Phytochemical analysis and antioxidant activity of endophytic fungi Curvularia aeria MTCC 12847 isolated from Tribulus terrestris L. leaf


 Objective: The objective of the present study is to perform phytochemical analysis and antioxidant activity of endophytic fungi isolated from Tribulus terrestris L. leaf.
Background: Curvularia aeria MTCC 12847 was isolated from Tribulus terrestris L. leaf and was used for phytochemical analysis which revealed the presence of phenol, flavonoids, terpenoids, steroids, alkaloids etc from different solvents extracts. The antioxidant activity was evaluated by nine separated methods: scavenging of free radical ABTS, DPPH, Hydrogen peroxide, Hydroxyl radical, Metal cheating, Nitric oxide, Superoxide radical, Reducing power and FRAP and the isolate Curvularia aeria MTCC 12847 showed good activity at low concentration.
Conclusion: Further work is needful to isolate the exact compound which is responsible for antioxidant activity and biophysical characterization will be done in the future.

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] .

Qualitative phytochemical analysis
It was done by following methods of [17, 18, and 19] .

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 (R 2 = 0.9907) (S1). The results were expressed atropine equivalents (µg of AE/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.

4g. Estimation of total tannins
The total tannins were determined using the method [29,30] with minor modification.
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 (R 2 = 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 (R 2 = 0.9982) (S9). The results were expressed as BSA equivalents (µg of BSA/mg of extract).

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 R f values were calculated by using the following formula: Retention factor (R f ) = 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 (K 2 S 2 O 8 ) 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 IC 50 (μg/ml). The inhibition percentage of ABTS radical was calculated using the following formula:

ABTS scavenging activity (%) = (A 0 -A 1 ) /A 0 × 100
Where, A 0 is the absorbance of the control and A 1 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-2picrylhydrazyl (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 (%) = (A 0 -A 1 ) /A 0 × 100 6c. Hydrogen peroxide scavenging (H 2 O 2 ) 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:

6d. Hydroxyl Radical Scavenging Assay
Hydroxyl radicals formed from FeSO 4 /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 = (A 0 -A 1 ) /A 0 × 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 FeCl 3 (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.

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% Na 2 HO 4 and 0.02% KH 2 PO 4 ) 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 = (A 0 -A 1 ) /A 0 × 100 6h. Metal chelating activity
It was estimated by method of [43] . Briefly, 0.1 mM FeSO 4 (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 = (A 0 -A 1 ) /A 0 × 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 (NBT 2+ ). All solutions were  Table 3 and Fig S13 to S19 shows ABTS, DPPH, Hydrogen peroxide, Hydroxyl radical, Nitric oxide, Metal chelating and Super oxide radical scavenging activity and S18 and S19 Contains Tables 4 and 5 for reducing power assay and FRAP assay which shows the OD values at different concentration.

Discussion
The endophytes have made greater interest in the use reservoir of natural bioactive compounds that the (host) produced [46] . Endophytic fungal species are now considered as exciting novel sources for obtaining new bioactive compounds and have been reported from several hosts [47] . Investigations on the phytochemical screening of the Tribulus terrestris L. revealed the presence of alkaloids, flavonoids, phenol, terpenoids etc. The presence of phytochemical within endophytes can be potential source for medicinal and industrial use. The presence of phytochemicals in the endophytes can be potential source of precursors in the development of synthetic drugs [48] .
The importance of compounds bearing antioxidant activity lies in the fact that they are highly effective against damage caused by reactive oxygen species (ROS) and oxygen derived free radicals, which contribute to a variety of pathological effects such as DNA damages, carcinogenesis, and cellular degeneration [49] . Antioxidants have been considered promising therapy for prevention and treatment of ROS linked diseases as cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer and Parkinson diseases), rheumatoid arthritis, and ageing [50].

Conclusion
Ethyl acetate extract of endophytic fungus Curvularia aeria MTCC 12847 significantly showed good result in phytochemical analysis and antioxidant activities. This finding confirms that endophytic fungi isolated from Tribulus terrestris L. was source of the potential bioactive substances. Thus, this plant appears to be an interesting plant which harbours active fungal isolates for development as pharmaceutical agents in the future.
The isolation of active metabolites which may be responsible for antioxidant activity may be investigated in future.

LIMITATIONS
The short duration of the study is an insufficiency of our work mainly related to a lack of funding.

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