Modulation of P450 enzymes by Cuban natural

Available online at www.sciencedirect.com

Chemico-Biological Interactions 172 (2008) 1–10

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes I. Rodeiro a , M.T. Donato b,c,d , A. Lahoz e , J.A. González-Lavaut a , A. Laguna f , J.V. Castell b,c,d , R. Delgado a , M.J. Gómez-Lechón c,d,∗ a

Departmento de Investigaciones Biomedicas, Centro de Qu´ımica Farmacéutica, 200 y 21, Atabey, Playa, P.O. Box 16042, Ciudad de la Habana, Cuba b Departamento de Bioqu´ımica y Biolog´ıa Molecular, Facultad de Medicina, Universidad de Valencia, Spain c Unidad de Hepatolog´ıa Experimental, Centro de Investigaci´ on, Hospital La Fe, Valencia, Spain d CIBERHEPAD, FIS, Spain e Unidad Mixta Hospital La Fe-Advancell, Valencia, Spain f Centro de Bioactivos Marinos, Loma 37, Vedado, P.O. Box 10400, Ciudad de la Habana, Cuba Received 21 September 2007; received in revised form 17 October 2007; accepted 18 October 2007 Available online 24 October 2007

Abstract This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50–1000 ␮g/mL) for 24 or 72 h. The results showed that 500 ␮g/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrations ≥200 ␮g/mL, whereas only moderate effects were induced by 1000 ␮g/mL Tt. Seven specific P450 activities were evaluated after 48 h exposure of cells to the products. MSBE reduced phenacetin Odeethylation (POD; CYP1A2) activity in a concentration-dependent manner (IC50 = 190 ␮g/mL). No decreases were observed in other activities. In contrast, mangiferin produced reductions in five P450 activities: IC50 values of 132, 194, >200, 151 and 137 ␮g/ml for POD (CYP1A2), midazolam 1 -hydroxylation (M1OH; CYP3A1), diclofenac 4 -hydroxylation (D4OH; CYP2C6), S-mephenytoin 4 -hydroxylation (SM4OH), and chlorzoxazone 6-hydroxyaltion (C6OH; CYP2E1), respectively. E. minutifolium, E. confusum and Tt extracts produced small reductions in SM4OH and C6OH activities, but no significant changes were noted in the other P450 activities. On the other hand, all the products increased the benzyloxyresorufin O-debenzylation (BROD; CYP2B1) activity, with MSBE, mangiferin or E. minutifolium showing the highest effects (about 2-fold over control). Our results showed in vitro effects of these natural products on P450 systems, possibly leading to potential metabolic-based interactions. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Cuban herbal products; Rat hepatocytes; Cytochrome P450; Polyphenols

1. Introduction ∗

Corresponding autor at: Avda de Campanar 21, 46009-Valencia, Spain. Tel.: +34 96 1973048; fax: +34 96 1973018. E-mail address: gomez [email protected] (M.J. Gómez-Lechón).

Herbal extracts have been used as traditional remedies for the treatment of diseases for almost 2000 years and are being increasingly used worldwide. Herbal remedies

0009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2007.10.004

2

I. Rodeiro et al. / Chemico-Biological Interactions 172 (2008) 1–10

are, however, not risk-free since herb–drug interactions and other safety issues have been reported. In fact, patients often combine prescription medications with herbal and dietary substances, thus affecting the disposition of conventional pharmaceuticals through the inhibition/induction of human cytochrome P-450 (P450) enzymes. It is well known that P450 is the most important phase I drug-metabolizing enzyme system responsible for the metabolism of a variety of xenobiotics, including drugs and endogenous substances such as steroids [1–3]. Approximately, 70% of human liver P450 is accounted for by CYP1A2, 2A6, 2B6, 2Cs, 2D6, 2E1 and 3A enzymes [4]. Among them, CYP3A4, 2D6, and 2Cs are highly responsible for the metabolism of most drugs in current use, whereas other P450s (CYP1A2, 2A6, 2B6, 2E1) are greatly involved in the metabolic detoxication/bioactivation of other xenobiotics [2,4,5]. Several examples have been reported in the literature that show herb–drug interactions after the co-administration of herb constituent(s) along with pharmaceutical drugs, which may lead to adverse drug interactions. These interactions occur by modulating the P450 system, including the induction or inhibition of specific P450 enzymes and the metabolic clearance of the drug [6,7]. A typical example is St. John’s wort, widely used for depressive disorders, which is a potent inducer of CYP3A4. Yet it also contains ingredients that inhibit CYP1A2, 2C9, 2C19 and 2D6 [8]. Another example is the Echinacea purpurea extract of well-established therapeutic usage in the protection of upper respiratory tract infections, and which has been described as a potent inhibitor of CYP3A4, 2D6 and 2C19 [9,10]. Finally, naturally occurring flavonoids, a group of phytochemicals displaying a wide range of biological activities, have also been shown to modulate the P450 system [11]. Therefore, special attention should be paid to the potential effects of herbal extracts, or their components, on P450 enzymes, and to the pharmaco-toxicological consequences when they are co-administered with other drugs. Herbal medicines have been used for many years in Cuba for the treatment of several diseases. In recent years, there is an increasing consumption of herbal extracts (Mangifera indica L., Thalassia testudinum, Erythroxylum minutifolium and E. confusum) that are often administered in combination with conventional therapeutic drugs. These extracts are used in the treatment of different pathologies, such as the prevention of age-associated oxidative stress in the elderly and to

improve patient welfare. Formulations of herbal extracts are used by patients suffering pain, inflammation or burns, and in the treatment of immunopathological disorders, including bronchial asthma, atopic dermatitis and other allergic diseases. Mango (M. indica L., Anarcadeaceae) stem bark aqueous extract (MSBE) has been developed in Cuba as a new natural product with a defined composition, whose brand name is Vimang® . Between 2001 and 2007, from 60 to 900 kg of Vimang has been sold annually as a nutritional supplement to improve the welfare of those patients suffering different types of pain and inflammation [12]. A topical preparation of the T. testudinum marine plant is registered and widely used in Cuba to protect burns and as an anti-aging cream [13]. At present, a new oral formulation has been developed as an antiinflammatory and hepatoprotective supplement, and as an aid for liver diseases. Finally, Erythroxylum extracts have been used as topical unguents for bacterial and/or viral infections of the skin, and decoctions are orally consumed by people for renal and respiratory affections. Chemical analysis of these extracts revealed that polyphenols are the main constituents responsible for their beneficial properties [12–16]. Plant polyphenols are an important group of chemicals with a recognized capacity to modulate P450 enzymes [17]. Thus, the aim of the present study was to investigate the potential inhibitory or inductive effects of these herbal extracts on the P450 system. The effects of mangiferin, the main polyphenolic component of M. indica L., were also analyzed. First, the cytotoxic effects of the test products on cultured rat hepatocytes were determined and then their effects on seven P450 activities were studied. 2. Materials and methods 2.1. Chemicals Collagenase and ␤-glucorinadase/arylsulfatase were obtained from Roche (Barcelona, Spain). Ham’s F-12 and Lebovitz L-15 medium and calf serum were acquired from Gibco (Madrid, Spain). Bovine serum albumin, benzoxyresorufin, resorufin, diclofenac, bufuralol, 1 -hydroxybufuralol, chlorzoxazone, S-mephenytoin and 4 -hydroxymephenytoin were purchased from Sigma Chemical Co. (Madrid, Spain). 4 -Hydroxydiclofenac, 6-hydroxychlorzoxazone, midazolam and 1 -hydroxymidazolam were supplied by Ultrafine (Manchester, UK). All other reagents used were of the purest grade available.


2.2. Plant material Table 1 summarizes the main characteristics of each product assayed. M. indica L. was collected in the region of Pinar del R´ıo, Cuba. Voucher specimens of the plant (Code: 41722) were deposited at the Herbarium of Academic of Sciences, Cuba. Stem bark aqueous extract of M. indica L. (MSBE) was prepared by decoction for 1 h. The extract was concentrated by evaporation and dried to obtain a fine brown powder. It melted at 210–215 ◦ C with decomposition. Mangiferin (1,3,6,7tetrahydroxyxanthone-C2-ß-d-glucoside) was isolated from the extract by methanol extraction with 90% purity and lyophilized for conservation purposes. The chemical composition of these products was performed using chromatographic methods, mass spectrometry and UV–vis spectrophotometry [12,14]. Leaves of two Erythroxylum species were collected in Pinar del R´ıo, Cuba, in April 2001. Specimens were deposited at the Herbarium of Instituto Pedagógico of Pinar del R´ıo (Erythroxylum confusum Britt: 9191 and E. minutifolium Griseb. var. minutifolium: 9200). Samples were dried in the shade for 20 days. Finally, leaves were triturated independently. For our studies, 10 g of

3

triturated leaves were weighed and submitted to reflux for 1 h with 100 mL of n-hexane. Extracts were filtered and the solid debris submitted to another reflux for 1 h with 100 mL of ethanol. Then, this procedure was repeated with 100 mL of distilled water. Maceration was obtained from ten grams of plant material from each species, and was then added with 100 mL of 70% ethanol. This was left to macerate for 3 days, and was then decanted. This procedure was repeated 3 times. The hydroalcoholic extracts of E. confusum and E. minutifolium were filtered, ethanol eliminated and the residue lyophilized. The chemical composition of the extracts was analyzed using a DR/4000U UV spectrophotometer (HACH, USA), and was further characterized by NMR (1 H and 13 C) with a DRX-600 analyzer (Bruker, Germany). It was then compared with the literature data [15]. The marine plant T. testudinum Banks and Soland ex. Koenig (T.t) was collected from the north Cuban coast at the “La Concha” beach (22◦ 05 45 N, 82◦ 27 15 W) in March 2004. Voucher specimens were deposited at the Cuban National Aquarium (IdO39). The plant material was washed, and leaves were dried and pulverized to obtain a fine powder. Then, the extract was eluted

Table 1 Cuban herbal extracts examined in the present study Product name

Chemical composition or structure

Ethnomedical and/or pharmacological properties

Mango (Mangifera indica L.) stem bark aqueous extract (MSBE)

Mangiferin and derivates as main components, gallic acid, 3-methyl gallate, propyl gallate, (+)-catechin, (−)-epicatechin, benzoic acid, 3,4-dihydroxy benzoic acid, propyl benzoate, d-glucopyranosyl, terpenes, polyalcohols, fatty acids, microelements and lignin

Antioxidant anti-inflammatory immunomodulatory anti-angiogenic hepatoprotective

Mangiferin, polyphenol isolated from MSBE Hydroalcholic extract from leaves of marine plant Thalassia testudinum (Tt)

E. minutifolium Griseb. var. ethanolic extract (E. minutifolium)

E. confusum Britt. ethanolic extract (E. confusum) [12,14,17].

1,3,6,7-Tetrahydroxanthone-C2-ß-d-glucoside Chrysoeriol 7-b-d-glucopyranosyl-2-sulfate (the main component), luteolin 7-b-d-glucopyranosyl-2-sulfate, apigenin 7-b-d-glucopyranosyl-2-sulfate, p-hydroxy-benzoic acids, proantocianinds, catechins, tannins, steroids, triterperns and saponinns Quercetin-3-rutinoside, ombuin-3-rutinoside and ombuin-3-rutinoside-5-glucoside (main components), reducing sugars and triterpenes Phenols/tannins and reducing sugars (main components), ombuin-3-rutinoside-5-glucoside, quercetin-3-rutinosoide, ombuin-3-rutinoside, coumarins and triterpenes/steroids

Antioxidant anti- HIV anticancer antidiabetic anti-inflammatory hepatoprotective immunomodulatory Antioxidant anti-inflammatory hepatoprotective and analgesic properties

Muscular, hepatic, renal and vesicular affections anti-inflammatory and anti-bacterial activities Anti-inflammatory, antibacterial, antifungal and anti-viral activities

4


with ethanol/H2 O 50:50 (v/v) for 7 days. Finally, it was filtrated, centrifuged, and evaporated under reduced pressure. The extract obtained was lyophilized for conservation purposes. The composition was characterized using chromatographic and spectroscopic techniques [13].

were tested because of the solubility of extracts in the medium. In order to study the effects on P450 activities, hepatocytes were treated for 48 h with range of sub-cytotoxic concentrations of the products. Control cells (only medium-treated cells) were included in all experiments.

2.3. Isolation and culture of hepatocytes

2.5. MTT assay

Hepatocytes were obtained from 200 to 300 g Sprague Dawley male rats by perfusion of the liver with collagenase, as described elsewhere [18]. Cell viability of the suspension, assessed by the trypan blue exclusion test, was higher than 85%. Cells were seeded at a density of 8 × 104 viable cells/cm2 in Ham’s F-12/Lebovitz L-15 (1:1) medium supplemented with sodium selenite (170 ␮g/mL), 2% calf serum, 0.2% bovine serum albumin, 50 mU/mL of penicillin, 50 ␮g/mL of streptomycin and 10 nM insulin. Cells were then incubated at 37 ◦ C in a 5% CO2 humidified atmosphere. Unattached cells were removed by changing the medium 1 h after plating. Cultures were shifted to serum-free medium supplemented with 10 nM insulin and dexamethasone after the first 24 h. Unless indicated, treatments with the products started 1 h after cell plating, and the medium (with or without products) was renewed daily. Cells were exposed to the products for 48 h.

After 24 and 72 h of exposure to the products, cytotoxic effects were measured by using MTT reduction, as previously described. Cell monolayers were washed with PBS, and 100 ␮L/well MTT reagent (5 mg/mL in medium) were added to each well. Then, plates were placed in the incubator for a further 4 h. The corresponding supernatants were discarded and cells were washed again. The dye was extracted with DMSO and optical density was read at 540 nm on a Microplate Reader. The percentage of inhibition of the succinic dehydrogenase reduction of MTT was calculated in relation to control cells in each experimental series (control cells were assumed to have 100% viability).

2.4. Treatment of hepatocytes The stock solutions of the extracts were prepared at a concentration of 1 mg/ml in culture medium, and conveniently diluted in culture medium to obtain the final desired concentrations. The products were re-added with each medium change. For cytotoxicity assays, hepatocytes cultured in 96-well plates were exposed 1 h after cell plating to several concentrations of extracts (50–1000 ␮g/mL) up to 72 h at 37 ◦ C. No higher doses

2.6. P450 activity assays Assays were performed by the direct incubation of the hepatocytes monolayer with a cocktail of substrates containing seven selective probes to measure the activity of individual P450 enzymes (Table 2). Dicumarol (10 ␮M) was added to the assay media to prevent further metabolism of the resorufin formed [21]. After 2 h incubation at 37 ◦ C, reactions were stopped by aspirating the incubation medium. Thereafter, medium samples were incubated with ␤-glucuronidase and arylsulfatase for 2 h at 37 ◦ C [21]. Metabolites formed during activity assays, and were quantified by using an HPLC-MS/MS (Micromass Quattro Micro; Waters, Milford MA, USA) in the electrospray ionization mode interfaced with an

Table 2 Rat P450 enzymes involved in the oxidation of model substrates Activity

Substrate

POD BROD D4OH B1OH C6OH SM4OH M1OH

Phenacetin Benzoxyresorufin Diclofenac Bufuralol Chlorzoxazone S-mephenytoin Midazolam

a b

Conc. (␮M)a 10 10 10 10 50 50 5

Reaction

Metabolite

Rat P450 enzymeb

O-Deethylation O-Debenzylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation

Acetaminophen Resorufin 4 -Hydroxydiclofenac 1 -hydroxybufuralol 6-Hydroxychlorzoxazone 4 -hydroxymephenytoin 1 -hydroxymidazolam

CYP1A2 (2C6) [19] CYP2B1 [20] CYP2C6 [20] CYP2D2 (2C6, 2C11) [20] CYP2E1 (1A2, 3A1) [19,20] Unknown CYP3A1 (3A2) [20]

Substrate concentrations during activity assay. Major P450 enzymes involved in the reaction. Minor contributions are indicated in parentheses.


Alliance 2795 HPLC (Waters Chromatography). LC was performed at 35 ◦ C. An aliquot (20 ␮l) was injected into a Teknokroma C18 column (50 mm × 2.1 mm, 3 ␮ particle size). The flow rate was 0.4 ml/min. The mobile phase was A 0.1% formic acid in acetonitrile and B 0.1% formic acid in water. The proportion of acetonitrile was increased linearly from 0 to 90% in 6 min, and then the injection column was allowed to re-equilibrate at initial conditions for 10 min. The eluted column was directed to an atmospheric pressure ionization interface without splitting, operating at 320 ◦ C using nitrogen as an auxiliary gas (50 L/min). MS spectrometer parameters were adjusted as described previously [22]. Activity values were referred to total cell protein, which was determined by the Lowry method adapted to 96-well plates [23]. Data were expressed as a percentage of the corresponding activity in control cultures (vehicle-treated cells only). To check the quality of cell preparations, the response of P450 activities to prototypical inducers (2 ␮M 3-methylcholanthrene, 1 mM phenobarbital, 50 ␮M rifampicin) was evaluated in each experiment. 2.7. Statistical analysis Each experimental procedure was performed in at least three cell preparations. Data were expressed as the mean ± S.D. using Microsoft Excel. Statistical analysis for processing cytotoxicity data was performed using the U Mann–Whitney test and the enzymatic activities data using the Student t-test. A P value less than 0.05 was considered statistically significant. Regression analysis was used to calculate the inhibitory concentration 50 (IC50 ), defined as the concentration of the product necessary to produce 50% inhibition of cell viability (cytotoxicity study) or 50% inhibition of enzyme activity (P450 activity assays). 3. Results 3.1. Evaluation of cytotoxicity of Cuban natural products Cytotoxicity was evaluated in cultured rat hepatocytes incubated in the presence of five Cuban natural products widely used in traditional medicine (M. indica L., T. testudinum, E. minutifolium and confusum extracts and mangiferin) (Table 1). Hepatocytes were exposed to a wide concentration range (50–1000 ␮g/mL) of the products for 24 and 72 h. As shown in Fig. 1, mangiferin did not show toxic effects, while the mangiferin-enriched MSBE extract showed a moderate cytotoxicity after 72 h of exposure at the higher concentrations evaluated

5

(500 and 1000 ␮g/mL). A moderate decrease (approx. 30%) of cell viability was induced by T. testudinum at 1000 ␮g/mL at an exposure of either 24 or 72 h. However, extracts obtained from the leaves of the two species of the Erythroxylum genus examined (E. minutifolium and E. confusum) produced a significant cytotoxic effect at concentrations higher than 100–200 ␮g/mL at both the incubation periods evaluated (Fig. 1). E. minutifolium was more cytotoxic (IC50 = 339 or 205 ␮g/mL at 24 or 72 h, respectively) than E. confusum (IC50 = 424 or 382 ␮g/mL at 24 or 72 h, respectively). A range of subcytotoxic concentrations (5–200 ␮g/mL) was selected for further investigation of the potential effects of the products on P450 activities. 3.2. Effects of Cuban natural products on P450 activities Fig. 2 summarizes the observed effects of the products assayed on seven P450 activities. In general, the 5 products were able to modulate P450 activities, although certain differences were found. MSBE reduced POD (CYP1A2) activity in a concentration dependent fashion (IC50 = 190 ␮g/ml) and did not produce any changes on M1OH (CYP3A1), B1OH (CYP2D2), D4OH (CYP2C6), SM4OH, and C6OH (CYP2E1) activities. In contrast, except for B1OH (CYP2D2), reductions in these P450 activities were found after the exposure of hepatocytes to mangiferin (Fig. 2), with IC50 values of 132, 194, >200, 151 and 137 ␮g/ml for POD, M1OH, D4OH, SM4OH, and C6OH activities, respectively. E. minutifolium, E. confusum and Tt extracts produced slight reductions in SM4OH and C6OH activities (IC50 > 200 ␮g/ml), but no significant effects on CYP1A2, 3A1, 2D2 or 2C6 enzymes were observed. On the other hand, treatment of rat hepatocytes with the different products resulted in a general increase in the BROD (CYP2B1) activity (Fig. 2). All the concentrations tested of MSBE, mangiferin and the E. minutifolium extract increased the BROD activity. The highest effects (about 2-fold over the control activity) were found in cells exposed to 100 ␮g/ml MSBE, or to 5–50 ␮g/ml mangiferin or E. minutifolium. These increases in BROD activity were similar to those observed in rat hepatocytes exposed to 1 mM phenobarbital (2-fold over control) or 50 ␮M rifampicin (1.9-fold over control), two prototypical inducers of CYP2B1, for the same period. Lower activity increases were observed at the highest concentrations of the products. Significant but lower increases were also found after hepatocyte exposure to E. confusum and Tt extracts (Fig. 2).

6


Fig. 1. Cytotoxic effects of Cuban natural products on rat hepatocytes. After 1 h of culture, hepatocytes were exposed to increasing concentrations of the products, and cell viability was determined 24 and 72 h later by the MTT test. Results are expressed as the percentage of cell viability in controls (untreated hepatocytes). Each point represents the mean ± S.D. of three experiments with six replicates. *p < 0.05 respect to control.

4. Discussion Plants used as natural products contain numerous classes of chemical constituents. In Cuba, the use of herbal mixtures for the treatment of different diseases is a common practice. Chemical studies of these natural products identified polyphenols as the main components of several of them and, at least in part, as being responsible for their biological properties [12–16]. Polyphenols are a family of compounds found in a variety of medicinal herbs, foods and food supplements used by humans. Strong antioxidant properties have been associated with these molecules [24–26]. In fact, antioxidant-rich dietary supplements are often recommended to preserve or regain good health. As a result, high dosages of these natural products are consumed by extensive populations to handle or prevent many oxidative stress-related diseases [26,27]. In vitro and in vivo reports have indicated that polyphenols can modulate the expression and/or activity of P450 enzymes [17]. Consequently, drug interactions can occur when herbal or natural products containing such molecules are used concomitantly

with conventional drugs, lending undesired effects such as impaired bioavailability of drugs with narrow therapeutic indices, altered plasma/tissue levels, and an enhanced bioactivation of drugs to reactive intermediates or toxic metabolites. Moreover, certain components of herbal complex mixtures may be metabolized to reactive metabolites, which could induce important adverse effects and clinical consequences. In this study we have investigated the potential effects of four natural extracts (MSBE, E. minutifolium, E. confusum and Tt) on major P450s involved in the metabolism of conventional drugs. Both increases and reductions in specific P450 activities were found after treating cultured rat hepatocytes with extracts. Moreover, marked effects of mangiferin, the main polyphenolic component of the MSBE, on P450 enzymes have been shown. MSBE is an extract obtained from the stem bark of M. indica L. (mango), which presents in vitro and in vivo antioxidant and anti-inflammatory activities [23,28–34]. In a previous study, we observed that a 48-h treatment of rat hepatocytes with MSBE increased pentoxyresorufin O-depentylation, a CYP2B1-dependent activity [23]. In


7

Fig. 2. Effects of Cuban natural products on P450 activities in rat hepatocytes. Cultured rat hepatocytes were incubated for 48 h with MSBE (Mangifera indica L. aqueous stem bark extract), mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-ß-D-glucoside), Minutifolium (hydroalcoholic extract from E. minutifolium leaves), Confusum (Hydroalcoholic extract from E. confusum leaves) or Tt (T. testudinum hydroalcohic extract). Then, P450 activities were assayed by the direct incubation of cell monolayers with a cocktail of selective substrates. The results are expressed as a percentage of the activity in control cells: 1.46 ± 0.07 (POD), 0.86 ± 0.08 (M1OH), 17.6 ± 2.1 (B1OH), 10.6 ± 1.0 (D4OH), 0.11 ± 0.06 (SM4OH), 0.023 ± 0.005 (BROD), and 6.4 ± 0.2 (C6OH) pmol/mg/min. Data are the mean ± S.D. of three independent experiments. *p < 0.05 in relation to control.

addition, the marked concentration-dependent decrease in ethoxyresorufin O-deethylation activity observed in hepatocytes incubated for short time periods (2 h) with the extract, revealed a potential direct interference of MSBE components with the CYP1A1 and/or 1A2 enzymes. Interestingly, no interactions of this natural product with other P450s (CYP2C, 2D2, 2E1, and 3A) were observed. The results of the present study are in agreement with these previous observations, and suggest that MSBE produces an induction of CYP2B1, as

well as an inhibition of CYP1A2, without changes in other P450s (Fig. 2). Mangiferin, like MSBE, produced a 2-fold increase in rat CYP2B1 activity and strongly inhibited CYP1A2 activity, showing an inhibitory potency even greater than that of the extract (IC50 132 vs. 190 ␮g/ml). Inversely, mangiferin also inhibited the activity of other P450s (CYP3A1, 2C6, 2E1 and SM4OH activities) (Fig. 2). This polyphenol is the main component of MSBE and is involved in the beneficial properties described for

8


the extract [12,35]. Interestingly, a marked inhibition of CYP1A2 and 2E1 by mangiferin could help to explain the chemoprotective properties attributed to this product [36,37] as both P450 enzymes are involved in the bioactivation of mutagens and carcinogens [3,4]. However, other potential mechanisms should not be ruled out. Moreover, the substrates selected for measuring the CYP1A2 and 2E1 activities (phenacetin and chlorzoxazone, respectively) are not exclusively metabolized by these P450 enzymes (see Table 2). Minor contributions of other P450s should be considered. Our results suggested that mangiferin could be responsible for the changes on the P450 activities observed in MSBEexposed hepatocytes. However, MSBE is a partially mangiferin-enriched extract, and other components such as phenolic acids, phenolic esters, flavan-3-ols and micronutrients (i.e. selenium) have been identified in its composition [12,14]. The final effect of this natural mixture on P450 enzymes must not be exclusively attributed to the action of its main active constituent as it is likely due to the combined action of the complex mixture [24,38]. Effects of other constituents or metabolites formed during the incubation of MSBE with hepatocytes could antagonize the inhibitory action of mangiferin on CYP3A1, 2C6, 2E1 and SM4OH activities. The genus Erythroxylum (Erythroxyceae) is widespread in tropical regions including Cuba, where there are 16 endemic species. It has been used in ethno-medical practices for muscular, liver, renal and vesicular afflictions as a diuretic, and in the treatment of venereal diseases because of its anti-inflammatory, anti-bacterial, tonic and stimulant properties [39,40]. In Erythroxylum, the prominent polyphenols are flavonols, where kaemferol, quercetin and ombuin are the main aglycones of Erythroxylum flavonols [41]. Studies conducted in Cuba with four endemic Erythroxylum species found high concentrations of flavonoids in leaves, and two bioactive extracts, E. minutifolium and E. confusum, were obtained and standardized [15]. In contrast to the other natural products assayed in our study, Erythroxylum mixtures induced cytotoxic effects on cultured rat hepatocytes (Fig. 1). A previous report using the Artemia salina assay considered E. confusum as a nontoxic product [42]. However, another study in Vero cells showed that the product become cytotoxic at 250 ␮g/mL (IC50 = 480 ␮g/mL) [15]. Granules and vacuoles were observed inside the cells, which increased in number and size, and were concentration-dependent. Our data show that E. minutifolium is even more toxic than the E. confusum extract (IC50 205 ␮g/mL vs. 382 ␮g/mL after a 72-h treatment). Both extracts produced slight decreases in CYP1A2, CYP2E1 and

SM4OH activities, whereas no changes were observed in CYP3A1, 2D2, and 2C6. However, different effects on the CYP2B1 enzyme were found. Treatment of rat hepatocytes with 5–50 ␮g/mL E. minutifolium increased catalytic activity (1.9-fold over control), and a reduced effect (1.4-fold increase) was observed at 100 ␮g/mL. In contrast, only the highest concentration of E. confusum extract increased CYP2B1 activity (1.5-fold over control). Although both extracts have a very similar composition, relative concentrations of reducing sugars and phenols/tannins differ [43], which could be a determinant of their different cytotoxicity of rat hepatocytes and their effects on CYP2B1 activity. T. testudinum is an aquatic plant distributed on the marine platform of Cuba with antioxidant, neuroprotective and hepatoprotective effects [44]. Our results indicate that the Tt extract shows reduced cytotoxicity in rat hepatocytes and produces few effects on the P450 system (slight inhibition of SM4OH and CYP2E1 activities and modest increases in CYP2B1). After comparison with the effects exhibited by the other natural products examined in this study, like MSBE, the Tt extract produced minor alterations of hepatocytes viability and functionality. These herbal extracts could be considered relatively safe products and possible candidates for further development as natural remedies. Obviously, more exhaustive in vitro and in vivo studies are required to confirm their pharmacological, toxicological and metabolic properties. Our results are the first to be reported on the potential interactions of these Cuban natural products with P450 enzymes, and they suggest that their intake may lead to herbal-drug interactions with pharmacokinetic and clinical consequences. New studies using other biological systems, such as human cells or in vivo models, are needed to determine the clinical relevance of these findings. Acknowledgments This work was supported by the Conselleria de Empresa, Universidad y Ciencia (grant 2005/0161 to Dr. Rodeiro) of the Generalitat Valenciana (regional government of Valencia) and with funds from the ALIVE Foundation and the European Commission (grant LSBCT-2005-037499). References [1] R. Meech, P.I. Mackenzie, Structure and function of uridine diphosphate glucuronosyltransferases, Clin. Exp. Pharmacol. Physiol. 24 (1997) 907–915.

I. Rodeiro et al. / Chemico-Biological Interactions 172 (2008) 1–10 [2] S. Rendic, F.J. Di Carlo, Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors, Drug Metab. Rev. 29 (1997) 413–580. [3] T. Shimada, Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons, Drug Metab. Pharmacokinet. 21 (2006) 257– 276. [4] F.J. Gonzalez, The 2006 Bernard B. Brodie award lecture. CYP2E1, Drug Metab. Dispos. 35 (2007) 1–8. [5] R.J. Bertz, G.R. Granneman, Use of in vitro and in vivo data to estimate the likelihood of metabolic pharmacokinetic interactions, Clin. Pharmacokinet. 32 (1997) 210–258. [6] A.P. Li, Screening for human ADME/Tox drug properties in drug discovery, Drug Discov. Today 6 (2001) 357–366. [7] J.H. Lin, A.Y. Lu, Interindividual variability in inhibition and induction of cytochrome P450 enzymes, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 535–565. [8] Y.N. Singh, Potential for interaction of kava and St. John’s wort with drugs, J. Ethnopharmacol. 100 (2005) 108–113. [9] J. Strandell, A. Neil, G. Carlin, An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies, Phytomedicine 11 (2004) 98–104. [10] M. Modarai, J. Gertsch, A. Suter, M. Heinrich, R. Kortenkamp, Cytochrome P450 inhibitory action of Echinacea preparations differs widely and co-varies with alkylamide content, J. Pharm. Pharmacol. 59 (2007) 567–573. [11] P. Hodek, P. Trefil, M. Stiborova, Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450, Chem. Biol. Interact. 139 (2002) 1–21. [12] A.J. Nún˜ ez, H. Castro, J. Agüero-Agüero, J. González, F. Naddeo, F. De Simona, L. Rastrelli, Isolation and quantitative analysis of phenolic antioxidants, free sugars and polyphenols from Mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as a nutritional supplement, J. Agric. Food Chem. 50 (2002) 762–766. [13] M. Rodr´ıguez, A. Laguna, E. L. Regalado, R. Menéndez, A. Garateix, A. Concepción, Producto obtenido a partir de la planta marina con propiedades estimuladoras del crecimiento del pelo, neuroprotectoras, hepatoprotectoras y protectoras de los rayos solares, as´ı como dermoregeneradoras, y su proceso de obtención, Patent Application (Cuba) No. 2007-0089 (2007). [14] A.J. Nún˜ ez, M.D. Durruthy, D. Rodr´ıguez, L. Nieto, L. González, V. Nicolais, L. Rastrelli, Comparison of major and trace elements concentrations in sixteen varieties of Cuban mango item bark (Mangifera indica L.), J. Agric. Food Chem. 55 (2007) 21–25. [15] J. González, H. Velez, K. González, A. Payo, J.A. GonzálezLavaut, J. Molina, S. Prieto, Flavonoid glycosides from Cuban Erythroxylum species, Biochem. Syst. Ecol. 34 (2006) 539–542. [16] M. Llanio, M.D. Fernández, B. Cabrera, M. Abad, M. Payá, M.J. Alcaraz, The marine plant Thalassia testudinum possesses antiinflammatory and analgesic properties, Pharmacologyonline 3 (2006) 594–595. [17] S. Zhou, Y. Gao, W. Jiang, M. Huang, A. Xu, J.W. Paxton, Interactions of herbs with cytochrome P450, Drug Metab. Rev. 35 (2003) 35–98. [18] M.J. Gómez-Lechón, J.V. Castell, Primary culture of human hepatocytes, in: J.B. Griffiths, A. Doyle, D.N. Newell (Eds.), Cell and Tissue Culture: Laboratory Procedures, John Wiley & Sons Ltd., Baffins Lane, England, 1998, ISBN 0471928526, pp. 151– 157.

9

[19] A.L. Minn, H. Pelczar, C. Denizot, M. Martinet, J.M. Heydel, B. Walther, A. Minn, H. Goudonnet, Y. Artur, Characterization of microsomal cytochrome P450-dependent monoxygenases in the rat olfactory mucosa., Drug Metab. Dispos. 33 (2005) 1229– 1237. [20] K. Kobayashi, K. Urashima, N. Shimada, K. Chiba, Substrate specificity for rat cytochrome P450 (CYP) isoforms: screening with cDNA-expressed systems of the rat, Biochem. Pharmacol. 63 (2002) 889–896. [21] M.T. Donato, M.J. Gómez-Lechón, J.V. Castell, A microassay formeasuring cytochrome P4501A1 and P4502B1 activities in intact human and rat hepatocytes cultured on 96-well plates, Anal. Biochem. 213 (1998) 29–33. [22] A. Lahoz, M.T. Donato, L. Picazo, M.J. Gómez-Lechón, J.V. Castell, Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry, Toxicol. In Vitro 21 (2007) 1247–1252. [23] M.J. Gómez-Lechón, X. Ponsoda, E. O’Connor, M.T. Donato, J.V. Castell, R. Jover, Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS, Biochem. Pharmacol. 66 (2003) 2155–2167. [24] I. Rodeiro, M.T. Donato, N. Jimenez, G. Garrido, R. Delgado, M.J. Gómez-Lechón, Effects of Mangifera indica L. aqueous extract (Vimang) on primary culture of rat hepatocytes, Food Chem. Toxicol. 45 (2007) 2526–2532. [25] C. Dani, S. Olibonils, R. Vanderlinde, D. Bonatto, M. Salvador, J.A. Henriques, Phenolic content and antioxidant activities of while and purple juices manufactured with organically or conventionally-produced grapes, Food Chem. Toxicol. (2007) 2574–2580. [26] G.L. Tipoe, T.M. Leung, M.W. Hung, M.L. Fung, Green tea polyphenols as an anti-oxidant and anti-inflammatory agent for cardiovascular protection, Cardiovasc. Hematol. Disord. Drug Targets 7 (2007) 135–144. [27] Y. Shukla, Tea and cancer chemoprevention: a comprehensive review, Asian Pac. J. Cancer Prev. 8 (2007) 55–66. [28] G. Mart´ınez, R. Delgado, G. Pérez, G. Garrido, A. Nún˜ ez, O. León, Evaluation of the in vitro antioxidant activity of Mangifera indica L. extract (Vimang), Phytother. Res. 14 (2000) 424–427. [29] G. Mart´ınez, E. Jalil, A. Giuliani, O. León, S. Ram, R. Delgado, A.J. Nún˜ ez, Mangifera indica L. extract (Vimang) reduces ischaemia-induced neuronal loss and oxidative damage in Gerbil brain, Free Rad. Res. 32 (2002) 1–7. [30] G. Garrido, R. Delgado, Y. Lemus, J. Rodriguez, D. Garcia, A.J. Nuñez, Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production on macrophages and microglia by a standard aqueous extract of Mangifera indica L. (Vimang). Role of mangiferin isolated from extract, Phytother. Res. 50 (2004) 165–172. [31] G. Garrido, D. González, Y. Lemus, D. Garc´ıa, L. Lodeiro, Y. Quintero, C. Delpote, A.J. Nuñez, R. Delgado, In vivo and in vitro anti-inflammatory activity of Mangifera indica L. extract (VIMANG), Pharmacol. Res. 50 (2004) 143–149. [32] D. Garcia, R. Delgado, F. Ubeira, J. Leiro, Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin, Int. Immunopharmacol. 2 (2002) 797–806. [33] D. Remirez, T. Shahrzard, R. Delgado, A. Sarandi, R. O’Brien, Preventing hepatocytes oxidative stress cytotoxicity with Mangifera indica L. extract (Vimang), Drug Metabol. Drug Int. 21 (2005) 19–29. [34] G. Pardo, S. Philip, A. Riaño, C. Sánchez, C. Viada, A.J. Nún˜ ez, R. Delgado, Mangifera indica L. (Vimang) protection against serum

10

[35]

[36]

[37]

[38]

[39]

I. Rodeiro et al. / Chemico-Biological Interactions 172 (2008) 1–10 oxidative stress in elderly humans, Arch. Med. Res. 37 (2006) 158–161. A.J. Nuñez, R. Delgado, G. Garrido, D. Garc´ıa, M. Guevara, G. Pardo, The paradox of natural products as pharmaceuticals. Experimental evidences of a mango stem bark extract, Pharmacol. Res. 55 (2007) 351–358. S. Guha, U. Ghosal, Chattopahyay, Antitumor, inmunomodulatoy and anti-HIV effect of mangiferin, a naturally ocurring glucoxylxanthone, Chemotherapy 42 (1996) 443–451. N. Yoshimi, K. Matsunaga, M. Katayama, Y. Yamada, T. Kuno, Z. Qiao, A. Hara, J. Yamahara, H. Mori, The inhibitory effects of mangiferin, a naturally ocurring glucosylxanthone, in bowel carcinogenesis of male F344 rats, Cancer Lett. 163 (2001) 163–170. I. Rodeiro, M.T. Donato, A. Lahoz, G. Garrido, R. Delgado, M.J. Gómez-Lechón, Interactions of polyphenols with the P450 system, Possible implications on human therapeutics, Mini Rev. Med. Chem., in press (November, 2007). J. Bisse, Arboles de Cuba, Editorial Cientifico-Tecnica, La Habana, Cuba, p. 146.

[40] H.J. Cano, G. Volpato, Herbal mixtures in the traditional medicine of aesten Cuba, J. Ethnopharmacol. 90 (2004) 293–316. [41] B.A. Bohm, T. Loo, K. Nicholls, T. Plowman, Flavonoid variation in Erythroxylum, Phytochemistry 25 (1988) 833– 837. [42] I. Mart´ınez, G. Quintero, L. Márquez, J.A. González-Lavaut, A. Reyes, A. Zarragoitia, Determinación de la citotoxicidad de extractos de Erythorxylum confusum Britt. Mediante el metodo de Artemia Salina, Acta Farm. Bonaerense 25 (2006) 429–431. [43] G. González, L. González, R. Pino, T. Garc´ıa, G. Carballo, A. Echemendia, T. Molina, G. Prieto, Phytochemical screening and in vitro anthierpetic activity of four Erytroxylum species, Acta Farm. Bonaerense 23 (2004) 5069–5071. [44] R. Nuñez, A. Garateix, A. Laguna, M.D. Fernández, E. Ortiz, M. Llanio, O. Valdés, A. Rodr´ıguez, R. Menéndez, Caribbean marine biodiversity as a source of new compounds of biomedical interest and others industrial applications, Pharmacologyonline 3 (2006) 111–116.