"A further family of aromatic compounds produced by lichens is derived from shikimic acid via phenylalanine and is exemplified by vulpinic acid (7.55), which was isolated from Letharia vulpina. Vulpinic acid has been synthesized by the lead tetra-acetate oxidation of polyporic acid." [ChemofFungi]

Vulpinic acid (Vulpinsaure) C₁₉H₁₄O₅
"Yellow prisms (EtOH), mp 148 °C"
"Deriv: Vulpinic acid methyl ether, yellow crystals (MeOH), mp 142-143 °C from vulpinic acid
with CHzNz in EtzO at 20 °C"
"StL: Letharia vulpina (1.) Hue" ^{[Siegfried ILS]}

Bryoria fremontii

"The greenish-yellow pigment in this and related species is a bitter, potentially toxic pulvinic acid derivative unique to lichens called vulpinic acid.... The harvested lichen was cleaned of debris, then soaked in running water and pounded in an attempt to remove the bitter, greenish vulpinic acid. It was then cooked in layers in underground steaming pits for as long as 24 hours. It could be eaten freshly cooked, or dried for winter use." ^{[Turner&Kuhnlein]}

Letharia vulpina

"There is no significant statistical correlation between light intensity and the overall concentration of either compound. For all lichens examined, the vulpinic acid content was greatest near the young branch tips (ranging from 3.7 to 5.7% by dry weight), and least in the old basal branches (0.087 to 1.7% by dry weight). The greatest range of vulpinic acid in a single thallus was that of a lichen which had 48 times more vulpinic acid by weight in its branch tips than in its base. Variability in vulpinic acid content of basal branch portions seemed to be related to the age of the thallus as a whole; a large lichen with a thick discolored base had the lowest vulpinic acid content, while a smaller more uniformly colored thallus had the highest." (stephenson1979)

"Concentrations of any compound in lichens will be greatest in old basal branches if branches of all ages produce the compound at a constant rate, assuming insignificant loss of the compound to factors such as weathering and turnover. Thus, a constant production of atranorin throughout the thallus of Letharia vulpina would bring about atranorin accumu- lation in older branches. In contrast, little vulpinic acid is found in basal branches. Because vulpinic acid is deposited in crystalline form on the surface of the fungal hyphae in the cortex, it is unlikely that the loss of this compound with age is due to its slow meta- bolism. Vulpinic acid is likely produced in large quantities by young branch tips, but production slows or stops as the branch gets older. Vulpinic acid concentration then decreases because: (a) the branches continue to thicken and thereby "dilute" the compound; and/or (b) vulpinic acid is leached from the thallus by rainwater. Vulpinic acid is at least four times more soluble in water than atranorin." (stephenson1979)

"The growing tips, therefore, produce copious amounts of toxic vulpinic acid in response to predation. Soredia, the vegetative reproductive bodies of L. vulpina, are also protected by large amounts of the compound; three samples of soredia from different populations contained 8.5, 9.8, and 14% vulpinic acid by air-dried weight. The same soredia contained 0.12, 0.006 and 0.11% atranorin, respectively. Vul- pinic acid is known to have some anti-bacterial properties [7], but these are probably second- ary to its anti-herbivore function." (stephenson1979)

"... enormous variation in the concentration of chemicals can occur within a single thallus. The gray base of a very large (13 cm diameter) Letharia vulpina thallus we examined contained 0.91% atranorin and only 0.005% vulpinic acid—one thousand times less vulpinic acid than is normally found in the growing branch tips of the lichen." (stephenson1979)

"Conventional detection methods carried out on this sample, such as thin-layer chromatography and microcrystal tests, would have detected atranorin but probably not vulpinic acid. The opposite would be true if the branch tips of the lichen had been sampled; vulpinic acid would have been detected, but not atranorin." (stephenson1979)

"A thallus collected in the San Gabriel Mountains of southern California in July 1977 contained no detectable atranorin by HPLC in the three youngest branch age classes, and only 0.025% atranorin overall, indicating that there may be wide geographic differences in the concentrations of compounds." (stephenson1979)

"We have also noted extreme variation of vulpinic acid concentration in populations of Alectoria fremontii Tuck. in California. The terminal 50 mm of thalli from Carmel Valley, California contained 0.23% vulpinic acid by air-dried weight compared to only 0.004% vulpinic acid in thalli from Sequoia National Park." (stephenson1979)

"Some chemical races reported to be lacking certain compounds may actually contain the compounds in concentrations too small to be detected by conventional methods." (stephenson1979)

"The concentrations of vulpinic and pinastric acids in thalli of Cetraria pinastri were found using HPLC to be approximately 1% (air dry weight) for vulpinic acid and approx- imately 2% (air dry weight) for pinastric acid. There is a certain amount of variability in concentrations when thalli of different ages are compared, a result that is consistent with results obtained by Stephenson and Rundel (1979) for Letharia vulpina (L.) Hue and L. columbiana (Nutt.) Thoms." (lawrey1983)

Antifeedant Properties

Letharia vulpina: "The suggested role of vulpinic acid as an anti-herbivore defense compound is supported by both this distribution of the compound and our observations that vulpinic acid acts as a feeding deterrent to certain invertebrates. No significant difference in the content of atranorin or vulpinic acid was found in lichens from microhabitats of different sunlight intensities" (stephenson1979)

Letharia vulpina: "Despite the anti-herbivore properties of vulpinic acid, we discovered the larva of a drug-store beetle, Stegobium paniceum LeConte (Coleoptera: Anobiidae), in the hollowed branch of a herbarium specimen. Many of the lichen branches had been tunnelled through and were packed with feces (presumably those of the larva), which contained 0.093% atranorin and 0.29% vulpinic acid by air-dried weight. The fact that the larva was eating mainly vulpinic acid-free medullary tissue and was able to pass out at least some vulpinic acid in its feces may account for its ability to develop successfully on Letharia vulpina." (stephenson1979)

Letharia vulpina: "Vulpinic acid is highly toxic to vertebrates (7, 8), the estimated LD₅₀ for the ingestion of Letharia vulpina by squirrels (a likely potential herbivore in its range) is 1.5 to 2.5 g dry weight of the lichen, assuming complete absorption of the ingested compound (1). Slüger et al. (9) noted that vulpinic acid also exhibits strong insecticidal activity. Aqueous extracts of L. vulpina are poisonous to the American cockroach, Periplanta americana, when injected in small quantities (10). In addition to exhibiting toxicity, vulpinic acid acts as a feeding deterrent to snails (Zopf, in (11)). Preliminary studies of our own show that the common garden snail Helix aspersa Müller (which has been observed in close association with, and almost certainly feeds on, lichens other than L. vulpina (12)) reduces its feeding rate drastically when vulpinic acid is added to its diet in concentrations corresponding to those found in the branch tips of L. vulpina. We have also observed that larvae of the variegated cutworm, Peridroma margaritosa Haw. (Lepidoptera: Noctuidae), an extreme generalist feeder, shun leaves coated with vulpinic acid. Letharia vulpina is free of significant predation in its natural habitat, despite its abundance and high nitrogen content." (stephenson1979)

"In more recent laboratory studies, vulpinic acid has been shown to elicit significant reductions in the feeding activity of several invertebrate grazers (Slansky 1979; Stephenson & Rundel 1979). Slansky's results, obtained using larvae of the polyphagous insect pest Spodoptera ornithogalli (Guenee), suggested that vulpinic acid functioned as a deterrent rather than a poison; when larvae were forced to consume food treated with vulpinic acid or starve, larval growth was not significantly different from that of control larvae. Even if vulpinic acid is not the potent toxin it was once believed to be, however, it appeared from Slansky's and other studies that vulpinic acid is at least a repellant or an inhibitor compound capable of discouraging generalist herbivore feeding." (lawrey1983)

"The mortality data show no contact toxic effect of the vulpinic acid on the slugs, despite the fact that more mortality might be expected in the higher con- centration treatments because the slugs were more starved. There is no evidence of toxicity by ingestion since mortality of slugs after eating treated seeds was lower in the higher concentration treatments. Lichen compounds have been used experimentally as antifeedants against the polyphagous insect pest Spodopteru ornithogalli (Lawrey, 1984), and no ef- fect on larval growth or survival was found. Vulpinic acid does have measurable toxicity however, its mouse LD50 is 150 mg kg-’ and it has reputedly been used in the past to poison wolves (Lawrey, 1984)" (clark1999)

"Experiments have shown that snails will feed on potatoes covered with cetraric, rhizocarpic and pinastrinie acids, poisonous to other animals, but will not feed on vulpinic acid which is recognized as poisonous to vertebrates." (George A. Llano)

"The low concentrations of vulpinic and pinastric acids, coupled with the results of slug preference tests, suggest that these compounds are frequently not able to function as defense compounds against lichen herbivores because they are not produced at sufficiently high concentrations. The concentration dependence of lichen compound-induced herbivore de- terrence is almost totally unknown. Slansky (1979) found that relatively low concentrations of vulpinic acid (0.6% dry weight) coated onto broccoli leaf surfaces reduced consumption of leaves by a polyphagous insect larva, Spodoptera ornithogalli. However, there was no apparent biological effect of vulpinic acid consumption by Spodoptera larvae; larvae forced to eat vulpinic acid-coated food exhibited the same growth and survivorship as control larvae. Furthermore, this herbivore does not normally consume lichens or lichen com- pounds. Herbivores like Pallifera varia that normally consume lichens of various species are more likely to be able to tolerate lichen compound consumption so long as the con- centration in the thallus is not too high." (lawrey1983)

"The antifeedant activity of vulpinic acid is much stronger than its repellent effect, and was dem- onstrated clearly in the leaf damage experiment. The plants were treated with only half the rate of vulpinic acid used for the time lapse video test because at 50 pg cm-2 there was evidence of a phytotoxic effect in the yellow patches which appeared on some of the leaves. However 25 pg cm-2 was enough to signifi- cantly reduce slug feeding on the plants, even in the more rigorous context of a no-choice test. There was no visible sign of leaf damage, such as yellowing of the leaves, at the lower rate. Phytotoxicity is fre- quently an obstacle to the widespread use of other- wise effective antifeedants as foliar sprays or seed treatments to control crop damage, especially by slugs (Airey et al., 1989)." (clark1999)

"Vulpinic acid is produced by non-lichen fungi as well as other lichens, some of which are eaten by molluscs (Lawrey, 1983) but in these experiments it was clearly capable, at the concentrations used, of deterring feeding in Derocerus reticulaturn", the field slug. (clark1999)

Photoprotection and Chemical Defense

The function of vulpinic acid as a photoprotective agent in lichens like Letharia vulpina (Wolf Lichen) is well-supported by recent physiological and chemical research. Studies demonstrate that this pigment specifically targets high-energy blue light and UV radiation to prevent damage to the lichen's photosynthetic partner.^{(Phinney et al. 2018)}

Blue Light Screening in Letharia vulpina

Research focusing on the "chartreuse" (yellow-green) color of Letharia vulpina has confirmed that vulpinic acid is a highly effective screen for photosynthetically active radiation (PAR), specifically in the blue spectrum.^{(Phinney et al. 2018)}

• Photosynthetic Protection: When vulpinic acid is removed from the lichen thalli, the underlying algae (photobiont) suffer significantly higher rates of photoinhibition—a reduction in photosynthetic capacity caused by excess light.^{(Phinney et al. 2018)}
• Efficiency: Experimental data using chlorophyll fluorescence indicates that vulpinic acid can screen up to 88% of blue light, effectively acting as a filter that prevents the photobiont from being overwhelmed by high-energy photons.^{(Phinney et al. 2018)}
• Ecological Trade-off: While this screening protects the lichen in exposed, high-light environments, it can slightly reduce photosynthetic efficiency in low-light conditions because it "competes" with the algae for blue light.^{(Beckett et al. 2021)}

UV Blocking and Photostability

Beyond the visible blue spectrum, vulpinic acid serves as a critical blocker for ultraviolet (UV) radiation, which is particularly intense in the alpine and sub-alpine habitats where Letharia is often found.

• UVB and UVA Absorption: Vulpinic acid has been identified as a potent UV blocker that is both photostable and non-cytotoxic to cells.^{(Legouin et al. 2017)} In species like Vulpicida pinastri, which also contains this acid, it works synergistically with other secondary metabolites like pinastric acid to enhance the overall UV-protective index.^{(Legouin et al. 2017)}
• Stability Mechanisms: The resilience of this pigment is bolstered by its environment. Within the lichen cortex, vulpinic acid interacts with exocellular polysaccharides, which enhance its photostability and prevent its chemical decomposition even under extreme solar stress.^{(Beckett et al. 2021)}

"In 1946 Bargellini (6) showed usnic and vulpinic acids to be active against Staph. aureus, C. diphtheriae and B. subtilis but not against Gram-negative bacteria." (Bustinza)

"Secondary metabolites of different species of lichen were tested for their activities against a variety of microbial species. While Gram-negative rods and fungi were not inhibited by these compounds, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and some anaerobic species (Bacteroides and Clostridium species) were susceptible at the concentrations tested. Vulpinic acid was generally less active than usnic acid, regardless of its stereochemistry. The susceptibility to usnic acid was not impaired in clinical isolates of S. aureus resistant to methicillin and/or mupirocin (Lauterwein et al. 1995)." [Rai MPBD]

"In this study, we found most of our lichen extracts inhibited the growth of the Gram-negative bacterium P. aeruginosa and extracts from three lichen species, L. columbiana, L. vulpina, and V. canadensis, were also effective against E. coli. Each of these three lichen species produces vulpinic acid as a principal secondary metabolite, suggesting that vulpinic acid may be active against E. coli. To our knowledge, there are no data available regarding the antibiotic effects of crude extracts of Letharia or Vulpicida species or purified vulpinic acid against E. coli except (Lauterwein et al., 1995). However, they were not able to document the antibiotic effects of vulpinic acid against E. coli at their highest concentration of 32 Ug/ml. In contrast, in our study, using higher concentrations of lichen extracts containing vulpinic acid (MIC = 125–250 Ug/ml), the growth of E. coli was inhibited. This variation in the results among different studies may be due to a combination of factors, including extraction of different lichen species, the solvent used for extraction, and the specific bacterial strain. Additional research is required to determine the specific factors influencing antimicrobial properties of lichen extracts." (shrestha2014)

"Alectosarmentin, (-)-usnic acid, physodic acid and 8’-O-ethyl-beta- alectoronic acid isolated from the alcoholic extract of the lichen Alectoria sarmentosa (Ach.) Ach. (Alectoriaceae) showed antimicrobial activity. Hypogymnia apinnata (atranorin), Letharia columbiana (vulpinic acid), Lobaria pulmonaria (Stictic acid, constictic acid, and norstictic acid) and Usnea filipendula (Usnic acid and salazinic acid) have been reported to have significant antibiotic activity against Micrococcus luteus, Staphylococcus aureus, Salmonella gallinarum and Serratia marcescens respectively." [HPEP]

Alleleopathy

[Dighton FEP]