217 ENDOLICHENIC FUNGI EXTRACTS
activity or to have negative effects such as mutagenicity and cytotoxicity.8,9 Therefore, there
has been a recent need to identify effective and safer tyrosinase inhibitors from natural
sources, especially for the cosmetic industry.10
Since it is known that UV increases ROS and that antioxidants prevent the damage caused
by ROS, antioxidants have recently come to the fore in the cosmetic industry. Antioxidants
play an active role in preventing, repairing, and reducing cellular damage caused by free
radicals.11 Recently, there is increased focus on taking precautions against photoaging and
pigmentation problems caused by UV damage. UV radiation is emphasized as one of the
main causes of photoaging and structural changes in the skin, and other forms of solar
radiation, including visible and infrared light, may also play a role in photoaging.12 UV
radiation increases the risk of long-term damage such as photoimmunosuppression and
photocarcinogenesis as well as photoaging.13 There has also been an increase in UV-induced
melanoma cases in various countries in recent years, and many health authorities are
working intensively on increasing the awareness of this issue and increasing the use of
sunscreen products.14,15 Therefore, the discovery of natural products with high antioxidant
activity is believed to be important.
The need for cosmetic products is increasing every year all over the world. While the
world cosmetics market had a market share of US $380.2 billion in 2019, it is expected to
reach US $463.5 billion with a compound annual growth rate of 5.3% (CAGR) from 2021
to 2027.16 Sun care products are especially among the products with the fastest growth
potential in the cosmetics market. The market for these products was valued at US $11.4
billion in 2021 and is expected to increase to US $17.6 billion by 2027 with a 7.3% CAGR
over 2022 to 2027.17 One of the main reasons for this growth is the increased awareness
of the harmful effects of UV rays. Sunscreens are widely used in daily life due to their
functions such as filtering, reflecting, and dispersing UV rays.18 In particular, consumers’
preference for organic-based products and the development of innovative and advanced sun
care products also contribute to the growth of this market.
Natural products have been the cornerstone of cosmetics and pharmaceutical production
in many parts of the world for years. Recently, there has been an increasing interest in
cosmetic products containing natural herbal extracts, especially for components such as
pigment removers and sunscreens, leading to a growing demand for natural extracts in
the global cosmetics market. Due to the increasing demand of consumers for healthy and
environmentally friendly products, the use of natural herbal ingredients has increased in
the cosmetics sector.19
Lichens are symbiotic associations of fungi (mycobionts), green algae and/or cyanobacteria
(photobionts). In addition to the fungal partner in this mutualism, lichens are also associated
with endolichenic fungi that reside within their thalli.20 Lichens producuse nearly 1,050
specific secondary metabolites that have antioxidant and antimicrobial effects. Despite the
unique biological activity of lichens, their slow growth rate, and limited numbers in nature
make their application in various industries difficult.21 There are also difficulties in the in
vitro production of lichens. However, ELFs are fungal species that live in the lichen thallus
and are easy to grow in lab scale in a shorter time and in large quantities. Therefore,
the importance of producing ELF with similar bioactivities that are not being used in
industries has emerged. ELF are considered promising biological resources for various
fields, given their ability to produce many valuable bioactive metabolites.22 Studies on the
bioactivities of ELF isolated from lichens are quite limited. Identifying and evaluating

218 JOURNAL OF COSMETIC SCIENCE
the composition and efficacy of ELF is particularly important in industries such as
pharmaceuticals and cosmetics. It is expected that the large-scale production of ELF, whose
bioactivity has been determined, will be used in various sectors, ensuring sustainability,
creating environmentally friendly products, and contributing economically.
In particular, consumers prefer sunscreen products with natural ingredients to take
precautions against various skin problems that may occur as a result of exposure to
UV rays. For this purpose, it was aimed to determine the in vitro cosmetic activities
of ELF extracts isolated from lichen thalli and to determine their potential use as a
potential source in the cosmetic industry. First, ELF isolation was performed from Usnea
sp., Lobaria pulmonaria, and Bryoria capillaris to determine in vitro the potential efficacy
of cosmetic potentials of lichens and isolated ELF extracts. Total antioxidant activities,
total phenolic and flavonoid contents, SPFs, and tyrosinase inhibitory activities were
determined.
MATERIALS AND METHODS
COLLECTION LICHEN SAMPLES AND ISOLATION OF THE ENDOLICHENIC FUNGAL SPECIES
The lichen samples of Usnea sp., Lobari pulmonaria, and Bryoria capillaris were collected
from the Aladağ region of Bursa.
For the isolation of endolichenic fungi, surface sterilization of Usnea sp., L. pulmonaria and
B. capillaris lichen samples was performed.23 For surface sterilization of lichen samples,
they were treated sequentially with 95% ethanol (1 minute), 10% sodium hypochlorite
(3 minutes), and 70% ethanol (1 minute) after washing with water, and then dried on
a sterile filter paper. Sterile lichen samples were then cut into small 1x1 cm pieces and
plated on potato dextrose agar. The plates were incubated at room temperature for 14 days.
After the incubation period, different numbers of ELF samples of each lichen species were
purified and pure ELF cultures were obtained.
The isolated and purified samples of ELF were labeled with the following codes: T04-P01,
T04-P03, and T04-P13 for Lobari pulmonaria T20-B02, T20-P07, T20-P10, T20-P26, and
T20-P27 for Bryoria capillaris and T22-P07 and T22-B07 for Usnea sp.
EXTRACTION OF LICHEN AND ELF SAMPLES
For extraction from lichen samples, they were first washed and dried. Lichen samples
were then placed in sterile bottles with acetone and left in the dark for 24 hours. After
24 hours, the samples were filtered through filter paper. Acetone was removed from the
lichen extracts using a rotary evaporator, and 1 mg of the lichen extracts was weighed and
dissolved in 1 mL of dimethyl sulfoxide (DMSO), adjusted to a concentration of 1 mg/
mL and stored at +4°C. Pure ELF isolates were incubated at 25°C for 21 days in 250 mL
Erlenmeyer flasks containing 150 mL of potato dextrose broth. Each fungal culture was
filtered to separate the filtrate from the molds. After filtration, an equal amount of ethyl
acetate was added to create an organic phase. The organic phase was then evaporated to
obtain dry extracts and the extracts were adjusted at a concentration of 1 mg/mL DMSO
and which were stored at +4°C.