Systematic Review of Usnic Acid Extraction from Wild-Grown Lichen Biomass

Abstract

Usnic acid (UA) is one of the most extensively studied specialized metabolites of lichens, attracting considerable interest due to its antimicrobial, anti-inflammatory, and cytotoxic properties. The efficiency of UA extraction from lichens depends on multiple interrelated biological and technological factors. This systematic review aims to synthesize and critically evaluate reported strategies for UA extraction from wild-grown lichen biomass, with particular emphasis on extraction efficiency, practicality, and application potential. This systematic literature review, based on the Scopus database was conducted by including original research articles reporting UA extraction from wild-growing lichens. The analysis covered species selection, sample pre-treatment, solvent type, and extraction methodology. A total of 117 studies were included. Due to the predominantly non-polar nature of UA, higher extraction efficiencies were generally achieved using solvents, including acetone, supercritical CO₂, vegetable oils, and lipophilic green solvent systems. Pre-treatment strategies such as grinding or flaking significantly enhanced extraction performance by improving mass transfer. Alongside conventional methods (maceration, reflux, Soxhlet), non-conventional techniques such as Supercritical Fluid Extraction (SFE), Ultrasound- (UAE), and Microwave-Assisted Extraction (MAE) enabled faster and more selective UA extraction with reduced solvent use. Notably, SFE have been reported as particularly promising in terms of selectivity, process control, and potential suitability for scale-up, with commercially available supercritical CO₂ extracts of Usnea species supporting the feasibility of this approach. This review provides a consolidated and application-oriented overview of UA extraction, highlighting strategies that balance efficiency, selectivity, sustainability, and practical implementation.

1. Introduction

Usnic acid, also known as usniacin or usninic acid (UA), is one of the most extensively studied specialized metabolites [1]. It is found in several genera of lichen as a yellow cortical pigment [2,3]. From the chemical point of view, it is a phenolic compound with dibenzofuran structure (Figure 1), which occurs in nature as enantiomers [4].

Since its first discovery and isolation in 1844 by Knop [5], research on UA evolved alongside the development of lichen chemistry as a distinct scientific discipline. Early systematic treatises, including Zopf’s Die Flechtenstoffe (1907) and later comprehensive monographs on lichen substances, established the chemical classification and analytical foundations that underpin modern extraction and isolation studies [5,6]. Numerous biological activities have been reported for this compound, including antimicrobial, antiviral, anti-inflammatory, analgesic, antiprotozoal, insecticidal, antitumor, antipyretic, antiproliferative, antioxidant, antiherbivoral, photoprotective, gastroprotective, cytoprotective, and immunostimulatory effects [2,7,8,9]. Both (+)- and (-)-UA enantiomers exhibit distinct biological activities, with the (-)-form showing selective herbicidal and antifungal effects, whereas the (+)-form is generally more potent in antimicrobial assays [10]. Considering the activities mentioned above, UA has found application in cosmetology, agriculture, pharmaceutical industry and dentistry [11]. It has been used in dietary supplements for weight loss as a result of the observed slimming effect in humans [12]. However, emerging cases of hepatotoxicity associated with intake of a dietary supplements containing this acid are raising safety concerns [13].

As the pharmaceutical potential of UA has received considerable attention, the development of effective methods for its isolation has also attracted great interest among researchers. The amounts of bioactive compounds in its natural matrix are often fairly low [14]. The reported UA content varies depending on the source and lichen species and has been estimated to range from approximately 2% to 8% of the dry thallus weight [12,15].

Although lichens are the primary source of UA, several studies have also reported its presence in plant, fungal and cultured lichen material. In plants, UA has been isolated from Cichorium intybus [16], Helicteres hirsuta [17], Canna indica [18], Daphne papyracea [19], Pterocarpus santalinus [20] and Euterpe oleracea fruit extract [21]. It has also been identified in several fungi, including Mycosphaerella sp. [22,23], Verruculina enalia [24] and Trochila sp. [25]. In Mycosphaerella nawae, the yield of the target compound was reported to be approximately 2.5% of the dry weight [22]; in Verruculina enalia, approximately 0.075% [24]; and in Mycosphaerella sp. about 0.17% of mass of the crude extract mass [23]. Additionally, attempts to produce UA in vitro have involved cultured lichen systems, such as symbionts or protoplast fusions of Usnea ghattensis [26,27], Usnea complanata [28] and species of Ramalina [29].

Numerous articles and scientific reports concerning activity of UA resulted in a large number of published reviews covering aspects of bioactivity with a large number of anticancer properties [30,31], hepatotoxicity [13], and nanotechnology applications [32]. Furthermore, reviews concerning the preparation and chemical transformation of the studied compound are accessible [15]. Despite the fact that UA could be prepared by chemical synthesis with high purity, low solubility in nontoxic solvents is the major limitation of its application as a pharmaceutical formulation [33].

To the best of our knowledge, no systematic review has comprehensively addressed the extraction conditions specific to UA. Given the multitude of factors influencing this process-including the choice of lichen species, sample pretreatment, extraction methodology, operational parameters (pressure, temperature, duration, repetition), and solvent characteristics-systematic optimization is clearly needed. Nevertheless, identifying universally efficient conditions is impractical, as published studies differ substantially in design and reporting. The heterogeneity and inconsistency of the available data further hinder direct comparisons and complicate the selection of an optimal extraction protocol. Therefore, this review aims to consolidate and critically examine the current literature on UA extraction, providing a systematic synthesis of methods, conditions, and outcomes to guide future research and applications.

2. Methods

This work was conducted as a systematic literature review in accordance with the PRISMA 2020 guidelines. The review protocol was registered retrospectively in the Open Science Framework (OSF): https://osf.io/7awc2/overview (accessed on 19 January 2026). The primary outcome of interest was quantitatively determined UA content, regardless of the units reported.

2.1. Eligibility Criteria

UA is most commonly extracted from wild-growing lichens [34]. Due to the wide occurrence of UA among lichen species, studies were eligible for inclusion if they investigated UA extraction from wild-grown lichen-derived material and reported quantitatively determined UA content, either directly or indirectly expressed in relation to the extracted material. Including data on plants, fungi, or cultured lichen-each influenced by additional and heterogeneous experimental factors-would introduce inconsistencies and complicate meaningful comparisons. Therefore, these studies were excluded, and are discussed only briefly in the introduction. Furthermore, review articles, clinical studies, studies without accessible full text, and studies lacking a description of the UA extraction process (e.g., solvent or extraction method) were excluded. Studies that did not report quantitative UA determination or UA isolation recovery expressed in relation to dry lichen biomass or dry extract weight were also excluded.

2.2. Literature Search

A systematic literature search was conducted in the Scopus database in December 2025. The following search query was applied:

TITLE-ABS-KEY (usnic AND acid AND (extract OR extraction OR extracted))

Records were screened based on titles and abstracts, followed by full-text assessment for eligibility according to the predefined criteria. Study selection was conducted by one reviewer. After screening, 117 studies were included in the final analysis. The study selection process is illustrated in the PRISMA flow diagram (Figure 2). The PRISMA 2020 checklist is provided in Table S1 (Supplementary Materials). A detailed list of excluded full-text articles with reasons is shown in Table S2.

2.3. Data Collection and Data Items

Data were extracted by one reviewer using a standardized data extraction form. For each included study, relevant information was collected directly from the full text. When necessary, Supplementary Materials were consulted to obtain missing methodological details. No automation tools were used in the data extraction process.

The following data items were extracted from each eligible study: lichen species, extraction method, solvent type, and operational parameters (temperature, pressure, extraction time, number of extraction cycles, liquid-to-solid ratio), as well as quantitatively determined UA content.

When reported, additional information such as origin of the raw material, sample pretreatment (e.g., grinding or flaking), and extraction yield (when provided by the authors) were also described. No assumptions were made for missing or unreported data.

The effect measure used in this review was the quantitatively determined UA content. For the purpose of comparative presentation, UA content reported in different units across the included studies (e.g., mg/g of dry lichen material, mg/g of extract, or percentage content) was converted to percentage content (%) and presented in this standardized form in Table 1.

Unit conversions were performed based solely on the information provided in the original publications, without imputing missing data or making additional assumptions.

2.4. Data Synthesis

Due to substantial heterogeneity in extraction protocols, solvents, and reporting formats, quantitative synthesis was not feasible. Data were therefore synthesized descriptively and summarized in tables and figures. Table 1 allows comparison of extraction methods and operational parameters in relation to reported UA content in the dry weight of the lichen (or dry extract). Figure 3 provides an overview of conventional (maceration, Soxhlet) and non-conventional (MAE, UAE, SFE) extraction techniques, as well as the major classes of extraction solvents, including polar/hydrophilic, non-polar/lipophilic, and “green” solvents (e.g., scCO₂, NADES). Key operational considerations, such as solvent demand, selectivity, simplicity, and scale-up perspective, are also indicated. The classification of extraction methods discussed in this review follows the framework proposed by [14]. Results from comparative analyses and optimization studies are also comprehensively presented. For optimization-focused investigations, either the center point [33] or the optimal extraction conditions reported in previous works [9,11,35] are included in Table 1.

Table 1. Summary of extraction and isolation methods for UA (% UA (dry lichen or extract basis, as reported) from lichen species.

Conditions (Temperature, Duration, Pressure, Flow Rate)		Lichen Species	Usnic Acid Content (Q/I, % or % (DE))	References
Conventional Methods
Maceration
Acetone
3 h; 50 °C; L:S = 5 mL/g; continuous stirring		Cetraria islandica	I: 0.4%	[36]
L:S = 20 mL/g; 3 h; number of extraction cycles = 5; room temperature		Cladonia incrassata	I: 0.013%	[37]
14 days; ~2 mL/g; room temperature; number of extraction cycles = 3		Cladonia pyxidata	I: ~0.1764%; ~1.8% (DE)	[38]
L:S = 2.5 mL/0.05 g; ice bath; number of extraction cycles = 4; 5 min/ 10 min		Cladonia arbusscula	Q: 2.1%	[39]
		Cladonia stellaris	2.4%
L:S = 10 mL/g; 15 min; number of extraction cycles = 5		Cladonia stellaris	Q: 0.482–3.08%	[10]
L:S = 15 mL/g; 15 min; stirring; 30 min; number of extraction cycles = 2			Q: 1.5–1.9%	[40]
60 min; room temperature; stirring; L:S = 7.5 mL/g			Q: 2.021 ± 0.087%	[33]
1 h; room temperature; L:S = 40 mL/g		Evernia prunastri Flavoparmelia caperata	Q: 3.24% (DE) 10% (DE)	[41]
5 min.; L:S = 30 mL/g; number of extraction cycles = 3		Flavoparmelia baltimorensis	Q: 2.3%	[42]
L:S = 200 mL/g; room temperature (20–22 °C)		Hypogymnia vittata H. physodes H. tubulosa	Q: 0.63 ± 0.03% 1.05 ± 0.00% 2.40 ± 0.00%	[43]
Room temperature; constant shaking; L:S = 25 mL/g; 5 h		Lethariella canariensis	Q: 0.01–0.06% (DE)	[44]
12 h; room temperature; L:S = 2.78 mL/g; number of extraction cycles = 3		Leprocaulon microscopicum	I: 1.2%	[45]
48 h; room temperature; L:S = 4.03 mL/g; number of extraction cycles = 3		Myelochroa leucotyliza	Q: 1.68% (DE) I: 0.075% (DE)	[46]
Room temperature; L:S = 200 mL/g		Parmelia flexilis	Q: 1.66 ± 0.10–5.13 ± 0.22%	[47]
L:S = 5 mL/g; room temperature; 72 h		Physconia venusta	Q: 0.85 ± 0.01%	[48]
4 h; room temperature; dark; L:S = 40 mL/g; stirring		Ramalina farinacea R. fastigiata R. fraxinea	Q: 0.022 ± 0.001% 0.028 ± 0.001% 0.014 ± 0.001%	[49]
1 h; room temperature (20–22 °C); L:S = 200 mL/g		Ramalina capitata R. fastigiata R. fraxinea R. pollinaria R. polymorpha	Q: 1.25 ± 0.29% 3.23 ± 0.16% 0.13 ± 0.01% 0.22 ± 0.01% 0.27 ± 0.02%	[50]
Ambient temperature		Ramalina peruviana	I: 0.00116%; 0.02185% (DE)	[51]
30 min.		Ramalina reticulata	I: 0.08%; 8% (DE)	[52]
24 h; number of extraction cycles = 3		Ramalina sp.	I: 0.0213%	[53]
1 h; room temperature (20–22 °C); L:S = 200 mL/g		Rhizoplaca chrysoleuca R. melanopthalma R. peltata	Q: 4 ± 0.07% 0.19 ± 0.01% 0.53 ± 0.04%	[54]
1 h; room temperature; L:S = 200 mL/g		Squamarina lentigera	Q: 2.47 ± 0.01%	[55]
Ambient temperature		Usnea baileyi	I: 0.0043%	[56]
L:S = 10 mL/g; 10 days; 20–22 °C; dark place; shaking 3–4 times/day → extract was made up to 100 mL (liquid extract)		Usnea barbata	Q: 2.119%	[57]
L:S = 10 mL/g			Q: 2.1153%	[58]
1 h; room temperature (20–22 °C); L:S = 200 mL/g		Usnea barbata U. florida U. hirta U. longissima U. rigida U. subflorida	Q: 2.16 ± 0.67% 2.36 ± 0.37% 0.68 ± 0.04% 1.12 ± 0.11% 0.22 ± 0.01% 6.49 ± 0.01%	[59]
5 min.; L:S = 30 mL/g; number of extraction cycles = 3		Usnea florida U. hirta	Q: 5.7% 5.8%	[42]
L:S = 40 mL/g; room temperature; dark; 4 h; stirring		Usnea filipendula U. fulvoreagens U. intermedia	Q: 0.110 ± 0.016% 0.119 ± 0.001% 0.097 ± 0.005%	[60]
Shaking for 2 h, then left overnight; L:S = 20 mL/g; number of extraction cycles = 3		Usnea ghattensis U. orientalis U. undulata	Q: 1.66 ± 3.2% (DE) 0.66 ± 4.1% (DE) 3.05 ± 3.9% (DE)	[61]
L:S = 1.5 mL/g; 14 days; number of extraction cycles = 3; room temperature		Usnea laevis	I: ~0.45%	[62]
48 h; room temperature		Usnea longissima	I: 0.5%; 11.5385% (DE)	[63]
Room temperature; L:S = 10 mL/g; number of extraction cycles = 3		Usnea steineri	I: 0.0139%	[64]
Overnight; room temperature; number of extraction cycles = 3		Usnea sp.	I: 20% (DE)	[65]
20 min; number of extraction cycles = 4		Vulpicida pinastri	Q: 1.86%	[66]
L:S = 15 mL/g; number of extraction cycles = 2; room temperature		Xanthoparmelia stenophylla	I: 0.22%	[67]
40 min; number of extraction cycles = 4			Q: 1.36%	[66]
3 days; room temperature; dark place; L:S = 10 mL/g; number of extraction cycles = 3; occasional shaking			Q: 4.447% (DE) I: 4% (DE)	[68]
Acetone:methanol (1:1)
L:S = 2 mL/g; room temperature; 3 days; number of extraction cycles = 3		Hypotrachyna enderythrae Xanthoparmelia farinosa Psiloparmelia distincta (rock) P. distincta (tree) Usnea durietzii	Q: 0.035 ± 0.00% 0.18 ± 0.01% 0.017 ± 0.00% 0.042 ± 0.00% 0.304 ± 0.02%	[69]
Chloroform
Stirring; room temperature (30 °C); 48 h; L:S = 10 mL/g		Parmelia erumpens	I: 2.69% (DE)	[70]
-		Usnea florida	Q: 3.58–4.21%	[71]
Chloroform:methanol
Chloroform:methanol (2:1); number of extraction cycles = 3		Usnea sp.	I: 0.6%	[2]
Chloroform:methanol (1:1 v/v); L:S = 2.5 mL/g; 3 days; room temperature		Ramalina asperula Thamnolia vermicularis subsp. solida Usnea sp.	Q: 1.01% 0.2% 1.07%	[72]
Dichloromethane
1 h; room temperature; L:S = 40 mL/g		Evernia prunastri Flavoparmelia caperata	Q: 7.02% (DE) Q: 58.9% (DE)	[41]
L:S = 4.4405 mL/g		Lecanora muralis	I: 0.0076%; 2.3497% (DE)	[73]
1 h; number of extraction cycles = 2; L:S = 3.53 mL/g		Protousnea poeppigii	I: 0.5731%; 9.36% (DE)	[74]
-		Ramalina capitata	Q: 25.8% (DE)	[75]
1,4-Dioxane
L:S = 40 (w/w); 70 °C; 30 min; 1,4-Dioxane 1,4-Dioxane:water 1:1		Usnea barbata	Q: 2.34% 2.08%	[76]
Ethanol
Ethanol 95%; room temperature; number of extraction cycles = 12; L:S = 1.07 mL/g		Alectoria sarmentosa	I: 2.3369%; 31.2143% (DE)	[77]
Ethanol: water (1:1)		Cladonia leptoclada	I: 0.8%	[78]
60 min; room temperature; stirring: L:S = 7.5 mL/g		Cladonia stellaris	Q: 0.612 ± 0.17%	[33]
24 h; room temperature; L:S = 20.83 mL/g		Parmotrema hypotropa	Q: 0.64% (DE)	[79]
4 h; room temperature; dark; L:S = 40 mL/g; stirring		Ramalina farinacea R. fastigiata R. fraxinea	Q: 0.024 ± 0.001% 0.022 ± 0.001% 0.015 ± 0.001%	[49]
L:S = 40(w/w); 70 °C; 30 min; Ethanol 40% Ethanol 70% Ethanol 96%		Usnea barbata	Q: 0.85% 1.24% 1.44%	[76]
Ethanol 70% (v/v); frequent shaking; 5 days; room temperature; L:S = 5 mL/g			Q: 0.00783%; 0.25 ± 0.00% (DE)	[80]
Ethanol 70% (v/v); 24 h; L:S = 6.39 mL/g			Q: 1.39% (DE)	[81]
Ethanol 96%; L:S = 10 mL/g; 10 days; 20–22 °C; dark place; shaking 3–4 times/day → extract was made up to 100 mL (liquid extract)			Q: 0.0025%	[57]
Ethanol 96%; L:S = 10 mL/g			Q: 0.2566%	[58]
L:S = 40 mL/g; room temperature; dark; 4 h; stirring		Usnea filipendula U. fulvoreagens U. intermedia	Q: 0.097 ± 0.003% 0.101 ± 0.001% 0.072 ± 0.002%	[60]
Ether
-		Ramalina capitata	Q: 4.4% (DE)	[75]
number of extraction cycles = 5; warm		Ramalina siliquosa	I: 0.12%	[82]
Ethyl acetate
1 week; L:S = 48.39 mL/g		Niebla homalea	I: 0.0048%	[83]
-		Ramalina capitata	Q: 8.2% (DE)	[75]
Heptane
number of extraction cycles = 2; 12 h + 3 h; room temperature; L:S = 3 mL/g		Ophioparma ventosa	I: 0.22%; 27.5% (DE)	[84]
Hexane
24 h; 20 °C; L:S = 16.67 mL/g; number of extraction repetitions = 3 (L:S = 1.67 mL/g)		Usnea dasopoga	Q: 0.15%	[85]
3 days; room temperature; dark place; L:S = 10 mL/g; number of extraction cycles = 3; occasional shaking		Xanthoparmelia stenophylla	Q: 58.733% (DE)	[68]
Methanol
Methanol 70%; 1 h; room temperature; L:S = 40 mL/g		Evernia prunastri	Q: 2.92% (DE)	[41]
		Flavoparmelia caperata	Q: 5.4% (DE)
L:S = 20 mL/g; 12 h; Water bath 45 °C		Evernia divaricata Ramalina fraxinea Usnea florida	Q: 5.071 ± 0.086% (DE) 1.621 ± 0.012% (DE) 9.941 ± 0.026% (DE)	[86]
Room temperature; constant shaking; L:S = 1.4286 mL/g		Lethariella canariensis	I: 0.0077%; 0.105% (DE)	[44]
48 h; room temperature; L:S = 4.03 mL/g; number of extraction cycles = 3		Myelochroa leucotyliza	Q: 0.90%	[46]
4 h; room temperature; dark; L:S = 40 mL/g; stirring		Ramalina farinacea R. fastigiata R. fraxinea	Q: 0.034 ± 0.003% 0.031 ± 0.002% 0.016 ± 0.001%	[49]
L:S = 20 mL/g; 24 h; number of extraction cycles = 2		Stereocaulon alpinum	I: 0.4518%; 3.8288% (DE)	[87]
Room temperature		Usnea aciculifera	I: 1.3267%; 4.975% (DE)	[88]
Q: L:S = 62.5 mL/g; 24 h; room temperature I: L:S = 8 mL/g; 3 days; number of extraction cycles = 3		Usnea cornuta	Q: 18.196 ± 0.038% (DE) I: 3.4%; 25% (DE)	[89]
L:S = 40 mL/g; room temperature; dark; 4 h; stirring		Usnea filipendula U. fulvoreagens U. intermedia	Q: 0.107 ± 0.009% 0.104 ± 0.007% 0.094 ± 0.004%	[60]
48 h; room temperature; stirring		Usnea misaminensis	I: 0.1936%; 2.3325% (DE)	[90]
3 days; room temperature; shaking		Usnea undulata	I: 2.7131% (DE)	[91]
1 h; L:S = 66.67 mL/g		Xanthoparmelia camtschadalis X. conspersa	I: 0.432 ± 0.06% 0.437 ± 0.07%	[92]
3 days; room temperature; dark place; L:S = 10 mL/g; number of extraction cycles = 3; occasional shaking		Xanthoparmelia stenophylla	Q: 4.852% (DE)	[68]
Methanol/water (1% NaCl)
Overnight; L:S = 8 mL/g; stirring		Ramalina implexa	I: 0.0056%	[93]
Methanol:dichloromethane (1:1)
12 h; 25 °C		Usnea florida	I: 2.4%; 12% (DE)	[94]
Petroleum ether
Room temperature		Usnea barbata	I: 1.052%	[95]
Vegetable oils
Cold sunflower oil; 3 months; room temperature, dark; L:S = 50 mL/g		Usnea barbata	Q: 8.04 ± 0.35%	[12]
Cold-pressed canola seed oil; 3 months; 21–22 °C; dark place; L:S = 24.72 mL/g; daily shaking			Q: 2.162%; 0.0915 ± 0.0018% (DE)	[96]
Water
L:S = 10 mL/g; 10 days; 20–22 °C; dark place; shaking 3–4 times/day → extract was made up to 100 mL (liquid extract)		Usnea barbata	Q: 0.0004%	[57]
L:S = 40 mL/g; 70 °C; 30 min			Q: 0.76%	[76]
L:S = 10 mL/g			Q: 0.0446%	[58]
Soxhlet extraction
Acetone
24 h; L:S = 25 mL/g		Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 1.96%; 56% (DE)	[97]
L:S = 20 mL/g; 12 h; 50 °C		Evernia divaricata Ramalina fraxinea Usnea florida	Q: 4.91 ± 0.002% (DE) 0.924 ± 0.004% (DE) 44.098 ± 0.025% (DE)	[86]
-		Hypogymnia physodes	I: 19% (DE)	[98]
-		Lethalia vulpina	I: 0.041%; 0.56% (DE)	[99]
-		Parmelia caperata	I: 38% (DE)	[100]
-		Usnea barbata	I: 19% (DE)	[101]
8 h; 70 °C			Q: 16.53 ± 6.53% (DE)	[102]
L:S = 10 mL/g; 8 h; 55–60 °C			Q: 1.3418%; 24.183 ± 0.0172% (DE)	[57]
			Q: 0.028278%	[103]
8 h; 55–60 °C			Q: 28.278% (DE)	[8]
-		Usnea subfloridana	Q: 0.7%; 13.91% (DE)	[11]
Acetone:dichloromethane (1:1)
3 h; L:S = 38.46 mL/g; number of extraction cycles = 2		Vulpicida pinastri	I: 1.07%; 16% (DE)	[104]
Chloroform
L:S = 1.5 mL/g		Cladonia substellata	I: 0.84%	[105]
Ethanol
24 h; L:S = 25 mL/g		Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 0.8432%; 13.6% (DE)	[97]
-		Usnea subfloridana	Q: 0.19%; 6.81% (DE)	[11]
Ethanol 70% (v/v); L:S = 10 mL/g		Usnea barbata	Q: 0.365%; 3.59 ± 0.38% (DE)	[80]
Ethanol 96%; L:S = 10 mL/g; 8 h; 75–80 °C			Q: 1.2125%; 10.8742 ± 0.0703% (DE)	[57]
Ethanol 96%; 8 h; 75–80 °C			Q: 12.721% (DE)	[8]
Ethanol 96%; ~90 min; 78.4 °C		Usnea laevis	Q: 13.3% (DE)	[106]
Ethyl acetate
8 h; 75–80 °C		Usnea barbata	Q: 37.673% (DE)	[8]
			I: 28.15% (DE)	[107]
Ethyl ether
-		Ramalina stenospora	I: 0.013%	[108]
L:S = 2 mL/g; 40 °C		Usnea longissima	I: 0.84%	[109,110]
Hexane
L:S = 50 mL/g; 45 min; number of extraction cycles = 3		Cladonia portentosa	I: 18.33% (DE)	[111]
L:S = 3.125 mL/g		Usnea sp.	I: ~0.125%	[112]
L:S = 6.67 mL/g		Usnea undulata	I: 0.827%; 16.96% (DE)	[113,114]
-		Asahinea chrysantha Asahinea scholanderi	I: 1.30% 0.01%	[115]
Methanol
8 h; 65 °C		Usnea barbata	Q: 13.760% (DE)	[8]
Petroleum ether
24 h; L:S = 25 mL/g		Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 0.252%; 12.6% (DE)	[97]
		Cladonia arbuscula	I: 0.0156%	[116]
-		Usnea subfloridana	Q: 0.57%; 31.65% (DE)	[11]
Sequential Soxhlet extraction
Acetone → Methanol	-	Parmotrema rampoddense	I: 1% (DE)	[117]
Ether → Ethanol 96.3% (v/v)	-	Usnea barbata	Q: 67.09% (DE) 2.43% (DE)	[81]
n-Hexane → dichloromethane → methanol	4 h for each solvent; L:S = 20 mL/g	Usnea aurantiacoatra	I: 84.466% (DE) 8.677% (DE) 1.075% (DE)	[118]
n-hexane → ethyl acetate → methanol	-	Cladonia convoluta	I: 0.3625% Q: 0.92%; 66% (DE) hexane; 16% (DE) ethyl acetate; 1.7% (DE) methanol	[119]
Petroleum ether → chloroform	12 h for each solvent	Usnea molliuscula	I: 1.62%	[120]
Reflux
Acetone	1 h; 90 °C; number of extraction cycles = 1	Cladonia arbuscula subsp. squarrosa	Q: 0.425 ± 0.08%	[9]
	1 h; L:S = 100 mL/g	Cladonia mitis	Q: 0.452 ± 0.054–2.158 ± 0.223% I: 0.998%	[121]
Dichloromethane	L:S = 7.14 mL/g; number of extraction cycles = 2	Usnea florida	I: 0.61%; 40% (DE)	[74]
Ethanol 70%	L:S = 16.67 mL/g; number of extraction cycles = 2	Usnea longissima	I: 0.0066%	[1]
Ethanol-water (95:5, v/v)	1 h; L:S = 15 mL/g; number of extraction cycles = 2	Usnea longissima	Q: 13.7% (DE)	[122]
	number of extraction cycles = 3 (2 h + 1 h + 1 h)		I: 0.0035%	[123]
	2 h; number of extraction cycles = 2	Cladonia alpestris	I: ~0.96%	[124]
Ethyl acetate	1.5 h; L:S = 4 mL/g; number of extraction cycles = 3	Usnea longissima	I: 3.74% (DE)	[125]
Decoction
Distilled water	12 min.; L:S = 10 mL/g; Boiling	Usnea longissima	Q: 1.97% (DE)	[126]
Non-conventional methods
Supercritical fluid extraction
CO2	40 min.; 35 MPa; 40 °C; 5 mL/min	Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 2.5%; 91% (DE)	[97]
	6 h; 50 °C; 350 barrs; 20 kg/h	Usnea barbata	I: 1.4783%; 71.3287% (DE)	[127]
	30 MPa; flow rate 0.3 kg/h; 40 °C 60 °C		Q: 0.219%; 36.49 ± 0.33% (DE) 0.226%; 59.48 ± 0.20% (DE)	[80]
	30 MPa; 40 °C; 1 ± 3 kg/h		Q: 42.3–63.45% (DE)	[128]
	50 MPa; 40 °C; 992 m3/kg		Q: 0.551–1.243%; 54.5–64.8% (DE)	[129]
	80 min; 85 °C; 150 atm.; 2 ml/min	Usnea subfloridana	Q: 0.45%	[11]
CO2 + 4.3% ethanol (co-solvent)	7.48 h; 300 bar; 5.5 mL/min; 42 °C	Usnea longissima	Q: 0.372 ± 0.022%	[35]
Subcritical CO2	30 MPa; 25 °C; 967 m3/kg	Usnea barbata	Q: 1.315%; 63.2% (DE)	[129]
Ultrasound-assisted extraction
Acetic Acid	20 °C; 25 min	Usnea amblyoclada	Q: 0.00153 ± 0.000113%–0.00234 ± 0.000197%	[130]
Acetone	30 min; 30–35 °C; number of extraction cycles = 4; L:S = 10.53 mL/g	Cladonia uncialis	Q: 52.52 ± 0.28% (DE)	[131]
	30 min; 35 °C; number of extraction cycles = 6; L:S = 20 mL/g		Q: 30.65% (DE)	[132]
	L:S = 40 mL/g; 30–35 °C; 35 kHz; 20 min		Q: 1.305 ± 0.0894%	[133]
	30 °C; L:S = 15 mL/g; number of extraction cycles =1 number of extraction cycles = 10		Q: 1.137% 1.45%	[134]
Camphor:Thymol (in molar ratio 0.3)	L:S = 60 mL/g; 30 min; number of extraction cycles = 1		Q: 1.193–1.496%
Acetone	25 °C; 30 min; number of extraction cycles = 3	Peltigera neckeri Peltigera canina Peltigera ponojensis	Q: 0.0000007% (DE) 0.0000012% (DE) 0.0000736% (DE)	[135]
Methanol		Peltigera neckeri Peltigera canina Peltigera ponojensis	0.0000006% (DE) 0.0000026% (DE) 0.0000038% (DE)
1,4-Dioxane 1,4-Dioxane:water Water	L:S = 40(w/w); 40 °C; 30 min; 18 kHz	Usnea barbata	Q: 2.40% 1.57% 0.65%	[76]
Heptane Diethyl ether Acetone Methanol	30 min; 30–35 °C; number of extraction cycles = 4; L:S = 10 mL/g	Cladonia uncialis	Q: 63.3 ± 3.0% (DE) 43.7 ± 1.0% (DE) 28.4 ± 3.0% (DE) 19 ± 1.0% (DE)	[7]
Heptane → Acetone	20 min; 35 °C; number of extraction cycles = 5; L:S = 4.6 mL/g		I: 0.28%; 33% (DE) heptane	[7]
Ethanol (80%)	L:S = 20 mL/g; 32.23 °C; 20 min	Evernia divaricata	Q: 0.01605 ± 0.00085%	[136]
	L:S = 20 mL/g; 37.02 °C; 20 min	E. prunastri	Q: 0.01886 ± 0.00013%
Ethanol 40% Ethanol 70% Ethanol 96%	L:S = 40 (w/w); 40 °C; 30 min; 18 kHz	Usnea barbata	Q: 1.07% 1.49% 1.54%	[76]
Methanol: dichloromethane (1:1, v/v)	30 min.; L:S = 185–200 mL/g; mixing	Dirinaria applanata	Q: 0.003989%	[137]
Methanol (75%)	30 min; L:S = 10 mL/g; number of extraction cycles = 3	Thamnolia subuliformis	Q: 0.8613% (DE)	[138]
Microwave-assisted extraction
Acetone	5 min; 80 °C; number of extraction cycles = 3; max 120 psi; max 100 W L:S = 20 mL/g	Cladonia foliacea	I: 0.15% Q: 1.173%	[4]
Ethanol	5 min; 80 °C; number of extraction cycles = 2; L:S = 20 mL/g; stirring; max 120 psi; max 100 W		Q: 1.351% I: 0.42%
Ethyl acetate			Q: 1.304%
Q: Limonene Ethyl lactate I: Ethyl lactate	L:S = 62.5 mL/g; 30 min; 100 °C; 20 W	Usnea cornuta	Q: 9.052 ± 0.001% (DE) 13.855 ± 0.020% (DE) I: 5.28%; 22% (DE)	[89]
Combined technique
Acetone-based
Maceration → silica impregnation → Soxhlet extraction with (1) hexane (2) benzene (3) chloroform		Cladina macaronesica	I: 0.018%; 0.067% (DE)	[139]
UAE: 30 min.; L:S = 50 mL/g; 40 kHz → Maceration: 23.5 h		C. mitis/arbuscula Cladina stellaris Flavocetraria cucullata F. nivalis	Q: 0.681 ± 0.345% 0.939 ± 0.466% 0.939 ± 0.486% 1.62 ± 0.069%	[140]
UAE: 30 min; L:S = 2000 mL/g → Maceration: 20 °C; overnight→ UAE: 30 min; L:S = 1000 mL/g		Xanthoparmelia chlorochroa	Q: 0.841 ± 0.367–1.813 ± 0.449%	[3]
Ethanol
Ethanol (70%, v/v) Percolation 2 h → 24 h after addition of the solvent; room temperature; L:S = 3 mL/g		Usnea barbata	Q: 0.00783%; 0.18 ± 0.02% (DE)	[80]

Q—quantitative determination; I—isolation; %—UA content expressed as % (w/w) of dry lichen thallus; % (DE)—UA content expressed as % (w/w) of dry extract. Species listed alphabetically within each study. Abbreviations: L:S—liquid-to-solid ratio, UAE—ultrasound-assisted extraction.

Table 1. Summary of extraction and isolation methods for UA (% UA (dry lichen or extract basis, as reported) from lichen species.

Conditions (Temperature, Duration, Pressure, Flow Rate)		Lichen Species	Usnic Acid Content (Q/I, % or % (DE))	References
Conventional Methods
Maceration
Acetone
3 h; 50 °C; L:S = 5 mL/g; continuous stirring		Cetraria islandica	I: 0.4%	[36]
L:S = 20 mL/g; 3 h; number of extraction cycles = 5; room temperature		Cladonia incrassata	I: 0.013%	[37]
14 days; ~2 mL/g; room temperature; number of extraction cycles = 3		Cladonia pyxidata	I: ~0.1764%; ~1.8% (DE)	[38]
L:S = 2.5 mL/0.05 g; ice bath; number of extraction cycles = 4; 5 min/ 10 min		Cladonia arbusscula	Q: 2.1%	[39]
		Cladonia stellaris	2.4%
L:S = 10 mL/g; 15 min; number of extraction cycles = 5		Cladonia stellaris	Q: 0.482–3.08%	[10]
L:S = 15 mL/g; 15 min; stirring; 30 min; number of extraction cycles = 2			Q: 1.5–1.9%	[40]
60 min; room temperature; stirring; L:S = 7.5 mL/g			Q: 2.021 ± 0.087%	[33]
1 h; room temperature; L:S = 40 mL/g		Evernia prunastri Flavoparmelia caperata	Q: 3.24% (DE) 10% (DE)	[41]
5 min.; L:S = 30 mL/g; number of extraction cycles = 3		Flavoparmelia baltimorensis	Q: 2.3%	[42]
L:S = 200 mL/g; room temperature (20–22 °C)		Hypogymnia vittata H. physodes H. tubulosa	Q: 0.63 ± 0.03% 1.05 ± 0.00% 2.40 ± 0.00%	[43]
Room temperature; constant shaking; L:S = 25 mL/g; 5 h		Lethariella canariensis	Q: 0.01–0.06% (DE)	[44]
12 h; room temperature; L:S = 2.78 mL/g; number of extraction cycles = 3		Leprocaulon microscopicum	I: 1.2%	[45]
48 h; room temperature; L:S = 4.03 mL/g; number of extraction cycles = 3		Myelochroa leucotyliza	Q: 1.68% (DE) I: 0.075% (DE)	[46]
Room temperature; L:S = 200 mL/g		Parmelia flexilis	Q: 1.66 ± 0.10–5.13 ± 0.22%	[47]
L:S = 5 mL/g; room temperature; 72 h		Physconia venusta	Q: 0.85 ± 0.01%	[48]
4 h; room temperature; dark; L:S = 40 mL/g; stirring		Ramalina farinacea R. fastigiata R. fraxinea	Q: 0.022 ± 0.001% 0.028 ± 0.001% 0.014 ± 0.001%	[49]
1 h; room temperature (20–22 °C); L:S = 200 mL/g		Ramalina capitata R. fastigiata R. fraxinea R. pollinaria R. polymorpha	Q: 1.25 ± 0.29% 3.23 ± 0.16% 0.13 ± 0.01% 0.22 ± 0.01% 0.27 ± 0.02%	[50]
Ambient temperature		Ramalina peruviana	I: 0.00116%; 0.02185% (DE)	[51]
30 min.		Ramalina reticulata	I: 0.08%; 8% (DE)	[52]
24 h; number of extraction cycles = 3		Ramalina sp.	I: 0.0213%	[53]
1 h; room temperature (20–22 °C); L:S = 200 mL/g		Rhizoplaca chrysoleuca R. melanopthalma R. peltata	Q: 4 ± 0.07% 0.19 ± 0.01% 0.53 ± 0.04%	[54]
1 h; room temperature; L:S = 200 mL/g		Squamarina lentigera	Q: 2.47 ± 0.01%	[55]
Ambient temperature		Usnea baileyi	I: 0.0043%	[56]
L:S = 10 mL/g; 10 days; 20–22 °C; dark place; shaking 3–4 times/day → extract was made up to 100 mL (liquid extract)		Usnea barbata	Q: 2.119%	[57]
L:S = 10 mL/g			Q: 2.1153%	[58]
1 h; room temperature (20–22 °C); L:S = 200 mL/g		Usnea barbata U. florida U. hirta U. longissima U. rigida U. subflorida	Q: 2.16 ± 0.67% 2.36 ± 0.37% 0.68 ± 0.04% 1.12 ± 0.11% 0.22 ± 0.01% 6.49 ± 0.01%	[59]
5 min.; L:S = 30 mL/g; number of extraction cycles = 3		Usnea florida U. hirta	Q: 5.7% 5.8%	[42]
L:S = 40 mL/g; room temperature; dark; 4 h; stirring		Usnea filipendula U. fulvoreagens U. intermedia	Q: 0.110 ± 0.016% 0.119 ± 0.001% 0.097 ± 0.005%	[60]
Shaking for 2 h, then left overnight; L:S = 20 mL/g; number of extraction cycles = 3		Usnea ghattensis U. orientalis U. undulata	Q: 1.66 ± 3.2% (DE) 0.66 ± 4.1% (DE) 3.05 ± 3.9% (DE)	[61]
L:S = 1.5 mL/g; 14 days; number of extraction cycles = 3; room temperature		Usnea laevis	I: ~0.45%	[62]
48 h; room temperature		Usnea longissima	I: 0.5%; 11.5385% (DE)	[63]
Room temperature; L:S = 10 mL/g; number of extraction cycles = 3		Usnea steineri	I: 0.0139%	[64]
Overnight; room temperature; number of extraction cycles = 3		Usnea sp.	I: 20% (DE)	[65]
20 min; number of extraction cycles = 4		Vulpicida pinastri	Q: 1.86%	[66]
L:S = 15 mL/g; number of extraction cycles = 2; room temperature		Xanthoparmelia stenophylla	I: 0.22%	[67]
40 min; number of extraction cycles = 4			Q: 1.36%	[66]
3 days; room temperature; dark place; L:S = 10 mL/g; number of extraction cycles = 3; occasional shaking			Q: 4.447% (DE) I: 4% (DE)	[68]
Acetone:methanol (1:1)
L:S = 2 mL/g; room temperature; 3 days; number of extraction cycles = 3		Hypotrachyna enderythrae Xanthoparmelia farinosa Psiloparmelia distincta (rock) P. distincta (tree) Usnea durietzii	Q: 0.035 ± 0.00% 0.18 ± 0.01% 0.017 ± 0.00% 0.042 ± 0.00% 0.304 ± 0.02%	[69]
Chloroform
Stirring; room temperature (30 °C); 48 h; L:S = 10 mL/g		Parmelia erumpens	I: 2.69% (DE)	[70]
-		Usnea florida	Q: 3.58–4.21%	[71]
Chloroform:methanol
Chloroform:methanol (2:1); number of extraction cycles = 3		Usnea sp.	I: 0.6%	[2]
Chloroform:methanol (1:1 v/v); L:S = 2.5 mL/g; 3 days; room temperature		Ramalina asperula Thamnolia vermicularis subsp. solida Usnea sp.	Q: 1.01% 0.2% 1.07%	[72]
Dichloromethane
1 h; room temperature; L:S = 40 mL/g		Evernia prunastri Flavoparmelia caperata	Q: 7.02% (DE) Q: 58.9% (DE)	[41]
L:S = 4.4405 mL/g		Lecanora muralis	I: 0.0076%; 2.3497% (DE)	[73]
1 h; number of extraction cycles = 2; L:S = 3.53 mL/g		Protousnea poeppigii	I: 0.5731%; 9.36% (DE)	[74]
-		Ramalina capitata	Q: 25.8% (DE)	[75]
1,4-Dioxane
L:S = 40 (w/w); 70 °C; 30 min; 1,4-Dioxane 1,4-Dioxane:water 1:1		Usnea barbata	Q: 2.34% 2.08%	[76]
Ethanol
Ethanol 95%; room temperature; number of extraction cycles = 12; L:S = 1.07 mL/g		Alectoria sarmentosa	I: 2.3369%; 31.2143% (DE)	[77]
Ethanol: water (1:1)		Cladonia leptoclada	I: 0.8%	[78]
60 min; room temperature; stirring: L:S = 7.5 mL/g		Cladonia stellaris	Q: 0.612 ± 0.17%	[33]
24 h; room temperature; L:S = 20.83 mL/g		Parmotrema hypotropa	Q: 0.64% (DE)	[79]
4 h; room temperature; dark; L:S = 40 mL/g; stirring		Ramalina farinacea R. fastigiata R. fraxinea	Q: 0.024 ± 0.001% 0.022 ± 0.001% 0.015 ± 0.001%	[49]
L:S = 40(w/w); 70 °C; 30 min; Ethanol 40% Ethanol 70% Ethanol 96%		Usnea barbata	Q: 0.85% 1.24% 1.44%	[76]
Ethanol 70% (v/v); frequent shaking; 5 days; room temperature; L:S = 5 mL/g			Q: 0.00783%; 0.25 ± 0.00% (DE)	[80]
Ethanol 70% (v/v); 24 h; L:S = 6.39 mL/g			Q: 1.39% (DE)	[81]
Ethanol 96%; L:S = 10 mL/g; 10 days; 20–22 °C; dark place; shaking 3–4 times/day → extract was made up to 100 mL (liquid extract)			Q: 0.0025%	[57]
Ethanol 96%; L:S = 10 mL/g			Q: 0.2566%	[58]
L:S = 40 mL/g; room temperature; dark; 4 h; stirring		Usnea filipendula U. fulvoreagens U. intermedia	Q: 0.097 ± 0.003% 0.101 ± 0.001% 0.072 ± 0.002%	[60]
Ether
-		Ramalina capitata	Q: 4.4% (DE)	[75]
number of extraction cycles = 5; warm		Ramalina siliquosa	I: 0.12%	[82]
Ethyl acetate
1 week; L:S = 48.39 mL/g		Niebla homalea	I: 0.0048%	[83]
-		Ramalina capitata	Q: 8.2% (DE)	[75]
Heptane
number of extraction cycles = 2; 12 h + 3 h; room temperature; L:S = 3 mL/g		Ophioparma ventosa	I: 0.22%; 27.5% (DE)	[84]
Hexane
24 h; 20 °C; L:S = 16.67 mL/g; number of extraction repetitions = 3 (L:S = 1.67 mL/g)		Usnea dasopoga	Q: 0.15%	[85]
3 days; room temperature; dark place; L:S = 10 mL/g; number of extraction cycles = 3; occasional shaking		Xanthoparmelia stenophylla	Q: 58.733% (DE)	[68]
Methanol
Methanol 70%; 1 h; room temperature; L:S = 40 mL/g		Evernia prunastri	Q: 2.92% (DE)	[41]
		Flavoparmelia caperata	Q: 5.4% (DE)
L:S = 20 mL/g; 12 h; Water bath 45 °C		Evernia divaricata Ramalina fraxinea Usnea florida	Q: 5.071 ± 0.086% (DE) 1.621 ± 0.012% (DE) 9.941 ± 0.026% (DE)	[86]
Room temperature; constant shaking; L:S = 1.4286 mL/g		Lethariella canariensis	I: 0.0077%; 0.105% (DE)	[44]
48 h; room temperature; L:S = 4.03 mL/g; number of extraction cycles = 3		Myelochroa leucotyliza	Q: 0.90%	[46]
4 h; room temperature; dark; L:S = 40 mL/g; stirring		Ramalina farinacea R. fastigiata R. fraxinea	Q: 0.034 ± 0.003% 0.031 ± 0.002% 0.016 ± 0.001%	[49]
L:S = 20 mL/g; 24 h; number of extraction cycles = 2		Stereocaulon alpinum	I: 0.4518%; 3.8288% (DE)	[87]
Room temperature		Usnea aciculifera	I: 1.3267%; 4.975% (DE)	[88]
Q: L:S = 62.5 mL/g; 24 h; room temperature I: L:S = 8 mL/g; 3 days; number of extraction cycles = 3		Usnea cornuta	Q: 18.196 ± 0.038% (DE) I: 3.4%; 25% (DE)	[89]
L:S = 40 mL/g; room temperature; dark; 4 h; stirring		Usnea filipendula U. fulvoreagens U. intermedia	Q: 0.107 ± 0.009% 0.104 ± 0.007% 0.094 ± 0.004%	[60]
48 h; room temperature; stirring		Usnea misaminensis	I: 0.1936%; 2.3325% (DE)	[90]
3 days; room temperature; shaking		Usnea undulata	I: 2.7131% (DE)	[91]
1 h; L:S = 66.67 mL/g		Xanthoparmelia camtschadalis X. conspersa	I: 0.432 ± 0.06% 0.437 ± 0.07%	[92]
3 days; room temperature; dark place; L:S = 10 mL/g; number of extraction cycles = 3; occasional shaking		Xanthoparmelia stenophylla	Q: 4.852% (DE)	[68]
Methanol/water (1% NaCl)
Overnight; L:S = 8 mL/g; stirring		Ramalina implexa	I: 0.0056%	[93]
Methanol:dichloromethane (1:1)
12 h; 25 °C		Usnea florida	I: 2.4%; 12% (DE)	[94]
Petroleum ether
Room temperature		Usnea barbata	I: 1.052%	[95]
Vegetable oils
Cold sunflower oil; 3 months; room temperature, dark; L:S = 50 mL/g		Usnea barbata	Q: 8.04 ± 0.35%	[12]
Cold-pressed canola seed oil; 3 months; 21–22 °C; dark place; L:S = 24.72 mL/g; daily shaking			Q: 2.162%; 0.0915 ± 0.0018% (DE)	[96]
Water
L:S = 10 mL/g; 10 days; 20–22 °C; dark place; shaking 3–4 times/day → extract was made up to 100 mL (liquid extract)		Usnea barbata	Q: 0.0004%	[57]
L:S = 40 mL/g; 70 °C; 30 min			Q: 0.76%	[76]
L:S = 10 mL/g			Q: 0.0446%	[58]
Soxhlet extraction
Acetone
24 h; L:S = 25 mL/g		Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 1.96%; 56% (DE)	[97]
L:S = 20 mL/g; 12 h; 50 °C		Evernia divaricata Ramalina fraxinea Usnea florida	Q: 4.91 ± 0.002% (DE) 0.924 ± 0.004% (DE) 44.098 ± 0.025% (DE)	[86]
-		Hypogymnia physodes	I: 19% (DE)	[98]
-		Lethalia vulpina	I: 0.041%; 0.56% (DE)	[99]
-		Parmelia caperata	I: 38% (DE)	[100]
-		Usnea barbata	I: 19% (DE)	[101]
8 h; 70 °C			Q: 16.53 ± 6.53% (DE)	[102]
L:S = 10 mL/g; 8 h; 55–60 °C			Q: 1.3418%; 24.183 ± 0.0172% (DE)	[57]
			Q: 0.028278%	[103]
8 h; 55–60 °C			Q: 28.278% (DE)	[8]
-		Usnea subfloridana	Q: 0.7%; 13.91% (DE)	[11]
Acetone:dichloromethane (1:1)
3 h; L:S = 38.46 mL/g; number of extraction cycles = 2		Vulpicida pinastri	I: 1.07%; 16% (DE)	[104]
Chloroform
L:S = 1.5 mL/g		Cladonia substellata	I: 0.84%	[105]
Ethanol
24 h; L:S = 25 mL/g		Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 0.8432%; 13.6% (DE)	[97]
-		Usnea subfloridana	Q: 0.19%; 6.81% (DE)	[11]
Ethanol 70% (v/v); L:S = 10 mL/g		Usnea barbata	Q: 0.365%; 3.59 ± 0.38% (DE)	[80]
Ethanol 96%; L:S = 10 mL/g; 8 h; 75–80 °C			Q: 1.2125%; 10.8742 ± 0.0703% (DE)	[57]
Ethanol 96%; 8 h; 75–80 °C			Q: 12.721% (DE)	[8]
Ethanol 96%; ~90 min; 78.4 °C		Usnea laevis	Q: 13.3% (DE)	[106]
Ethyl acetate
8 h; 75–80 °C		Usnea barbata	Q: 37.673% (DE)	[8]
			I: 28.15% (DE)	[107]
Ethyl ether
-		Ramalina stenospora	I: 0.013%	[108]
L:S = 2 mL/g; 40 °C		Usnea longissima	I: 0.84%	[109,110]
Hexane
L:S = 50 mL/g; 45 min; number of extraction cycles = 3		Cladonia portentosa	I: 18.33% (DE)	[111]
L:S = 3.125 mL/g		Usnea sp.	I: ~0.125%	[112]
L:S = 6.67 mL/g		Usnea undulata	I: 0.827%; 16.96% (DE)	[113,114]
-		Asahinea chrysantha Asahinea scholanderi	I: 1.30% 0.01%	[115]
Methanol
8 h; 65 °C		Usnea barbata	Q: 13.760% (DE)	[8]
Petroleum ether
24 h; L:S = 25 mL/g		Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 0.252%; 12.6% (DE)	[97]
		Cladonia arbuscula	I: 0.0156%	[116]
-		Usnea subfloridana	Q: 0.57%; 31.65% (DE)	[11]
Sequential Soxhlet extraction
Acetone → Methanol	-	Parmotrema rampoddense	I: 1% (DE)	[117]
Ether → Ethanol 96.3% (v/v)	-	Usnea barbata	Q: 67.09% (DE) 2.43% (DE)	[81]
n-Hexane → dichloromethane → methanol	4 h for each solvent; L:S = 20 mL/g	Usnea aurantiacoatra	I: 84.466% (DE) 8.677% (DE) 1.075% (DE)	[118]
n-hexane → ethyl acetate → methanol	-	Cladonia convoluta	I: 0.3625% Q: 0.92%; 66% (DE) hexane; 16% (DE) ethyl acetate; 1.7% (DE) methanol	[119]
Petroleum ether → chloroform	12 h for each solvent	Usnea molliuscula	I: 1.62%	[120]
Reflux
Acetone	1 h; 90 °C; number of extraction cycles = 1	Cladonia arbuscula subsp. squarrosa	Q: 0.425 ± 0.08%	[9]
	1 h; L:S = 100 mL/g	Cladonia mitis	Q: 0.452 ± 0.054–2.158 ± 0.223% I: 0.998%	[121]
Dichloromethane	L:S = 7.14 mL/g; number of extraction cycles = 2	Usnea florida	I: 0.61%; 40% (DE)	[74]
Ethanol 70%	L:S = 16.67 mL/g; number of extraction cycles = 2	Usnea longissima	I: 0.0066%	[1]
Ethanol-water (95:5, v/v)	1 h; L:S = 15 mL/g; number of extraction cycles = 2	Usnea longissima	Q: 13.7% (DE)	[122]
	number of extraction cycles = 3 (2 h + 1 h + 1 h)		I: 0.0035%	[123]
	2 h; number of extraction cycles = 2	Cladonia alpestris	I: ~0.96%	[124]
Ethyl acetate	1.5 h; L:S = 4 mL/g; number of extraction cycles = 3	Usnea longissima	I: 3.74% (DE)	[125]
Decoction
Distilled water	12 min.; L:S = 10 mL/g; Boiling	Usnea longissima	Q: 1.97% (DE)	[126]
Non-conventional methods
Supercritical fluid extraction
CO2	40 min.; 35 MPa; 40 °C; 5 mL/min	Mixture of species: Cladonia stellaris, Cladonia arbuscula, Cladonia rangiferina	Q: 2.5%; 91% (DE)	[97]
	6 h; 50 °C; 350 barrs; 20 kg/h	Usnea barbata	I: 1.4783%; 71.3287% (DE)	[127]
	30 MPa; flow rate 0.3 kg/h; 40 °C 60 °C		Q: 0.219%; 36.49 ± 0.33% (DE) 0.226%; 59.48 ± 0.20% (DE)	[80]
	30 MPa; 40 °C; 1 ± 3 kg/h		Q: 42.3–63.45% (DE)	[128]
	50 MPa; 40 °C; 992 m3/kg		Q: 0.551–1.243%; 54.5–64.8% (DE)	[129]
	80 min; 85 °C; 150 atm.; 2 ml/min	Usnea subfloridana	Q: 0.45%	[11]
CO2 + 4.3% ethanol (co-solvent)	7.48 h; 300 bar; 5.5 mL/min; 42 °C	Usnea longissima	Q: 0.372 ± 0.022%	[35]
Subcritical CO2	30 MPa; 25 °C; 967 m3/kg	Usnea barbata	Q: 1.315%; 63.2% (DE)	[129]
Ultrasound-assisted extraction
Acetic Acid	20 °C; 25 min	Usnea amblyoclada	Q: 0.00153 ± 0.000113%–0.00234 ± 0.000197%	[130]
Acetone	30 min; 30–35 °C; number of extraction cycles = 4; L:S = 10.53 mL/g	Cladonia uncialis	Q: 52.52 ± 0.28% (DE)	[131]
	30 min; 35 °C; number of extraction cycles = 6; L:S = 20 mL/g		Q: 30.65% (DE)	[132]
	L:S = 40 mL/g; 30–35 °C; 35 kHz; 20 min		Q: 1.305 ± 0.0894%	[133]
	30 °C; L:S = 15 mL/g; number of extraction cycles =1 number of extraction cycles = 10		Q: 1.137% 1.45%	[134]
Camphor:Thymol (in molar ratio 0.3)	L:S = 60 mL/g; 30 min; number of extraction cycles = 1		Q: 1.193–1.496%
Acetone	25 °C; 30 min; number of extraction cycles = 3	Peltigera neckeri Peltigera canina Peltigera ponojensis	Q: 0.0000007% (DE) 0.0000012% (DE) 0.0000736% (DE)	[135]
Methanol		Peltigera neckeri Peltigera canina Peltigera ponojensis	0.0000006% (DE) 0.0000026% (DE) 0.0000038% (DE)
1,4-Dioxane 1,4-Dioxane:water Water	L:S = 40(w/w); 40 °C; 30 min; 18 kHz	Usnea barbata	Q: 2.40% 1.57% 0.65%	[76]
Heptane Diethyl ether Acetone Methanol	30 min; 30–35 °C; number of extraction cycles = 4; L:S = 10 mL/g	Cladonia uncialis	Q: 63.3 ± 3.0% (DE) 43.7 ± 1.0% (DE) 28.4 ± 3.0% (DE) 19 ± 1.0% (DE)	[7]
Heptane → Acetone	20 min; 35 °C; number of extraction cycles = 5; L:S = 4.6 mL/g		I: 0.28%; 33% (DE) heptane	[7]
Ethanol (80%)	L:S = 20 mL/g; 32.23 °C; 20 min	Evernia divaricata	Q: 0.01605 ± 0.00085%	[136]
	L:S = 20 mL/g; 37.02 °C; 20 min	E. prunastri	Q: 0.01886 ± 0.00013%
Ethanol 40% Ethanol 70% Ethanol 96%	L:S = 40 (w/w); 40 °C; 30 min; 18 kHz	Usnea barbata	Q: 1.07% 1.49% 1.54%	[76]
Methanol: dichloromethane (1:1, v/v)	30 min.; L:S = 185–200 mL/g; mixing	Dirinaria applanata	Q: 0.003989%	[137]
Methanol (75%)	30 min; L:S = 10 mL/g; number of extraction cycles = 3	Thamnolia subuliformis	Q: 0.8613% (DE)	[138]
Microwave-assisted extraction
Acetone	5 min; 80 °C; number of extraction cycles = 3; max 120 psi; max 100 W L:S = 20 mL/g	Cladonia foliacea	I: 0.15% Q: 1.173%	[4]
Ethanol	5 min; 80 °C; number of extraction cycles = 2; L:S = 20 mL/g; stirring; max 120 psi; max 100 W		Q: 1.351% I: 0.42%
Ethyl acetate			Q: 1.304%
Q: Limonene Ethyl lactate I: Ethyl lactate	L:S = 62.5 mL/g; 30 min; 100 °C; 20 W	Usnea cornuta	Q: 9.052 ± 0.001% (DE) 13.855 ± 0.020% (DE) I: 5.28%; 22% (DE)	[89]
Combined technique
Acetone-based
Maceration → silica impregnation → Soxhlet extraction with (1) hexane (2) benzene (3) chloroform		Cladina macaronesica	I: 0.018%; 0.067% (DE)	[139]
UAE: 30 min.; L:S = 50 mL/g; 40 kHz → Maceration: 23.5 h		C. mitis/arbuscula Cladina stellaris Flavocetraria cucullata F. nivalis	Q: 0.681 ± 0.345% 0.939 ± 0.466% 0.939 ± 0.486% 1.62 ± 0.069%	[140]
UAE: 30 min; L:S = 2000 mL/g → Maceration: 20 °C; overnight→ UAE: 30 min; L:S = 1000 mL/g		Xanthoparmelia chlorochroa	Q: 0.841 ± 0.367–1.813 ± 0.449%	[3]
Ethanol
Ethanol (70%, v/v) Percolation 2 h → 24 h after addition of the solvent; room temperature; L:S = 3 mL/g		Usnea barbata	Q: 0.00783%; 0.18 ± 0.02% (DE)	[80]

Q—quantitative determination; I—isolation; %—UA content expressed as % (w/w) of dry lichen thallus; % (DE)—UA content expressed as % (w/w) of dry extract. Species listed alphabetically within each study. Abbreviations: L:S—liquid-to-solid ratio, UAE—ultrasound-assisted extraction.

Assessment of reporting bias and certainty of evidence was not performed, as the review focused on methodological studies reporting quantitative UA content. A potential source of bias arises from the exclusion of studies for which full-text articles were not accessible. Eleven records could not be retrieved in full text and were therefore excluded from the final analysis. While this limitation may have influenced the completeness of the evidence base, the relatively small proportion of excluded full-text articles suggests that its impact on the general trends identified in this review is likely limited.

All studies meeting the eligibility criteria were included in a single descriptive synthesis, as no predefined subgroups or separate syntheses were planned. Potential sources of heterogeneity among the included studies were explored descriptively by comparing extraction methods, solvents, and operational parameters in relation to the reported UA content.

3. Results

3.1. Non-Extraction Factors

3.1.1. Influence of the Environment

The concentration of UA in lichens is strongly influenced by environmental conditions, including seasonal fluctuations, temperature, light availability, precipitation, and habitat characteristics [8,47]. Solar radiation has been identified as a key stimulatory factor, with experimental work demonstrating that UV radiation induces UA biosynthesis in Xanthoparmelia stenophylla [66]. Further ecophysiological evidence indicate that UA is not solely constitutively accumulated in lichen thalli but can also be actively resynthesized in response to UV-B radiation following chemical depletion. After acetone rinsing to remove pre-existing secondary metabolites, exposure to UV-B radiation induced a significant resynthesis of UA, supporting an inducible and environmentally regulated biosynthetic response rather than a purely constitutive accumulation. This effect was most pronounced in the exposed apical parts of Cladonia arbuscula and C. stellaris [39].

Seasonal variability can result in substantial differences in UA content, even within the same species. For example, extracts of Cladonia uncialis collected in different years exhibited marked variability in UA levels (52.52%, 28.4% and 30.65%), which has been attributed to both fluctuating weather conditions during the vegetation period as well as minor differences in extraction parameters [7,131,132]. Similarly, lower UA concentrations were reported in Flavocetraria nivalis harvested in late spring and early summer, with precipitation showing an inverse correlation with UA content [47]. Comparable seasonal effects were observed in acetone extracts of Lethariella canariensis, in which UA levels were approximately six-fold higher in winter than in summer [44]. Significant site- and time-dependent variation was likewise demonstrated in extracts analyzed by Dailey et al. [3].

Beyond temporal factors, the geographical origin of samples exerts a major influence on the secondary-metabolite profile [3,9,121]. Field studies on Cladonia stellaris demonstrate pronounced site-specific variability in UA content despite sampling across a broad latitudinal range. The absence of a significant latitude effect, combined with high within-region variability, suggests that local environmental conditions rather than geographical position per se are the primary determinants of UA accumulation [10]. In Cladonia mitis, the difference between the lowest and highest UA content in samples collected across Norway, Sweden, and Poland exceeded a fourfold range [121]. Similarly, Cladonia stellaris collected across Finland exhibited more than sixfold variation [10].

Elevation is widely recognized as a relevant determinant of UA accumulation, although reported trends remain inconsistent. Popovici et al. proposed an optimal elevation range of 700–1500 m for UA accumulation [141]. In contrast, Neupane et al. observed a decrease in UA content in Parmelia flexilis with increasing altitude in the Himalayas [47], whereas Galanty et al. reported the opposite pattern in low-elevation (50–500 m) Cladonia mitis populations [142]. On the other hand, Rahman et al. found no significant relationship between UA accumulation and altitude across five collection sites [40]. These discrepancies likely reflect differences in temperature regimes among studies as well as variation in the elevational ranges examined.

Recent field-based research on Usnea amblyoclada further supports the interpretation that temperature, rather than altitude per se, is the primary driver of UA variability. UA concentrations decreased significantly with elevation, with pronounced differences observed between 900 and 2100 m a.s.l. The absence of significant microsite-related effects (e.g., sun-exposed rock aspects) suggests that broad-scale thermal gradients outweigh small-scale habitat heterogeneity [130]. Similar conclusions were drawn from studies reporting low UA levels in lichens collected at very high elevations (3356–4109 m) [69].

Another important factor influencing UA quantification is the specific thallus region selected for analysis. In Usnea subfloridana, lateral branches contained higher UA concentrations than the central chord under identical extraction conditions [11]. This observation is consistent with the proposed photoprotective role of UA and its preferential accumulation in more exposed thallus regions [2]. Furthermore, in Psiloparmelia distincta, UA content differed between specimens growing on rock and tree substrates [69].

In recent studies, detailed metadata on sample collection—including altitude, collection season, and exact geographical coordinates—have been routinely reported [4,7,8,10,33,37,38,39,40,43,45,46,47,49,53,54,55,57,58,59,60,61,62,63,64,66,69,72,74,76,79,81,83,84,85,87,88,95,96,97,98,100,101,105,107,111,113,114,117,119,122,125,126,130,131,132,133,134,136,137,140]. Such transparency in reporting enhances comparability among datasets and supports more accurate interpretation of secondary metabolite variability.

Given the high natural variability in secondary metabolite production, some researchers have explored the cultivation of lichen tissues under controlled conditions to enhance UA synthesis [26,27,28,29]. These studies indicate that selected nutritional and environmental factors can positively influence UA production, as previously noted in the Introduction.

3.1.2. Species

The selection of lichen species is a critical factor influencing UA yield. Comparative studies have demonstrated that UA concentration depends strongly on the species investigated. For example, Tas et al. applied two extraction protocols across multiple lichen species and reported pronounced species-dependent differences. While Letharia vulpina and Lobaria pulmonaria contained no detectable UA, Evernia divaricata and Ramalina fraxinea yielded higher concentrations when extracted with methanol in a water bath. In contrast, acetone extraction using a Soxhlet apparatus provided optimal results for Usnea florida [86]. These findings underscore the necessity of tailoring extraction parameters to the biological characteristics of each species.

Several genera have been consistently identified as rich sources of UA, including Alectoria, Lecanora, Evernia, and Parmelia with Usnea, Ramalina, Cladonia, and Xanthoparmelia being among the most UA-abundant [11,35,97]. Quantitative analyses expressed relative to lichen dry mass indicate that the highest UA concentrations are found in Usnea barbata (8.04%) [12], U. hirta and U. florida (5.8% and 5.7%, respectively) [42], Remototrachyna flexilis (5.13%) [47], as well as Usnea subfloridana, Omphalodina chrysoleuca, and Ramalina fastigiata (6.49%, 4.0%, and 3.23%, respectively) [50,54,59].

All reviewed genera and species, along with their family affiliations, are summarized in

Table 2. Lichen species with detected usnic acid concentrations.

Family	Species
Caliciaceae	Dirinaria applanata
Cladoniaceae	Cladonia arbuscula, C. confusa (s.: C. leptoclada), C. foliacea (s.: C. convoluta), C. incrassata, C. macaronesica (s.: Cladina macaronesica), C. mitis (s.: Cladina mitis), C. portentosa (s.: C. alpestris), C. pyxidata, C. rangiferina (s.: Cladina rangiferina), C. substellata, C. stellaris (s.: Cladina stellaris), C. uncialis
Icmadophilaceae	Thamnolia subuliformis, T. vermicularis
Lecanoraceae	Omphalodina chrysoleuca (s.: Rhizoplaca chrysoleuca), Protoparmeliopsis muralis (s.: Lecanora muralis), Rhizoplaca melanopthalma, R. peltata
Leprocaulaceae	Leprocaulon microscopicum
Ophioparmaceae	Ophioparma ventosa
Parmeliaceae	Alectoria sarmentosa, Asahinea chrysantha, A. scholanderi, Cetraria islandica, Dolichousnea longissima (s.: Usnea longissima), Eumitria pectinata (s.: Usnea misaminensis), E. baileyi (s.: Usnea baileyi), Evernia prunastri, E. divaricata, Flavoparmelia caperata (s.: Parmelia caperata), F. baltimorensis, Hypogymnia physodes, H. tubulosa, H. vittata, Hypotrachyna enderythraea, Lethalia vulpina, Lethariella canariensis, Myelochroa leucotyliza, Nephromopsis cucullata (s.: Flavocetraria cucullata), N. nivalis (s.: Flavocetraria nivalis), Notoparmelia erumpens (s.: Parmelia erumpens), Parmotrema rampoddense, P. hypotropum (s.: P. hypotropa), Protousnea poeppigii, Psiloparmelia distincta, Remototrachyna flexilis (s.: Parmelia flexilis), Usnea aciculifera, U. amblyoclada, U. angulata (s.: U. undulata), U. aurantiacoatra, U. barbata, U. cornuta, U. dasopoga, U. durietzii, U. filipendula, U. florida, U. fulvoreagens, U. ghattensis, U. hirta, U. laevis, U. molliuscula, U. orientalis, U. quasirigida (s.: U. rigida), U. steineri, U. subflorida, Vulpicida pinastri, Xanthoparmelia camtschadalis, X. chlorochroa, X. conspersa, X. farinosa X. stenophylla (s.: X. somloensis)
Peltigeraceae	Peltigera canina, P. neckeri, P. ponojensis
Physciaceae	Poeltonia venusta (s.: Physconia venusta)
Ramalinaceae	Niebla homalea, Ramalina asperula, R. capitata, R. farinacea, R. fastigata, R. fraxinea, R. implexum (s.: R. implexa), R. menziesii (s.: R. reticulata), R. peruviana, R. pollinaria, R. polymorpha, R. siliquosa, R. stenospora
Stereocaulaceae	Squamarina lentigera, Stereocaulon alpinum

Data presented in Table 2 were compiled from the Global Biodiversity Information Facility (GBIF: https://www.gbif.org/; accessed on 13 February 2026), the USDA Plants Database, and the National Center for Biotechnology Information (NCBI). Species names follow GBIF Backbone Taxonomy (GBIF Secretariat, 2023; https://doi.org/10.15468/39omei; accessed on 13 February 2026) [143]. Taxa reported by the authors under synonymous names are indicated as synonyms and marked with the abbreviation “s”.

.

Within the analyzed taxa, species of the genus Usnea-notably Usnea barbata, U. florida, and Dolichousnea longissima-have been the most extensively studied.

The UA concentration in Usnea barbata has been reported by multiple sources to be approximately 2% of the dry lichen mass [8,57,58,59,81,96,127,129]. The lowest yields were obtained using maceration in 70% ethanol or water, or percolation in 70% ethanol [57,80], whereas the highest values (8.04%) were achieved through long-term cold maceration in sunflower oil [12]. With respect to isolation, the average amount of purified UA typically reached about 1% of the dry lichen weight [95,127]. Notably high UA concentrations have been reported for SFE extracts [80] as well as Soxhlet ether and acetone extracts [81,141]. The distribution of Usnea barbata is shown in Figure 4.

The UA content in Dolichousnea longissima varied markedly depending on the extraction procedure applied, with reported concentrations ranging from 0.37% to 1.12% of the dry lichen weight [35,59]. The yield of isolated UA also differed substantially, ranging from as high as 0.84% of the dry matter [109,110] to as low as 0.0035% [123].

The UA content in extracts obtained from Usnea florida is relatively high when expressed per dry thallus mass. In the study by Cansaran et al., which quantified UA across several Usnea species, acetone extracts of U. florida exhibited the second-highest concentration of the compound, exceeded only by U. subflorida [59]. Comparable results were reported by Goga et al., where acetone macerates of Parmeliaceae species showed the highest UA levels in Usnea hirta, followed closely by U. florida [42].

Analysis of the collected studies further revealed that several lichen species did not yield detectable amounts of UA under the extraction and analytical conditions applied by the respective research groups. Species in which UA was not extracted or identified are summarized in

Table 3. Lichen species lacking detectable or isolable usnic acid.

Family	Species	Reference
Cladoniaceae	Cladonia chlorophaea	[69]
Graphidaceae	Cryptotheciasp.	[137]
Lethariaceae	Letharia columbiana	[73]
	Letharia vulpina	[86]
Lobariaceae	Lobaria pulmonaria	[86,136]
Pannariaceae	Parmelia birulae	[115]
Parmeliaceae	Bryoria fuscescens	[136]
	Evernia prunastri, Hypogymnia physodes	[133]
	Hypogymnia tubulosa	[66]
	Parmotrema tinctorum, P.robustum, Pleurosticta acetabulum	[42]
	Parmotrema grayana	[137]
	Punctelia graminicola	[69]
	Pseudoevernia furfuracea	[41,133,136]
	Platismatia glauca	[41]
Physciaceae	Heterodermia obscurata, Physcia sorediosa, Pyxine cocoes	[137]
	Physcia adscendens (s.: Physcia ascendens)	[133]
Stereocaulaceae	Stereocaulon vesuvianum	[73]
Teloschistaceae	Gyalolechia bassiae (syn.: Caloplaca bassiae)	[137]
	Xanthoria parietina	[133]
Umbilicariaceae	Lasallia pustulata (syn. Umbilicaria pustulata), Umbilicaria crustulosa	[41]
	Umbilicaria aff. calvenses	[69]

, grouped by family.

As discussed above, the detectability and quantification of UA depend strongly on the extraction conditions, analytical methods, and detection techniques employed. This variability is exemplified by Cladonia rangiferina, for which one study reported no detectable UA, whereas another confirmed its presence [140,144].

3.1.3. Pre-Treatment

The degree of grinding is a key parameter in lichen pre-treatment prior to extraction, as it affects not only particle size and the contact area between the solvent and the solid, but also the internal structure of the lichen thallus, thereby influencing solvent accessibility, mass transfer, and overall extraction efficiency [9,129]. Most studies describe the degree of fragmentation of lichen thallus as grinding, crushing, or milling to a fine powder [8,33,48,51,56,57,64,68,70,86,89,93,96,102,103,125,137]. In some cases, the degree of comminution is further specified by reporting the sieve number or particle size range [11,57,68,72,81,103,117,125,134].

In wet homogenization approaches, high-shear disintegration of lichen thalli is performed directly in the extraction solvent, enabling simultaneous mechanical disruption and rapid solvent penetration. Compared to dry grinding, this approach minimizes diffusion-limited release of UA and has been reported as an effective alternative pretreatment-extraction strategy [39].

Ivanovic et al. compared different pretreatment methods prior to supercritical fluid extraction (SFE), including flaking, impact plus shearing, and cutting plus grinding. More intensive mechanical disruption was shown to enhance extraction yields, whereas milder pretreatment may favor extraction selectivity and preserve biological activity, suggesting a trade-off between yield and extract composition. Their results indicated that flaking was the most effective pretreatment, while impact plus shearing combined with rapid gas decompression resulted in the lowest UA content. Application of the optimal pretreatment increased the extraction yield and UA content by up to 160% and 50%, respectively, compared to the least effective method. In the context of supercritical CO₂ extraction, these structural modifications translate into changes in matrix density, particle packing, and solvent diffusivity, which govern extraction kinetics and overall process performance and, consequently, significantly influence SFE efficiency and the content of target compounds [128,129].

Consequently, pretreatment should be regarded as a combined structural and physicochemical modification step rather than solely a mechanical size-reduction process.

Post-isolation milling has also been explored as a strategy to enhance the aqueous solubility of UA. Mechanical treatment induces changes in surface morphology and crystal lattice organization, which can improve the physicochemical properties and solubility of the compound [112].

3.2. Type of Solvent

Solvent-based extraction is employed in all studies reviewed for UA. Among the key extraction parameters-including extraction time and liquid-to-solid ratio (L:S)-the choice of solvent has consistently been identified as the most influential factor determining extraction efficiency [33]. According to fundamental solubility principles, the extraction efficiency of UA, a predominantly non-polar compound, decreases with increasing solvent polarity [14,81].

Among conventional solvents, acetone and ethyl acetate are the most frequently reported as effective and versatile extractants, providing high UA recovery across multiple extraction techniques [8,9,11,33,141]. Non-polar solvents such as hexane and dichloromethane may yield even higher UA concentrations in selected cases [41,68,75]; however, their applicability is often limited by toxicity and environmental concerns.

Ethanol generally results in lower UA concentrations, particularly when compared with acetone [97], but remains attractive due to its safety profile [33]. Its performance can be improved through process intensification strategies such as microwave-assisted extraction [4]. In this context, ethanol and methanol are often preferred despite lower UA selectivity, owing to their high overall extraction yields and capacity to solubilize a broad spectrum of biologically active compounds [8]. Although acetone is generally considered the most efficient solvent for UA extraction, comparative studies have demonstrated that this trend is not universal. In selected Usnea species, methanol has been reported to yield slightly higher UA concentrations than acetone [60]. Beyond commonly used alcohols, certain aprotic solvents have also shown high extraction efficiency. In Usnea barbata, 1,4-dioxane produced higher UA concentrations than ethanol–water mixtures and pure ethanol extracts under both maceration and ultrasound-assisted extraction conditions further emphasizing the strong influence of solvent polarity and solvation properties on UA recovery [76].

In recent years, increasing attention has been directed toward non-conventional and environmentally benign solvents as alternatives to petrochemical extractants [89,134]. Supercritical carbon dioxide (scCO₂), employed in supercritical fluid extraction (SFE), offers several advantages, including non-toxicity, non-flammability, high selectivity, and mild operating conditions [35]. Early studies on UA extraction using supercritical fluids demonstrated that scCO₂ outperformed conventional Soxhlet extraction under all tested conditions, particularly when small amounts of polar co-solvents, such as ethanol or polar aprotic acetone, were added [97]. Subsequent optimization studies confirmed that increasing co-solvent concentration enhances UA recovery, with optimal levels typically remaining below 5% [35,97].

Other green extraction approaches include the use of bio-based solvents such as ethyl lactate, limonene, vegetable oils, and natural deep eutectic solvents (NADES). Ethyl lactate, evaluated as a sustainable alternative, exhibited higher extraction efficiency than methanol when applied in microwave- and ultrasound-assisted extraction, whereas methanol was used in conventional maceration [89]. Lipophilic solvents, particularly sunflower oil, have demonstrated exceptionally high extraction efficiency under mild, long-term maceration conditions, further confirming the strong relationship between UA recovery and solubility in non-polar media [12]. Similarly, lipophilic NADES systems based on camphor and thymol exhibited enhanced efficiency compared with acetone under ultrasound-assisted extraction conditions [134].

From an industrial perspective, the choice of extraction solvent represents a critical trade-off between extraction efficiency and process safety. Several solvents reported for UA extraction, including chloroform, dichloromethane, 1,4-dioxane, hexane, and methanol, belong to ICH Class 2 and should be limited in pharmaceutical applications due to their inherent toxicity. In contrast, acetone-one of the most frequently used and efficient laboratory-scale solvents-is classified as an ICH Class 3 and is generally considered acceptable under controlled conditions, although its high flammability necessitates strict safety measures and solvent recovery at larger scales [145]. Importantly, recent studies indicate that safer and more sustainable alternatives, such as supercritical CO₂ extraction, NADES, and bio-based solvents (e.g., vegetable oils, limonene, ethyl lactate), can provide comparable extraction efficiency while substantially reducing toxicological and environmental concerns. Overall, solvent selection should consider not only UA yield but also the intended application of the extract, balancing extraction efficiency, compound selectivity, safety considerations, and biological activity.

3.3. Extraction Method

Previous studies have demonstrated that the extraction technique substantially influences both the overall extraction yield and UA content [80]. Across all extraction approaches, key process parameters-including temperature, pressure, extraction time, number of extraction cycles, and the liquid-to-solid (L:S) ratio-play a decisive role in determining extraction efficiency.

In general, increasing temperature and extraction time enhances extraction efficiency; however, excessively high temperatures may result in solvent losses, co-extraction of impurities, and degradation of thermolabile compounds [14]. Experimental evidence indicates that UA remains chemically stable at temperatures up to 70 °C under the investigated conditions, suggesting that thermal degradation is not a limiting factor in its extraction [4]. Nevertheless, elevated temperatures are not universally optimal. For example, in a design-of-experiments study employing camphor:thymol NADES, temperature (20–35 °C) had no significant effect on UA extraction efficiency [134].

The L:S ratio is particularly important in methods lacking continuous solvent flow, such as maceration, reflux, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE). Higher L:S ratios generally increase extraction yield; however, excessive solvent volumes may reduce process efficiency and prolong solvent removal.

Similarly, extraction efficiency typically increases with time until solute equilibrium between the solid matrix and solvent is reached [14]. However, excessively prolonged extraction may lead to reduced yields, as reported in [33].

3.3.1. Conventional Method

Conventional extraction techniques-including heat reflux, Soxhlet extraction [9], and maceration [4]-have historically been the most widely applied approaches for UA extraction. Maceration is particularly common and is frequently combined with shaking or stirring to enhance mass transfer [4,33,41,57,68,93,96,103].

Acetone-based maceration generally provides higher UA content compared to methanol-based or acetone Soxhlet extraction [46,57]. Extraction efficiency depends strongly on the L:S ratio, number of extraction cycles, and duration, with 30–60 min often identified as optimal for shaking-assisted maceration [9,33]. Although repeated extraction cycles are relatively common [42], shaking-assisted repetitions were shown not to significantly improve UA recovery compared with a single extraction process [9].

Long-term maceration using lipophilic oils (e.g., sunflower or canola oil) has demonstrated exceptionally high UA recovery (~8% of dry lichen), exceeding commercially available SFE extracts of Usnea barbata, which contain approximately 2% UA in dried lichen [12,96]. However, this approach requires prolonged extraction times and results in complex extract matrices, potentially complicating analytical applications. Reported L:S ratios in maceration processes range widely, from 2 mL/g [69] to 200 mL/g [47].

In certain cases, heat reflux extraction provides higher UA yields than maceration or UAE, likely due to enhanced solubility at elevated temperatures; however, this may increase the risk of co-extracting undesired compounds [9,141].

Soxhlet extraction, particularly with 70% ethanol or acetone, remains an effective method for UA isolation. Nevertheless, SFE frequently outperforms Soxhlet extraction in terms of yield and selectivity, especially when cleaner extracts are required [80,97].

3.3.2. Non-Conventional Method

In recent years, increasing attention has been directed toward alternative and green extraction technologies, particularly UAE and SFE. This shift is driven by the limitations of conventional methods, including their reliance on elevated temperatures and large volumes of potentially toxic organic solvents [35].

The influence of extraction duration on UA recovery in SFE has been investigated in several studies. Brovko et al. reported that extending extraction time from 40 to 60 min did not result in a significantly increase UA content or overall yield when using supercritical CO₂ [97]. Similarly, Atíla Dínçer et al. evaluated extraction durations of 5–9 h and observed that extraction time had a less pronounced effect on UA recovery compared with parameters, such as temperature and co-solvent concentration [35]. In contrast, Boitsova et al., employing a multifactorial experimental design, identified an optimal extraction time of 80 min for maximizing UA content under elevated temperature (85 °C) and moderate pressure (15 MPa). This duration exceeded the baseline experimental level applied in that study (55 min at 250 atm and 60 °C) for Usnea subfloridana [11].

Temperature effects in SFE have also been extensively examined. Brovko et al. studied temperatures of 40, 60, and 80 °C at pressures between 10 and 35 MPa. At low pressure (10 MPa), increasing temperature from 40 to 80 °C reduced CO₂ density, resulting in decreased extraction yield and lower UA content. However, at higher pressures (20–35 MPa), increasing temperature enhanced both yield and UA concentration [97]. Comparable results were reported by Atíla Dínçer et al., who observed increased UA recovery per dry lichen weight within the investigated temperature range of 35–45 °C [35]. These findings indicate that single factor optimization is insufficient for defining optimal SFE conditions.

Pressure significantly modulates temperature effects and strongly influences extraction performance. In the study by Boitsova et al., although maximal UA recovery was achieved at 23.2 MPa, a lower pressure of 15 MPa was selected as optimal from a technological perspective, considering both extraction efficiency and process feasibility [11]. Similarly, Brovko et al. demonstrated that pressure determines whether temperature increases enhance or reduce extraction efficiency, underscoring the interdependence of these parameters in SFE [97].

Despite relatively low total extraction yields reported in some studies, SFE provides high UA extraction efficiency in terms of UA concentration in the dry extracts [80]. Moreover, SFE has been identified as one of the most economically favorable non-conventional extraction techniques due to its relatively low energy consumption [35].

Compared with conventional methods, SFE can be performed as a single-step process without the use of organic solvents, yielding dry extracts free of solvent residues and minimizing degradation of thermolabile compounds [11]. The high selectivity of supercritical CO₂ toward UA, attributed to the limited co-extraction of other phenolic compounds, further enhances the value of this technique [97]. Consequently, SFE consistently produces UA-enriched extracts compared with conventional extraction methods [80,81]. Commercially available supercritical carbon dioxide extracts of Usnea barbata have been reported to contain higher UA concentrations than extracts obtained using conventional techniques such as Soxhlet extraction or maceration [81]. This findings highlight the potential of SFE as an efficient and selective technique for UA extraction and demonstrate its feasibility beyond laboratory scale.

UAE offers rapid extraction with reduced solvent consumption. Sonication time is critical, as prolonged exposure (>30 min) may lead to UA degradation [9]. UAE is often combined with overnight maceration or repeated extraction cycles to enhance recovery [3,7,9,132,140]. Under the tested conditions, ultrasound-assisted extraction produced approximately twofold lower UA content (0.233 ± 0.017%) compared with heat reflux extraction (0.425 ± 0.008%) [9]. Notably, under the investigated conditions, UAE performed at lower temperatures yielded higher UA concentrations in ethanolic and 1,4-dioxane extracts of Usnea barbata compared with classical maceration conducted at elevated temperatures [76].

MAE enables rapid extraction with high reproducibility and relatively low operational costs. An optimized MAE protocol, based on acetone and ethanol, allows complete extraction in less than 20 min and is suitable for scale-up in biological, pharmaceutical, or commercial applications [4]. Comparative analyses of green solvents in MAE indicated that increasing extraction temperature did not significantly affect UA recovery. Among the tested conditions, ethanolic extracts exhibited the highest UA concentrations [89].

3.4. Extraction Efficiency

Extraction efficiency in lichen studies depends strongly on the selected species, extraction method, solvent, and process conditions. Comparative analyses indicate that conventional Soxhlet extraction using ethanol often provides the highest yields (10.18 ± 0.02%), whereas supercritical CO₂ extraction may result in substantially lower yields (0.38–0.60%) [80]. Notably, exceptions to this trend have been reported, with supercritical CO₂ extraction achieving both the highest overall yield and UA concentration in the extract under specific experimental conditions [97].

Importantly, higher total extraction yields do not necessarily correspond to higher UA content. In several studies, increased extraction yield coincided with lower concentrations of the target compound [7,80]. For example, in UAE, methanol yielded the highest extraction yield (5.41%) but the lowest UA concentration, whereas heptane and diethyl ether produced low yields (0.67% and 0.76%) with relatively higher UA content [7]. However, in studies focused on U. barbata, a decrease in extraction yield obtained with the same solvent was accompanied by a proportional decrease in UA content expressed per gram of dried lichen, a trend also observed in other investigations conducted under comparable conditions [57]. Similarly, in MAE experiments, extraction efficiency and UA recovery exhibited a positive but non-linear relationship, with the highest UA yield achieved at the second-highest rather than the maximum total extraction yield [4].

Collectively, these findings indicate that a direct correlation between total extraction efficiency and UA content in dry lichen is not consistently observed across comparative studies [11,80,97]. Further evidence of this inconsistency was provided in a study demonstrating that extraction efficiency and UA content varied independently; conditions leading to higher total extract yields did not necessarily enhance selectivity toward UA [35].

Acetone, a commonly used solvent, generally produces lower extraction yields compared to ethanol (0.35–7.97% vs. 0.62–12.52%) across various studies [8,57,97,102]. Nevertheless, some researchers have reported that acetone outperforms methanol and hexane (5.05%, 2.11%, and 0.91%, respectively) [68], a trend also described by Do et al., and Ojha et al. [46,113].

Species-specific differences further influence extraction outcomes. Acetone maceration of Usnea steineri and U. baileyi yielded 6.15% and 10%, respectively, while Ramalina sp. reached 4.26% [53,56,64]. In Soxhlet successive solvent extraction of Usnea aurantiacoatra, yields obtained with hexane, dichloromethane, and methanol were 1.04 ± 0.26%, 0.73 ± 0.15%, and 4.58 ± 1.02%, respectively [118]. For Cladonia portentosa, corresponding yields with hexane, ethyl acetate, and methanol were 1.36%, 0.98%, and 7.56% [111]. In another study on Usnea undulata, successive Soxhlet extraction yielded 4.87% for n-hexane, while the subsequently applied acetone extraction provided the highest yield (6.10%), followed by methanol (3.81%) [113,114]. These data underscore the influence of both lichen species and solvent polarity on extraction efficiency.

Methanol maceration yielded higher UA levels than certain non-conventional approaches employing green solvents; however, the opposite trends were observed for total extraction efficiency. When green solvents were used, MAE resulted in higher extraction efficiency than UAE (limonene: 20.7% vs. 19.0%; ethyl lactate: 24.8% vs. 15.3%), whereas methanol maceration yielded 13.5% [89].

In summary, optimization of UA extraction requires careful consideration of the extraction method, solvent, and lichen species selection. Non-conventional approaches, particularly MAE using green solvents, demonstrate considerable potential for achieving high extraction efficiency while maintaining bioactive compound content.

4. Conclusions

This review demonstrates that UA extraction efficiency from wild-grown lichens is govern by complex and interrelated biological and technological factors rather than by a single dominant parameter. Among non-extraction variables, lichen species selection- strongly influenced by taxonomic affiliation and geographic origin-plays a critical role in determining UA availability and ultimately limits achievable extraction yields.

Pre-treatment of lichen material, particularly flaking, significantly enhances extraction efficiency by improving solvent–solid contact and mass transfer. Optimized pre-treatment often exerts a stronger influence on UA recovery than prolonged extraction times or repeated extraction cycles, underscoring the importance of this frequently underestimated step.

Among extraction-related parameters, solvent selection remains the most decisive factor. Due to the non-polar nature of UA, higher extraction efficiencies are generally achieved using solvents of low to intermediate polarity with strong affinity for hydrophobic compounds, including acetone, ethyl acetate, supercritical CO₂, vegetable oils, and lipophilic NADES. While conventional organic solvents continue to provide high recovery, green and non-conventional solvent systems represent promising alternatives and, in some cases, may outperform traditional approaches.

From a practical perspective, conventional methods remain attractive due to their simplicity and low equipment requirements. In contrast, non-conventional techniques-particularly SFE and MAE-offer superior selectivity, minimal solvent residues, cleaner extracts, and greater potential for controlled and scalable processing. The availability of commercially produced SFE extracts confirms that selected UA extraction strategies have already progressed toward application-oriented implementation.

Overall, this review provides a consolidated reference on the key factors affecting UA extraction. Future research should prioritize systematic, multi-factorial optimization and standardized analytical protocols, as inconsistencies in reported extraction parameters remain a major limitation. An additional challenge in the utilization of wild-grown biomass is the pronounced environmental variability of UA content. Seasonal fluctuations, geographical origin, and local climatic conditions may result in several-fold differences in UA accumulation within the same species, complicating standardization and reproducibility of extraction outcomes. Therefore, controlled comparative studies integrating environmental and methodological parameters are essential to better define the determinants of metabolite variability. Although more sustainable solvents and extraction techniques are increasingly being developed, future research should focus on cultured lichens and other environmentally responsible sources of UA, thereby reducing reliance on wild biomass and promoting fully sustainable production strategies.