The human lung cancer (NCI-H460) and colon cancer (Caco-2) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] was purchased from Sigma Chemical (St. Louis, MO, USA) and RPMI-1640 medium was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). The EnzChek^® Elastase Assay kit (E-12056) was purchased from Molecular Probes (Eugene, OR, USA), and mushroom tyrosinase, tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ethanol (EtOH), ethyl acetate (EtOAc), dimethylsulfoxide (DMSO) and sodium dodecyl sulfate (SDS) were purchased from Merck KGaA (Darmstadt, Germany).

Marigold (Tagetes erecta L.) flowers were collected from Chiang Mai, Thailand in the winter of 2008 and authentication was performed by Dr Kongkanda Chayamarit in the Forest Herbarium (Forest Research Office, Royal Forest Department, Bangkok, Thailand). Voucher specimens have been deposited at the Herbarium Section (Northern Research Center for Medicinal Plants, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand).

Dried powder of the marigold flower (250 g) was separately extracted by Soxhlet’s apparatus (Sigma-Aldrich, Gillingham, UK) with two different solvents, ethyl acetate and ethanol. Each extract was subsequently filtered through Whatman No.4 paper and evaporated under a vacuum. The residues obtained from the ethyl acetate and ethanol extracts were named MF_EtOAc (yield, 10.26%) and MF_EtOH (yield, 23.83%), respectively. MF_EtOAc and MF_EtOH extracts were employed for further study.

The TPCs of MF_EtOAc and MF_EtOH extracts were determined using the Folin-Ciocalteau method as previously described by Kähkönen et al(13). Briefly, 1 ml of the appropriately diluted sample was mixed with 5 ml Folin-Ciocalteau reagent and 4 ml sodium carbonate (7% w/v). The mixture was fully agitated and allowed to stand for 30 min in the dark at room temperature. The absorbance was then measured at 765 nm using a spectrophotometer (Lambda 25; Perkin Elmers, Waltham, MA, USA). The TPC value of each extract was determined in comparison with standard gallic acid and expressed as milligram gallic acid equivalents in 1 g of dried extract (mg GAE/g).

H460 cells were cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were grown in a humidified atmosphere of 5% CO₂ at 37°C until >60% confluence was reached. The cells were then passaged every other day using a solution containing 0.05% trypsin and 0.5 mM EDTA.

Caco-2 cells were cultured in Dulbecco’s modified Eagle medium to which 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine were added. The cells were incubated under the same conditions as the H460 cells and subcultured every week using 0.5% trypsin and 0.5 mM EDTA solution.

The cytotoxicity of the MF_EtOH and MF_EtOAc extracts was assessed using the MTT assay. Mitochondrial respiration, an indicator of cell viability, reduced MTT from a yellow water-soluble dye to a purple insoluble formazan product by succinate dehydrogenase (14). Since MF_EtOH and MF_EtOAc extracts are partially water-soluble, DMSO was employed to enhance their solubilities in an aqueous medium. It was found that 0.5% DMSO solution did not significantly affect the cell viability compared with that of the control group (P<0.05). Therefore, test solutions of MF_EtOH and MF_EtOAc extracts were prepared in 0.5% DMSO aqueous solution. The toxicity of the MF_EtOH extract was studied at three concentrations (10, 50 and 100 μg/ml), while the toxicity of the MF_EtOAc extract was studied at two concentrations (10 and 50 μg/ml) due to the lower solubility.

Caco-2 and H460 cells were cultured separately in 96-well plates for 48 and 24 h at seeding densities of 5×10⁴ and 2×10⁴ cells/well in 100 μl medium, respectively. Prior to testing, the cell culture medium was removed and cells were washed with phosphate-buffered saline (PBS). Experiments were initiated by adding 100 μl MF_EtOH or MF_EtOAc solution to each well. After 24 h of incubation, cells were washed with PBS and 100 μl MTT solution (0.5 mg/ml) was added to each well. Following a further 4 h of incubation, 100 μl SDS solution (10% w/v) was added to each well and the cells were incubated overnight. Absorbance was then measured at a wavelength of 590 nm using a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Untreated cells (0.5% DMSO) were used as the control group.

The elastase inhibition activities of MF_EtOH and MF_EtOAc extracts were assayed using the EnzChek^® Elastase Assay kit. This kit consisted of DQ™ elastin from bovine neck ligaments that was labeled with fluorescent BODIPY^® FL conjugate, elastase from pig pancreas, N-methoxysuccinyl-ala-ala-pro-val-chloromethyl ketone (elastase inhibitor) and reaction buffer.

The principle was based on the digestion of non-fluorescent elastin substrate (BODIPY^® FL conjugated with DQ™ elastin) to highly fluorescent fragments by elastase. The digestion products were subsequently measured using a fluorescence microplate reader (Thermomax; Molecular Devices).

DMSO solution (5% v/v) was employed to enhance the solubility of MF_EtOH and MF_EtOAc extracts, respectively. The elastase inhibition activity of MF_EtOH was studied at three concentrations (50, 125 and 250 μg/ml), while two concentrations of MF_EtOAc extract (50 and 125 μg/ml) were studied due to the limited solubility of MF_EtOAc. The test was performed in 96-well plates that were hidden from the light. DMSO (5%) and elastase inhibitor were used as a negative and a positive control, respectively.

Following 2 h of incubation at 25°C, fluorescence absorbance was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm, in accordance with the instructions supplied with the EnzChek^® Elastase Assay kit and described by Leu et al(15). The percentage of elastase inhibition activity was calculated using the following equation (1):

Where ‘A’ was enzyme activity in absence of the inhibitor and ‘B’ was enzyme activity in the presence of the inhibitor.

The tyrosinase inhibition activities of MF_EtOH and MF_EtOAc extracts were determined using the commercially available mushroom tyrosinase as previously described (16). L-DOPA and kojic acid were utilized as the substrate and positive control, respectively. Briefly, 40 μl L-DOPA (0.85 μM) solution was mixed with 40 μl extract solution or kojic acid. A total of 40 μl tyrosinase solution (520 U/ml) was then added to the mixture and the enzyme reaction was evaluated by the absorbance measurement of dopachrome formation at 492 nm using a Thermomax microplate reader (Molecular Devices).

The percentage of tyrosinase inhibition activity was calculated using the following equation (2):

Where ‘A’ was enzyme activity in absence of the inhibitor and ‘B’ was enzyme activity in the presence of inhibitor. The concentration giving 50% tyrosinase inhibition activity (IC₅₀) was determined by interpolation of the dose-response curves.

Results are presented as the mean ± standard deviation. An unpaired Student’s t-test (two-tailed) was used to test the significance of the difference between two mean values. One-way analysis of variance was used when more than two means were compared. P<0.05 was considered to indicate a statistically significant difference.

The TPCs of MF_EtOH and MF_EtOAc extracts, expressed as gallic acid equivalents, are shown in Fig. 1. Different extraction solvents differed in polarities and therefore resulted in different TPC values of the two extracts. As a result, there was a significant difference between the TPC values of the MF_EtOAc extract (318.05 mg GAE/g) compared with that of the MF_EtOH extract (17.78 mg GAE/g). The TPC value of the MF_EtOAc extract was found to be ~18-fold higher than that of the MF_EtOH extract.

To investigate whether the MF_EtOH and MF_EtOAc extracts affected the viability of H460 and Caco-2 cells, the MTT assay was performed. The cytotoxicity of the MF_EtOH and MF_EtOAc extracts is presented as a percentage of cell viability relative to the control group (100%). The mean and standard deviation of each experiment were calculated from 10 replicates.

The viability of the H460 cells following treatment with different concentrations of MF_EtOH or MF_EtOAc extracts ranged between 87.78 and 126.2% (Fig. 2). No significant differences were identified among the H460 cell viabilities of the treated and control groups (P>0.05).

The cytotoxicity studies of MF_EtOH and MF_EtOAc extracts on Caco-2 cells obtained similar results to the H460 cells (Fig. 3). The Caco-2 cell viabilities following treatment with different concentrations of MF_EtOH and MF_EtOAc extracts were found to range between 91.3 and 101.9%, with no significant differences among the data groups (P>0.05).

As shown in Fig. 4, the elastase inhibition activities of MF_EtOH and MF_EtOAc extracts were found to be significantly higher than that of the negative control at concentrations of 250 and 125 μg/ml, respectively. When these two extracts were applied at lower concentrations, no significant differences were identified in elastase inhibition activities compared with that of the negative control (data not shown). At a concentration of 125 μg/ml, the elastase inhibition activity of MF_EtOAc extract was observed to be significantly higher than that of 250 μg/ml MF_EtOH extract (P=0.004). It may be inferred that the MF_EtOAc extract possessed stronger elastase inhibition activity than that of the MF_EtOH extract. The positive control showed 82.20% inhibition of elastase activity.

The in vitro inhibition effects of various concentrations of MF_EtOH and MF_EtOAc extracts on tyrosinase activities are shown in Fig. 5. The results demonstrated that MF_EtOH and MF_EtOAc extracts exhibited potent inhibitory effects against mushroom tyrosinase. The IC₅₀ values of MF_EtOH and MF_EtOAc extracts against tyrosinase were 1,078 and 1,467 μg/ml, respectively. These observations suggested that the MF_EtOH extract exerted a more marked tyrosinase inhibition compared with that of the MF_EtOAc extract. However, at a concentration of 5,000 μg/ml, MF_EtOAc extract resulted in a higher tyrosinase inhibition of 90.6%, while the MF_EtOH extract exhibited 68.5% inhibition.

Due to the aforementioned tyrosinase inhibition activity, kojic acid was selected as the positive control in this study. The IC₅₀ values of MF_EtOH and MF_EtOAc extracts were 23- and 31-fold higher than that of kojic acid (IC₅₀=47 μg/ml).