Five kilograms Taiwan Good Agriculture Practice (TGAP) certified burdock leaves were obtained from Kang-Li burdock farm in the Gueilai region of Pingtung in Taiwan. Gallic acid, chlorogenic acid (CHA), 1,1-diphenyl-2-pycrylhydrazyl (DPPH), L-3,4-dihydroxyphenylalanine (L-DOPA), N-Suc-(Ala)₃-nitroanilide (SANA), mushroom tyrosinase, porcine pancreatic elastase and Folin-Ciocalteu reagent were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Liquid chromatography grade methanol and glacial acetic acid were purchased from Macron Fine Chemicals (Avantor Performance Materials, Center Valley, PA, USA). A Cosmosil 5C₁₈-MS-II reverse-phase high performance liquid chromatography (HPLC) column (4.6×250 mm internal diameter; Nakalai Tesque, Inc., Kyoto, Japan) was used for HPLC. Ethanol and phosphoric acid were of analytical reagent grade.

Fresh burdock leaves were dried through four methods: Sun drying, shadow drying, oven drying and freeze-drying. For sun drying, fresh leaves were exposed directly to sunlight for 16 h. In shadow drying, fresh leaves were dried at an ambient temperature of 27°C and a relative humidity of 39% for 72 h. For oven drying, fresh leaves were dried in an oven at 60°C for 16 h. In freeze-drying, a lyophilizer (cat no. 8530023; FD-series, Panchun, Taipei, Taiwan; http://www.panchum.com.tw/product_show.asp?id=1038) was used to dry the burdock leaves for 72 h at −80°C. The four types of BLE (30 g of each) were separately added to 600 ml of ethanolic aqueous solution [30, 50 or 95% ethanol (EtOH)] and refluxed for 3 h in a reflux extraction apparatus (cat no. 203–330; Angu, Kaohsiung, Taiwan). Subsequently, the extract solution was filtered and the total polyphenol content was prescreened, as described below. The solution from each drying method with the highest amount of total antioxidant components was lyophilized to obtain the BLE at −80°C for 24 h, which was stored in an Eureka Auto Dry Box at 25°C and 40% relative humidity (AD-88S; Taiwan Dry Tech Corp., Taipei, Taiwan) until required for analysis.

The total polyphenol content was estimated spectrophotometrically using Folin-Ciocalteu reagent based on a colorimetric oxidation/reduction reaction, as described previously (22,29) with minor modifications. Briefly, BLE solution (20 µl) obtained through the different drying methods, 100 µl 0.2 N Folin-Ciocalteu reagent and 80 µl 7.5% Na₂CO₃ were added to each well of a 96-well microplate and mixed thoroughly. The solution was left to stand for 30 min at room temperature and the absorbance was measured at 765 nm using a microplate reader. The total polyphenol content was calculated as the gallic acid equivalent (GAE) on the basis of a calibration curve of gallic acid standard and expressed in mg/ml, as previously described (22).

BLE antioxidant activity was evaluated on the basis of the scavenging capacity toward a stable DPPH free radical as described previously (22,30,31). A stock solution of BLE (1 mg/ml) was prepared and diluted with methanol to a suitable concentration at 0.5 mg/ml. An aliquot of 50 µl of each dilution was transferred into the wells of a 96-well microplate. A working solution of DPPH (250 µM) in methanol was freshly prepared and an aliquot of 150 µl was added to each well. After incubation for 30 min at 25°C, the remaining DPPH was measured at 490 nm using an ELISA reader. The antioxidant activity was expressed as the DPPH scavenging percentage, as previously described (22,31).

The expression of CHA (which is an antioxidant component) in BLE was measured using an HPLC assay as described previously (10). Briefly, HPLC analyses were performed using an HPLC system equipped with an L2130 HPLC pump, an L2450 photodiode detector, an L2200 autosampler (Hitachi, Ltd., Tokyo, Japan) and a Cosmosil 5C₁₈-MS-II reverse-phase HPLC column. A mixture of methanol/water/glacial acetic acid (30:69:1, v/v; Sigma-Aldrich; Merck KGaA) was used as the mobile phase. A wavelength of 320 nm was used for monitoring the phenolic compounds and CHA. The flow rate and injection volume were set to 1 ml/min and 10 µl, respectively.

The effect of BLE on porcine pancreatic elastase activity was estimated spectrophotometrically using SANA as the substrate according to the method described by Moon et al (4) with slight modifications. The release of p-nitroaniline was monitored for 15 min at 25°C by measuring the absorbance at 410 nm on a microplate reader. The reaction was performed in a mixture containing 0.2 M Tris-HCl buffer (pH 8.0), 10 µg/ml elastase, 5 mM SANA and the test sample (5 mg/ml) in a 96-well microplate. Each test sample was preincubated for 10 min at 25°C; the reaction commenced upon adding the substrate. The inhibition of elastase was calculated as follows: Inhibition (%) = [(A-B)/A] ×100, where A and B were the absorbance at 410 nm without and with the test sample, respectively.

The tyrosinase inhibition activity of BLE was determined by spectrophotometrically measuring the rate of dopachrome formation using L-DOPA as the substrate according to the method described by Choi et al (32) with slight modifications. Briefly, L-DOPA (15 mM; 40 µl), phosphate buffer (pH 6.8; 67 mM; 80 µl) and 40 µl of either the same buffer or the test sample (5 mg/ml) were added to each well of a 96-well microplate. Subsequently, 40 µl mushroom tyrosinase (125 units) was added to the reaction mixture. After incubation at 37°C for 30 min, this reaction mixture was evaluated for the formation of dopachrome using a microplate reader, by measuring the linear increase in the optical density at 492 nm. The tyrosinase inhibition was calculated as follows: Inhibition (%) = [(A-B)/A] ×100, where A and B were the absorbance at 492 nm without and with the test substance, respectively.

The results are presented as the mean ± standard deviation (SD) of triplicate experiments. The statistical difference between the results of different groups was analyzed using one-way analysis of variance and a post hoc Duncan's new multiple range test. SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

Four drying treatments for fresh burdock leaves were assessed; shadow drying, oven drying, sun drying and freeze-drying, and the total polyphenol content of the resulting BLEs was analyzed. As illustrated in Fig. 1, the total polyphenolic content following extraction with 95% EtOH was highest using the freeze-drying method, followed by the oven drying, shadow drying and sun drying methods. The polyphenolic content obtained from the freeze-drying method was significantly (~3 times) increased compared with that obtained through the other methods tested (P<0.05; Fig. 1). The effect of the concentration of ethanolic solvent used for extraction on the polyphenol content of BLE was investigated in the present study. As depicted in Fig. 2, freeze-drying the BLEs following extraction using 95% EtOH yielded a significantly lower polyphenolic content (GAE) compared with solvents extracted using 30 and 50% EtOH (P<0.05), indicating that an increase in the aqueous ratio results in a higher polyphenol content.

A DPPH-scavenging assay was used to measure the antioxidant capacity of the BLE obtained using different concentrations of EtOH as presented in Fig. 3. A similar effect was observed, with BLE extracted using 30 and 50% EtOH yielding a higher DPPH savaging ability compared with BLE extracted using 95% EtOH.

CHA is an antioxidant component of burdock leaves (26,27). As observed in Fig. 4, freeze-drying BLE following extraction using 50 and 95% EtOH resulted in a significantly lower CHA content compared with solvents extracted using 30% EtOH (P<0.05). This indicates that an increase in the ratio of ethanol negatively affects CHA extraction. The trend observed in the CHA content in the BLE using different concentrations of EtOH for extraction was only marginally similar to those for polyphenolic content and antioxidant activity. This suggests that polyphenolic components other than CHA contribute to polyphenolic content and antioxidant activity.

As depicted in Fig. 5, the BLEs produced following the use of 30, 50 and 95% EtOH exhibited a marked tyrosinase inhibitory effect (>25% inhibition). There was no significant difference in tyrosinase inhibition between the groups. In addition, the BLEs obtained using 30 and 50% EtOH notably inhibited elastase activity (>60%), and this inhibition was significantly increased, compared with that if the BLE obtained using 95% EtOH (P<0.05; Fig. 6). These results indicate that the antielastase activity of BLE is higher compared with its antityrosinase activity.