1. Isolation of endophytic fungi
Visakhapatnam (Location 17°40'48.32''N, 83°12'5.8''E.) is situated between the Eastern Ghats and the Bay of Bengal Tribulus terrestris L. was collected from the Campus, Andhra University. Leaf samples were surface sterilized by dipping in 70% ethanol (v/v) for 1 min and 3.5% NaOCl (v/v) for 3 min, rinsed thrice with sterile water and dried. Bits of 1.0 X 1.0 cm size were excised with the help of a sterile blade. Two hundred segments of Tribulus terrestris L. leaf segments were placed on the water agar (16%) (WA) medium supplemented with Streptomycin (100 mg/l; Sigma, St. Louis, MO, USA) were used for the isolation of endophytic fungi. The Petri dishes were sealed using parafilm and The Petri dishes were incubated at 25-27°C till the mycelia start growing from the samples [15]. The plant material was also deposited in Botany Department herbarium (AUV), Andhra University with Voucher specimen numbers – 22294.
2. Secondary metabolites extraction
The endophytic fungus EF1 i.e. Curvularia aeria MTCC-12847 was cultured in 1-l Erlenmeyer flasks containing 500 ml of optimized culture media (PDB) under optimized parameters (pH: 6-6.5, Temperature: 27°C-28°C, Incubation days: 8-9 day) under static conditions. The culture broth was then filtered to separate the culture filtrate and mycelium. Culture filtrate was properly blended and centrifuged at 4,000 rpm for 5 min. The supernatant was transferred to a separating funnel to which was added the same volume of ethyl acetate. An ethyl acetate solution containing the fungal metabolite was 98% concentrated in a Buchi R-300 Rotavapor (India) at 50°C and stored at 4°C until its use [16].
3. Qualitative phytochemical analysis
It was done by following methods of [17, 18, and 19].
4. Quantitative phytochemical analysis
4a. Estimation of total Alkaloids
Total alkaloid content was estimated by the method of [20, 21] with minor modification. The absorbance for test and standard solutions were determined against the reagent blank at 470 nm with an UV-Spectrophotometer (SHIMADZU-1800, Japan). Absorbance = 0.004 Atropine (µg/ml) - 0.05 (R2 = 0.9907) (S1). The results were expressed atropine equivalents (µg of AE/mg extract).
4b. Total flavonoid content
Total flavonoid content was estimated by a colorimetric method reported [22, 23] by with minor modification.
Absorbance = 0.005 Quercetin (µg/ml) + 0.101(R2 = 0.9989) (S2). The results were expressed as Quercetin equivalents (µg of QE/mg of extract).
4c. Determination of total phenolic content
Total phenolic content was determined by using the Folin-Ciocalteu [24, 25] method of with minor modification.
Absorbance = 0.010 Gallic acid + 0.173 (R2 = 0.9938) (S3). The results were expressed gallic acid equivalents (µg of GAE/mg extract).
4d. Estimation of total terpenoids
Estimation of total terpenoids in the crude extract was determined by the method of [26] with minor modification.
Absorbance = 0.001 linalool + 0.032 (R2 = 0.9912) (S4). The results were expressed linalool equivalents (µg of linalool/mg extract).
4e. Estimation of total sterol
Estimation of total sterols in the crude extract was done by the method of [27] with minor modification.
Absorbance = 0.0026 Beta-Sisterol (µg/ml) - 0.047 (R2 = 0.9986) (S5). The results were expressed as Beta-Sisterol equivalent (µg of Beta-Sisterol/mg of extract).
4f. Estimation of Total saponins
Total saponin content was analyzed spectrophotometerically following the method of [28] with minor modifications.
Absorbance = 0.0005 Diosgenin (µg/ml) - 0.042 (R2 = 0.9983) (S6). The results were expressed as diosgenin equivalents (µg of DE/mg crude extract).
4g. Estimation of total tannins
The total tannins were determined using the method [29, 30] with minor modification.
Absorbance = 0.0192 tannic acid + 0.015 (R2 = 0.9961) (S7). The results were expressed as tannic acid equivalents (µg of Tannic acid/mg of extract).
4h. Estimation of total carbohydrates
The total carbohydrate content was estimated by the method of [31].
Absorbance = 0.0092 glucose + 0.063 (R2 = 0.9953) (S8). The results were expressed as glucose equivalents (µg of Glucose/mg of extract).
4i. Estimation of total protein
The total protein content was estimated using the method of [32].
Absorbance = 0.0098 BSA + 0.015 (R2 = 0.9982) (S9). The results were expressed as BSA equivalents (µg of BSA/mg of extract).
5. TLC (Thin layer chromatography profile)
For the separation of different phytochemical compounds in the endophytic fungal extract TLC was performed by the method of [33, 34]. After the separation of phytochemical constituents, the plates were viewed under UV light at 254 nm and 366 nm and bands were marked and Rf values were calculated by using the following formula:
Retention factor (Rf) = Distance travelled by the solute
Distance travelled by the
6. Antioxidant activity
6a. Free radical-scavenging ability by the use of a stable ABTS radical cation
The free radical-scavenging activity was determined by ABTS radical cation decolorization assay described by [35]. The ABTS.+ cation radical was produced by the reaction between 5 ml of 14 mM ABTS solution and 5 ml of 4.9 mM potassium persulfate (K2S2O8) solution, stored in the dark at room temperature for 16 hr. Before use, this solution was diluted with ethanol to get an absorbance of 0.700 ± 0.020 at 734 nm. The endophytic fungal extract of different concentrations with 1 ml of ABTS solution and absorbance were recorded at 734 nm. Ethanol was run in each assay, and all measurements were taken after at least 6 min. Similarly, the reaction mixture of standard group was obtained by mixing 950 μl of ABTS. + solution and 50 μl of BHT. As for the antiradical activity, ABTS scavenging ability was expressed as IC50 (μg/ml). The inhibition percentage of ABTS radical was calculated using the following formula:
ABTS scavenging activity (%) = (A0 – A1) /A0 × 100
Where, A0 is the absorbance of the control and A1 is the absorbance of the sample.
6b. DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity
The antioxidant activity of the extract were determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay by the method of [36, 37] with some modifications. Freshly prepared 1ml of DPPH (0.004 % in methanol) solution was added in each of these test tubes containing extracts (100 to 500 µg/ml) and after 30 min incubation in the dark, the absorbance was taken at 517 nm. BHT was used as a positive control. The DPPH solution was used as blank. Scavenging of the DPPH free radical was measured using the following equation:
DPPH radical scavenging (%) = (A0 – A1) /A0 × 100
6c. Hydrogen peroxide scavenging (H2O2) assay
The ability of the extracts to scavenge hydrogen peroxide was estimated by following the method of [38]. A solution of hydrogen peroxide (40 mmol/L) was prepared in phosphate buffer (50 mmol/L, pH 7.4). The concentration of hydrogen peroxide was determined by absorption at 230 nm using a spectrophotometer. Extract (1 mg/mL) in distilled water were added to hydrogen peroxide and absorbance at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. Ascorbic acid was used as a positive control.
The percentage of hydrogen peroxide scavenging was calculated as follows:
Scavenged H2O2 (%) = (A0 – A1) /A0 × 100
6d. Hydroxyl Radical Scavenging Assay
Hydroxyl radicals formed from FeSO4/hydrogen peroxide can be noticed by their attribution to hydroxylate salicylate thus forming hydroxylated salicylate complex which can be observed at wavelength 562 nm[39]. Several concentrations of extract were added to 6 mM hydrogen peroxide (0.7 ml), 1.5 mM FeSO4 (1 ml), 20 mM sodium salicylate (0.3 ml), were used as the reactive mixture (3 ml). Absorbance of hydroxylated salicylate complex was determined at wavelength 562 nm after 1 hr of incubation at 37°C. Again, ascorbic acid was used as positive control.
The scavenging activity was calculated by the given formula below:
% scavenging activity = (A0 – A1) /A0 × 100
6e. Reducing power assay
The reductive potential of the extract was determined according to the method of[40] with minor modification. Different extracts and standard (1 mg/mL) in 1 mL of distilled water were mixed with phosphate buffer (2.5 mL, 0.2 mol/L, pH 6.6) and potassium ferricyanide (2.5 mL, 1% w/v) and was then centrifuged for 10 min at 3000 rpm/min. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1% w/v) and the absorbance was measured at 700 nm in a spectrophotometer. High absorbance value of the reaction mixture indicates greater reductive potential.
6f. FRAP assay
The total antioxidant potential of a sample was determined using the ferric reducing ability of FRAP assay by [41] as a measure of antioxidant power.
Briefly, FRAP reagent 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 HCl) and 2.5 mL FeCl3 (20 mM) at 10:1:1 (v/v/v). Extract at different concentrations (100 to 500 μg/ml) were then added to 3 ml of FRAP reagent and the reaction mixture was incubated at 37°C for 30 min. The increase in absorbance at 593 nm was measured.
6g. Nitric oxide Scavenging Activity
Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide (NO), which interacts with oxygen to produce nitrite ions, which can be estimated using Griess Illosvosy reaction by[42]. The absorbance of these solutions was measured at 540 nm against the corresponding blank solution.
Extract at different concentrations were mixed with the 100 mM sodium nitropusside (2.5 ml) and PBS (0.02% KCl, 0.88% NaCl, 0.115% Na2HO4 and 0.02% KH2PO4) to make up to maximum of 3 ml for the reaction mixture and was incubated at 25°C for 15 min, after that 0.5 ml from the reaction mixture was replaced with 0.5 ml of Griess reagent (2% phosphoric acid, 1% sulphanilamide and 0.1% naphthyl ethylenediamine dihydrochloride).
Percentage inhibition was calculated as:
% Scavenging of Nitric oxide = (A0 – A1) /A0 × 100
6h. Metal chelating activity
It was estimated by method of [43]. Briefly, 0.1 mM FeSO4 (0.2 ml) and 0.25 mM ferrozine (0.4 ml) were added subsequently into extract (0.2 ml). After leaving to stand for 10 min, the absorbance was read at wavelength 562 nm. EDTA was used as a positive control.
Chelating activity was calculated using the following formula:
Metal chelating activity = (A0 – A1) /A0 × 100
6i. Superoxide Radical Scavenging Activity (SOD)
Superoxide radicals were generated by method of Beauchamp [44, 45] with slight modification. Superoxide radicals were generated in riboflavin, methionine, illuminate and assayed by the reduction of NBT to form blue formazan (NBT2+). All solutions were prepared in 0.05 M phosphate buffer (pH 7.8). The photo-induced reactions were performed using fluorescent lamps (20 W). The total volume of the reactant mixture was 3 mL and the concentrations of the riboflavin, methionine and NBT were 1.33 x 10-5, 4.46 x 10-5 and 8.15 x 10-8 M, respectively. The reactant was illuminated at 25°C for 40 min. The photochemically reduced riboflavin at different concentrations was added to the reaction mixture, in which O2 was scavenged, thereby inhibiting the NBT reduction. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity and the readings were taken at 560nm. The inhibition percentage of superoxide anion generation was calculated by using the following formula:
O.-2 scavenging effect (%) = (A0 – A1) /A0 × 100