510, Delegación Coyoacán, México, D.F., México. This plant, popularly known as black radish, belongs to the E-mail: [email protected] cruciferous family, which includes cabbage, cauliflower, red Received 16 March 2012 Revised 27 January 2013 Copyright © 2013 John Wiley & Sons, Ltd. Accepted 01 February 2013 I. G. CASTRO-TORRES ET AL. radishes, spinach and broccoli (Sipos et al., 2002). The medicine, called ezetimibe, which inhibits the expression origin of black radish is not known exactly; nevertheless, of the Niemann-Pick type C1 type 1 (NPC1L1) protein in many Asian and African regions, it is used for treating and thereby diminishing the rate of intestinal absorption different problems associated with the gastrointestinal, of cholesterol (de Bari et al., 2012). Ezetimibe inhibits hepatic and biliary systems (Vargas et al., 1999). Its effects the formation of cholesterol gallstones in experimental have not been scientifically demonstrated; they are only models (Wang et al., 2008; Zúñiga et al., 2008); regrettably, known from ethnobotanical information. In Mexico, the there are no specific clinical studies that can evaluate plant is used to treat pigmented and cholesterol gallstones the effect of NPC1L1 expression in the prevention (Castro-Torres et al., 2012). We know that treatment is of gallstones. The pharmacological treatment has its a different concept from prevention; in the search limitations; the medications used as prophylactics are: for curative and preventive treatments for cholesterol ursodeoxycholic acid, ezetimibe and statins (Di Ciaula gallstones, the essential thing is to diminish the concen- et al., 2010); it is important to mention that when tration of intestinal, hepatic and biliary cholesterol, and gallstones are already formed, these therapeutic also, although less importantly, of plasmatic cholesterol. agents present a low capacity to dissolve them, Black radish is capable of diminishing serum lipids ursodeoxycholic acid being the most used medicine for in C57BL/6 mice, an important species that develops it, but, in order to work, gallstones should be strictly of gallstones (Castro-Torres and Naranjo-Rodríguez, cholesterol, and they must be within a gallbladder with 2012); the decrease of serological cholesterol can be a adequate motility. The search for a therapeutic target is factor associated with the development of gallstones, but focused on biliary secretion and the intestinal absorption it does not necessarily indicate that there is a constant of cholesterol, but it is very important to consider biliary lipid secretion; nevertheless, black radish can also the production of reactivate oxygen species, which dissolve cholesterol gallstones in mice (Castro-Torres are produced by cholesterol loading and that cause et al., 2012), which demonstrates the strong effect it secretion rates to exceed normal levels. Considering has on cholesterol that is already crystallized and inside these species to be target molecules and knowing a matrix of glycoproteins. Moreover, if it can disintegrate that they can be produced in both hepatocytes and in gallstones, it is probable that it can diminish biliary the vesicular epithelium, the glucosinolates and their cholesterol concentration; these factors are indeed essential degradation products (with antioxidant capacity) that for avoiding the formation of lithiasis. No toxicity related are present in black radish might impede the formation to the different therapeutic effects of black radish has of gallstones in the gallbladder and at the same time been reported, but, with respect to cholesterol gallstones diminish biliary cholesterol secretion by reducing treatment, we have reported that the DL50 of black radish hepatic cholesterol. juice is over 5000 mg/kg (Castro-Torres et al., 2012), which allow us to think that the natural product is not toxic, that it is effective and that it is not necessary to look for a lethal Glucosinolates dose at higher values than the one we analyzed because therapeutic effects produced with minimal doses are Glucosinolates are biologically active secondary metabolites preferred. In Mexico, there are also ethnobotanical uses that play an interspecific and intraspecific role in the of the plant for the treatment of pigment gallstones, which Brassicaceae family of plants (Sønderby et al., 2010). have a pathophysiology different from that of cholesterol Chemically, they are anionic glycosides (Fig. 1), and gallstones; calcium salts of bilirubin abound in the their content changes depending on the species, the part first ones, crystallized cholesterol in the second ones of the plant and climatic conditions (Agerbirk and (van Erpecum, 2011); it would be important to start new Olsen, 2012). Glucosinolates possess structural diversity, studies in order to analyze the effect of black radish in reflecting the fact that their biogenetic precursors pigmented gallstones, which do not have pharmacological are tyrosine, phenylalanine and tryptophan (Sønderby treatment, only surgical. et al., 2010). These secondary metabolites are inside vacuoles; myrosinase is the enzyme responsible for hydrolyzing them into different degradation products, Cholesterol gallstones such as nitriles, isothiocyanates and thiocyanates (Peñas et al., 2011). The strong flavor that glucosinolates and Cholesterol gallstones are one of the most frequent their derivatives give to cruciferous plants is associated gastroenterological affections, representing a gallbladder with a defense mechanism against herbivorous organisms disease (Portincasa et al., 2006); its prevalence in Western (Mithöfer and Boland, 2012). Among the most studied countries is 14–16%, caused by obesity and a high-fat diet. glucosinolates, there are two compounds, glucoraphasatin In Latin America, Chile and Mexico are the countries and glucoraphanin, found mainly in radishes and broccoli. with the highest prevalence of cholesterol gallstones; in Glucoraphasatin has demonstrated antioxidant effects in these regions, the genetic background is a determining cancer models in situ, as has its degradation product, an factor (Stinton et al., 2010). There are numerous risk isothiocyanate called raphasatin (Hanlon et al., 2007). factors for the development of cholesterol gallstones: Glucoraphanin and its immediate product, sulforaphane, female sex, obesity, sedentary lifestyle, high-fat diet, also have antioxidant properties and can diminish the levels advanced age, hypercholesterolemia, among others of plasmatic cholesterol (Rodríguez-Cantú et al., 2011; (Stokes et al., 2011). The pathophysiology of gallstones Boddupalli et al., 2012). The red radish (Raphanus sativus is complex and polygenic; nevertheless, there are two L.) is a cruciferous rich in isothiocyanates; its administra- processes related to cholesterol transport and use that tion to C57BL/6 J mice under a hypercholesterolemic are highly relevant for preventing the formation of diet can reduce serum triglycerides and cholesterol levels gallstones: biliary secretion and intestinal absorption (Balasinska et al., 2005). The metabolism of cholesterol (Portincasa and Wang, 2012). Nowadays, there is a and triglycerides is strongly associated with gallstones

Figure 1. Major glucosinolates and their degradation products. formation (Smelt, 2010), and C57BL/6 J mice are susceptible The diet administered to Wistar rats works as a lithogenic models for their development. The ethnobotanical uses of diet in mice; the diet produces cholesterol gallstones radishes and broccoli include diminishing cholesterol in mice, while rats cannot produce them because they levels and as antioxidants in, but there is no current wide lack a gallbladder. The treatment with black radish scientific support for this, and we have a long way ahead juice had significant antioxidant effects in rats, the before determining the active secondary metabolites and same that can be expected in mice, which have the ability their action mechanism. to prevent the formation of gallstones. Black radish, in contrast to other cruciferous plants, is used in traditional medicine to prevent and to dissolve cholesterol Glucoraphasatin and antioxidant effects and pigmented gallstones, aspects that reinforce the scientifically demonstrated therapeutic effects and Glucoraphasatin and its hydrolysis product, raphasatin, that are associated to biliary lithiasis prevention. After are known for their antioxidant properties, which have assessing the antioxidant properties of black radish been related to cancer prevention (Fahey et al., 2012); with in vitro models, the investigations continued with nevertheless, these properties have not been extensively in situ models. explored with respect to other pathologies. The absence Antioxidant enzymes can prevent the formation of of antirust nutrients and an imbalance in glutathione reactive oxygen species, which generate problems with homeostasis, the main antioxidant molecule of the human the gallbladder’s motility; this is one of the main factors organism, are typical in cholesterol gallstones disease that predispose to the development of gallstones. It is (Worthington et al., 2004). Glucoraphasatin is the main feasible to consider the study of metabolites that are glucosinolate of black radish, identified in high concentra- capable of activating the expression of antioxidant enzymes tions in aqueous extracts (approximately 30 mmol of in order to avoid gallstones formation; for example, glucoraphasatin for each gram of dry vegetable material). raphasatin (isolated from black radish) is a powerful In animal models, the levels of glutathione peroxidase in enzymatic detoxification inductor. In (HepG2) hepatic cell erythrocytes are increased by the black radish juice; lines and in a concentration of 10 mM, this metabolite the possible active metabolite might be glucoraphasatin induces messenger RNA expression of important phase II (Lugasi et al., 2005). In Hungary, a study was carried out to detoxification enzymes: quinone reductase, hemoxigenase evaluate the juice extracted from the root of black radish; and thioredoxin reductase (Hanlon et al., 2007). A study they used Wistar male rats fed with a hyperlipidaemic diet carried out in the United States demonstrated that the (cholesterol 2%, colic acid 0.5% and fat 20%). Later, aerial parts of black radish (stems and leaves), in contrast these rats were treated with black radish juice for 9 days, to the root, more effectively induce the expression of administered to them on demand and diluted 10 times in hepatic detoxification phase I and II antioxidant enzymes water. The rats that received only a hyperlipidaemic diet (Hanlon et al., 2009). This analysis, carried out in had high serological concentrations of primary and cell lines, demonstrates a significant antioxidant effect. secondary lipid peroxidation products, while in the animals In biliary lithiasis conditions, reactive oxygen species treated with black radish juice, these peroxidation products can act at the hepatic and biliary level; in the gallbladder, diminished significantly. Lipid-rich diets also alter the they produce cellular infiltration, edema, granular blood antioxidant system; therefore, in rats treated with a hyperplasia and hypersecretion of mucin, which facili- high fat diet, the concentration of glutathione peroxidase tates the formation of biliary sludge and accelerates diminished significantly, as opposed to what happened the development of gallstones (Koppisetti et al., 2008). to rats treated with black radish juice, which showed a Also, these species facilitate cholesterol crystallization, higher level of this enzyme (Lugasi et al., 2005). This study while enzymes with antioxidant capacity can prevent many is crucial, associated with cholesterol gallstones prevention. pathophysiological disorders related to gallstones.

Glucoraphanin and cholesterol-lowering effects The SREBP genes are transcription factors that inter- vene in cholesterol homeostasis and biosynthesis inside Glucoraphanin is the second major glucosinolate in the hepatocyte (Khesht and Hassanabadi, 2012). In black radish; its more important degradation product is C57L/J mice, susceptible to biliary lithiasis, the abnormal sulforaphane, which has demonstrated to be an effective regulation of the SREBP-2 gene increases biliary metabolite for diminishing hepatic cholesterol levels. cholesterol secretion; therefore, if we consider that One of the imbalances necessary for the formation of glucosinolates inhibit this gene, we would expect a cholesterol gallstones is the biliary hypersecretion of decrease in cholesterol biliary secretion. SREBP-2 is also this molecule towards the gallbladder (van Erpecum, an important modulator of the NPC1L1 protein, which is 2011), a product of the high concentration of cholesterol in charge of transporting biliary cholesterol and inhibiting in the hepatocytes; therefore, the inhibition of hepatic its intestinal absorption (Pramfalk et al., 2010). These pre- cholesterol is a key target for preventing biliary lithiasis. cedents are an important factor, which must be investigated A study evaluated glucoraphanin and sulforaphane further in order to describe the action mechanism of in Syrian hamsters fed with a hypercholesterolemic diet glucosinolates and their degradation products with the for 7 weeks; these secondary metabolites diminished same detail of other transport proteins and, at the same hepatic cholesterol levels, an effect that was associated time, to promote the search of a preventive treatment that with the inhibition of the genetic expression of Sterol generates the least number of adverse effects. Regulatory Element-binding Protein (SREBP-1 and SREBP-2) and Fatty Acid Synthase (Rodríguez-Cantú et al., 2011), important molecules that regulate cholesterol and plant sterol homeostasis. The Syrian hamsters are CONCLUSION important models for the study of cholesterol gallstones, because their bile has very similar components to that Black radish is an important cruciferous plant that has of human beings; consequently, the demonstrated demonstrated hypocholesterolemic, antilithiasic and effects indicate that under a lithogenic diet, both antioxidant properties in different biological models; glucoraphanin and sulforaphane might prevent the these effects can prevent the formation of cholesterol formation of gallstones, since there is a decrease of gallstones, an increasingly prevalent gastrointestinal hepatic cholesterol which could mean less secretion towards affection. The effects of black radish associated with the bile; nevertheless, in the study, a hypercholesterolemic the prevention of cholesterol gallstones may be due to diet was administered that does not necessary cause the two main glucosinolates, and to their degradation formation of gallstones. The possible action mechanism products: glucoraphasatin (raphasatin) and glucoraphanin reported is critical, since in the study of gallstones the (sulforaphane), but biological and phytochemical canalicular membrane proteins of the hepatocytes investigations must continue in order to complement (ABCG5 and ABCG8) are the ones that have been the existent contributions. more thoroughly analyzed; they are in charge of transporting the cholesterol from the liver towards the biliary canaliculus, but the cholesterol carriers Conﬂict of Interest and regulators inside the hepatocyte have been poorly investigated. The authors have declared that there is no conﬂicts of interest.

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