Reactive oxygen species (ROS), including hydroxyl radicals, hydrogen peroxide, superoxide radicals, and nitrite oxide, are produced as a consequence of the normal metabolism of living organisms . ROS can readily react with most cellular biomolecules, including carbohydrates, proteins, lipids, and DNA, thus causing tissue damage or cell death . Because removing ROS is important for the protection of living systems, antioxidant agents that can slow or prevent the oxidation process by removing free radical intermediates are needed. However, several strong synthetic antioxidants have been shown to be highly toxic [3,16]. Therefore, the antioxidant activity of natural product extracts has been studied by measuring the free radical scavenging activity.
Undaria pinnatifida, commonly called “miyeok” in Korea and “wakame” in Japan, is a common edible brown seaweed, which is plentiful on the shores of the Korean peninsula and Japan and along some regions of the coastlines of Australia and New Zealand . In Oriental medicine, U. pinnatifida has been used for blood purification, intestinal strength, and menstrual regularity . In addition, in Korea, it is popularly consumed by post-partum women, as it contains a high content of calcium and iodine, important nutrients for nursing mothers .
The Cordyceps species, which encompasses many Chinese medicinal mushrooms, are entomopathogenic fungi belonging to the Clavicipitaceae and Ascomycotina. The major bioactive compound of C. militaris is cordycepin, which displays many biological and pharmacological activities, such as immunological stimulating, antivirus, and anticancer activities [1,5,7,22,28].
In this study, we fermented U. pinnatifida using C. militaris mycelia to examine changes in bioactivity. In addition, we performed radical scavenging activity assays to investigate the antioxidant activity of U. pinnatifida fermented with C. militaris mycelia (FUCM). Various assays have been used to examine the antioxidant properties of natural compounds, including DPPH, hydroxyl and alkyl radical scavenging activity, ABTS radical scavenging activity, and ferric reducing antioxidant power (FRAP).
We found that FUCM displayed a higher bioactivity than either U. pinnatifida or C. militaris mycelia alone. These findings indicate that FUCM may potentially be used as a dietary supplement.
Materials and Methods
U. pinnatifida was obtained from a local market (Wando, Korea). C. militaris was generously provided by Dr. Jeong. A voucher specimen (UT-13-011) has been deposited at the Korea National University of Transportation, Chungju, Korea. We obtained 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 2,2-azobis(2-amidinopropane) hydrochloride (AAPH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of the highest commercially available grade.
Preparation of Fermented U. pinnatifida Extract
U. pinnatifida was washed 2–3 times with tap water and drained after the residual saltiness was checked with an Electro Dialyzer (Changjo, Seoul, Korea). Oryza sativa was immersed in water for 12 h and drained. U. pinnatifida with 5% of O. sativa was sterilized and inoculated with C. militaris mycelia. These were cultured at 25℃ for approximately 20 days. To prepare extracts, fermented U. pinnatifida was boiled for 2 h in 10 volumes of distilled water. The extracts were filtered with No. 41 paper, evaporated, and lyophilized. FUCM extracts were vacuum dried and stored at -20℃ until used. The extraction yields from FUCM are shown in Table 1.
Table 1.FUCM (fermented U. pinnatifida with C. militaris mycelia), ORAC (oxygen radical absorbance capacity). Values represent means ± SD (n = 3). Means marked with the same letter are not significantly different (p > 0.05).
Various Radical Scavenging Activities Measured by Electron Spin Resonance (ESR)
DPPH radical scavenging activity. DPPH radical scavenging activity was measured as described by Nanjo et al. . Briefly, 60 µl of each sample at various concentrations was added to 60 µl of DPPH (60 µM) in a methanol solution. After the solution was mixed vigorously for 10 sec, it was transferred into a 100 µl Teflon capillary tube, and the scavenging activity of each extract, with regard to DPPH radicals, was measured using an ESR spectrometer. The spin adduct was measured by the ESR spectrometer exactly 2 min later. The experimental conditions were as follows: central field, 3475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave power, 5 mW; gain, 6.3 × 105 ; and temperature, 298 K.
Hydroxyl radical scavenging activity. Hydroxyl radicals were generated by an iron-catalyzed Haber-Weiss reaction (i.e., a Fenton-driven Haber-Weiss reaction), and the generated hydroxyl radicals rapidly reacted with nitrone spin-trap DMPO. The resultant DMPO-OH adduct was detected using an ESR spectrometer. Briefly, 0.2 ml of each sample at various concentrations was mixed with 0.2 ml of DMPO (0.3 M), 0.2 ml of FeSO4 (10 mM), and 0.2 ml of H2O2 (10 mM) in a phosphate buffer solution (pH 7.2), and the resulting solution was transferred into a 100 µl Teflon capillary tube. After 2.5 min, the ESR spectrum was recorded on a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The experimental conditions were as follows: central field, 3,475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave power, 1 mW; gain, 6.3 × 105 ; and temperature, 298 K.
Alkyl radical scavenging activity. Alkyl radicals were generated by AAPH. The PBS (pH 7.4) reaction mixtures, which contained 0.1 ml of 10 mM AAPH, 0.1 ml of 10 mM 4-POBN, and 0.1 ml of the indicated concentrations of the tested samples, were incubated at 37℃ in a water bath for 30 min. Th e samples were th en transferred to 100 µl Teflon capillary tubes. The spin adduct was recorded on an ESR spectrometer. The measurement conditions were as follows: central field, 3,475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave power, 1 mW; gain, 6.3 × 105 ; and temperature, 298 K.
Ferric Reducing Antioxidant Power Assay
To measure the antioxidant capacity of the different FUCM extracts, the FRAP method was performed as described previously .In these experiments, a 3 ml aliquot of the FRAP reagent, a mixture of 0.3 M acetate buffer, 10 mM TPTZ in 40 mM HCl, and 20 mM ferric chloride (10:1:1 (v/v/v)) were combined with 1 ml of the FUCM extract. To determine the antioxidant capacity of the samples, the absorbance values were compared with those obtained from the standard curves of FeSO4 (0–10 mM). The antioxidant capacity values were expressed as mM FeSO4 equivalent per mg extract (mM FeSO4 eq./mg extract).
ABTS Radical Scavenging Activity
The total antioxidant activities of the FUCM extract were measured using the ABTS.+ radical cation decolorization assay . ABTS was dissolved in water to a concentration of 7 mM, and the ABTS radical cation (ABTS.+ ) was produced from the ABTS stock solution with the addition of 2.45 mM potassium persulfate (K2S2O8 ). The mixture was then incubated at RT in the dark for 14 h. To determine the scavenging activity, 0.9 ml of ABTS reagent was mixed with 0.1 ml of extracts and the absorbance was measured at 734 nm. The antioxidant activities of the extracts were expressed by Trolox equivalents antioxidant capacity (TEAC), as mM Trolox equivalents/mg extract.
Oxygen Radical Absorption Capacity (ORAC) Assay
The ORAC assay was based on a modified method of Ou et al. . Samples and Trolox solutions were made in 75 mM phosphate buffer (pH 7.4). Fifty microliters of blank, Trolox standard, or extract was mixed with 50 µl of fluorescein (7.8 µM) solution and incubated for 15 min at 37ºC; each condition was performed in triplicate. Upon injection of 25 µl of 221 mM AAPH, the fluorescence was measured every 5 min for about 120 min (excitation wavelength 485 nm, emission wavelength 535 nm) using a fluorescence microplate reader (SpectraMax M2/M2e, CA, USA). The final ORAC values of the samples were calculated using the net area under the decay curves (AUC) and were expressed as µmol Trolox equivalent (TE) per milligram extract (µmol TE/mg extract).
For analysis of alginate, U. pinnatifida, C. militaris mycelia, and FUCM extract were subjected to the analytical HPLC (Ultimate 3000, Thermo Scientific) system. The YMC-Pack Ph column (5 µm, 250 mm × 4.6 mm) was operated in the system (injection volume 20 µl) at a flow rate of 0.7 ml/min and detection wavelength of 200 nm. The mobile phase was 0.05% phosphoric acid in water (pH 7.0) for 15 min. Standard alginate was purchased from Sigma-Aldrich for the comparative analytical and experimental studies. For analysis of cordycepin, U. pinnatifida, C. militaris mycelia, and FUCM extract were subjected to an analytical HPLC system equipped with a UV detector. An Inspire C18 (5 µm, 250 mm × 4.6 mm) column was used. Elution was performed at a flow rate of 0.8 ml/min, using water (A) and meth anol (B) as mobile ph ases. The solvent gradient changed according to the following condition: a linear gradient of 17% (B) in 0-30 min, and 17-100% (B) in 30-40 min. Chromatograms were acquired at UV 260 nm. Standard cordycepin was purchased from Sigma-Aldrich for the comparative analytical and experimental studies.
Data are reported as the mean ± standard deviation for triplicate determinations. Analysis of variance (ANOVA), accompanied by Tukey’s tests (GraphPad Prism 5), was conducted to identify the significant differences between samples (p < 0.05).
Extraction Yields of U. pinnatifida, C. militaris Mycelia, and FUCM Extracts
Twenty days after inoculation, we confirmed the fermentation product by visual observation. We found that C. militaris mycelia grew thoroughly on the interior of the product (Fig. 1). The yields of the U. pinnatifida, C. militaris mycelia, and FUCM extracts were 15.22%, 2.15%, and 51.40%, respectively (Table 1). FUCM extracts showed much higher extraction yields than the U. pinnatifida or C. militaris mycelia extracts.
Fig. 1.Observation of the exterior (A) and interior (B) of fermented U. pinnatifida with C. militaris mycelia.
Radical Scavenging Activity Assessed by ESR Measurement
DPPH is a stable free radical and has been widely used as a tool to measure the free radical scavenging activities of antioxidants . The FUCM extracts scavenged DPPH radicals in a dose-dependent manner (Fig. 2). As previously determined, the lower the IC50 value, the higher the antioxidant capacity. The FUCM extracts (IC50 , 0.022 ± 0.002 mg/ml) showed higher DPPH radical scavenging activities than U. pinnatifida (IC50 , 0.773 ± 0.032 mg/ml) or C. militaris mycelia (IC50 , 0.318 ± 0.021 mg/ml) (Table 1). The FUCM extracts were significantly (p < 0.05) increased up to 35 times that of U. pinnatifida extracts on DPPH radical scavenging activities.
Fig. 2.DPPH radical scavenging activity of FUCM extracts (A) and ESR spectra (B). The mean ± SD is shown for triplicate experiments.
The alkyl radical spin adduct of the 4-POBN/free radical was generated from AAPH, and a decrease in ESR signals was observed with an increase in the FUCM extract dose (Fig. 3). The FUCM extracts (IC50 , 0.023 ± 0.001 mg/ml) showed higher alkyl radical scavenging activities than U. pinnatifida (IC50 , 0.238 ± 0.003 mg/ml) or C. militaris mycelia (IC50 , 0.372 ± 0.011 mg/ml) (Table 1). The FUCM extracts showed significantly (p < 0.05) increased alkyl radical scavenging activities up to 16 times that of C. militaris mycelia extracts.
Fig. 3.Alkyl radical scavenging activity of FUCM extracts (A) and ESR spectra (B). The mean ± SD is shown for triplicate experiments.
Hydroxyl radicals generated in the Fe2+ /H2O2 system can be trapped by a DMPO forming spin adduct, detectable by an ESR spectrometer (Fig. 4). The FUCM extracts (IC50 , 0.475 ± 0.018mg/ml) showed higher hydroxyl radical scavenging activities than U. pinnatifida (IC50 , 7.752 ± 0.221 mg/ml) or C. militaris mycelia (IC50 , 0.515 ± 0.016 mg/ml) (Table 1). Hydroxyl radical scavenging activities of FUCM extracts were significantly (p < 0.05) increased to up to 16 times that of U. pinnatifida extracts.
Fig. 4.Hydroxyl radical scavenging activity of FUCM extracts (A) and ESR spectra (B). The mean ± SD is shown for triplicate experiments.
These combined results demonstrate that the FUCM extracts had an obvious effect on various radical scavenging activities, and the scavenging activity was higher than that of the original samples.
Ferric Reducing Antioxidant Power Assay
The FRAP assay measures the antioxidant effects of any substance in the reaction medium based on its reducing ability. The reducing properties are generally associated with the presence of reducing agents, which exert antioxidant activity by donating a hydrogen atom and, therefore, breaking the free radical chain reaction . The FRAP value of the extract increased in a dose-dependent manner (Fig. 5). The results of this assay showed that the FUCM extract (0.443 ± 0.052 mM FeSO4 eq./mg extract) had a 4-times higher FRAP value than U. pinnatifida extracts (0.125 ± 0.040 mM FeSO4 eq./mg extract) or C. militaris mycelia extracts (0.109 ± 0.010 mM FeSO4 eq./mg extract) at 5.0 mg/ml. FUCM extracts exhibited a significant (p < 0.05) FRAP value, compared with U. pinnatifida extracts or C. militaris mycelia extracts. Our results show that FUCM extracts had a much higher antioxidant ability after fermentation.
Fig. 5.Antioxidant activity by FRAP value. The FRAP values were expressed as mM FeSO4 equivalent per mg extract. Data are presented as the mean ± SD (n = 3) with one way ANOVA followed by Tukey’s test. Different letters represent statistically significant differences (p < 0.05).
ABTS Radical Scavenging Activity
The scavenging activity of the sample on ABTS radicals generated by potassium persulfate was compared with a standard amount of Trolox. The TEAC value of the FUCM extract (0.396 ± 0.002 mM Trolox eq./mg extract) was similar to that of the U. pinnatifida extracts (0.413 ± 0.006 mM Trolox eq./mg extract) and higher than that of C. militaris extracts (0.204 ± 0.002 mM Trolox eq./mg extract) at 5.0 mg/ml (Fig. 6).
Fig. 6.Antioxidant activity by ABTS radical scavenging. TEAC values were expressed as mM Trolox equivalent per mg extract. Data are presented as the mean ± SD (n = 3) with one way ANOVA followed by Tukey’s test. Different letters represent statistically significant differences (p < 0.05).
Oxygen Radical Absorbance Capacity Assay
On the other hand, the ORAC method is based on hydrogen atom transfer reactions, which scavenge peroxyl radicals through the decomposition of azo compounds [4,9,17]. The ORAC values of FUCM, U. pinnatifida, and C. militaris extracts were 63.19 ± 2.12, 56.09 ± 1.19, and 52.55 ± 2.57 µM TE/mg, respectively (Table 1).
Standard Fingerprint Profiling
Fingerprints of U. pinnatifida, C. militaris mycelia, and FUCM were performed using high-performance liquid chromatography and compared with the alginate and cordycepin standards. The retention time for alginate and cordycepin was 2.433 and 12.813 min respectively under optimum chromatographic conditions. The processed channel linear range for alginate and cordycepin was 0.25-2.0 and 0.625-0.5 mg/ml at 200 and 260 nm, respectively. Based on these chromatographic conditions, we established the standard curves for alginate and cordycepin.
The regression equations of calibration curves and their coefficients were calculated using Chromeleon 6.8 software (Thermo Scientific, Sunnyvale, CA, USA). The HPLC analytical results showed the contents of alginate in U. pinnatifida and FUCM to be 0.66 ± 0.02 and 0.93 ± 0.01 mg/mg, respectively (C. militaris mycelia, ND) (Fig. 7A). The contents of cordycepin in the C. militaris mycelia and FUCM were 0.16 ± 0.01 and 0.3 ± 0.02 µg/mg (U. pinnatifida, ND), respectively (Fig. 7B). After fermentation of U. pinnatifida with C. militaris mycelia, the contents of alginate and cordycepin were increased to 40% and 87.5% compared with non-fermentation of U. pinnatifida and C. militaris mycelia.
Fig. 7.High-performance liquid chromatographic fingerprints of U. pinnatifida, C. militaris mycelia, and fermented U. pinnatifida with C. militaris mycelia (FUCM). (A): alginate (2.433 min); (B): cordycepin (12.813 min).
Our results indicate that U. pinnatifida fermented with C. militaris mycelia exhibited enhanced contents of alginate and cordycepin compared with U. pinnatifida and C. militaris fermented alone.
ROS decrease the normal defense systems of organisms, leading to various abnormalities, including myocardial ischemia, inflammatory disease, carcinogenesis, and Alzheimer’s disease, by attacking proteins, lipids (including those in cellular membranes), and DNA . Antioxidants are vital substances, possessing the ability to protect the body from damage by free-radical-induced oxidative stress .
In a previous study, Je et al.  reported that brown seaweed, U. pinnatifida, exhibited excellent antioxidant activity against DPPH, hydroxyl, and alkyl radicals. The hydrolysis of proteins and carbohydrates increases solvent exposure of residue side-chain groups, which facilitates reactions between peptides and free radicals and ROS and transitional metal ions, leading to increased antioxidant activities . Several authors found a strong correlation between antioxidant activities and DPPH, ABTS, and FRAP assays [2,8]. Fermentation is a useful method for producing biological materials, being a metabolic process that converts sugar to acids, gases, and/or alcohol using yeast or bacteria instead of Cordyceps militaris that uses a protein instead of sugar for the generation of energy . Therefore, fermentation methods using mushroom mycelia are a promising alternative for obtaining useful and potent substances.
We hypothesized that fermentation of U. pinnatifida with C. militaris mycelia could change the molecular weight during fermentation through decomposition, which may improve the antioxidant activities of the extract. Their antioxidative effects were evaluated in different free radical scavenging assays, including DPPH, hydroxyl, and alkyl radicals using an ESR spectrophotometer, ABTS, FRAP, and ORAC assays.
Interestingly, our results indicate that FUCM had higher radical scavenging activities than either U. pinnatifida or C. militaris mycelia alone. The combined results of this study indicated that FUCM displays effective radical scavenging activity. Therefore, FUCM could be an important natural and bioavailable antioxidant agent. However, further studies are required to determine the antioxidative compounds present in FUCM and the molecular and biochemical mechanisms of its biofunctional activities.