Lichen Depsides and Tridepsides: Progress in Pharmacological Approaches

Depsides and tridepsides are secondary metabolites found in lichens. In the last 10 years, there has been a growing interest in the pharmacological activity of these compounds. This review aims to discuss the research findings related to the biological effects and mechanisms of action of lichen depsides and tridepsides. The most studied compound is atranorin, followed by gyrophoric acid, diffractaic acid, and lecanoric acid. Antioxidant, cytotoxic, and antimicrobial activities are among the most investigated activities, mainly in in vitro studies, with occasional in silico and in vivo studies. Clinical trials have not been conducted using depsides and tridepsides. Therefore, future research should focus on conducting more in vivo work and clinical trials, as well as on evaluating the other activities. Moreover, despite the significant increase in research work on the pharmacology of depsides and tridepsides, there are many of these compounds which have yet to be investigated (e.g., hiascic acid, lassalic acid, ovoic acid, crustinic acid, and hypothamnolic acid).


Introduction
Using the traditional definition of lichens, these organisms are a symbiotic association consisting of a mycobiont (Ascomycota and Basidiomycota phylum) and a photosynthetic partner (which is an algae or a cyanobacterium). The rise of "Omics" technologies such as genomics, transcriptomics, proteomics, and metabolomics allows us to deeply study the symbiotic partnership in lichens. Photobiont and mycobiont are not the only members of this symbiosis. Specific bacterial microbiomes, such as Alphaproteobacteria communities and lichenicolous fungi, have also been identified and characterized on lichens [1][2][3]. Between 17,000 and 20,000 lichen species that inhabit diverse ecosystems have been identified [4]. The nutritional uses as food or flavoring agents, the spiritual uses in religious ceremonies, the industrial uses as natural dyes, and the environmental ones as biomonitors of pollution have made lichens a significant resource for different economic activities [5]. Like higher plants, lichens have been used for their therapeutic properties in many traditional medicine systems, such as those of Ayurvedic and Unani medicine, for, e.g., bronchitis, asthma, amenorrhea, stomach disorder, and vomiting [6]. For example, the Usnea species has been used in many cultures around the world for its antiseptic, wound healing, antibacterial, and anti-inflammatory properties. Traditional knowledge and confirmed activity studies led to the improvement of its pharmacological potential by new pharmaceutical formulations. Popovici et al. have developed bioadhesive oral films with Usnea barbata extract in Canola oil as an effective oral formulation [7]. However, the usefulness of lichens in the pharmaceutical industry goes far beyond this. The biological synthesis of nanoparticles (NPs) has become an active line of research. The reducing or stabilizing capacity of different natural sources, including lichens, is used in a simple, non-toxic, eco-friendly process known as green synthesis [8]. Lichenan from Usnea longissima was used to decorate selenium nanoparticle surfaces, showing great stability and studies presented atranorin as a weak antioxidant, as revealed in a beta-carotene-linoleate model system (14% antioxidant activity at 200 µg/mL) [48].
Conversely, a TBARS assay showed that atranorin induced lipoperoxidation at 0.1 to 100 µg/mL, increased NO production (only at high concentrations), and enhanced H 2 O 2 formation. This compound acted as a superoxide scavenger, and hydroxyl radical/nitric oxide scavenging activity was not observed [47].
Cytotoxic effects on different cell lines (cancer and normal) were also investigated. Atranorin has demonstrated moderate activity against breast cancer cells such as MDA MB-231 and MCF-7, with IC 50 values of 5.36 µM and 7.55 µM, respectively, by the downregulation of the Bcl-2, Akt, Bcl-w, and Bcl-xL proteins and the induction of Bax and caspase-3 expression. In silico studies confirmed the high interaction between the depside and the oncoproteins [49]. Cytotoxicity was also observed in human lung cancer cell lines (A549), epithelial carcinoma cell lines (SKHep1 and Huh-7), primary cancer cell lines (SNU-182), melanoma cell lines (HTB-140), prostate cancer cell lines (DU-145 and PC-3), and murine leukemia cell lines (P388) [50][51][52][53]. The tumorigenesis reduction and antimigratory activity against human lung cancer was mediated by the downregulation of activator protein 1 (AP-1), Wnt, and signal transducer and activator of transcription (STAT) pathways, as well as the inhibition of RhoGTPase activity [50]. Moreover, atranorin showed effects against hepatocellular carcinoma tumorigenesis by reducing cell proliferation (at 80 µg/mL), attenuating the cell cycle (G2/M phase cell cycle arrest), inducing cell death through necrosis, and diminishing metastatic potential by the suppression of cell migration and invasion [51]. Additionally, atranorin was found to be effective in inhibiting the cancer cell proliferation, migration, and actin cytoskeleton organization in melanoma cell lines (HTB-140) and prostate cancer lines (DU-145 and PC-3) [52]. The killing effect of atranorin on gastric cancer was also studied using complexes formed by superparamagnetic iron oxide nanoparticles (SPION) and atranorin. In vitro results on gastric cancer stem cells showed a reduction in proliferation, invasion, and tumorigenicity by reducing the expression of members of the Xc-/GPX4 axis and their mRNA 5-hydroxymethylcytidine modification and by inducing ferroptosis [54].
Moreover, this compound revealed cytotoxicity against all the cell lines (A2780, HCT-116 p53+/+ and HCT-116 p53−/−, SK-BR-3, HL-60, HT-29, Jurkat, and MCF-7) except HeLa, highlighting its activity against HL-60 cells. The clonogenic ability for the inhibition of all the tested tumor cells and the effects on the cell cycle at 200 µM (accumulation in S-phase at expense of G1/G0-phase) was also observed. In addition, atranorin seems to be effective as a pro-apoptotic agent with a p53-dependent action [55]. In particular, the studies on A2780 cancer cells and HT-19 also showed that atranorin caused cell death by reactive oxygen species/reactive nitrogen species (ROS/RNS) overproduction, caspase-3 activation, phosphatidylserine externalization, and mitochondrial membrane potential loss [56].
In vivo experiments were also conducted. Atranorin administration to BALB/c mice with T1-induced cancer disease was related to longer survival time, reduced tumor size, and higher numbers of apoptotic 4T1 cells. Comparing the effects of atranorin on normal mammary epithelial NMuMG cells and 4T1 cancer cells, it was observed that 4T1 cells were more sensitive to atranorin, reducing the clonogenic ability of carcinoma (75 µM), inducing apoptosis mediated by caspase-3 activation and poly ADP ribose polymerase (PARP) cleavage, and enhancing the depletion of Bcl-xL protein [57]. Moreover, atranorin SPION complexes showed tumorgenicity reduction in NOD-scid mice [54].
Previous studies focused on the effects of atranorin alongside irradiation (360-366 nm), as evidenced in the inhibition of 8-methoxypsoralen (MOP)-human serum albumin (HSA), photobinding by 20.1%, and also as revealed in the significant hemolysis in red cell suspension; it acted as a photoprotective and photohemolytic agent, respectively [63,64].

Barbatic Acid
Barbatic acid showed antiparasitic activity against the adult worms and larval stages of Schistosoma mansoni by causing death (IC 50 value of 99.43 µM), affecting mobility, as was reflected in movements being presented only in the extremities, and triggering tegumentary damage [18,67]. Moreover, this depside was active against Staphylococcus aureus and Enterococcus faecalis (both the commercial and the clinic strains), with MIC values of 7.8 to 31.3 µg/mL [68]. This compound also displayed molluscicidal activity against Biomphalaria glabrata at 20 and 25 µg/mL [67].
Furthermore, this secondary metabolite was a potent cytotoxic agent against the lung cancer A549 cell line, with an IC 50 value of 1.78 µM, by inducing apoptosis, as shown in the cell cycle arrest in the G0/G1 phase, the cleavage of PARP, and the activation of caspase-3 activity [69]. Conversely, this compound was found to have little effect in inhibiting the tumor promoter-induced Epstein-Barr virus (EBV), with an IC 50 value higher than 100 µM [70]. Finally, in silico studies revealed that barbatic acid is a weak diuretic agent on the active site of the with-no-lysine kinase 1 (WNK1) domain [71].

Diffractaic Acid
The antimicrobial activities of diffractaic acid were studied against bacteria and fungi. The inhibition zones in the Staphylococcus aureus and Escherichia coli cultures were 17.25 mm and 12.75 mm, respectively, at concentrations of 1000 ppm, exhibiting a strong activity (less than the amoxicillin control) [72]. This compound exhibited strong activity against Fusarium fujikuroi (MIC value of 16 × 10 −3 mg/mL). This activity was higher than that of the flucytosine, clotrimazole, and ketoconazole drugs and similar to that of amphotericin B and posaconazole [73]. Furthermore, diffractaic acid was a potent antimycobacterial agent, with an MIC value of 15.6 µg/mL [31].
Diffractaic acid has also been investigated for its analgesic, antipyretic, and antiinflammatory properties. This compound, at a dose of 200 mg/kg, exerted a moderate analgesic effect and a hypothermic effect on normal body temperature in male ddY mice. However, diffractaic acid did not suppress the fever in mice with lipopolysaccharide(LPS)induced hyperthermia [14]. Moreover, diffractaic acid inhibited the formation of LTB4 in polymorphonuclear leukocytes, with an IC 50 value of 8 µM via a nonspecific redox mechanism [41].
This secondary metabolite showed a protective effect against indomethacin-induced gastric ulcers in Wistar rats via increasing the antioxidant capacity (augmented enzyme activities and reduced glutathione levels and decreased lipid peroxidation) and via suppressing neutrophil infiltration. As an index of the neutrophil infiltration, myeloperoxidase (MPx), and nitric oxide synthase (NOS) activities were used. In gastric mucosal lesions, the activities of MPx and inducible nitric oxide synthase (iNOS) were increased. Diffractaic acid reduced MPx and iNOS activities and increased constitutive nitric oxide synthase (cNOS) activity [74].
Several works have focused on studying the cytotoxic activity of diffractaic acid in different types of tumor cells. This secondary metabolite showed moderate cytotoxicity in the cells of the nervous system (IC 50 values of 122.26 mg/L in neurons and IC 50 values of 35.67 mg/L in glioblastoma multiforme cells) [75]. Moreover, this depside exhibited a cytostatic effect through antiproliferation activity in the human keratinocyte HaCaT cell line, with an IC 50 value of 2.6 mM [76]. Furthermore, diffractaic acid demonstrated a significant cytotoxicity against human breast cancer (MCF-7 cell line), human epithelial carcinoma (HeLa cell line), and human lung cancer (NCI-H460 cell line) at a concentration of 100 µg/mL [77]. In another study, this lichen compound displayed a strong proliferative action against the colon carcinoma HCT-116 cell line (IC 50 value of 42.2 µM) and moderate activity against the breast adenocarcinoma MCF-7 cell line and the cervix adenocarcinoma HeLa cell line (IC 50 values of 93.4 µM and 64.6 µM, respectively) [78]. In addition, diffractaic acid was found to be a moderate inhibitor of thioredoxin reductase [79]. Likewise, this compound was found to have little effect in inhibiting the tumor promoter-induced Epstein-Barr virus (IC 50 value of >100 µM) [70].
All these studied activities led the investigation of new formulations that reduce the cytotoxicity of the treatment with diffractaic acid. The encapsulation of the compound with 2-hydroxypropyl-B-cyclodextrin on PLC microspheres improved its solubility and reduced the cytotoxicity in monkey kidney fibroblasts (Vero cells) [80].

Divaricatic Acid
Divaricatic acid has resulted in being an effective antimicrobial agent against Grampositive bacteria, with MIC values ranging from 7.0 µg/mL for Bacillus subtilis to 64.0 µg/mL for Staphylococcus aureus, highlighting its therapeutic role in methicillinresistant Staphylococcus aureus (MRSA) infections. Moreover, this compound showed anti-Candida activity (MIC value of 20 µg/mL) [81]. Divaricatic acid, which is the major compound of the ether extract of the lichen Ramalina aspera, showed molluscicidal activities against Biomphalaria glabrata and cercaricidal activities against Schistosoma mansoni [82]. In another study, Silva et al. also demonstrated the antiparasitic properties of divaricatic acid against Schistosoma mansoni worms by affecting motility and viability (IC 50 100.6 µM). Indeed, this compound resulted in being not cytotoxic against human peripheral blood mononuclear cells, suggesting that it is safe for humans [83].
Cytotoxic activities were also investigated in UACC-62 human and B16-F10 murine melanoma cancer cells and NIH/3T3 fibroblasts using a sulforhodamine B assay. This depside exhibited strong activity against UACC-62 (GI 50 7µM) and B16-F10 cells (GI 50 11.3 µM), being more selective against melanoma cells than 3T3 normal cells (GI 50 [84]. In deepening the mechanism of these effects, it was observed that the virulence of the Gram-negative opportunistic pathogen Pseudomonas aeruginosa was reduced by inhibiting quorum sensing on diverse Pseudomonas aeruginosa strains (54% of gfp expression of lasB-gfp and 50% of rhlA-gfp at a concentration of 116 µM). These genes are essential in the process because they encode the virulence factors elastase and rhamnolipids [23]. In another study, this depside diminished the maturation and growth of Candida albicans biofilms (Minimal Biofilm Inhibition Concentration (MBIC 50 ) ≤ 12.5 µg/mL)) [85]. Moreover, this lichen compound displayed activity against the liver stage of the malaria parasite Plasmodium, targeting the fatty acid synthesis (FAS)-II pathway [86].
In another study, Férnandez-Moriano et al. investigated the neuroprotective activity of evernic acid, based on its antioxidant properties, in a model of oxidative stress, which was hydrogen peroxide-induced in astrocytes and neurons. This compound increased cell viability and reduced/oxidized the glutathione (GSH/GSSG) ratio and antioxidant enzymes expression. Moreover, evernic acid reduced lipid peroxidation, intracellular ROS overproduction, protein carbonyls content, and caspase-3 activity. The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway contributes to this neuroprotection [87]. Neuroprotective effects were also shown in an MPTP-induced Parkinson's disease model. Hence, evernic acid inhibited apoptosis and mitochondrial dysfunction, and it reduced oxidative stress in primary neurons. Moreover, a reduction was demonstrated in motor dysfunction and in dopaminergic neuronal death and astroglia activation using a C57BL/6 mouse model [88].
In terms of cytotoxic activities, evernic acid showed low cytotoxicity for the malignant mesothelioma cell line (MM98), the vulvar carcinoma cell line (A431), and the human keratinocyte cell line (HaCaT). There was also no effect on the stimulation of cell migration as measured by scratch healing assays in HaCaT [89]. Contrary to these data, evernic acid demonstrated strong cytotoxic activity at 25 and 50 µg/mL concentrations in a HeLa cell line and a reduction in A549 cancer cell proliferation (at 12.5, 25, 50, and 100 µg/mL). These depside concentrations were studied in healthy HUVEC cells with no toxic results, making this a good candidate for cancer treatment [90,91]. Evernic acid was also investigated against glioblastoma multiforme (GBM) cancer using A-172 and T98G cells, with moderate activity in A-172 cultures (IC 50 33.2 µg/mL). Multiple targets play a significant role in brain tumors, such as an immunosuppressive environment, inflammation, the degradation of hyaluronic acid, oxidative stress, and acetylcholine cholinesterase. Evernic acid also showed an inhibitory activity against indoleamine-2,3 dioxygenase 1 (IDO1), COX-2, hyaluronidase, and butyrylcholinesterase [92]. It should be also noted that evernic acid is a moderate inhibitor of tumor promoter-induced Epstein-Barr virus activation (64.6% of an inhibitory effect at a concentration of 50 µM) [71]. Finally, regarding the toxicity of this compound, in silico prediction tests showed no mutagenic effects, no tumorigenic effects, no reproductive alterations, and no irritant effects [84].

Isolecanoric Acid
Isolecanoric acid has shown a prolonged antioxidant action. Based on the antioxidant properties, de Pedro et al. investigated its protective role in two neurodegenerative diseases models (L-BMAA for the Amyotrophic lateral sclerosis model and rotenone for the Parkinson's disease model) in the human dopaminergic neuroblastoma SH-SY5Y cell line. Pretreatments with 10 and 25 µM of isolecanoric acid prevented mitochondrial dysfunction by decreasing the mitochondrial membrane potential (∆Ψm), reduced oxidative stress by attenuating the ROS production, attenuated early and late apoptosis, and inhibited glycogen synthase kinase-3 beta (GSK3β) and casein kinase I (CK1) [93].

Lecanoric Acid
Lecanoric acid is of interest as an antimicrobial and antihelmintic agent since it inhibits 100% of Gram-negative bacteria Aliivibrio fischeri, and it causes 80% mortality in nematode Caenorhabditis elegans at 100 µg/mL [4]. Moreover, lecanoric acid was active as an antimicrobial agent against a wide variety of bacteria and fungi, with MIC values of 0.5 to 1 mg/mL [24].
The antioxidant properties of this compound are controversial according to the studies. Therefore, according to Ristic et al., lecanoric acid showed a slight antioxidant capacity, as evidenced in the DPPH assay (IC 50 value of 424.5 µg/mL) and the reducing power assay (0.0165 to 125 µg/mL) [24]. Jayaprakasha et al. also showed that this compound had weak-moderate antioxidant activity (36% antioxidant activity at 500 µg/mL using the betacarotene-linoleate model system) [48]. However, according to Thadhani et al., lecanoric acid has good antioxidant activity compared to other lichen substances [superoxide radical (SOR) test (IC 50 value of 91.5 µmol), DPPH (IC 50 value of 34 µmol), and nitric oxide radical (NOR) test (IC50 value of 53.5 µmol)] [94].
Studies on the cytotoxic activity of lecanoric acid showed that this depside has moderate activity against colon HCT116 cancer cells and reduced cell colony formation by decreasing Axin2 expression and M phase arrest (downregulation of CDK1, upregulation of cyclinB1 and pH3) [95,96]. Lecanoric acid also exhibited slight activity against human larynx carcinoma Hep-2 cells, human breast carcinoma MCF7 cells, human kidney carcinoma 786-0 cells, murine melanoma B16-F10 cells (IC 50 values > 50 µg/mL), Hela cells (IC 50 value of 124 µg/mL), and A549 cells and LS174 cells (IC 50 value of 200 µg/mL) [24,97]. Moreover, lecanoric acid was an effective thioredoxin reductase inhibitor for cancer therapy, even more effective than the common antitumoral drugs such as doxorubicin and cisplatin [79].

Methyl Evernate
Methyl evernate has displayed antimicrobial activity against bacteria and fungi and is especially active against Bacillus cereus (MIC value of 0.125 mg/mL) and Candida albicans (MIC value of 0.25 mg/mL). Furthermore, methyl evernate had low DPPH radical scavenging activity (IC 50 value of 391.57 µg/mL) and higher reducing power than acetone extracts of Ramalina spp. [24]. Moreover, this depside inhibited the cancer cell growth of the human epithelial carcinoma Hela cell line (IC 50 value of 46.45 µg/mL), the human lung carcinoma A549 cell line (IC 50 value of 76.84 µg/mL), and the human colon carcinoma LS174 cell line (IC 50 value of 161.37 µg/mL).

Olivetoric Acid
Olivetoric acid has antimicrobial activity against a wide range of bacteria, yeast, and fungi. This compound was active against 12 of 15 species of bacteria and yeast (it was inactive in Klebsiella pneumoniae, Pseudomonas aeruginosa, and Pseudomonas syringae) and against 7 of 11 species of fungi (it was inactive against Alternaria citri, Alternaria tenuissima, Aspergillus niger, and Gaeumannomyces graminis) [100].
Olivetoric acid showed slight to moderate antioxidant properties in cultured human amnion fibroblasts (total antioxidant capacity value of 20.79 mmol Trolox equivalent/L) and in cultured human lymphocytes (HLs) (total antioxidant capacity value of 3.79 mmol Trolox equivalent/L) [16,101]. Finally, olivetoric acid induced cytotoxicity and genotoxicity against the glioblastoma multiforme U87MG cell line (IC 50 value of 17.55 mg/L) and primary rat cerebral cortex (PRCC) cells (IC 50 value of 125.71 mg/mL) via oxidative stressinduction, as evidenced by the lactate dehydrogenase (LDH) activity and oxidative DNA damage [102]. Moreover, concentrations of 100-400 mg/L of olivetoric acid showed activity against human hepatocellular carcinoma cells (HepG2) and the upregulation of the proapoptotic genes, BAK, CASP6, CASP7, CASP8, FADD, FAS, and FASLG [103]. Furthermore, this depside has resulted in being of interest as anti-angiogenic agent, as evidenced by its ability to prevent rat adipose tissue endothelial cell (RATECs) cell proliferation by disrupting microtubules and inhibiting actin polymerization [104].

Perlatolic Acid
Perlatolic acid has good antimicrobial properties against methicillin-resistant Staphylococcus aureus strains, with an MIC 90 value of 32 µg/mL, and it showed a synergic action with gentamicin and an antagonism action with levofloxacin [105].
This compounds also exerted neurobiological processes such as neuroprotection, neurotrophicity, and neurogenesis. Hence, this depside acts as a neurotrophic and proneurogenic agent (125.34 µm in neurite outgrowth at 0.5 µM) by inducing the upregulation of neurotrophic genes (BDNF and NGF). This neurotrophic activity is also related to the increased histone acetylation of H3 and H4 protein in a mouse neuroblastoma (Neuro2A) cell line. Moreover, this secondary metabolite was a potent acetylcholinesterase (AChE) inhibitor (IC 50 6.8 µM) [63]. Moreover, in silico, in vitro, and in vivo studies have shown that perlatolic acid is a potent anti-inflammatory compound by inhibiting microsomal prostaglandin E2 synthase-1 (IC 50 0.4 µM), 5-lipoxygenase (IC 50 1.8 µM for cell-based assay and IC 50 0.4 µM for purified enzyme), tumor necrosis factor alpha-induced nuclear factor kappa B (IC 50 7 µM), and leukocyte recruitment [106,107]. Furthermore, perlatolic acid showed slight to moderate immune-modulating properties in cultures of peritoneal macrophage cells from mice, as evidenced in a significant increase in hydrogen peroxide release and a slight increase in nitric oxide (NO) release activity [108].

Ramalic Acid/Obtusatic Acid
Ramalic acid/Obtusatic acid was active as an antimicrobial agent against five bacteria and ten fungal species with MIC values from 0.125 to 1 mg/mL [24]. Moreover, this depside showed slight to moderate antioxidant activity (DPPH radical scavenging activity with IC 50 value of 324.61 µg/mL and reducing power of 0.0142 at 125 µg/mL) [24]. Furthermore, ramalic acid/obtusatic acid showed weak to moderate cytotoxic activity against the human epithelial carcinoma (Hela) cell line, the human lung carcinoma (A549) cell line, and the human colon carcinoma (LS174) cell line with IC 50 values of 43.24 µg/mL, 93.98 µg/mL, and 74.28 µg/mL [24]. On the other hand, this secondary metabolite was inactive as an inhibitor of LTB4 production via non-mediation by redox reactions and as an antiproliferative agent against the human keratinocyte HaCaT cell line [41,76].
In vitro

Antimicrobial
Active against all bacteria and yeast except K. pneumoniae, P. aeruginosa, and P.
syringae. Active against all tested fungi except A. citri, A. tenuissima, A.niger, and G. graminis.

Tridepsides
The biological activities and tridepside chemical structures have been gathered in Table 2 and Figure 2.

Gyrophoric Acid
Gyrophoric acid is a potent antimicrobial agent against a wide range of bacteria and fungi, with MIC values from 0.019 mg/mL for B. subtilis [117]. Moreover, the antimicrobial activity for this tridepside was also demonstrated by Candan et al., highlighting its effect against the bacteria Bacillus cereus and Bacillus subtilis and the fungi Candida albicans and Candida glabrata [118]. Furthermore, gyrophoric acid showed larvicidal activity against the second and third instar larvae of the mosquito Culiseta longiareolata, with LC (50) and LC (90) values of 0.41 ppm and 1.93 ppm, respectively [39].
Gyrophoric acid has also demonstrated potent antioxidant properties. Hence, the IC 50 values for DPPH and superoxide anion scavenging were 105.7 µg/mL and 196.6 µg/mL, respectively, and its reducing power value was 1.32 at 1000 µg/mL [117].
Gyrophoric acid has been investigated for its role as a cytotoxic agent against different cancer cells. This compound reduced the cell viability of human ovarian carcinoma (A2780 cells), human promyelocytic leukemia (HL-60 cells), human T cell lymphocyte leukemia (Jurkat cells), malignant melanoma (Fem-x cells), and chronic myelogenous leukemia (K562 cells) [55,117]. In particular, the studies on A2780 cancer cells revealed that gyrophoric acid caused the accumulation of these cells in the G2/M phase at the expense of the G0/G1 phase. Moreover, this compound reduced the percentage of Fem-x cells and K562 cells in the G0/G1 and S-G2/M phases of the cell cycle [117]. Furthermore, this secondary metabolite inhibited the clonogenic ability of breast SK-BR-3 cancer cells [55]. Additionally, gyrophoric acid caused the cell death of human cervix carcinoma (HeLa) by oxidative stress and apoptosis pathways, as evidenced in the ROS overproduction, DNA oxidative damage, and caspase-3 activation [17]. A study on lichen compounds that interact with DNA revealed that gyrophoric acid was able to inhibit topoisomerase I activity at a concentration of 25 µM [61]. However, this tridepside has resulted in being inactive as an apoptotic agent, as revealed by its ineffectiveness as a caspase-3 activator on hepatocytes [119], and it has low activity against A375 melanoma cancer cell line even at the highest concentrations tested [58].
Other studies, based on the antiproliferative capacity of gyrophoric acid, have investigated its effect on skin cells for therapeutic purposes for psoriasis. This tridepside significantly inhibited the growth of the human keratinocyte HaCaT cell line, with the IC 50 value of 1.7 µM by a cytostatic mechanism [76]. Moreover, gyrophoric acid exerted a photoprotective effect on HaCaT cells with a sun protection factor (SPF) of (SPF > 5) [120,121]. Anti-aging effects were also investigated on ultraviolet A (UVA)-treated dermal fibroblasts, showing upregulated mRNA levels of COL1A1/COL3A1/SOD2 genes and type I collagen protein levels [122].
Gyrophoric acid has been shown to have antihypertensive properties by acting as an angiotensin II type-1 receptor (AT1) antagonist by interacting with residues ARG167, TRP84, and VAL108 [123]. Moreover, gyrophoric acid has been identified as a non-competitive PTP1B inhibitor, with an IC 50 value of 3.6 µM, making it a drug candidate for type 2 diabetes and obesity [99]. Finally, gyrophoric acid is of interest for its properties as a healing agent, especially when combined with usnic acid, that promotes tissue regeneration [89].

Tenuiorin Acid
Tenuiorin acid showed weak to moderate antiproliferative action against the human cancer breast T-47D cell line (ED 50 152.6 µM), the human cancer colon WIDR cell line (ED 50 95.9 µM), and the human cancer pancreas PANC-1 cell line (ED 50 87.9 µM), which seems to be related to its ability to inhibit 5-lipoxygenase activity [22]. Using a Thioflavin T (ThT) fluorescence assay, tenuiorin acid was a potent neuroprotective agent which acted as a tau inhibitor (IC 50 100 µM) [124].

Trivaric Acid
The tridepside trivaric acid has resulted in being a promising antidiabetic agent. In silico and in vitro studies revealed that this compound inhibited protein tyrosine phosphatase 1B (PTP1B) by blocking its active site with an IC 50 value of 173 nM. Moreover, this tridepside improved the insulin-stimulated glucose uptake through the insulin receptor (IR)/IRS/Akt/GLUT2 pathway in the human liver HepG2 cancer cell line. Furthermore, in vivo studies demonstrated the beneficial effects of trivaric acid as an antidiabetic agent at doses of 5 mg/kg and 50 mg/kg through significantly improving lipid and glycemic profiles [125,126].
In another study, trivaric acid exerted a potent anti-inflammatory action by significantly inhibiting human leukocyte elastase (IC 50 value of 1.8 µM) [127].
Tenuiorin acid showed weak to moderate antiproliferative action against the human cancer breast T-47D cell line (ED50 152.6 μM), the human cancer colon WIDR cell line (ED50 95.9 μM), and the human cancer pancreas PANC-1 cell line (ED50 87.9 μM), which seems to be related to its ability to inhibit 5-lipoxygenase activity [22]. Using a Thioflavin T (ThT) fluorescence assay, tenuiorin acid was a potent neuroprotective agent which acted as a tau inhibitor (IC50 100 μM) [124].

Trivaric Acid
The tridepside trivaric acid has resulted in being a promising antidiabetic agent. In silico and in vitro studies revealed that this compound inhibited protein tyrosine phosphatase 1B (PTP1B) by blocking its active site with an IC50 value of 173 nM. Moreover, this tridepside improved the insulin-stimulated glucose uptake through the insulin receptor (IR)/IRS/Akt/GLUT2 pathway in the human liver HepG2 cancer cell line. Furthermore, in vivo studies demonstrated the beneficial effects of trivaric acid as an antidiabetic agent at doses of 5 mg/kg and 50 mg/kg through significantly improving lipid and glycemic profiles [125,126].
In vitro

Conclusions and Prospects
Most of the works on the pharmacological activity of depsides and tridepsides have been published in the last 10 years, which shows the growing therapeutic interest in the secondary metabolites of lichens. Most of these works are in vitro studies, with the occasional in silico and in vivo studies.
Lichens have been investigated for their ability to inhibit bacterial growth. The most common Gram-positive bacteria genera studied on depsides and tridepsides are Bacillus and Staphylococcus, followed by Mycobacterium, Streptococcus, and Enterococcus. Among the Gram-negative bacteria, the genera Escherichia and Proteus were the most investigated. In general terms, depsides and tridepsides showed weak to moderate antimicrobial activity, being more potent against Gram-positive bacteria. In addition, the antifungal activity of several of these compounds (e.g., atranorin, divaricatic acid, gyrophoric acid, lecanoric acid, and methyl evernate) has mainly been studied against Candida spp. Moreover, some depsides displayed good antiparasitic activity against Plasmodium falciparum and Schistosoma mansoni.
Moreover, antioxidant activity has also been widely investigated using different in vitro techniques, such as DPPH assay, hydroxyl radical scavenging activity, superoxide radical scavenging activity, and reducing power assay. Depsides and tridepsides have a phenolic structure that provides antioxidant properties. The compounds with the greatest capacity to scavenge free radicals are sekikaic acid and atranorin.
The cytotoxic activity of depsides and tridepsides has been extensively investigated in in vitro studies. The mechanisms involved in the cytotoxicity of these compounds imply oxidative stress induction (ROS overproduction), apoptosis induction (caspase-3 activation, Bcl2/Bax signaling pathway), cell-cycle arrest, and 5-lipoxygenase antagonist therapy.
Despite the interesting activities that have been compiled in this review, the information seems to stop after the in vitro assays with diverse cell lines. Only a few studies have continued with the in vivo model. The great variety of activities indicate low specificity, which must be deepened with regard to structure-activity studies and toxicological studies. Preclinical and clinical studies should focus on identifying the molecular targets for the action and the nontoxic doses in humans. Furthermore, there are also several compounds for which there is no study of pharmacological activity, such as hiascic acid, lassalic acid, ovoic acid, crustinic acid, and hypothamnolic acid, all being potential metabolites to be investigated. On the other hand, new technology advances will allow the improvement of growing yields and compound extraction, solving the current problem that limits the study with these interesting compounds

Conflicts of Interest:
The authors declare no conflict of interest.