2,2-Diphenyl-1-picryl-hydrazyl (DPPH), Folin-Ciocalteu reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), propidium iodide (PI), RPMI-1640 medium, fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The SOD assay kit-WST, Cell counting kit-8 and Hoechst 33258 solution were purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Gallic acid was purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

The DLD-1 human colon cancer cell line was obtained from the Cell Resource Center for Biomedical Research, Aging and Cancer, Tohoku University, Japan. It was grown in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin. The culture was maintained at 37°C in a humidified 5% CO₂ atmosphere (ESPEC CO₂ Incubator). The cells were cultured for 2–3 days to reach the logarithmic phase and were then used for the experiment.

The roots of Actinidia kolomikta were collected from the northern part of Indochina. The scheme of the extraction procedure is presented in Fig. 1. For the high temperature extraction, the roots were extracted with distilled water at 100°C for 1 h at a ratio of 1:10 (w/v). The operation was repeated twice. The aqueous extract was centrifuged at 6,000 rpm for 15 min and filtered through a filter paper (GF/A, 47 mm; Whatman, UK). The filtrate was concentrated and lyophilized to obtain the water extraction (WE) fraction. The WE fraction was redissolved in distilled water and was added to four volumes of 99.5% ethanol and then stored at 4°C overnight for precipitation. The precipitate and supernatant were separated and collected by centrifugation at 8,000 rpm for 15 min. The supernatant was concentrated and lyophilized to obtain the water extraction-ethanol supernatant (WE-ES) fraction. The precipitate was washed with 80% ethanol and dissolved in distilled water. After the centrifugation, the supernatant was concentrated and lyophilized to obtain the water extraction-ethanol precipitate (WE-EP) fraction.

The low temperature extraction operation was the same as the high temperature treatment, but distilled water at 40°C was used.

The content of total carbohydrates in the extracts was determined by the phenol-sulfuric acid method (11,12), with glucose as a standard. First, 1 ml of the extraction fraction was collected in glass tubes and 1 ml of 5% phenol solution and 5 ml of concentrated sulphuric acid were added. After mixing for 2 min at room temperature, the tubes were boiled at 100°C for 15 min in a hot water bath. Subsequently, the absorbance was read at 486 nm with a spectrophotometer (HACH, DR/4000 U).

The total polyphenol content was determined using the Folin-Ciocalteu method (3). A volume of 7.9 ml distilled water, 0.1 ml extract fraction and 0.5 ml Folin-Ciocalteu reagent (1:1 with distilled water) were added and mixed in a tube for 1 min. Then, 1.5 ml of sodium carbonate (20 g/100 ml) was added, mixed and allowed to stand for 2 h at room temperature in the dark. The absorbance was read at 765 nm. The total polyphenol content was determined using gallic acid as a standard.

The levels of SOD-like activity in the extracts were measured using the SOD assay kit-WST according to the technical manual provided by Dojindo Molecular Technologies. Briefly, in a 96-well plate, sample solution (20 μl) was added to each sample and blank 2-well, and 20 μl double-distilled water was added to each blank 1-and blank 3-well. Then, 200 μl of WST working solution was added to each well. After mixing, 20 μl of dilution buffer was added to each blank 2- and blank 3-well, and 20 μl of enzyme working solution was added to each sample- and blank 1-well. The plate was incubated at 37°C for 20 min, and the optical density (OD) was determined at 450 nm, using a microplate reader (Model 550; BioRad, USA). The SOD-like activity was calculated using the following equation: SOD activity (inhibition rate %) = {[(A_blank1-A_blank3)-(A_sample-A_blank2)]/(A_blank1-A_blank3)} × 100.

A_blank1, A_blank2, A_blank3 and A_sample are the absorbances of blank 1-, blank 2-, blank 3- and sample-wells. The SOD activity (1 unit) was defined as the amount of enzyme having a 50% inhibitory effect on WST-1.

The DPPH scavenging activity was measured according to Nakajima et al (13) and Yang et al (14). Volumes of 80 μl of 0.5 mM DPPH in MeOH solutions and 80 μl of 0.1 M MES buffer in 50% MeOH (pH 6.0) were added to a 96-well plate, and an aliquot of sample solution was added for a total volume of 200 μl. After 10 min of reaction, the OD was measured at 570 nm with a microplate reader. The DPPH radical-scavenging activity was calculated using the following equation: DPPH-scavenging activity (%) = (1-A_sample/A_control) × 100.

The scavenging activity of the sample is expressed as a 50% effective concentration (EC₅₀), which represents the sample concentration (μg/ml) inhibiting 50% of the DPPH radical activity.

The DLD-1 cells were grown in RPMI-1640 medium at 37°C in a 5% CO₂ atmosphere to logarithmic phase. The cells were harvested, and an aliquot (100 μl) of the DLD-1 cell suspension (5×10⁴ cells/ml) was dispensed into a 96-well plate and pre-incubated at 37°C in a 5% CO₂ atmosphere for 24 h. The cells were then exposed to various concentrations (25, 50, 100, 200 and 400 μg/ml) of the extracts for 12, 24 and 48 h. After drug exposure, 10 μl of Cell Counting reagent solution was added, and incubation was carried out at 37°C for 4 h. The cell numbers were quantitated by reading the OD at 450 nm.

The flow cytometric assay was performed according to Zhang et al (15) with some modifications. DLD-1 cells (1×10⁵ cells/ml) were incubated in a 6-well plate with the extracts at concentrations of 25 and 100 μg/ ml for 48 h. The cells were harvested and washed with cold PBS (-) and then fixed in 70% ethanol at 4°C for 4 h. Cells were strained with PI solution (20 μg/ml) at 4°C for 30 min. DNA histograms were generated by flow cytometry (BD LSR; BD Biosciences). Data from 10,000 cells per sample were collected, and the percentage of apoptotic cells was obtained with the CellQuest software (Becton Dickinson).

DLD-1 cells (1×10⁵/ml) were incubated in 6-well plate with the extracts at concentrations of 25 and 100 μg/ml for 48 h. The cells were subsequently harvested and stained with Hoechst 33258 (10 μg/ml) for 10 min. Nuclear morphological changes were observed using a Leica fluorescence microscope.

Experiments were conducted in triplicate, and the results were expressed as the mean ± SD. Statistical significance was calculated by a two-tailed Student’s t-test (16). p<0.05 was considered significant.

The extraction yields are shown in Fig. 2. Yields of WE, WE-EP and WE-ES produced using the 100°C procedure were 10.9, 5.89 and 3.35%, while the yields of the fractions produced using the 40°C procedure reached only 5.53, 3.28 and 2.5%. Thus, the extraction yields produced using the 100°C procedure were higher than those produced using the 40°C extraction procedure.

The total carbohydrate contents are shown in Fig. 3. The total carbohydrate contents of the WE and WE-EP fractions produced using the 100°C extraction procedure were 249.6 and 226.6 mg/g, respectively, which were significantly higher than the total carbohydrate contents of WE (175.1 mg/g) and WE-EP (183.0 mg/g) produced using the 40°C extraction procedure. The results also revealed that the total carbohydrate content in the WE-EP fraction was higher than that in the WE-ES fraction.

The total polyphenol contents are shown in Fig. 4. The polyphenol contents of the WE, WE-EP and WE-ES fractions produced using the 100°C extraction procedure were higher than the polyphenol contents of the fractions produced using 40°C. Moreover, the polyphenol contents of WE and WE-ES produced using the 100°C extraction procedure were significantly higher than those of the extracts produced using the 40°C extraction procedure (p<0.05). The polyphenol content of the WE-ES fraction was higher than that of WE-EP. Data revealed that the polyphenol content of WE-ES produced using the 100°C treatment consisted of 201.9 mg gallic acid/g, which was the highest content compared to all of the other fractions.

As shown in Fig. 5, compared to the WE and WE-EP fractions, the WE-ES fraction exhibited a higher antioxidant activity; the SOD-like activities of the WE-ES fraction produced using the 100°C and 40°C extraction procedures were 790.3 and 742.6 U/mg, respectively, while the WE-EP fraction exhibited the lowest SOD-like activities (377.9 and 374.1 U/mg, respectively, for the WE-EP fractions produced using the 100°C and 40°C extraction procedures).

The DPPH radical-scavenging activity is shown in Fig. 6. The positive control Trolox revealed the strongest DPPH radical-scavenging activity (EC₅₀, 31.7 μg/ml), and the WE-ES fraction produced using the 100°C extraction procedure had the second strongest radical-scavenging activity (EC₅₀, 87.4 μg/ml). Moreover, all of the fractions (WE, WE-EP and WE-ES) produced using the 100°C extraction procedure revealed significant higher DPPD radical-scavenging activities compared to the fractions produced at 40°C (p<0.05).

The WE-EP and WE-ES fractions were used for the experiment of anti-proliferation on the DLD-1 cells (Fig. 7). In this experiment, the extracts at various concentrations (25, 50, 100, 200 and 400 μg/ml) were used. The results revealed that, the higher the concentration of the extract, the lower the cell survival rate. Treatment at the concentration of 400 μg/ml revealed the strongest inhibitory effect on the DLD-1 cells. This suggests that the WE-EP and WE-ES fractions (produced using the 100°C and 40°C extraction procedures) exhibited anti-proliferative effects on DLD-1 cells in a dose-dependent manner. Moreover, the WE-ES fraction inhibited DLD-1 cell proliferation in a time-dependent manner, compared to WE-EP, for which a slight recovery of DLD-1 cell proliferation at 48 h was noted. When DLD-1 cells were treated with the WE-EP and WE-ES fractions for 12 h, a decrease in DLD-1 cell proliferation was noted. However, after the 48-h treatment, the cell survival rates increased in the DLD-1 cells exposed to WE-EP. For example, when the DLD-1 cells were treated with the extracts at the concentration of 400 μg/ml for 24 h, the cell survival rates induced by WE-EP and WE-ES (produced using the 100°C extraction procedure) were 30.7 and 27.0%, respectively. At 48 h, the cell survival rates were 36.5 and 17.35%, respectively. The same trend was observed when cells were treated with fractions produced using the 40°C extraction procedure. Based on these results, the anti-proliferative effect of the WE-ES fraction was stronger than that of WE-EP.

Results of the apoptosis assay are shown in Fig. 8. When the DLD-1 cells were treated with the WE-EP fraction (produced using the 100°C extraction procedure) at concentrations of 25 and 100 μg/ml for 48 h, the percentage of apoptotic cells decreased from 1.24 to 0.8% while the percentage increased from 0.94 to 1.72% upon treatment of WE-EP produced using the 40°C extraction procedure). These data did not reveal a significant increase. In comparison with the WE-EP treatment, when DLD-1 cells were exposed to WE-ES (produced using the 100°C extraction procedure) at concentrations of 25 and 100 μg/ml for 48 h, the percentage of apoptotic cells increased from 3.26 to 25.68% while the percentage of apoptotic cells increased from 6.09 to 24.25% when treated with WE-ES produced at 40°C. This suggests that WE-ES induces DLD-1 cell apoptosis in a dose-dependent manner.

The criteria used to identify pro-apoptosis includes nuclear shrinkage and chromatin condensation (17). As shown in Fig. 9, nuclear fragmentation was clearly noted when the DLD-1 cells were exposed to WE-ES (25 and 100 μg/ml) for 48 h, while upon treatment with WE-EP at 25 and 100 μg/ml for 48 h, apoptotic cells were hardly observed.