219 ENDOLICHENIC FUNGI EXTRACTS
ANALYSIS OF TOTAL ANTIOXIDANT, TOTAL PHENOLIC AND TOTAL FLAVONOID CONTENT
The total antioxidant activity of lichen and ELF extracts (1 mg/mL) was determined using
the 96-well plate methods.24 First, 100 µL of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) in
methanol (40 µg/mL) and 50 µL of 1 mg/mL extract in DMSO were added to a 96-well
plate. The mixture was incubated in the dark for 30 minutes. After incubation, absorbance
was measured at 517 nm against the blank using a Cytation-3 microplate reader. L-Ascorbic
acid (1 mg/mL) was used as a positive control. The percentage of radical scavenging activity
was calculated using the following formula:
%(A DPPH scavening activity A
C S C =-[]×100 )/A
• A
C :Absorbance of control
• A
S :Absorbance of sample
Total phenolic content (TPC) of lichen and ELF extracts (1 mg/mL). Briefly, 40 µL of lichen and
ELF extracts, 100 µL of Folin-Ciocalteu reagent and 75 µL of 7.5% Na2CO3 were added
to each well and incubated. After incubation, absorbance was measured at 750 nm versus
blank using a Cytation-3 microplate reader. Samples were measured in triplicate and a
standard curve was constructed using gallic acid starting at a concentration of 1 mg/mL.
Phenolic content was expressed as gallic acid equivalents.25
The total flavonoid (TFC) content was determined according to the method of Yang et al.26
75 µL of sample solutions (1 mg/ml) and 75 µL of 2% AlCl
3 were mixed in a 96-well plate.
After 15 minutes of incubation, the absorbance was measured against the blank at 435 nm
using a Cytation-3 device. A standard graph was prepared from a stock solution of rutin
at a concentration of 1 mg/mL. The flavonoid content was expressed as rutin equivalents.
IN VITRO DETERMINATION OF SPF
In vitro SPF values of lichen and ELF extracts were calculated according to the method of
Mansur et al.27 A measure of 1 mg of each extract was dissolved in 1 mL of methanol and
analyzed by UV spectrophotometry from 290 to 320 nm. Methanol was used as the blank.
Rutin was used as a positive control. After the measurements, SPF values were calculated
according to the following formula:
SPF CF×Σ320 EE(λ I(λ)× ABS(λ) =-× 290× )
• CF =10 (correction factor)
• EE(λ) =erythematogenic effect
• I(λ) =sun intensity
• ABS(λ) =absorbance
TYROSINASE INHIBITORY ACTIVITY ASSAY
In the tyrosinase inhibition activity, tyrosinase enzyme was used as the enzyme and
l-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate.28 Lichen and ELF extracts were
prepared homogeneously in DMSO at 1 mg/mL. To the wells on the microplate, 150 µL
of phosphate buffer (0.05 M, pH =6.8), 10 µL of 1 mg/mL extracts, and 20 µL of enzyme
solution were added. The microplate was shaken for 3 minutes in a Cytation-3 device, and
ANALYSIS OF TOTAL ANTIOXIDANT, TOTAL PHENOLIC AND TOTAL FLAVONOID CONTENT
The total antioxidant activity of lichen and ELF extracts (1 mg/mL) was determined using
the 96-well plate methods.24 First, 100 µL of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) in
methanol (40 µg/mL) and 50 µL of 1 mg/mL extract in DMSO were added to a 96-well
plate. The mixture was incubated in the dark for 30 minutes. After incubation, absorbance
was measured at 517 nm against the blank using a Cytation-3 microplate reader. L-Ascorbic
acid (1 mg/mL) was used as a positive control. The percentage of radical scavenging activity
was calculated using the following formula:
%(A DPPH scavening activity A
C S C =-[]×100 )/A
• A
C :Absorbance of control
• A
S :Absorbance of sample
Total phenolic content (TPC) of lichen and ELF extracts (1 mg/mL). Briefly, 40 µL of lichen and
ELF extracts, 100 µL of Folin-Ciocalteu reagent and 75 µL of 7.5% Na2CO3 were added
to each well and incubated. After incubation, absorbance was measured at 750 nm versus
blank using a Cytation-3 microplate reader. Samples were measured in triplicate and a
standard curve was constructed using gallic acid starting at a concentration of 1 mg/mL.
Phenolic content was expressed as gallic acid equivalents.25
The total flavonoid (TFC) content was determined according to the method of Yang et al.26
75 µL of sample solutions (1 mg/ml) and 75 µL of 2% AlCl
3 were mixed in a 96-well plate.
After 15 minutes of incubation, the absorbance was measured against the blank at 435 nm
using a Cytation-3 device. A standard graph was prepared from a stock solution of rutin
at a concentration of 1 mg/mL. The flavonoid content was expressed as rutin equivalents.
IN VITRO DETERMINATION OF SPF
In vitro SPF values of lichen and ELF extracts were calculated according to the method of
Mansur et al.27 A measure of 1 mg of each extract was dissolved in 1 mL of methanol and
analyzed by UV spectrophotometry from 290 to 320 nm. Methanol was used as the blank.
Rutin was used as a positive control. After the measurements, SPF values were calculated
according to the following formula:
SPF CF×Σ320 EE(λ I(λ)× ABS(λ) =-× 290× )
• CF =10 (correction factor)
• EE(λ) =erythematogenic effect
• I(λ) =sun intensity
• ABS(λ) =absorbance
TYROSINASE INHIBITORY ACTIVITY ASSAY
In the tyrosinase inhibition activity, tyrosinase enzyme was used as the enzyme and
l-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate.28 Lichen and ELF extracts were
prepared homogeneously in DMSO at 1 mg/mL. To the wells on the microplate, 150 µL
of phosphate buffer (0.05 M, pH =6.8), 10 µL of 1 mg/mL extracts, and 20 µL of enzyme
solution were added. The microplate was shaken for 3 minutes in a Cytation-3 device, and
