Endophytic fungi were isolated from a medicinal plant named as Teucrium stocksianum taken from district Bajaur, Khyber Pakhtunkhwa, Pakistan. Stem, leaves and wood of plant were placed under running tap water for 15 minutes and then cut into small pieces of 0.2-0.5 µm using sterile scalpel. The sample were sterilized with 70 % ethanol solution for 3 minutes then with aqueous sodium hypo chloride (4 % available chlorine) for 3-5 minutes followed by rinsing with 70 % ethanol for 2-10 seconds and finally washed with double distilled water in laminar flow hood. Wood, leaves and roots sample were placed on sterilized SDA plates which are then incubated at 28 ℃ for 7 days. After that the appeared colonies were purified on SDA plates separately at the same conditions. Purified strains were preserved on SDA slants placed at 4 ℃ for short term preservation.
Screening for pigment production
Different strains were isolated from medicinal plant but only one strain named as N11 produced brown pigment on SDA plate. It was then inoculated in 200 ml of Sabouraud dextrose broth. A mycelium plug of 5 mm in diameter was cut from the freshly grown plate with the help of sterilized scalpel and inoculated in 200 ml of SDB in 500 ml Erlenmeyer flask after being autoclaved at 121 ℃ for 15 minutes. The inoculated flask was cultured in dark at 28 ℃ for ten to fourteen days with shaking at 150 rpm in shaking incubator.
Identification of fungal strain
Conventionally morphological characters were used for tentative identification of fungal strains. Colony characters such as aerial mycelium, density and pigment production were recorded. Microscopic features of the fungus were observed by using a light microscope.
Further molecular identification was done using 18S rRNA sequencing and homology analyses from the Database.
Optimization of fungal pigment production
Fungal cultivation and pigment yield estimation
Fungal mycelial plugs (3-5 mm in diameter) obtained from the margin of the fresh colony on SDA at 28 °C for 1 week was transferred into a 100 ml of liquid medium in 250 ml Erlenmeyer flask after being autoclaved at 121 °C for 15 min. Cultivation was performed in the dark at 28 °C with shaking at 150 rpm on a shaking incubator for ten to fourteen days. After cultivation the media was filtered, and pigment was estimated by using spectrophotometric analysis. The Optical density was measured at 660 nm (wavelength which represents the absorption maxima for orange-brown color). The pigment yield was estimated by converting OD into AU with method explained by (Palacio-Barrera et al. 2019). The following formula was used for conversion of optical density into AU
Formula for Absorbance Units:
AU= [Abs x Vfl] / Vr
Where AU =Absorbance units
Vfl = Filtered volume and Vr = Volume read in spectrophotometer
After cultivation the mycelium was harvested by using pre weighed Whatman No. 1 paper and then dried at room temperature for 48 hours. After drying the dry cell weight of mycelium was measured and expressed as mg/ml (Visalakchi and Muthumary 2010).
Effect of liquid culture medium
Three different media (Sabouraud Dextrose Broth (SDB), Potato Dextrose broth (PDB) and rice medium) were used for cultivation of fungal strain. After inoculation flasks were incubated in shaking incubator (150 rpm) at 28 ℃ for 21 days. The culture liquid medium that presented the highest yield of the pigment was selected for further experiments.
Effect of temperature
Inthis experiment, a fungal culture was inoculated in the selected liquid medium that had been obtained from previous experiments. A pH value of 6.0 was adjusted by using 1 N HCl and 1 N NaOH and the culture was incubated at 20, 25, 30, 35 °C for 21 days with shaking at 150 rpm. The temperature that presented the highest yield of the pigment was selected for further experiments.
Effect of initial pH
The initial pH of the selected suitable components of the medium that was from previous experiments was adjusted from 5.0 to 8.0 with the help of 1 N HCl and 1 N NaOH in each flask before being autoclaving. After inoculation, cultures were incubated at temperature that presented the highest pigment yield for 21 days. The initial pH of culture media that presented the highest yield of the pigment was selected for further experiments.
Large scale cultivation and extraction
For this experiment 2000 ml Erlenmeyer flasks containing 1000 ml of optimized medium and pH was autoclaved. Five fungal mycelium plugs of 3-5 mm obtained from the edges of growing fungal colony on SDA were transferred to the autoclaved medium and the flask was incubated at optimized temperature with 150 rpm rotation in a rotary shaker.
After fermentation the medium was filtered through Whatman No. 1 paper and the filtered medium was extracted using two volumes of ethyl acetate according to the method described by (Suwannarach et al. 2019a). After extraction the ethyl acetate fraction was evaporated in a rotary evaporator and crude extract obtained was saved for further experimentation.
Fungal pigment purification by Normal phase column chromatography
The purification of crude pigment was carried out by using normal phase column chromatography followed by Thin Layer Chromatography (TLC). The crude pigment was dissolved in ethyl acetate and mixed with silica gel which is used as a stationary phase. The column (column size 45 x 2.6 cm) was filled with prepared silica gel and the pigment was eluted by using different solvents (N-hexane, ethyl acetate, methanol, acetone and acetonitrile) in different ratios. The eluted fractions were examined by TLC in pre-coated TLC plates in the same mobile phase to check the purity of fractions. Retention factor (Rf) value of each sample was calculated as described by (Gupta et al. 2019).
All the fractions collected were subjected to TLC and the fractions having same Rf values were combined, and the solvent was evaporated to obtain the purified pigments in dried form.
Fourier-transform infrared spectroscopy (FT-IR) analysis of the purified pigment
Orange-brown pigments isolated from Aspergillus sp. N11 were analyzed by FTIR (Bruker, Tensor 27, equipped with ZnSe ATR) spectrophotometer in the range of 400−4000 cm−1 wavelength. Approximately 2 μg of pigments were placed on a diamond window of the spectrophotometer. The FTIR spectrum was used to identify different functional groups present in the pigments.
Determination of Antimicrobial activity of purified pigment
The antimicrobial activity of purified pigment was evaluated against gram positive Staphylococcus aureus (ATCC 25923), gram negative Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC 10231)strains were tested by using agar well diffusion method described earlier by (Walia et al. 2020). Bacterial test strains were inoculated in nutrient broth and incubated for 12-18 hours at 37 ℃ and 150 rpm in shaking incubator while fungal strains were inoculated in Sabouraud dextrose broth and incubated at 30 ℃for 48-72 hours at 150 rpm in a shaking incubator. Inoculum equal to 0.5 McFarland of each strain was prepared. Bacterial inoculum was spread on Muller Hinton plates and fungal inoculum was spread on SDA plates by using sterile swabs. Wells of 6 mm diameter were made aseptically by using a sterile cork borer. 100 µl of purified pigmented fractions having a concentration of 1 mg/ml were added in wells aseptically along the solvent ethyl acetate as negative control, levofloxacin (10 µg) for bacterial strains and nystatin (50 µg) for fungal strains as positive control. Then plates were incubated at 37 ℃ for bacteria and at 30 ℃ for fungi. when incubation period was completed, each plate was examined for zone of inhibition. The zone of inhibition was reported in milli- meters (mm).
Determination of antioxidant activity by DPPH assay
DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stable free radicle having red color in solution form. The antioxidants neutralize this free radicle and change its color to yellow. This method is used widely to check the scavenging of free radicles (Devi 2018). Purified pigment was dissolved in methanol to attain the conc. of 200 µg/ml and added to 1 ml of 3 mM DPPH solution and incubated at 37 ℃ for 30-45 minutes. Ascorbic acid (1000 µg/ml) was taken as positive control while methanol and DPPH solution was taken as negative control. After incubation OD was taken at 517 nm and %inhibition was calculated by using the formula given below:
%Inhibition = (Ac-As/Ac) ×100
Where Ac = absorbance of control and As = absorbance of pigment