C. americana, K. ramosissima, T. diffusa and J. mollis specimens were collected in the Mexican states of Coahuila and Nuevo León during the spring and summer of 2006. The plants were authenticated and voucher specimens of each plant (UAN-17624, UAN-4850, UAN-8272 and UAN-17854, respectively) were deposited in the institutional herbarium located in the Facultad de Ciencias Biológicas of the Universidad Autónoma de Nuevo León.

Absolute anhydrous ethyl alcohol was obtained from Mallinckrodt Baker (México City, México). Si-libinin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and carbon tetrachloride (CCl4, 99.9%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide was purchased from ACS Research Organics (Cleveland, OH, USA). Thiobarbituric acid reactive substances were purchased from Kit OXItek (Buffalo, NY, USA). Dulbecco's modified Eagle's medium, fetal bovine serum trypsin 0.25% (1x), penicillin G (100 μg/mL), streptomycin (100 pg/mL) and phosphate-buffered saline were purchased from Gibco Invitrogen (Carlsbad, CA, USA).

C. americana samples were separated into flower and stem/leaf portions and both were used; the aerial parts of K. ramosissima and T. diffusa were used, and J. mollis samples were separated into nut, leaf and bark portions and were used. Each portion was dried at room temperature for two weeks and then ground finely. The extracts were obtained by shaking 50 g of dried material in a 300 mL volume of ethanol and water (90:10) for 30 minutes. Three extracts of each sample were prepared. The extracts were filtered and concentrated under reduced pressure at 37 °C, dried in an oxygen-free environment at 37 °C, and stored at -80 °C until used.

The antioxidant activity of the hydroalcoholic extracts was evaluated using the DPPH radical scavenging method.13 A 1,000 μg/mL extract and a 0.2 mg/mL solution of DPPH in pure ethanol were prepared. Five hundred microliters of each solution were then mixed and allowed to stand at room temperature in the dark for 30 minutes. Absorbance at 517 nm was then measured using a Beckman DU 7500 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). Quercetin was used as a positive control. Three analytical replicates were carried out on each sample.

The reduction percentage was estimated using the following equation: Scavenging effect (%) = (A-B) × 100/A, where A = absorbance in the presence of DPPH alone (blank) and B = absorbance in the presence of DPPH and the extract.

Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with heat-inactivated fetal bovine serum (10%), penicillin G (100 IU/mL), streptomycin (100 μg/mL) and 1% nonessential amino acids at 37 °C in a humidified atmosphere containing 95% O2 and 5% CO2. At 80-90% confluence, the cells were trypsinized and plated (1 × 106 cells per well). The cells were used after attachment.

The cytotoxicity of plant extracts on Huh7 cells were evaluated using cell viability and levels of aspartate aminotransferase (AST) and malondialdehyde (MDA) in the medium after exposure to extracts at concentrations of 10, 100, and 1,000 μg/ mL in phosphate-buffered saline for one hour. Silibinin was used as a control. Each assay was performed in triplicate and experiments were repeated three times.

Cell viability was assessed using the MTT reduction assay with slight modifications.14 This colorimetric assay involves the conversion of MTT to a purple formazan derivative by mitochondrial succinate dehydrogenase, which is present only in viable cells. Huh7 cells (1 μ 106) were treated with each plant extract (10, 100 or 1000 ×g/mL in phosphate-buffered saline) for one hour. After removal of the extracts from the wells, the cells were washed with phosphate-buffered saline. The cells were then incubated with 150 μL of MTT (0.5 mg/mL) for two hours, after which the medium was removed and 560 μL of dimethyl sulfoxide was added to dissolve the formazan crystals that had formed. Absorbance was measured in an ELISA microplate reader at 570 nm (Multiskan Ex; Thermo Labsystems, Vantaa, Finland). Viability was defined as the ratio (expressed as a percentage) of absorbance of treated cells to untreated control cells.

AST activity in the supernatant after exposure of cells to various extract concentrations of plant extracts for one hour was determined. The supernatant was stored at -80 °C until analysis. AST activity was determined using slides in a biochemical autoanalyzer (Vitros DTII Systems Chemistry, module DTSCII; Johnson & Johnson Ortho-Clinical Diagnostics, New Brunswick, NJ, USA).

Lipid peroxidation was measured by quantifying thiobarbituric acid reactive substances in supernatants after exposure of cells to various extract concentrations for one hour according to the method of Kit OXItek. The supernatants were stored at -80 °C until analysis. Fluorescence intensity was determined using a 530 nm excitation filter and a 550 nm emission filter in an LS45 spectrophotometer (Perkin Elmer, Waltham, MA, USA). The results were expressed as MDA equivalents.

Cells were treated with various concentrations of plant extracts (10, 100 and 1,000 μg/mL in phosphate-buffered saline) for one hour, followed by the addition of 40 mM CCl4 in 0.05% dimethyl sulfoxide and subsequent incubation for two hours. The supernatant medium was then collected and stored at 80 °C until quantification of AST and MDA levels. We incubated Huh7 cells with silibinin (100 μg/mL) under the same conditions as a hepatoprotective positive control.

All variables were measured in triplicate and experiments were repeated three times. Results are expressed as the mean ± standard deviation. Data were analyzed using Student’s t-test, a multivariate linear model and Pearson’s correlation method. SPSS software (v.15.0; SPSS Inc., Chicago, IL, USA) was used for all analyses. A P < 0.05 was considered significant.

At a concentration of 1,000 μg/mL, the flower extract of C. americana, the aerial extract of T. diffusa and the leaf and bark extract of J. mollis exhibited DPPH free radical scavenging activity (79 ± 1.6%, 82.4 ± 4.6%, 80 ± 2%, 76.8 ± 1.2%, respectively), followed by the nut extract of J. mollis (68 ± 3%), the C. americana stem/leaf extract (62.5 ± 4.8%) and the K. ramosissima extract (53 ± 1.78%). Quercetin exhibited a DPPH free radical scavenging activity of 90.08 ± 0.9% at a concentration of 1,000 μg/mL.

The cytotoxicity of plant extracts on Huh7 cells is listed in Table 1. The plant extracts were toxic when there was a decrease in cell viability of more than 60% compared with untreated cells, an AST level > 50 IU/L or an MDA level greater than that of the control. According to these parameters, 1,000 μg/mL of the C. americana stem/leaf extract, the aerial extract of T. diffusa, the nut extract of J. mollis and all concentrations of the aerial extract of K. ramosissima were toxic. All the other extracts were used to evaluate hepatoprotective activity.

The effects of the six plant extracts on cells treated with CCl4 are shown in Figure 1. AST levels were significantly elevated after treatment with CCl4. Treatment with 100 μg/mL silibinin reduced CCl4-induced elevation of AST level at all concentrations of C. americana flower extract, C. americana stem/leaf extract (10 μg/mL), J. mollis leaf extract (100 and 1000 μg/mL) and J. mollis bark extract (10 and 100 μg/mL) (P < 0.001) (Figure 1A).

Figure 1.

Hepatoprotective effects of various concentrations of extracts of the flower or stem/leaf portions of C. americana, Aerial part of T.diffusa, Nut or Leaf or Bark portions of J.mollis on Huh7 cells exposed to CCl4. AST: Aspartate aminotransferase. MDA: Malondialdehyde equivalents. (A) AST (IU/L) and (B) MDA (nmol/mL). Values are expressed as the mean ± standard deviation for nine experiments. *P<0.001; **P=0.001; #, P=0.05.

(0.25MB).

Cells exposed to CCl4 showed a significant increase in MDA level. All extracts prevented lipid peroxidation when added at levels of 10 pg/mL, 100 pg/mL and 1000 pg/mL, except for the 10 pg/mL J. mollis leaf extract, which resulted in an increase in MDA level greater than that induced by CCl4 (Figure 1B).