The endophytic fungi of Salvia miltiorrhiza Bge.f. alba are a potential source of natural antioxidants

Plant materials

Nine healthy Salvia miltiorrhiza Bge. f. alba were randomly collected in July 2010 from Salvia miltiorrhiza cultivation zone at three sites in Taishan Medical University, Shandong Province, China. The current-year roots (4–6 cm × 2–3 cm, length × diameter), stems (8–12 cm × 1–2 cm, length × diameter), leaves and flowers were placed in sterile plastic bags and stored at 4°C until isolation procedure was started.

Isolation of fungal endophytes

Plant tissues were surface-sterilized using the procedure described by Fisher et al. ([1993]) with minor modification. The root, stem, leaf and flower segments were cleaned under running tap water. After air-drying, the cleaned stems and roots were cut into small pieces, and then all the tissues were surface sterilized by immersion in 75% ethanol for 1 min, 5% sodium hypochlorite solution for 3 min and then washed three times in sterile distilled water for 1 min each time. The surface-sterilized samples were cut into small pieces using a sterile blade and placed on sterile potato dextrose agar (PDA) at 28°C. The hyphal tip of endophytic fungus growing out from the plant tissue was cut by a sterile pasture pipette and transferred to PDA plate supplemented with ampicillin (150 mg/L) and streptomycin (100 mg/L) to inhibit the bacterial growth until the mycelium or colony appeared surrounding the segments. The efficiency of surface sterilization procedure was checked for each sterilized plant segment following the imprint method. In addition, to check the presence of surface associated fungi, plant samples without surface sterilization were cultured in the same condition as negative controls. Colonization frequency (CF) was calculated as the total number of plant tissue segments colonized by an endophyte, divided by the total number of incubated segments.

Endophytic fungi from the roots, stems, leaves and flowers of Salvia miltiorrhiza Bge. f. alba have been numbered and codified as SaR1- SaR7, SaS1, SaL1 and SaF1- SaF5. All the isolated endophytic fungi have been stored on PDA slants at 4°C and kept at College of Life Sciences, Taishan Medical University, Shandong Province, China.

Identification of fungal endophytes

The identification procedure of endophytic fungi was based on morphology and molecular methods. The morphological characters included culture characteristics and the morphology of conidia. The molecular method was carried out to characterize some non-sporulating group using the ribosomal internal transcribed spacer (ITS) sequence. Fungal genomic DNA was extracted from fresh mycelia using an SDS extraction protocol described by Plaza et al. ([2004]). The primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) were used to amplify the ITS region. The PCR products were subsequently purified and sequenced in two directions on an ABI 3700 automated sequencer. The resulting sequences were subjected to BLAST searches of the NCBI GenBank database to determine the identity of the fungi.

Fermentation and preparation of crude extracts

Each isolated endophytic fungus strain was inoculated in potato dextrose liquid medium with 150 rpm shaking at 25°C for 5–7 days. Mycelia and broths were separated by filteration through two layers of cheesecloth. Mycelia were thoroughly washed with sterile distilled water, air-dried in an oven at 60°C, and ground into fine powder. The dried mycelia sample (2 g) was extracted with 50 mL of 95% ethanol for three times. Culture filtrates was treated by rotary evaporation under vacuum and then extracted thrice with a threefold of the volume of 95%(v/v) ethanol. The obtained extracts were filtered by Whatman No.1 filter paper and then concentrated under vacuum at 45°C to yield the crude extracts. As a control, 2 g of plant root power was extracted with 50 mL of 95% ethanol and stored at 4°C till further process.

Phytochemical screening

Preliminary phytochemical analysis of the crude extracts of fungi and plant root was carried out for the presence of the following metabolites such as saponins, phenol, flavonoids, cardiac glycosides, steroids, tannins, alkaloids, anthroquinone and terpenoids according to standard methods (Devi et al. [2012]; Edeoga et al. [2005]; Maobe et al. [2013]).

Determination of total phenolic content

Total phenolic contents from endophytic fungi and plant root were respectively measured by Folin-Ciocalteu’s colorimetric method (Taga et al. [1984]) with some modification. Briefly, 100 mg of different ethanol extracts was added to 5 mL of 0.3% HCl in methanol/deionised water (60:40, v/v) respectively. The resulting mixture (100 μL) was added to 2 mL of 2% aqueous sodium carbonate. Then the mixture was incubated for 2 min at room temperature. 100 μL of 50% Folin-Ciocalteu’s reagent was added to treated mixture and incubated for 30 min at room temperature, and absorbance was measured at 750 nm with the spectrophotometer against blank. The total phenol content was calculated on the basis of the standard curve of gallic acid. Phenol contents were expressed as mg of gallic acid equivalents (GAEs) per g of extract.

Determination of total flavonoid content

Total flavonoid content was estimated by a colorimetric method reported by Barros et al. ([2007]). The extract (250 μL) was mixed with distilled water (1.25 mL) and sodium nitrite solution (5%, 75 μL). After 5 min incubation at room temperature, aluminum chloride solution (10%, 150 μL) was added. After 6 min, sodium hydroxide (1 M, 500 μL) and distilled water (275 μL) were added to the mixture. The solution was mixed well and incubated at 25°C for 30 min. Absorbance was measured at 510 nm against blank. The content of flavonoid was calculated on the basis of the standard curve of quercetin and the results were expressed as mg of quercetin equivalents per g of extract.

Determination of antioxidant activity

Antioxidant activity of examined extracts was measured using ferric ion reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay.

FRAP assay

FRAP reagents was freshly prepared by mixing 25 mL acetate buffer (300 mM, pH 3.6), 2.5 mL 2,4,6-tris (2-pyridyl)-S-triazine (TPTZ) solution (10 mM TPTZ in 40 mM/L HCl) and 2.5 mL FeCl₃ (20 mM) water solution. Each sample (150 μL) (0.5 mg/mL) dissolved in methanol was added to 4.5 mL of freshly prepared FRAP reagent and stirred. After 5 min, absorbance was measured at 593 nm, using FRAP working solution as blank (Szőllősi and Szőllősi Varga [2002]; Tomic et al. [2009]). A calibration curve of ferrous sulfate (100–1000 μmol/L) was used and results were expressed in μmol Fe²⁺/mg dry weight extract. The relative activity of the samples was compared with the standards ascorbic acid and butylated hydroxytoluene (BHT).

DPPH radical assay

The free radical scavenging activities of different extracts were carried out by using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay (Braca et al. [2001]). Crude extract (0.1 mL) was mixed with 3 mL of a 0.004% methanol solution of DPPH. The mixture were reacted in the dark condition for 30 min and the absorbance was determined at 517 nm. The percentage inhibition activity was calculated using the following equation:

DPPH scavenging effect (The free radical scavenging ) = [(Abs_control–Abs_sample)/Abs_control] × 100, Where Abs_control is the absorbance of the control reaction, Abs_sample is the absorbance of the extract/standard.

Statistical analysis

Analysis of variance was performed by ANOVA. Means between treatment groups were compared for significance by using Duncan new multiple-range test. P values < 0.05 were considered to be significant and P values < 0.01 to be very significant. All experiments were performed in triplicate (n = 3) and results were reported as means ± standard deviation (SD).

Isolation and identification of endophytic fungi

Preliminary phytochemical screening

Determination of total phenol content and total flavonoid content

Antioxidant capacity analysis

The antioxidant capacity of the ethanol extracts of four selected endophytes and plant extract were assessed for their ability to reduce TPRZ-Fe (III) complex to TPTZ-Fe (II) (Table 4). The reducing ability of the ethanol extracts was in the range of 512.25 to 1682.21 μmol Fe (II)/mg. The FRAP values for the extracts of F. proliferatum SaR-2 and A. alternata SaF-2 were significantly higher than those of ascorbic acid and BHT, while the FRAP values for the extracts of S. commune SaR-3 and T. hirsuta SaR-6 were significantly lower than that of ascorbic acid but higher than that of BHT.

The antioxidant activities of the extracts of endophytic fungi and plant root, as compared with the standards ascorbic acid and BHT, were determined by the capability to scavenge DPPH free radicals. Figure 1 showed the dose response curve of DPPH radical scavenging activity of all samples. It was observed that the ethanol extracts of F. proliferatum SaR-2 and A. alternata SaF-2 had higher radical scavenging activity than that of plant root. At a concentration of 0.1 mg/mL, the scavenging activity of the plant root extract reached 80.23%, while the scavenging activities of F. proliferatum SaR-2 and A. alternata SaF-2 were 90.14% and 83.25% respectively. Even though the DPPH radical scavenging abilities of the extract of F. proliferatum SaR-2 (90.14%) was lower than those of the standards ascorbic acid (95.43%) and BHT (94.14%) at 0.1 mg/mL, it still reached at high concentration. Thus the study showed that F. proliferatum SaR-2 could be a promising resource of natural antioxidants.