Comparison in antioxidant and antitumor activities of pine polyphenols and its seven biotransformation extracts by fungi

Preparation of pine polyphenols

Because of annual and renewable availability, pine cones (without pine nuts) were applied in the study. Pine polyphenols (PPs) were perpetrated according to our previous method (Li & Wang, 2015). In order to increase the purity of polyphenols, the chromatographic column of D101 macroporous resins was used to enrich polyphenols again. 40% (v/v) ethanol solution was used to elute polyphenols. Finally, a 57.7% purity of polyphenols was achieved.

Strains used

Aspergillus niger, Aspergillus oryzae, Aspergillus carbonarius, Aspergillus candidus, Trichodermas viride, Mucor wutungkiao and Rhizopus sp were preserved in our laboratory. Fungal strains were inoculated on Potato Dextrose Agar (PDA), composed of (g/L) peeled potatoes 200, dextrose 20, and agar 15, incubated at 28 °C in darkness. To obtain pure cultures, mycelium was repeatedly transferred onto plates with Basal Medium (BM, g/L) (Contreras-Domínguez et al., 2006) composed of dextrose 20, (NH₄)₂SO₄ 3, KH₂PO₄ 1.3, Na₂HPO4 0.12, MgSO₄⋅5H₂O 0.3, CaCl₂ 0.02 and agar 15.

Biotransformation conditions and products enriched

The liquid medium employed in biotransformation was BM broth, tween-80 (0.1%, v/v) and trace elements (1 mL/L). Trace elements are composed of (g/L) FeSO₄⋅7H₂O 5, ZnSO₄⋅7H₂O 1.4, MnSO₄⋅H₂O 1.6, CuSO₄⋅5H₂O 0.3 and CoCl₂⋅6H₂O 3.7, which were adjusted to pH 6.0 with 6 N HCl before autoclaving. Media was sterilized by autoclaving at 121 °C for 20 min. Microorganisms were cultivated in two stages in a medium (Sanchez-Gonzalez & Rosazza, 2006). The first phase cultivation was used to start the second phase. In first phase, 10⁷ fungal spores were administered to 500 mL conical flasks with 200 mL liquid media, and grew for 48 h at 28 °C, 150 rpm in a SHA-C incubator (Jiangsu, China). Then, in the second phase, 10 mL of concentrated sterile polyphenols solution was added to cultivation to a final medium concentration of 0.4 mg/mL, and continued to cultivate for 48 h at 28 °C, 150 rpm. Finally, filter paper was used to filter medium to remove mycelia. The final liquid was the biotransformation liquid. Biotransformation liquid or cultures without polyphenols was loaded into the D101 macroporous resin chromatographic column; 60% (v/v) ethanol solution eluted products. The elution of 2 BV was gathered and concentrated by removing the ethanol using a rotary evaporator device (RE52AA; Shanghai Huxi Instrument Co., Changsha, China) at 50 °C. Finally, biotransformation extracts were freeze-dried by vacuum (Zirbus, Bad Grund, Germany) in −50 °C  for 24–48 h.

Determination of polyphenols content

Polyphenols content of the PPs and seven biotransformation extracts was determined by Folin-Ciocalteu method with some modifications (Lin et al., 2012a; Lin et al., 2012b). Sample solution of 0.5 mL was transferred to a 10 mL test tube, to which 0.5 mL of 1 M Folin-Ciocalteu reagent was added. After 5 min, 1.0 mL of 10% (w/v) Na₂CO₃ was added and the volume was added to 3 ml with deionized water. After 2 h of incubation at room temperature, the absorbance was measured at 760 nm in a SPECORD^®200 Plus UV/VIS Spectrophotometers. Water (for PPs) or cultures without polyphenols (for biotransformation extract, respectively) were used the blank. Polyphenols content was then expressed as mg of gallic acid equivalents/mg of samples. The regression line was y = 0.01235x + 0.01302 (R² = 0.9994, n = 6), where y is OD_760nm of gallic acid and x is gallic acid concentration (0–50 µg/mL).

Determination of yield of polyphenols in biotransformation extract

According to the following formula, yield of polyphenols in seven biotransformation extracts was calculated.

Y  = Polyphenols content in biotransformation extracts/Polyphenols content before biotransformation 100%.

Where Y is yield of polyphenols in biotransformation extract.

Trolox equivalent antioxidant capacity (TEAC)

Trolox equivalent antioxidant capacity of the PPs and seven biotransformation extracts was carried out (Wang et al., 2013). Briefly, an ABTS solution (7 mM) was mixed with potassium persulfate (140 mM) with a ratio of 62.5:1 for 16 h in darkness at room temperature to produce ABTS radical cation (ABTS^⋅+) stock solutions. The ABTS^⋅+ stock solution was diluted to an absorbance of 0.70 ± 0.05 at 734 nm as a working solution. An aliquot (5 µL) of aqueous solution containing 1 mg/mL sample was mixed thoroughly with the ABTS^⋅+ solution (200 µL) and after 6 min in darkness at room temperature; the absorbance was read at 734 nm in a gene 5 microplate reader. Different levels (0, 7.8125, 15.625, 31.25, 62.5, 125, 250, and 500 µg/mL) of Trolox standard solutions were prepared and assayed under the same condition. Water (for PPs) or cultures without polyphenols (for biotransformation extract, respectively) were used the blank. Results were taken into account in Trolox equivalent, i.e., µg Trolox/mg samples. The regression line was y = 0.0853x − 0.3306 (R² = 0.9992, n = 6), where y is ABTS^⋅+ radical scavenging activity and x is Trolox concentration (µg/mL).

DPPH free radical scavenging activity

DPPH free radical scavenging activity of the PPs and seven biotransformation extracts was measured by means of the absorbance of DPPH at 517 nm (Xia et al., 2014). Briefly, 1.0 mL sample solution was added to 1.0 mL of DPPH solution (0.2 mM in methanol). The decrease in the solution absorbance at 517 nm was measured after 30 min of incubation. Water (for PPs) or cultures without polyphenols (for biotransformation extract, respectively) were used the blank. DPPH free radical scavenging activity was calculated using the following formula: ${\displaystyle \text{Scavenging activity}\left(\text{\%}\right)=\left(1-A_1∕A_0\right)\times 100}$ where A₀ is the absorbance of DPPH free radicals without sample, and A₁ is the absorbance of DPPH free radicals with samples. The efficient concentration of samples that scavenged 50% of DPPH free radical (EC₅₀) was calculated and expressed as µg/mL.

Superoxide anion radical scavenging activity

The superoxide anion radical scavenging activity of the PPs and seven biotransformation extracts was established by monitoring the competition of those with NBT for the superoxide anion generated by the PMS–NADH system (Biswas, Chatli & Sahoo, 2012). The reduction mixture contained 150 µL NBT (100 µM), 450 µL NADH (100 µM) and a sample solution 200 µL. Total volume was made up to 1 mL with distilled water and then 1.9 mL of Tris–HCl buffer (0.02 M, pH 8.0) was added. The reaction was started by adding 100 µL of PMS (100 µM) and then the change in absorbance (A) was recorded at 560 nm after 1 min at 37 °C. Water (for PPs) or cultures without polyphenols (for biotransformation extract, respectively) were used the blank. The superoxide anion free radical scavenging activity was calculated with the following equation: ${\displaystyle \text{Scavenging activity}\left(\text{\%}\right)=\left(1-A_1∕A_0\right)\times 100}$ where A₀ is the absorbance of superoxide anion radicals without sample; and A₁ is the absorbance of superoxide anion radicals with sample. The efficient concentration of samples that scavenged 50% of the superoxide anion radicals (EC₅₀) was calculated and expressed as µg/mL.

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity of the PPs and seven biotransformation extracts was investigated using Fenton’s reaction (Fe²⁺ + H₂O₂ → Fe³⁺ + OH −  + OH⁻) (Jiang, Chen & Shi, 2013). The reaction mixture containing 1 mL sample solution was incubated at 37 °C  for 2 h with 1 mL of 9 mM salicylic acids, 1 mL of 9 mM FeSO₄, and 1 mL of 8.8 mM H₂O₂, and then the absorbance was read at 510 nm. Water (for PPs) or cultures without polyphenols (for biotransformation extract, respectively) were used the blank. The hydroxyl radical scavenging activity was calculated from the following equation: ${\displaystyle \text{Scavenging activity \%}=\left[1-\left(A_{\mathrm{i}}-A_{\mathrm{j}}\right)∕A_0\right]\times 100}$ where A₀ is the absorbance of the hydroxyl radical with a treated control. A_i and A_j are the absorbances of the hydroxyl radical with the treated sample, and the absorbance of the hydroxyl radical with the non-treated sample. The efficient concentration of samples that scavenged 50% of the hydroxyl radicals (EC₅₀) was calculated and expressed as µg/mL.

Reducing power (RP) assay

The RP assay of the PPs and seven biotransformation extracts was conducted (Seo et al., 2012). A freshly prepared sodium phosphate buffer (1.0 mL, 0.2 M, pH 6.6) and 1% K₃Fe (CN)₆ (1.0 mL) were added to the sample solution (1.0 mL). After incubating the mixtures at 50 °C for 20 min, 10% trichloroacetic acid (1.0 mL) was added. The resulting mixtures were centrifuged at 3,000 rpm for 10 min at 4 °C. The upper layer (1.0 mL) was diluted with water (1.0 mL), and 0.1 % FeCl₃ (0.5 mL) was hereby added. The absorbance of the resultant solution was measured at 700 nm. Water (for PPs) or cultures without polyphenols (for biotransformation extract, respectively) were used the blank. The efficient concentration of samples that were of 0.5 absorbance value (RP_0.5) was calculated and expressed as µg/mL.

Cell lines and culture conditions

LoVo (human colon adenocarcinoma cell), HeLa 60 (human cervical carcinoma cell) and BxPC-3 (human pancreatic carcinoma cell) cells were preserved in our laboratory and were cultured in RPMI 1640 medium (HyClone, San Angelo, TX, USA), supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin at 37 °C and 5% CO₂ in a incubator.

MTT assay

Cells were plated in a 96-well plate at a density of 5× 10³ cells/well and then treated with samples at various concentrations (0.l, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL). PBS (for PPs) or cultures without polyphenols (for biotransformation extract, respectively) were used the blank. 48 h later, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well and placed in an incubator for 4 h. Then, the supernatant was removed, and the formazone crystals were dissolved using DMSO. The absorbance was then measured using a microplate reader at a wavelength of 490 nm. The results were expressed as a percentage (%) of inhibition rate calculated with the following equation: ${\displaystyle \text{Inhibition rate}\left(\text{\%}\right)=\left(1-A_1∕A_0\right)\times 100}$ where A₀ is the absorbance without sample; and A₁ is the absorbance with sample. The efficient concentration of samples that inhibited 50% of cells (IC₅₀) was calculated and expressed as µg/mL.