Microbial transformation can strengthen the antioxidant and antitumor activities of polyphenols. Polyphenols contents, antioxidant and antitumor activities of pine polyphenols and its biotransformation extracts by
The main ingredient of pine barks extracts is proanthocyanidins, followed by catechin-based flavonoids and phenolic acids. These proanthocyanidins and catechin-based flavonoids have high potential values of physiological activities, such as antioxidants (
The natural polyphenols have definite limitations in applications because of some shortcomings such as low bioavailability. The
Microorganisms for biotransformation mainly originate in fermented foods, such as fermented soy, wine and yoghurt. Lactic acid bacteria, bacillus, yeast and aspergillums are mainly representative microorganisms (
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Phenazine methosulphate (PMS), 3-(4,4-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2, 2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Nitrotetrazolium blue chloride (NBT) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma (St. Louis, MO, USA); Nicotinamide adenine dinucleotide (NADH) was purchased from Roche, Switzerland; 1 M Folin-Ciocalteu reagent was obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China); D101 macroporous resins were obtained from the Chemical Plant of Nankai University (Tianjin, China); Food grade 95% ethanol was purchased from a local reagents corporation.
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 (
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 (
Polyphenols content of the PPs and seven biotransformation extracts was determined by Folin-Ciocalteu method with some modifications (
According to the following formula, yield of polyphenols in seven biotransformation extracts was calculated.
Where
Trolox equivalent antioxidant capacity of the PPs and seven biotransformation extracts was carried out (
DPPH free radical scavenging activity of the PPs and seven biotransformation extracts was measured by means of the absorbance of DPPH at 517 nm (
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 (
The hydroxyl radical scavenging activity of the PPs and seven biotransformation extracts was investigated using Fenton’s reaction (Fe2+ + H2O2 → Fe3+ + OH − + OH−) (
The RP assay of the PPs and seven biotransformation extracts was conducted (
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.
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:
The samples were analyzed using UPLC-ESI-Q-TOF-MS (Agilent 6520 Accurate Mass Q-TOF/MS; Agilent, Santa Clara, CA, USA) in positive ion mode. Ions were generated using an electrospray ion source. The UPLC apparatus used (Waters Acquity; Waters, Bilford, MA, USA) consists of a binary pump, a quaternary pump, a solvent degasser, an autosampler and a thermostat column compartment. Samples were carried on a C18 column (Waters Acquity BEH C18, 50 mm × 2.1mm, 1.7 µm; Waters, Bilford, MA, USA) at a column temperature of 40 °C. The binary mobile phase consists of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The 13-min-long gradient was as follows: 0–2 min, 5–10% B linear, 2–4 min, 10–30% B linear, 4–8 min, 30–80% B linear, 8–10 min, 80–100% B linear, 10–11 min, 100% B isocratic, 11–12 min, 100–5% B linear, followed by 1 min of re-equilibration of the column before the next run. The flow rate was maintained at 0.25 mL/min.
The nitrogen pressure and flow rate on the nebulizer were 25 psi and 10 L.min−1, respectively, with a drying gas temperature of 330 °C. The capillary voltage was 4 kV. The scan range was set at m/z 50–1,500. The fragmentor voltage was fixed at 100 V. UPLC-ESI-TOF-MS data were acquired under positive ion mode using Mass Hunter (Agilent) software.
All tests were performed in triplicate and the results were presented as mean ± standard deviation (SD). Differences between mean values were compared by the Tukey
Polyphenols contents of the PPs and seven biotransformation extracts were investigated and illustrated in
Each value represents mean ± SD (
Yield of polyphenols in seven biotransformation extracts was investigated and shown in
Each value represents mean ± SD (
The TEAC of the PPs and seven biotransformation extracts was investigated and illustrated in
* represent value significantly different from the PPs (
DPPH free radical scavenging activities of the PPs and seven biotransformation extracts were investigated and shown in
Each value represents mean ± SD (
Fungi | EC50 (µg/mL) | RP0.5 (µg/mL) | ||
---|---|---|---|---|
DPPH⋅ | O |
OH⋅ | ||
PPs | 324.0 ± 13.52 | 117.1 ± 6.13 | 519.9 ± 25.91 | 363.2 ± 5.34 |
265.7 ± 8.33 |
84.2 ± 4.04 |
310.4 ± 9.72 |
232.2 ± 9.12 |
|
322.9 ± 10.41 | 122.3 ± 8.12 | 628.8 ± 36.94 | 231.6 ± 7.53 |
|
425.9 ± 15.84 | 292.5 ± 23.32 | 435.0 ± 26.94 | 297.7 ± 16.21 |
|
243.5 ± 6.16 |
97.0 ± 2.84 | 649.0 ± 32.65 | 594.1 ± 17.54 | |
226.6 ± 4.27 |
72.7 ± 3.06 |
367.7 ± 19.02 |
271.3 ± 10.67 |
|
213.3 ± 4.43 |
85.5 ± 3.97 |
633.9 ± 36.73 | 248.2 ± 13.42 |
|
390.5 ± 13.44 | 95.7 ± 3.02 | 381.5 ± 15.91 |
388.6 ± 5.29 |
Each value represents mean ± SD (
Represent value significantly different from the PPs (
Represent value very significantly different from the PPs (
EC50 (µg/mL) values are calculated from the regression lines using six different concentrations (50–1,200 µg/mL) in triplicate and their data are presented as 50% scavenging activity.
RP0.5 values are presented by the sample concentrations at 0.5 of absorbance value at 700 nm.
Superoxide anion radical scavenging activities of the PPs and seven biotransformation extracts were investigated and depicted in
Each value represents mean ± SD (
Hydroxyl radical scavenging activities of the PPs and seven biotransformation extracts were investigated and depicted in
Each value represents mean ± SD (
Reducing power assays of the PPs and seven biotransformation extracts were investigated and illustrated in
Each value represents mean ± SD (
Microbial fermentation can strengthen the food antioxidant activity. The biotransformation of green tea polyphenols by
The cytotoxicities of the PPs and seven biotransformation extracts against LoVo, HeLa-60 and BxPC-3 cell lines were evaluated. The PPs and seven biotransformation extracts inhibit tumor cells proliferation in a dose-dependent manner at concentrations of 0.1–0.6 mg/mL. The IC50 values were calculated and depicted in
Fungi | IC50 (µg/mL) | ||
---|---|---|---|
LoVo | Hela60 | BxPC-3 | |
PPs | 312.5 ± 15.33 | 348.0 ± 16.71 | 348.8 ± 15.91 |
306.9 ± 13.82 | 311.8 ± 12.82 | 297.0 ± 14.92 | |
301.6 ± 18.85 | 323.1 ± 16.14 | 370.8 ± 18.55 | |
474.2 ± 28.42 | 522.5 ± 29.53 | >600 | |
303.3 ± 13.91 | 558.4 ± 24.26 | >600 | |
285.0 ± 8.53 | 322.5 ± 14.64 | 452.1 ± 22.76 | |
258.8 ± 10.81 |
371.1 ± 19.78 | 363.9 ± 16.54 | |
346.1 ± 16.32 | >600 | 488.0 ± 21.93 |
Each value represents mean ± SD (
Represent value significantly different from the PPs (
IC50 (µg/mL) values are calculated from the regression lines using six different concentrations (100–600 µg/mL) in triplicate and their data are presented as 50% inhibitory activity.
Correlation coefficients of polyphenols contents, antioxidant and antitumor of the PPs and seven biotransformation extracts were investigated and the results were shown in
Antioxidant | Antitumor | |||||||
---|---|---|---|---|---|---|---|---|
TEAC | DPPH⋅ | O |
OH⋅ | RP | LoVo | Hela60 | BxPC-3 | |
Polyphenols | 0.9130 |
0.6843 |
0.7313 |
0.0001 | 0.0555 | 0.8021 |
0.5093 | 0.0427 |
Represent value significantly different (
Represent value very significantly different (
All components of the PPs and the BAC were analyzed by HPLC-ESI-TOF-MS. Postive mode was employed in MS detection.
In this study, there are eleven polyphenols identified in the PPs and the BAC, and the results showed that the main reactions acted in the PPs by
Aspergillus, Mucor and Ttrichoderma can express and secrete polyphenol oxidase, including laccase and tyrosinase, to the outside of the cell (
Microbial biotransformation of the PPs can affect antioxidant and antitumor activities against human cancer cells. Microbial biotransformation is a new drug discovery with low pollution, low by-products, and low toxic effects. Fungi can be directly used for the biotransformation of polyphenols, screening for functional products. In this study,
Pine polyphenols
Biotransformation extract of
Biotransformation extract of
Biotransformation extract of
Biotransformation extract of
Biotransformation extract of
Biotransformation extract of
Biotransformation extract of
2,2-Diphenyl-1-picrylhydrazyl
3-(4,4-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
2, 2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
We would like to thank Dr Tao Yuan for the help with microbial cultures.
The authors declare there are no competing interests.
The following information was supplied regarding data availability:
The raw data has been supplied as a