Improving Antioxidant Activity of Ophioglossum thermale Kom . by Fermentation with Talaromyces purpurogenus M 18-11

Ophioglossum thermale Kom. was fermented with several fungi. The total phenolic and flavonoid contents (TPCs and TFCs) and antioxidant activities of fermented and non-fermented O. thermale (FOT and NFOT) were investigated. The results showed that Talaromyces purpurogenus M18-11 fermented O. thermale possessed significantly improved TPC and TFC and exhibited significantly stronger 1,1-diphenyl-2-picrylhydrazyl (DPPH) (half maximal inhibitory concentration (IC50) = 75.7 ± 2.1 μg mL) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (IC50 = 20.52 ± 1.68 μg mL) free-radical scavenging activities, ferric reducing antioxidant power (0.585 ± 0.045 mmol g), and reducing power (half maximal effective concentration (EC50) = 30.52 ± 1.91 μg mL) than original material. The determination of the contents of representative flavonoids and their glucosides revealed that the improvements are attributed to the hydrolysis of homoisoflavonoid and flavonoid glucosides, glycometabolism, as well as fungal metabolites. This paper is the first to report the fermentation of O. thermale with pure strains and T. purpurogenus is an effective strain to process O. thermale for improving the antioxidant activity.


Introduction
The genus Ophioglossum (Ophioglossaceae) is a small terrestrial plant that is distributed worldwide. 1hytochemical researches revealed that the herb of Ophioglossum contains massive fatty acids and their esters, 2,3 amino acid, 4 flavonoids, [5][6][7][8][9][10][11] and polysaccharoses. 12,13phioglossum thermale Kom., "Yizhijian" in traditional Chinese medicine (TCM), is a plant of the genus Ophioglossum, which is frequently used as a herbal medicine for clearing heat and detoxicating.Its main chemical constituents are flavonoids 2,14,15 which possess significant antioxidant activity.7 Herbal fermentation processing began approximately 4000 years ago in China, which is frequently used to produce secondary metabolites from domestic plants in bulk by utilizing the metabolic mechanisms of microorganisms.Solid-state natural fermentation (SSF) was very popular in the processing of herbal medicines in ancient China, such as Semen Sojae Praeparatum, Mass Galla chinesis et camelliae Fermentata, Massa Medicata Fermentata, and Rhizoma Pinelliae Fermentata, etc.With the development of microbial technology, pure strain fermentation has become increasingly acceptable and reliable for processing herbal medicines in modern times.In recent years, some fermented traditional medicines (FTMs) have been reported.FTMs exhibit stronger biological activities or higher bioavailability in the human body compared with raw materials.For example, Hsu et al. 16 reported that Bacillus subtilis fermented Radix astragali could stimulate the biosynthesis of type I procollagen in a dose-dependent manner in both aged and young human dermal fibroblast cells.Aspergillus oryzae-fermented Curcuma longa L. could effectively prevent CCl 4 -induced hepatic damage in rats.17 Wang et al. 18 improved bioactivities of polyphenol extracts from Psidium guajava L. leaves by fermentation of Monascus anka and Saccharomyces cerevisiae.

Plant material
The herbs of O. thermale were collected from Wenshan, Yunnan, China, in February 2014, and were identified by Assistant Professor Shu-Da Yang from School of Pharmacy, Kunming Medical University, Kunming, China.A voucher specimen (2014-0t-01) has been deposited in School of Chemical Science and Technology, Yunnan University, Kunming, China.
Potato dextrose agar (PDA; 1 L water, 200 g potato, 20 g dextrose, and 15 g agar) slant culture mediums were inoculated with fungi above and incubated in a constant temperature incubator at 28 °C for 5 days.Five grams of O. thermale were added to a 100 mL Erlenmeyer flask to function as the fermentation culture medium.After being infiltrated with 15 mL water and sterilized at 121 °C for 30 min, the mature slants culture mediums were added and incubated at 28 °C for 30 days.

Antioxidant activities DPPH radical-scavenging activity
The DPPH free radical-scavenging activity was estimated by the method described previously. 19DPPH radical-scavenging activity of the sample was calculated as follows: (1) where is the absorbance of sample solution mixed with DPPH and is the absorbance of the blank solution (ethanol).Rutin was used as a positive control.All tests were performed in triplicate.The half maximal inhibitory concentration (IC 50 ) value was defined as the effective concentration at which the DPPH radical was scavenged by 50%.Anti-radical power (ARP) was defined as 1/IC 50 .ARP i and ARP 0 were represented as the ARP of each sample and ROT, respectively.

ABTS assay
ABTS antioxidant capacity was measured using a previous ABTS method. 19,20ABTS radical-scavenging activity was calculated as follows: (2) where is the absorbance of sample solution mixed with ABTS and is the absorbance of the blank solution.The IC 50 value was defined as the effective concentration at which the ABTS radical was scavenged by 50%.ARP was defined as 1/IC 50 .ARP i and ARP 0 were represented as the ARP of each sample and ROT, respectively.

FRAP
The FRAP assay measures the reduction of ferric iron to the ferrous form in the presence of antioxidants.The assay protocol followed a previously reported procedure. 21he antioxidant capacity of the sample was expressed as μmol ferrous sulfate per g extract (μmol g -1 ), which was calculated from the standard graph obtained with the ferrous sulfate solution.

Reducing power
Reducing power was tested using a reported method. 22,2300 μL of sample at proper concentration was made up to 0.75 mL with phosphate buffer (300 mM, pH 6.6), and 1.5 mL of 1% (m/v) K 3 Fe(CN) 6 was added.The mixture was shaken vigorously and left to stand for 20 min at 50 °C.After the addition of 1.5 mL of 10% (m/v) trichloroacetic acid, the mixture was centrifuged at 3000 rpm for 10 min.A 1.5 mL of supernatant was mixed with distilled water (1.5 mL), and 0.3 mL of 0.1% (m/m) FeCl 3 was added before the absorbance was determined at 700 nm.Half maximal effective concentration (EC 50 ) was defined by the concentration at absorbance (A) = 0.50; antioxidant reducing power (ARP) was defined as 1/EC 50 .ARP i and ARP 0 were represented as the ARP of each sample and ROT, respectively.

Determination of total phenolic content
The total phenolic content was measured using the Folin-Ciocalteau method. 24Folin-Ciocalteu's reagent was prepared according to the method of GB/T 23527-2009 (CN). 25Fifty microliters of extract solution was added to 2.25 mL of Folin-Ciocalteu's reagent, which was pre-diluted ten-fold with distilled water.Five minutes later, 1.5 mL of Na 2 CO 3 (7.5%,m/v) solution was added, and the mixture was allowed to stand for 30 min at ambient temperature.Absorbance was measured at 765 nm.The TPC was expressed as mg gallic acid equivalent per g extract (mg GAE g -1 ) and calculated according to the calibration curve obtained from the standard solution of gallic acid at various concentrations.

Determination of total flavonoid content
The total flavonoid content was determined by a colorimetric assay described previously. 23,26The total flavonoid content was expressed as mg rutin equivalent per g (mg RE g -1 ).

HPLC analysis
The determination and quantification of main flavonoids compounds were measured using an Agilent 1260 Infinity Series HPLC system, equipped with an Agilent G1315D DAD detector, an Agilent G1311C quaternary gradient pump, and Agilent Zorbax SB-C18 (250 × 4.6 mm i.d., 5 μm), and coupled to an Agilent OpenLab data-processing station.A gradient elution system consisting of solvent A (water containing 6 mM acetic acid) and B (acetonitrile) was used for the analysis, and the gradient program was as follows: 0-25 min, 15-65%B; 25-30 min, 65-100%B; 30-35 min, 100%B.The peaks were confirmed by the UV absorptions at 320 and 355 nm and the retention times while the flow rate was 1.0 mL min -1 .The column temperature was set at 35 °C, and the injection volume was 20 μL.Flavonoids were identified by using standard addition method, and quantified according to standard curves.

Results and Discussion
After fermentation of O. thermale with several fungi, a preliminary experiment by thin layer chromatography (TLC) was measured.Four fermented samples were selected for further experiments.

Antioxidant activities
Oxidation is ubiquitous and has pernicious effects on both food quality and human health.Previous research indicated that oxidative damage can cause browning and off-flavors, change the nutrient value of food, disrupt cellular function and attenuate the formation of compounds used to fight aging and cardio-vascular disease. 27Zhang et al. 7 reported that the extract of O. thermale possesses antioxidant activity.In the present study, therefore, the antioxidant activity of ROT, BFOT, and FOT1-4 were tested.
DPPH is a stable free radical that has been used widely to determine the free radical-scavenging activity of natural antioxidants.The DPPH radical scavenging activities shown in Figure 1 revealed that FOT1-3 exhibited more significant activities than ROT, BFOT, and FOT4, in particular, FOT2 possessed 2-fold more antioxidant activity with an IC 50 value of 75.7 ± 2.1 μg mL -1 than ROT.
ABTS is another free radical that is frequently used to study free radical-scavenging activity.Notably, the ABTS radical-scavenging activity assay is an important indicator of total antioxidant activity (TAA).As shown in Figure 2, FOT1-3 showed stronger ABTS radicalscavenging activities with the IC 50 values of 18.61 ± 1.32, 20.52 ± 1.68, and 19.20 ± 0.92 μg mL -1 , than FOT and BFOT.
Reducing power of the natural plant extracts might be strongly correlated with their antioxidant activity.It is necessary to discuss the reducing power of a natural plant extract to elucidate the relationship between its antioxidant effect and reducing power.The results shown in Figure 3 suggest FOT2 exhibits significantly improved reducing power with an EC 50 value of 30.52 ± 1.91 μg mL -1 .The FRAP is often used as an indicator of phenolic antioxidant activity as important as reducing power.The antioxidant potential of sample was estimated by their abilities, which is to reduce Fe III -TPTZ to Fe II -TPTZ.Similar to reducing power, FOT2 possessed improved antioxidant activity (0.585 ± 0.045 mmol g -1 , Figure 4).

Total flavonoid content (TFC)
As the main constituents of O. thermale are flavonoids including quercetin, 3-methoxyquercetin, ophioglonol and their esters, the TFCs of the fermented and non-fermented materials are indicators for evaluating the effects of fermentation.The TFCs of ROT, BFOT, and FOT1-4 are shown in Figure 5. FOT2 and FOT3 possessed obvious improved TFCs with the values of 120.35 ± 0.84 and 107.94 ± 4.22 mg RE g -1 , respectively, whereas FOT1, FOT4, and BFOT exhibited TFC with same grade as ROT.
These results suggest that T. purpurogenus might be an effective strain for processing O. thermale for improving the TPC, TFC, and antioxidant activity.The improvement of antioxidant activity might be caused by the changes of flavonoids, as well as fungal metabolites produced by fungi during fermentation.

HPLC analysis
To clarify the flavonoids, it was performed positive antioxidant activities in the fermented and non-fermented O. thermale.HPLC was employed to identify the changes of main flavonoids isolated from O. thermale.Six main flavonoid compounds isolated from O. thermale, luteolin (1), quercetin (2), 3-methoxyquercetin (3), ophioglonol (4), ophioglonol 4'-O-α-D-glucopyranoside (5), and pedunculosumoside B (6) (Figure 6) were used to identify the peaks in chromatograms of ROT, BFOT, and T. purpurogenus FOT.As shown in Figure 7, flavonoid glucoside 5 could be not detected in T. purpurogenus FOT, suggesting that flavonoid glucosides are hydrolyzed by T. purpurogenus-fermentation. The peaks at 30-35 min observed in ROT disappeared after fermentation, suggesting that some low-polarity constituents are metabolized leading to the enrichment of the flavonoids.The contents of three main flavonoids 3-5 were determined by HPLC.The results in Table 1 show that the contents of flavonoid aglycones 3 and 4 were enriched.This might be caused by the hydrolysis of flavonoid glucosides and glycometabolism.

Conclusions
The present study is the first to report that pure strain fermentation processing is effective in improving the TPC, TFC, and antioxidant activity of O. thermale.Fermentation with T. purpurogenus could be an innovative approach to process O. thermale.TFC and antioxidant activity enhancements with T. purpurogenus might be attributed to hydrolysis of flavonoid glycosides, decomposition of glycoside and other low-polarity constituents, and fungal metabolites.