PHYTOTHERAPY RESEARCH, VOL.

11, 254256 (1997)

SHORT COMMUNICATION

Evaluation of the In Vitro Antioxidant Activity in Extracts of Uncaria tomentosa (Willd.) DC.
C. Desmarchelier1 , E. Mongelli1, J. Coussio2 and G. Ciccia1
1 C tedra de Biotecnologa y Microbiologa Industrial; Facultad de Farmacia y Bioqumica, Universidad de Buenos Aires, Junn 956 a (1113) Buenos Aires, Argentina 2C tedra de Farmacognosia, IQUIMEFA-CONICET; Facultad de Farmacia y Bioqumica, a Universidad de Buenos Aires, Junn 956 (1113) Buenos Aires, Argentina

Different extracts of U. tomentosa were tested in vitro for their antioxidant activity utilizing tert-butylhydroperoxide-initiated chemiluminescence in rat liver homogenates. Methanol fractions of both stem-bark and roots were capable of exerting antioxidant activity by this technique. The presence of different concentrations of bark and root methanol extracts also prevented TBARS production and free radical-mediated DNA-sugar damage. 1997 by John Wiley & Sons, Ltd. Phytother. Res. 11, 254256, 1997
(No. of Figures: 0. No. of Tables: 2. No. of Refs: 16.)

Keywords: Uncaria tomentosa; antioxidant activity; chemiluminescence; thiobarbituric-acid-reactive substances; superoxide-mediated toxicity; DNA damage.

INTRODUCTION Uncaria tomentosa (Willd.) DC. is a vine belonging to the family Rubiaceae that grows in the Amazon forests of Peru, and is locally known as u a de gato. The aqueous extract n and decoction of the bark of this plant are widely used in traditional Peruvian medicine for the treatment of cancer, arthritis, diabetes and as a potent antiinammatory (Yepez et al., 1991). Previous pharmacological work has demonstrated antiinammatory activity in rat paw oedema assays (Aquino et al., 1991), while protective antimutagenic effects have been determined in vitro against induced photomutagenesis (Rizzi et al., 1993). However, the mechanisms by which these extracts and fractions exert their activity is not yet fully understood. It has been determined that active oxygen molecules play an important role in the inammation process (Yuda et al., 1991). On the other hand, DNA damage related to mutagenic processes may also be induced by oxygen radical toxicity (Imlay and Linn, 1988). In this study, different extracts of bark and roots of U. tomentosa were tested in vitro for their antioxidant activity utilizing tert-butyl-hydroperoxide-initiated chemiluminescence, production of thiobarbituric acid reactive-substances (TBARS) in rat liver homogenates and oxidative DNA sugar damage induced by Fe(II) salts.

Plant extracts. Infusions were prepared by macerating 5 g of powdered plant material for 20 min with 100 mL of boiling water, ltering the mixture and freeze-drying the ltrate (aqueous extract) in a Chriss lyophilizer (Germany). Decoctions were prepared by boiling 10 g of dry plant material in 100 mL of distilled water for 20 min. The mixture was ltered and lyophilized as described above. The resulting powder was considered as the decoction. Dichloromethane extracts were prepared by extracting 5 g of dry powdered plant material for 24 h with 50 mL of dichloromethane (Dorwill, Argentina). The extract was ltered and concentrated under reduced pressure at 43 C in a Savant Speed Vac Plus SC210A concentrator. The marc was extracted with methanol (Mallinckrodt, USA), in the same conditions as described for the dichloromethane extract (Anonymous, 1995). DMSO (Merck, Argentina) was used to pre-solubilize dichloromethane and methanol extracts (nal concentration, 5% v/v). Preparation of rat liver homogenates. Adult Wistar rats of 180200 g fed on a standard laboratory diet and water ad libitum were used. The livers were excised, perfused and homogenized with 120 mM KCl, 50 mM phosphate buffer, pH 7.4, (1:10 w/v). The samples were centrifuged at 700 g for 10 min at 0 4 C. The supernatant fraction was kept at 20 C until use. Protein was measured by the Lowry et al. (1951) method. Hydroperoxide-initiated chemiluminescence. Hydroperoxide initiated chemiluminescence of liver homogenates (Gonzalez Flecha et al., 1991) was measured in a LKB Wallac 1209 Rackbeta liquid scintillation counter in the outof-coincidence mode, at 30 C. Rat liver homogenates adjusted to nal protein concentration of 0.5 mg/mL in 120 mM KCl, 50 mM phosphate buffer, pH 7.4 and 2 mL nal volume were placed in 10 35 mm glass tubes, which were allocated in low potassium 25 50 mm glass vials. Plant extracts, solubilized in distilled water, were added to
Accepted (revised) 22 October 1996

MATERIALS AND METHODS

Plant material. Plant material was kindly provided by Dr

Fernando Cabieses, of the Instituto Nacional de Medicina Tradicional, in Lima, Peru.

Correspondence to: C. Desmarchelier. CCC 0951418X/97/03025403

1997 by John Wiley & Sons, Ltd.

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255

the reaction medium at nal concentrations of 10, 100 and 1000 g/mL. Chemiluminescence was initiated by addition of 3 mM t-butyl hydroperoxide (t-BOOH) (Sigma, USA), and was expressed as cps/mg protein. Chemiluminescence inhibition values are expressed as the ratio of the maximum emission in the presence of plant extracts to that in their absence (control). Catechin (Sigma, USA) was used as a positive control or standard. Finneys (1971) statistical method of probit analysis was used to calculate the IC50 (concentration that inhibit 50% of the chemiluminescence) with the 95% condence interval.
Thiobarbituric acid-reactive substances (TBARS) assay.

into 2 mL n-butanol. After centrifugation, the uorescence of the butanol layer was measured as described above. The iron (II)-dependent DNA damage inhibition values are expressed as the ratio of TBA reacting species in the presence of plant extracts to that in their absence (control). Catechin was used as a standard. RESULTS AND DISCUSSION Different extracts of bark and roots of Uncaria tomentosa were tested for their in vitro antioxidant activity using the hydroperoxide-initiated chemiluminescence assay in rat liver homogenates. This assay determines the free radical trapping activity in a chain reaction that occurs both in the hydrophilic and the hydrophobic domains of the biological membranes (Fraga et al., 1987b). Table 1 shows the effects of three concentrations of the plant extracts on chemiluminescence. This effect is expressed as a percentage of inhibition evaluated at maximum emission (Cadenas and Sies, 1982), and negative inhibitions mathematically expressed indicate an increase in chemiluminescence. Clearly, methanol extracts of both bark and root only were capable of inhibiting the light emission at all concentrations. The bark aqueous extract and decoction were only effective at higher concentrations (100 and 1000 g/mL). The antioxidant activity of methanol extracts was further assayed by determining TBARS production in rat liver homogenates and the inhibition of free radical-mediated DNA-sugar damage (Table 2). The former assay determines the production of malondialdehyde and related compounds which are by-products of lipid peroxidation, a free radical process in the hydrophobic core of biomembranes. The extracts assayed were moderately active in inhibiting TBARS production. However, hydroperoxide-initiated chemiluminescence was more sensitive than TBARS production in determining changes in lipid peroxidation, as reported previously (e.g. Lavagno et al., 1991). Oxygen radicals may attack DNA either at the sugar or the base, giving rise to a large number of products. Attack at a sugar ultimately leads to sugar fragmentation, base loss, and a strand break with a terminal fragmented sugar residue (Imlay and Linn, 1988). Rizzi et al. (1993) pointed out that the in vitro antimutagenic power of U. tomentosa may be

TBARS were determined as in Fraga et al. (1987a). Rat liver homogenates, adjusted to 10 mg protein/mL in 120 mM KCl, 50 mM phosphate buffer, pH 7.4, were incubated with 10, 100 and 1000 g dry weight/mL of plant extract at 37 C for 15 min. Sodium dodecyl sulphate (Sigma, USA) (0.2 mL of 3% (w/v) and 0.05 mL of BHT (Sigma, USA) 4% in ethanol were added. After mixing, 2 mL of 0.1N HCl, 0.3 mL of 10% (w/v) phosphotungstic acid (Sigma, USA) and 1 mL of 0.7% (w/v) 2-thiobarbituric acid (Sigma, USA) were added. The mixture was heated for 60 min in boiling water, and TBARS were extracted into 5 mL n-butanol (Merck, Argentina). After centrifugation, the uorescence of the butanol layer was measured at 515 nm excitation and 555 nm emission using an Hitachi F-3010 uorescence spectrophotometer. The values are expressed as the ratio of the amount of TBARS formed in the presence of plant extracts compared with control. Catechin was used as a standard.
Assay of DNA sugar damage. The DNA-sugar damage was

assayed by the method of Halliwell and Gutteridge (1981) with modications. The reaction mixture in a total volume of 1.2 mL contained 0.5 mL calf thymus DNA (Sigma, USA) (1 mg/mL of 0.15 M NaCl), 0.5 mL phosphate buffer (0.1 M, pH 7.4), 0.2 mL ferrous ammonium sulphate (Merck, USA) (4.8 mM) and 1000, 100 and 10 g/mL of the methanol plant extract. The reaction mixture was incubated for 1 h at 37 C in a water bath shaker. After incubation, 1 mL TBA 1% (w/v) plus 1 mL 2.8% (w/v) trichloroacetic acid were added to the reaction mixture and kept in a boiling water bath for 15 min. TBA reacting species were extracted

Table 1. Antioxidant and prooxidant activities in extracts of U. tomentosa using the hydroperoxide-initiated chemiluminescence assay: LC50 and 95% condence interval
Extract Inhibition of chemiluminescence (%) 1000 g/mL 100 g/mL 10 g/mL IC50 and 95% condence interval ( g/mL)

Catechina Bark Aqueous Decoction Dichloromethane Methanol Root Aqueous Decoction Dichloromethane Methanol

85 58 46 0 86 0 31 87 65

81 30 24 0 64 16 61 167 44

73 19 47 0 22 0 105 4 9

1 (0.012.7) NDb ND ND 56 (3683) ND ND ND 259 (167436)

Inhibition of chemiluminescence was calculated by [1-(Etm-Etp)/(EcmEcp)] 100; Etm and Etp: emission intensity of test samples at maximum emission, Ecm and Ecp: emission intensity of the control at maximum emission. Catechin was used as a standard. a Data of inhibition of chemiluminescence for concentrations below 10 g/mL, not show. b ND, not determined.

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Table 2. Inhibition of free radical-mediated DNA-sugar damage and TBARS production by methanol extracts of Uncaria tomentosa
System + extract ( g) TBARS DNA-sugar damage

Catechin ( g/mL) 1 10 100 Bark ( g/mL) 1 10 100 Root ( g/mL) 1 10 100

30.27 45.56 55.79 5.95 10.70 25.95 2.20 8.12 20.49

61.0 72.7 79.0 35.4 46.7 60.7 19.5 27.9 49.7

The TBARS and iron (II-dependent DNA damage inhibition values are expressed as the ratio of TBA reacting species in the presence of plant extracts to that in their absence (control). Catechin was used as a standard.

due to the antioxidant property of bark components acting to quench the oxyradicals generated in DNA by UV irradiation. Table 2 also shows the effect of U. tomentosa methanol extracts on iron (II)-dependent DNA-sugar damage. The presence of various concentrations (1, 10 and 100 g/mL)

of stem-bark and root extracts in the reaction mixture prevented the free radical-mediated DNA-sugar damage. Thus, these results suggests that the extracts could contain compounds which may reduce free radical-mediated damage to the DNA-deoxyribose. Antioxidant activity in extracts of U. tomentosa was determined using different bioassays, and the free radical scavenger capacity was observed both against lipid-peroxidation and DNA damage in methanol extracts of bark and roots. Since U. tomentosa extracts and fractions previously showed signicant antiinammatory (Aquino et al., 1991) and antimutagenic activities (Rizzi et al., 1993), it may be proposed that their efcacy could be partially due to their free radical scavenging activity. Bioactivity in U. tomentosa has often been shown to be higher in extracts than in isolated active principles (Wagner et al., 1985). Recent studies on the synergistic effects of plant constituents have shown that the isolation of an active compound may not always account for all the qualitative or quantitative activity of a total extract containing an identical amount (Houghton, 1995), and this may be the case for U. tomentosa. However, future studies are needed to identify the compounds responsible for the antioxidant activity and to evaluate the in vivo activity in animal models of lipid peroxidation and oxidative damage to DNA.

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