Journal of Ethnopharmacology 102 (2005) 3236

Antibacterial and antioxidant activities of ethanol extract from Paullinia cupana Mart.
Adriana Basile a, , Lydia Ferrara b , Marisa Del Pezzo c , Guido Mele d , Sergio Sorbo e , Paola Bassi f , Domenico Montesano b
a Department of Biological Sciences, Section of Plant Biology, Federico II University, via Foria 223, 80139 Naples, Italy Department of Pharmaceutical and Toxicological Chemistry, Federico II University, via D. Montesano 49, 80131 Naples, Italy c Department of Cellular and Molecular Biology and Pathology, Federico II University, via S. Pansini 5, 80131 Naples, Italy d Department of Experimental Pharmacology, Federico II University, via D. Montesano 49, 80131 Naples, Italy Centro Interdipartimentale di Servizio per la Microscopia Elettronica CISME, Federico II University, via Foria 223, 80139 Naples, Italy f Department of Plant Biology, La Sapienza University, Piazzale Aldo Moro 5, Rome, Italy b

Received 27 January 2004; received in revised form 29 April 2005; accepted 20 May 2005 Available online 22 July 2005

Abstract The antibacterial and antioxidant activity of the ethanol extract from Paullinia cupana var. sorbilis Mart. (Sapindaceae) seeds, commonly called guarana, was assessed towards selected bacteria as well as in different antioxidant models. The extract, at a concentration between 16 and 128 g/ml, showed a signicant antibacterial effect expressed as minimum inhibitory concentration (MIC) against both Gram-negative and Gram-positive bacteria. In particular, Pseudomonas aeruginosa (MIC = 16 g/ml), Proteus mirabilis (MIC = 32 g/ml), Proteus vulgaris (MIC = 32 g/ml) and Escherichia coli (MIC = 32 g/ml) were the most inhibited. The antioxidant activity was determined by the malonyldialdehyde (MDA) test, measuring the MDA concentration in 3T3-L1 cells after induced cellular damage using ferric ammonium citrate (FAC). The reduction of lipid peroxidation was 62.5% using a guarana extract with a concentration of 2 g/ml. This effect was dose/dependent. The ethanol extract from Paullinia cupana seeds was analysed by spectrophotometry to determine the concentration of catechol substances after treatment of the extract with p-aminophenol. The total phenolics content in the ethanol extract was also determined spectrophotometrically according to the FolinCiocalteu procedure and calculated as gallic acid equivalents (GAE). The concentration of catechol equivalent was 6.06 0.13 mg/g (mean S.D.), while the total phenolic content was 8.43 0.21 mg/g (mean S.D.). The correlation index between antioxidant activity and catechol content was 0.96. 2005 Published by Elsevier Ireland Ltd.
Keywords: Paullinia cupana; Ethanol extract; Antibacterial activity; Antioxidant activity; Sapindaceae

1. Introduction Paullinia cupana var. sorbilis Mart. (Sapindaceae), commonly called guarana, is a cultivated variety of the wild type var. cupana Ducke found in the upper Amazon basin (Radkofer, 1895).

Corresponding author. Tel.: +39 081 2538508; fax: +39 081 450165. E-mail address: adbasile@unina.it (A. Basile).

The rst chemical examination of guarana seeds was performed in the 18th century by the German botanist Theodore von Martius, who isolated a bitter, white crystalline substance with remarkable physiological properties. This substance was named guaranine, and was later renamed caffeine. For many years Paullinia cupana (guarana) has been the focus of attention due to the presence of caffeine and its pharmacological activity as a stimulant (Henman, 1982; Belliardo et al., 1985; Bempong and Houghton, 1992). Some Amazonian tribes have used guarana mainly as a stimulant,

0378-8741/$ see front matter 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2005.05.038

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astringent and to treat chronic diarrhoea. Other past and present tribal uses of guarana are: as a painkiller, febrifuge, to treat hypertension, migraine, neuralgia and dysentery. It is also reputed to be a cardiovascular drug and to prevent atherosclerosis (Bydlowski et al., 1988). Modern uses of guarana do not differ much from traditional ones, but some noticeable additions include: aphrodisiac and mind-expanding properties (Smit and Rogers, 2002). In Latin America it has incited interest in the eld of bromatology because it is used both as a avouring agent in beverages such as cola drinks (in soft drink beverages) and guarana champagne, as well as in some kinds of alcoholic drinks such as vinos guaranados and in liqueurs. More recently, guarana has been treated as a diet product for slimming in the form of powder, tablets and jams, pure or in association. Results and chromatographic proles performed by Carlson and Thompson (1998) for 14 commercial products in solid dosage form showed that a number of these products might not contain authentic guarana as an active ingredient or contain less than the declared quantity. Commercial guarana from central Amazon comes from the seed kernels of Paullinia cupana Mart., which is a woody vine of the Sapindaceae. Studies determining the effects of guarana extracts on mood and mental performance show activities that can be summarised with the terms alerting, revitalising, awakening and providing mental energy (Smit and Rogers, 2002). Guarana administration was able to partially reverse the amnesic effect of scopolamine in mice (Espinola et al., 1997). Furthermore, some authors describe the effects of guarana on exercise in normal and epinephrine-induced glycogenolytic mice (Miura et al., 1998). Furthermore, guarana was demonstrated to be protective against gastric lesions induced by ethanol and indomethacin in rats (Campos et al., 2003). In addition, guarana content in polyphenols (tannic-, caffeic- and gallic-acid) may well account for its use as a digestive tonic, but it also raises some concerns about its use as tannins are increasingly recognised antinutrients and dietary carcinogens (Ferguson, 2001; Morton, 1992). Its chemical composition features xanthic bases such as caffeine (25,00075,000 ppm), theophylline (500750 ppm) and theobromine (300500 ppm) (Belliardo et al., 1985), the latter two being found in guarana bark, owers and leaves, but not in the seeds (Henman, 1982). Purine alkaloids were found (Weckerle et al., 2003) with a differential seed alkaloid distribution: the seed kernel (embryo with bulky cotyledons) and seed coat (testa) accumulate much caffeine (i.e., 4.28 and 1.64%, respectively), whereas the aril is virtually alkaloid free (Baumann et al., 1995). The absence of toxicity was demonstrated in vivo (Espinola et al., 1997; Mattei et al., 1998) and in vitro at low concentrations (Santa Maria et al., 1998). The aim of this study was to evaluate the in vitro antibacterial activity of the ethanol extract from Paullinia cupana Mart. seeds against Gram-positive and Gram-negative bacteria and its antioxidant activity on 3T3-L1 cells after induced cellular damage, using ferric ammonium citrate (FAC). Antioxidant

activity was discussed in relation to catechol and to polyphenol content.

2. Materials and methods 2.1. Plant material Seeds from Paullinia cupana var. sorbilis Mart. (Sapindaceae), commonly called guarana, were purchased from Brisighello (Verona, Italy) and authenticated by Prof. Adriana Basile, Department of Biological Sciences, Section of Plant Biology, Federico II University of Naples (Italy). Voucher specimens were deposited at the Herbarium of the Botanical Garden of Naples (Italy). 2.2. Extraction Solvents (analytical grade) for extraction were purchased from SigmaAldrich (San Diego, California); p-aminophenol and catechol were purchased from Carlo Erba Reagents (Italy). The water used was MilliQ grade. A double beam UV/vis Helios Alpha Spectrophotometer (Thermo Optek, Milan, Italy), coupled to a PC, was used for the absorbance measurements. An Naviglio Extractor (Depurex 88, Padua, Italy), a dynamic solidliquid extractor, was used to perform the extraction of guarana seeds. Twenty grams of powdered guarana seeds were extracted using 2 l of ethanol as solvent in a Naviglio Extractor apparatus for 8 h under pressure (8 atm) and at room temperature. At the end of the extraction time the solution was automatically ltered (Naviglio et al., 2001). After evaporation of the solvent under reduced pressure at 35 C, the w/w yield from dry starting material was 14.9% (2.98 g). 2.3. Antimicrobial assay Ten bacterial strains, obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA), were employed. They included the Gram-positive bacteria: Staphylococcus aureus (ATCC 13709), Staphylococcus epidermidis (ATCC 10875), Streptococcus faecalis (ATCC 14428) and the Gram-negative bacteria: Proteus mirabilis (ATCC 7002), Proteus vulgaris (ATCC 12454), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 11229), Salmonella typhi (ATCC 19430), Enterobacter cloacae (ATCC 10699), and Bacillus subtilis (ATCC 10774). Bacterial strains were grown on Mueller Hinton (MH) agar plates (DIFCO, Detroit, MI, USA) and suspended in MH broth (DIFCO). The MIC values against bacterial strains were performed using the Ericcson and Sherris (1971) brothdilution method (MH broth). Inoculum suspensions were prepared from 6 h broth cultures and adjusted to 0.5 McFarland turbidity equivalents. The extract was sterilized by Millipore ltration (0.45 m) and added to MH broth medium. The MIC determination was performed as reported previously

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A. Basile et al. / Journal of Ethnopharmacology 102 (2005) 3236

Table 1 Antibacterial activity of guarana ethanol extract expressed as minimum inhibitory concentrations (MICs) in g/ml Extract Escherichia coli Salmonella typhi Staphylococcus epidermidis Enterobacter cloacae Staphylococcus aureus Bacillus subtilis Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Streptococcus faecalis 32 64 128 64 64 R 32 32 16 R Cefotaxime 0.1 0.5 0.1 R 2 32 0.03 2 16 R Penicillin 64 4 0.03 R 0.03 32 4 R R 2 Tetracycline 4 1 0.1 4 2 2 32 4 32 8

enzyme immunoassay system according to the suppliers manual (Enzymatic test, Roche Molecular Biochemicals). The results are presented as picomoles of malonyldialdehyde/mg of cell protein, determined by the Lowry method. 2.5. Catechol determination For the calibration curve, the p-aminophenol solution was prepared at 1 mM using HCl 0.1 M aqueous solution. Catechol standard solution was prepared at 0.01 M in 95% ethanol. The calibration curve was prepared by loading, separately, in six 10 ml volumetric asks 3.0 ml of 2% (w/v) NaOH solution, 3.0 ml of p-aminophenol solution and 0.0, 10.0, 25.0, 50.0, 75.0 and 100 l of catechol standard solution. The nal volume was completed with ethanol and the absorbance was measured at 586 nm 1 min after adding catechol (Magna et al., 2003). As for catechol determination, we used the standard addition method because of the interference of the matrix. Three ml of 2% (w/v) NaOH solution and 3.0 ml of p-aminophenol were added to six 10 ml volumetric asks. Then 1 ml of guarana ethanol extract and the adequate catechol standard solution volume 0.0, 10.0, 25.0, 50.0, 75.0 and 100 l were added. The nal volume was completed with ethanol and the absorbance was measured at 586 run 1 min after the addition of catechol (Magna et al., 2003). The quantitative analysis of catechol in guarana seeds was performed by a particular spectrophotometric method (Magna et al., 2003): the reaction of p-aminophenol with catechol forms indophenol dye-like species. We performed 10 determinations (n = 10). 2.6. Determination of total phenolics Total phenol content of guarana ethanol extract was determined using the FolinCiocalteu technique (Singleton and Rossi, 1965) Briey, a 50 l aliquot of ethanol extracts was assayed with 250 l Folin reagent and 500 l sodium carbonate (20%, w/v). The mixture was vortexed and diluted with water to a nal volume of 5 ml. After incubation for 30 min at room temperature, the absorbance was read at 765 nm in a cuvette of 1 cm and total phenols in the guarana ethanol extract were expressed as gallic acid equivalents (GAE), using a calibration curve of a freshly prepared gallic acid solution. We performed 10 determinations (n = 10). For the gallic acid, the curve absorbance versus concentration is described by the equation y = 0.0012x 0.0345 (R2 = 0.9997). 2.7. Statistics Data were evaluated by means of analysis of variance (signicance of difference was accepted at P < 0.05) and a subsequent Dunnett test, using the PISM program (Graph Pad Software, San Diego, CA). Data on antioxidant activity and catechol content underwent a correlation test.

Reference antibiotics: cefotaxime: Na-cefotaxime; penicillin: benzyl penicillin sodium; tetracycline: tetracycline. R = absence of inhibition at 1000 g/ml.

(Basile et al., 1997). The bacterial suspensions were aerobically incubated for 24 h at 37 C. The extract was tested in triplicate. The experiment was performed 4 times and the results are shown in Table 1. The MIC was dened as the lowest concentration able to inhibit any visible bacterial growth. Control cultures, containing only sterile physiological Tris-buffer, were also prepared. In addition, MIC values for tetracycline hydrochloride (Pharmacia, Milan, Italy), benzylpenicillin sodium (Cynamid, Catania, Italy) and cefotaxime sodium (Roussel Pharmacia, Milan, Italy) were determined. 2.4. Antioxidant assay To prepare cytoplasmic extract, 3T3-L1 cells were washed in phosphate buffer solution (PBS). Then cells were treated with lysis buffer (10 mM HEPES, pH 7.5, 3 mM MgCl2 , 40 mM KC1, 5% glycerol, 1 mM dithiotreitol) at 4 C. Cell debris and nuclei were pelleted by centrifugation at 7000 g for 5 min at 4 C, and supernatants were stored at 80 C. Lipid peroxidation products from mature adipocytes were measured by the thiobarbituric acid colorimetric assay. Briey, after hemin or apoferritin treatment, cells were washed three times with 1 PBS, incubated with 20 g/ml ferric ammonium citrate (FAC) for 2 h at room temperature, then washed once more and scraped in 1 PBS containing 0.5 mM EDTA and 1.13 mM butylated hydroxytoluene. Cell lysis was performed by means of six cycles of freezing and thawing. One milliliter of 10% (w/v) trichloroacetic acid was added to 450 l of cellular lysate. After centrifugation at 1500 g for 10 min, 1.3 ml 0.5% (w/v) thiobarbituric acid was added, and the mixture was heated at 100 C for 20 min. After cooling, malonyldialdehyde formation was recorded (A530 nm and A550 nm ) in a Perkin-Elmer LS-5B spectrouorimeter. The ferritin content was determined using a uorimetric

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Fig. 1. Antioxidant activity of guarana ethanol extract expressed as picomoles of malonyldialdehyde/mg of cell protein. Individual values represent mean S.E.M. from triplicate measurements (*** P < 0.001): (1) control (absence of ferric ammonium citrate and guarana extract); (2) presence of ferric ammonium citrate and absence of guarana extract; (35) presence of ferric ammonium citrate and guarana extract at 0.5, 1.0 and 2.0 g/ml, respectively. Inhibition percentage: (3) 17.8% (0.5 g/ml extract); (4) 44.6% (1.0 g/ml extract); (5) 65.2% (2.0 g/ml extract) versus ferric ammonium citrate (FAC).

3. Results The antibacterial activity results are shown in Table 1. Our ndings showed that the ethanol extract from Paullinia cupana seeds had interesting activity against both Gramnegative and Gram-positive bacteria. The guarana extract proved to be active against eight out of the 10 bacterial strains used and was particularly active against Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis and Escherichia coli (MIC values of 16 g/ml for the rst and 32 g/ml for the others, respectively). As for Salmonella typhi, Enterobacter cloacae and Staphylococcus aureus an MIC value of 64 g/ml was found, while Staphylococcus epidermidis was the least affected with an MIC value of 128 g/ml. Bacillus subtilis and Streptococcus faecalis were not inhibited. The results of the antioxidant assay demonstrated that the ethanol extract from guarana considerably reduced lipid peroxidation. The malonyldialdehyde test (MDA test) showed that the reduction of cellular damage was 65.2% using a guarana extract at a concentration of 2 g/ml. This effect was dose/dependent (see Fig. 1). Following the above-described analytical procedure we recorded a concentration of catechol equivalent to 6.06 0.13 mg/g (mean S.D.), while the total phenolic content, determined using FolinCiocalteau procedure, was 8.43 0.21 mg/g (mean S.D.). The correlation index between antioxidant activity and catechol content was 0.96.

4. Discussion and conclusions Our ndings on antibacterial activity of guarana could justify some ethnopharmacological uses such as against diarrhoea and dysentery because we demonstrated strong activity

of this plant against some pathogens of the digestive tract. On the other hand, the antioxidant activity could explain the ethnopharmacological uses against atherosclerosis. Due to the continuous emergence of antibiotic-resistant strains there is continual demand for new antibiotics. In many developing countries about 80% of available drugs come from medicinal plants and in industrialized countries plants make up the raw material for processes, which synthesize pure chemical derivatives (Penso, 1980). The ethanol extract from guarana seeds showed an inhibiting activity on disease causing Gram-negative and Gram-positive bacteria, the most inhibited being Pseudomonas aeruginosa. This is particularly interesting from a medical point of view because this microbial agent is responsible for severe opportunistic infections. These ndings were also shown in other plant extracts (Castaldo Cobianchi et al., 1988; Basile et al., 1992, 1997, 1999, 2000; Vuotto et al., 1999). Cytotoxic activity of guarana was reported in prokaryotic cells (Fonseca et al., 1994). The growing interest in the substitution of synthetic food antioxidants with natural ones has fostered research on plant sources and screening of raw materials to identify new antioxidants. Interest in oxidation reactions is not conned to the food industry, as antioxidants are widely needed to prevent deterioration of other perishable goods, such as cosmetics, pharmaceuticals and plastics. In addition, other biological properties such as anticarcinogenicity, antimutagenicity, antiallergenicity and antiaging activity have been reported for natural and synthetic antioxidants (Moure et al., 2001). Our results showed interesting antibacterial and antioxidant activity of the ethanol extract from guarana. This is probably due to polyphenol substances present in the extract. On the other hand, phenolic antimicrobial activity is well documented (Karamanoli, 2002). Polyphenols are the major plant compounds with antioxidant activity, although they are not the only ones. The antioxidant activity of phenolic compounds is reported to be mainly due to their redox properties (Galato et al., 2001; Zheng and Wang, 2001), which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. Our results are in agreement with Mattei et al. (1998) who reported that a concentration of 1.2 g/ml of guarana seed powder results in an antioxidant effect in vitro on inhibiting the process of lipid peroxidation. The high correlation index value (0.96) indicated that the antioxidant activity is probably due to catechol content. It is feasible that polyphenol substances play a role as an iron cytoprotective agent by limiting the reactivity of intracellular iron on lipids in adipocytes themselves, where lipids are biosynthesised. It has been demonstrated that cultured endothelial cells briey pulsed with heme became highly resistant to oxidant-mediated injury and to the accumulation of endothelial lipid peroxidation products (Eisenstein et al., 1991; Balla et al., 1992). The contemporary presence of antibacterial and antioxidant activities in the ethanol extract from guarana suggests

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