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Review of the biological properties and

toxicity of usnic acid
a

A. A. S. Arajo , M. G. D. de Melo , T. K. Rabelo , P. S. Nunes , S.

a

L. Santos , M. R. Serafini , M. R. V. Santos , L. J. Quintans-Jnior

& D. P. Gelain
a

Federal University of Sergipe, So Cristvo, Brazil

Federal University of Rio Grande do Sul, Porto Alegre, Brazil

Federal University of Sergipe, Lagarto, Brazil

Published online: 24 Feb 2015.

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To cite this article: A. A. S. Arajo, M. G. D. de Melo, T. K. Rabelo, P. S. Nunes, S. L. Santos, M.
R. Serafini, M. R. V. Santos, L. J. Quintans-Jnior & D. P. Gelain (2015): Review of the biological
properties and toxicity of usnic acid, Natural Product Research: Formerly Natural Product Letters,
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Natural Product Research, 2015

http://dx.doi.org/10.1080/14786419.2015.1007455

Review of the biological properties and toxicity of usnic acid

A. A. S. Araujoa, M. G. D. de Meloa, T. K. Rabelob, P. S. Nunesa, S. L. Santosa, M. R. Serafinic*,
M. R. V. Santosa, L. J. Quintans-Juniora and D. P. Gelainc
a
Federal University of Sergipe, Sao Cristovao, Brazil; bFederal University of Rio Grande do Sul, Porto
Alegre, Brazil; cFederal University of Sergipe, Lagarto, Brazil

Downloaded by [Mairim Serafini] at 02:11 02 March 2015

(Received 3 October 2014; final version received 7 January 2015)

EXTRACTION

LICHEN

analgesic

Antiinfl
ory
inflammatory

healing

Antimicrobial

Antioxidant

REVIEW
antiprotozoal

Live injury

Antiviral
Larvicidal

UV
p
protection

Since its first isolation in 1844, usnic acid [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl1,3(2H,9bH)-dibenzo-furandione] has become the most extensively studied lichen
metabolite and one of the few that are commercially available. Lichens belonging to
usnic acid-containing genera have been used as crude drugs throughout the world.
There are indications of usnic acid being a potentially interesting candidate for such
activities as anti-inflammatory, analgesic, healing, antioxidant, antimicrobial,
antiprotozoal, antiviral, larvicidal and UV protection. However, some studies reported
the liver toxicity and contact allergy. Thus, further studies are needed to establish the
efficacy and safety of usnic acid
Keywords: usnic acid; lichen; biological activity; antioxidant; antimicrobial

1. Introduction
Lichens are formed through the symbiosis between a fungal and a photosynthetic partner such as
algae or cyanobacteria. More than 17,000 species and over 800 lichen products are known.
Polysaccharides, proteins and secondary metabolites produced by lichens have attracted the
attention of investigators due to their biological activities (Lisci et al. 2003; Melo et al. 2011;
Rabelo et al. 2012). Components such as usnic acid (UA) (Figure 1) are used for perfumery and
for medicinal purpose (Kohlhardt-Floehr et al. 2010). Since its first isolation in 1844, UA [2,6diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione] has become the most
extensively studied lichen metabolite and one of the few that are commercially available. This
natural compound has showed different biological and physiological activities that might have a
great relevance in pharmacology and clinics (Campanella et al. 2002; Manojlovic et al. 2012).

*Corresponding author. Email: maiserafini@hotmail.com

q 2015 Taylor & Francis

A. Araujo et al.

Figure 1. UA structure (Ingolfsdottir 2002).

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Many biological properties of this drug are known; however, despite its importance, there are no
recent reviews on these properties and on the toxicity of UA.
2. Antioxidant and pro-oxidant activities
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the
pathogenesis of numerous diseases such as cancer, inflammatory diseases and neurodegenerative
disorders (Seifried et al. 2007). Under normal physiological conditions, ROS/RNS participate as
intracellular messengers and regulatory molecules. They are tightly regulated by the balancing
systems formed by different antioxidants, antioxidant enzymes and proteins (Kowaltowski et al.
2009). Main actions of secondary metabolites in biological systems have also been linked to
their redox properties (Melo et al. 2011; Rabelo et al. 2012).
Some studies have showed antioxidant properties of UA in gastric mucosal. (Odabasoglu
et al. 2006) demonstrated that UA exhibits antioxidant effect when used as therapy against
indomethacin-induced gastric ulcers in rats. In these studies, it was observed that gastric lesions
were significantly reduced by all doses of UA as compared with the indomethacin-treated
group. Moreover, UA induced a significant inhibition of the formation of reactive species, a
decrease in lipid peroxidation and an increase in antioxidant enzyme activities, such as
glutathione peroxides and superoxide dismutase (Halici et al. 2005; Odabasoglu et al. 2006).
Several studies have showed that the redox activity associated with natural antioxidants is
attributed to total content of phenolic compounds (Rice-Evans et al. 1995; Scalbert et al. 2005;
Halliwell 2008). Antioxidant and pro-oxidant biochemical agents have presented an important
role to design strategies for the prevention and/or management of oxidative damage. Recent
studies demonstrate that UA displays variable redox-active properties, acting as an antioxidant
and pro-oxidant agent, according to different system conditions and/or cellular environment.
The antioxidant effect of UA is certainly associated with the capacity of this lichenic secondary
metabolite to perform peroxyl radical scavenging, quench hydroxyl radicals and to reduce the
production of nitrite. On the other hand, UA presented a pro-oxidant capacity in a lipid-rich
system, enhancing TBARS formation induced by AAPH incubation. In addition, UA decreased
cell viability of neuron-like cells (SH-SY5Y) in culture. This effect is associated with the ability
of UA to increase intracellular ROS production in these cells (Rabelo et al. 2012).
In other studies, UA showed a pro-oxidant and also an antioxidant behaviour. The UA
extracted from Xanthoparmelia farinosa (Vainio) was used in a human lymphocyte cell line
(Jurkat-cells) under UVB irradiation, causing lethal damaging effects on cell membranes and
reducing cell metabolism when presented in a high concentration. However, in low
concentrations and under physiological UVB intensity, UA exhibits an antioxidant function
(Kohlhardt-Floehr et al. 2010).
Regarding the antiproliferative activity, both (2 ) and ( ) isomers of UA showed moderate
to strong cytotoxicity against a wide variety of murine and human cancer cell lines (Takai et al.

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1979; Kumar and Muller 1999a). The (2 )-UA also induced apoptosis of the murine leukemia
L1210 cells in a dose- and time-dependent manner (Bezivin et al. 2004). Other studies have also
reported the cytotoxic effect of UA and atranorin against various in vitro cancer models: A2780
(ovarian carcinoma), MCF-7 (breast adenocarcinoma), HT-29 (colon adenocarcinoma), HL-60
(promyelocytic leukaemia), Jurkat (T-cell lymphocyte leukaemia) HeLa (cervix adenocarcinoma), SK-BR-3 (breast adenocarcinoma) HCT-116 p53 / (colon carcinoma) and HCT-116
p53 2 /2 (wild-type p53 colon carcinoma, as well as p53-null). In this report, cell proliferation
induced by UA or atranorin was found to be more efficient at equitoxic doses and correlated
more strongly with a higher apoptotic index (Backorova et al. 2011).
In recent studies used to assess the cytotoxic activity of purified lichen metabolites in three
human cancer cell lines, MCF-7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and
HCT-116 (colon carcinoma), UA in the concentrations higher than 25 mM showed the highest
cytotoxic activity against all cancer cell lines analysed when compared with the other lichen
metabolites (Brisdelli et al. 2012).
3. Antimicrobial and antiprotozoal activity
Carvalho et al. for the first time have described the effects of UA on the protozoan Trypanosoma
cruzi. Ultrastructural analysis of treated epimastigotes showed damage to mitochondria, with a
marked increase in kinetoplast volume and vacuolation of the mitochondrial matrix. Intense lysis
of bloodstream trypomastigotes was observed with all drug concentrations tested. Besides
mitochondrial and kinetoplast damage, trypomastigotes also presented enlargement of the
flagellar pocket, as well as intense cytoplasm vacuolation. Treatment of infected macrophages
with 40 or 80 mg/mL UA induced marked cytoplasm vacuolation in intracellular amastigote
forms, with disorganisation of parasite kinetoplast and mitochondria, but with no significant
ultrastructural damage to the host cells (De Carvalho et al. 2005).
The activity of UA against Candida orthopsilosis and Candida parapsilosis on planktonic
and biofilm conditions was investigated by Pires et al. (2012). The results presented in this study
were the first report of UA showing in vitro inhibitory and fungicidal activity against
environmental isolates of C. orthopsilosis and C. parapsilosis. UA exhibited an anti-Candida
effect, with IC50 of 1.95 mg/mL and IC80s of 7.8 and 15.6 mg/mL (Pires et al. 2012).
The effects of treatments with ( ) UA by oral, subcutaneous or intralesional routes during
the course of infection of BALB/c mice infected with Leishmania amazonensis was described by
Fournet et al. (1997). In this work, it was observed that subcutaneous and oral treatments with
UA did not produce any effect, but by intralesional administration, we observed a significant
effect that reduced by 43.34% of the weight lesions and by 72.28% of the parasite loads in
infected footpads (Fournet et al. 1997).
Elo et al. (2007), reported a study on the antimicrobial activity of UA and its sodium salt
against clinical isolates of Vancomycin-resistant enterococci (VRE) (using strains with both the
van A and the van B genotypes) and methicillin-resistant Staphylococcus aureus (MRSA)
in vitro. The UA and, especially, the sodium salt had a potent antimicrobial activity against all
clinical isolates of VRE and MRSA studied (Elo et al. 2007).
The in vitro antimicrobial activities of UA were evaluated in combination with five
therapeutically available antibiotics, using checkerboard microdilution assay against
methicillin-resistant clinical isolates strains of S. aureus by Segatore et al. (2012).
A synergistic action was observed in combination with gentamicin, while antagonism was
observed with levofloxacin. The combination with erythromycin showed indifference, while
variability was observed for clindamycin and oxacillin (Segatore et al. 2012).
A study published by Lira et al. (2009), was designed to evaluate the in vitro release profile,
cytotoxicity and antimycobacterial activity of UA encapsulated into liposomes against

A. Araujo et al.

Mycobacterium tuberculosis H37Rv. The results indicated a strong interaction between

liposomes and J774 macrophages, thereby facilitating UA penetration into cells and
considerably improving its activity against the M. tuberculosis. Ramos and Silva (2010)
evaluated the antimicrobial activity of UA against susceptible and resistant clinical isolates of
M. tuberculosis, and against four species of non-tuberculous mycobacteria (NTM).
In this study, UA showed activity against both resistant and susceptible strains, allowing one
to infer that there is no cross resistance with isoniazid, rifampicin and streptomycin, when the
molecular basis to resistance is mutation of the loci KatG S315T, RpoB S531L and RpsL K43R.
According to the authors, this is a pertinent point because these drugs are the basis of the current
therapy for tuberculosis, and the molecular alteration observed in these strains is responsible for
the resistance of these strains studied (Ramos and da Silva 2010).

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4. Antiviral activity
Campanella et al. (2002), studied the effect of UA on the proliferation of mouse polyomavirus in
3T6 cells. The results showed that polyomavirus DNA replication was severely inhibited at noncytotoxic concentration of UA. According to the authors, UA acts as a generic repressor of RNA
transcription (Campanella et al. 2002). A recent study investigated the antiviral activity of UA
and its derivatives against the pandemic influenza virus A(H1N1) pdm09. A total of 26
compounds representing ( ) and (2 ) isomers of UA and their derivates were tested for
cytotoxicity and anti-viral activity in MDCK cells through microtetrazolium test and virus yield
assay, respectively. Absolute configuration was shown to have critical significance for the antiviral activity. With minor exceptions, in the pair of enantiomers, (2 )-UA was more active
comparing with ( )-isomer, but its biological activity was reversed after the UA was chemically
modified (Sokolov et al. 2012).
5. Larvicidal activity
Dengue is a viral disease caused by a Flavivirus transmitted by the mosquito Aedes aegypti. UA
exhibited LC50 of 6.61 (6.16 7.06 ppm) demonstrating that it possessed efficacy against
A. aegypti. However, it was toxic to brine shrimps, a reference organism in assays to evaluate the
potential toxicity hazard to invertebrates in ecosystems (Bomfim et al. 2009).
6. Anti-inflammatory activity and healing
Inflammation is a protective host response to foreign antigenic challenge or tissue injury that, if
unopposed, could lead to loss of tissue structure as well as function (Riella et al. 2012).
Vijayakumar and co-workers demonstrated that UA, isolated from the lichen Roccella
montagnei, showed a dose-dependent anti-inflammatory activity when tested on rats, employing
acute and chronic models. These findings might be related to the UA biological properties,
which may be involved in the inhibition of the prostaglandin synthesis, similarly to non-steroidal
anti-inflammatory drugs (Vijayakumar et al. 2000). Su et al. (2014) noted that UA protects LPSinduced acute lung injury in mice by attenuating inflammatory responses and oxidative stress.
The evaluation of the anti-inflammatory activity indicated that UA attenuated the expression of
tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), interleukin-8 (IL-8) and macrophage
inflammatory protein-2 (MIP-2). The improved level of interleukin-10 (IL-10) in the
bronchoalveolar lavage fluid (BALF) was also observed.
Huang et al. (2011) studied the anti-inflammatory effect and mechanism of UA by
lipopolysaccharide (LPS) stimulated by the RAW264.7 cell line. They found that UA has a
dose-dependent activity against cytokines and pro-inflammatory mediators, leading to a
reduction excretion of TNF-a, IL-6, interleikin 1 beta (IL-1b) and inducible nitric oxide

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synthase (iNOS) to cyclooxygenase 2 (COX-2) via suppression of factor nuclear kappa B (NFkB). Another observation was the dose-dependent activity of UA in the increased production of
IL-10 and heme oxygenase 1 (HO-1).
The wound-repairing properties of UA enamines (compounds 2 11), obtained through
nucleophilic attack of amino acids or decarboxyamino acids at the acyl carbonyl of the enolised
1,3 diketone, were evaluated using in vitro and in vivo assays by Bruno et al. (2013). This study
attributed significant wound-healing properties to single UA derivatives. The results of in vitro
and in vivo assays were quite consistent, showing lowest cytotoxicity combined to highest
healing performance for 8 and 9, which in most cases were preferable to their precursor UA.
Furthermore, the study suggests the possible use of these compounds in the promotion of wound
healing or anti-aging skin preparations. A recent study showed that collagen-based films
containing liposome-loaded UA are quite useful in improving burn healing. UA has been shown
to be involved with the modulation of some biological events in this process, such as the
inflammatory response, epithelisation and collagen formation (Nunes et al. 2011).

7. UV protection
Natural substances extracted from plants and lichens have been recently considered as potential
sunscreens thanks to their absorption on the UV region and also to their antioxidant power. Thus,
the potential antioxidant and pro-oxidant activity of UA extracted from X. farinosa (Vainio)
using a human lymphocyte cell line (Jurkat-cells) under UV-B-irradiation was reported. Cell
survival and cell metabolism were determined using different conditions such as UA
concentration and UVB dose. Compared with the controls, the cells incubated with UA in
concentrations of 1 1028 and 1 1026 M showed a higher cell survival and a normal
metabolism under low doses of UVB-light up to 0.1 J/cm2. When both higher UVB doses (up to
14 J/cm2) and higher concentrations of UA (1 1024 M) were used, the opposite effect was
observed. It is concluded that such effects are due to bi-functional (a switch of) anti-oxidative
and pro-oxidative behaviour of UA under UV-B-irradiation (Kohlhardt-Floehr et al. 2010).
In another study published, UA was tested in vivo and in vitro as possible UV-light filters and
the protection factor was compared with that found for the references: Nivea sun Spray LSF 5,
octylmethoxycinnamate (OMC) and 4-tert.-butyl-49-methoxy dibenzoylmethane (BM-DBM).
In conclusions, UA resulted in being the best UVB filter, with an in vivo protection factor similar
to Nivea sun Spray LSF 5. The protection factor, as well as the good UV-light absorption,
suggests that UA may be useful as new filters in sun-screen preparations (Rancan et al. 2002).

8. Live injury
Toxic injury occurs in the liver more often than any other organ. That can be attributed to the fact
that virtually all ingested substances that are absorbed are first presented to the liver and also that
the liver is responsible for the metabolism and elimination of many substances (Al-Bekairi et al.
1991).
UA is the normal component of lichen cells and is one of the most common and abundant
lichen metabolites. This natural compound has shown a great relevance in pharmacology and
clinics and is well known as an antibiotic. Also, it is endowed with several biological and
physiological activities including antiparasitic, antimitotic, antiproliferative, anti-inflammatory,
analgesic and antipyretic (Al-Bekairi et al. 1991; Lauterwein et al. 1995; Okuyama et al. 1995;
Cardarelli et al. 1997; Kumar and Muller 1999b; Vijayakumar et al. 2000; Campanella et al.
2002; Cocchietto et al. 2002). Nevertheless, the compound has been associated with severe liver
damage (hepatotoxicity) when taken as a dietary supplement for the purpose of weight loss.

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A. Araujo et al.

The relatively high doses of UA required to achieve weight reduction can result in serious
side-effects. For example, the Food and Drug Administration received at least 21 reports of
hepatotoxicity in consumers who ingested dietary supplements containing UA or sodium usniate
for weight loss, thereby raising safety concerns. These hepatotoxicities resulted in 1 death, 1
liver transplant, 7 individuals with liver failure, 10 cases of chemical hepatitis and 4 cases of
mild hepatic toxicity (Favreau et al. 2002; Neff et al. 2004).
Several mechanistic studies of UA-related hepatotoxicity have been performed. In cultured
primary mouse hepatocytes, UA caused mainly necrosis with no apparent apoptosis (Han et al.
2004. In animal studies, UA induced extensive liver necrosis in mice (Ribeiro-Costa et al. 2004;
da Silva Santos et al. 2006) but appeared less toxic to rats, although mitochondrial swelling and
changes in endoplasmic reticulum were observed in rat liver (Pramyothin 1986; Lira et al. 2009).
The proposed mechanisms for UA-related liver injury include uncoupling of oxidative
phosphorylation, inhibition of oxidative phosphorylation, increased oxidative stress, lipid
peroxidation and depletion of glutathione (GSH). The working model is that the disruption of
mitochondrial respiratory function and oxidative stress conspires to undermine cell viability.
Evidence is rapidly accumulating and suggests that the disruption of mitochondrial function is a
general mechanism that underlies an increasing variety of organ toxicities (Boelsterli and Lim
2007; Dykens and Will 2007; Dykens et al. 2008; Joseph et al. 2009).
The toxic effects of UA were reported on human hepatoblastoma HepG2 cells (30), in
order to evaluate the interactions, if any, of low non-toxic concentrations of UA and LPS, a
potential contaminant of food, leading to toxicity in HepG2 cells (Sahu et al. 2012).
The liver performs a multitude of functions including the regulation of carbohydrate, fat, and
protein metabolism, the detoxification of endo- and xenobiotics, and the synthesis and secretion
of plasma proteins and bile (Bissell et al. 2001). The liver, located between the absorptive
surface of the gastrointestinal tract and drug targets throughout the body, is central to the
metabolism of virtually every foreign substance (Bissell et al. 2001). This hepatic
biotransformation involves oxidative pathways, primarily through the cytochrome P-450
enzyme system. After further metabolic steps, which usually include conjugation to a
glucuronide, a sulfate or a glutathione, the hydrophilic product is exported into plasma or bile by
transport proteins located on the hepatocyte membrane, and it is subsequently excreted by the
kidney or the gastrointestinal tract (Lee 2003).
Severe hepatotoxicity has been associated with the use of weight loss diet supplements
containing UA. This compound is a dibenzofuran metabolite produced by many lichens, a
symbiosis between a wide variety of fungal species and photosynthetically active algae or
cyanobacteria. It has been reported that UA has multiple biological effects. Because of their
reported beneficial biological characteristics, this natural compound has been used worldwide to
treat a number of ailments (Rafanelli et al. 1995; Ingolfsdottir 2002).
Favreau et al. (2002) reported on seven patients who developed acute hepatitis after using
LipoKinetix. This dietary supplement contains sodium usniate, norephedrine, yohimbine, 3-5diiodothyronine and caffeine; both UA and ephedra alkaloids.
Neff et al. retrospectively reviewed the records of 12 patients who had hepatotoxicity
supposedly related to the ingestion of herbal weight loss compounds from various ingredients,
including ma huang and UA. Data recorded on each patient include the duration of therapy, time
to the presentation from the last ingestion, and the determination of the contribution of other
possible underlying diseases or medical conditions (Neff et al. 2004).
Durazo et al. (2004) reported on a healthy 28-year-old woman who developed acute liver
failure within one month after commencing UA (Pure Usnic acid, Industrial strength; AAA
Services, USA) 500 mg/day for 2 weeks (Durazo et al. 2004).
Han et al. (2004) evaluated in primary cultured murine hepatocytes that the UA treatment
(5 mM) resulted in 98% necrosis within 16 h (no apoptosis was detected). UA treatment was

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Natural Product Research

associated with the early inhibition and uncoupling of the electron transport chain in
mitochondria of cultured hepatocytes. This inhibition of mitochondria by UA corresponded to a
fall in ATP levels in hepatocytes. In isolated liver mitochondria, UA was observed to directly
inhibit and uncouple oxidative phosphorylation. Oxidative stress appears to be central in UAinduced hepatotoxicity based on the following findings: (1) pre-treatment with antioxidants
(butylated hydroxytoluene Vitamin E) decreased UA-induced necrosis by nearly 70%; (2)
depletion of mitochondrial GSH with diethylmaleate increased susceptibility of hepatocytes to
UA; (3) UA treatment was associated with the increase in free radical generation, measured
using the fluorescent probe, dichlorodihydrofluorescin. This study suggested that the mechanism
by which UA induces hepatotoxicity may be similar to rotenone (rotenone treatment has been
shown to induce necrosis in primary cultured hepatocytes mediated by reactive oxygen species
generation) (Han et al. 2004).
Pramyothin et al. (2004) evaluated treatment in rats with a high dose of ( )-UA (200 mg/kg
per day, i.p.) for 5 days, and there was no significant change in serum transaminase activity
(serum AST and ALT) while the electron micrographs showed apparent morphological damage
of mitochondria and endoplasmic reticulum. ( )-UA at a high dose (1 mM) as well as carbon
tetrachloride (CCl4, the reference hepatotoxin), carbon tetrachloride is metabolised by the
cytochrome P450 system, especially CYP 2E1, to CClz3, a free radical that induces cell
membrane injury and disturbance of Ca2 homeostasis, and resulting in cell death. Increase in
lipid peroxidation, decrease in glutathione (GSH) content and increase in aniline hydroxylase
activity (CYP 2E1) were also found. The hepatotoxic effect of high-dose ( )-UA may involve
its reactive metabolite(s), causing loss of integrity of membrane-like structures, resulting in the
destruction of mitochondrial respiration and oxidative phosphorylation (Pramyothin 1986).
Santos et al. (2006) evaluated the effects of the nanoencapsulation of UA (an attempt to
improve antitumour activity and reduce hepatotoxicity) and observed that UA hepatotoxicity
was substantially reduced when animals were treated with UA-loaded nanocapsules.
Haematology, biochemical and histopathological analyses demonstrated that UA into PLGAnanocapsules reduced hepatotoxicity (Santos et al. 2006). The findings showed that the
encapsulation of UA into PLGA-nanocapsules was able to maintain and improve its antitumour
activity and considerably reduce the hepatotoxicity of this drug.
Sanchez et al. (2006) reported the development of severe hepatotoxicity in two young
patients, a husband and a wife, (both 38 years of age) who were bodybuilders taking the multiingredient health supplement UCP-1 (BDC Nutrition, USA) for 3 months. UCP-1 contains UA
(150 mg), L -carnitine (525 mg) and calcium pyruvate (1050 mg) per capsule. The wife developed
fulminant hepatic failure requiring liver transplantation. The husband experienced submassive
necrosis but did not require liver transplantation. They were taking a multi-ingredient
preparation containing UA, L -carnitine and calcium pyruvate (Sanchez et al. 2006).
Foti et al. (2008) related that UA is a weak inhibitor of cytochrome CYP2D6 and a potent
inhibitor of cytochrome CYP2C19. Based on the potent inhibition of CYP2C enzymes, UA has
significant potential to interact with other medications (Foti et al. 2008).
Krishna et al. (2011) described a case of a young healthy woman that presented fulminant
hepatic failure requiring emergent liver transplantation caused by a dietary supplement and fat
burner containing UA, green tea and guggul tree extracts (Yellapu et al. 2011).
Sonko et al. (2011) evaluated UA-associated hepatotoxicity in vitro using isolated rat
hepatocytes. They measured cell viability and ATP content to evaluate UA-induced cytotoxicity
and applied 13C isotopomer distribution measuring techniques to gain a better understanding of
the glucose metabolism during cytotoxicity. The observed increase in oxidative phosphorylation
at 1 and 5 M UA may be an attempt by the cells to compensate for diminished mitochondrial
function as evidenced by altered carbon dioxide, lactate, glucose and glutamate isotopomer

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A. Araujo et al.

labeling patterns. The isotopomer distribution results show that oxidative phosphorylation and
gluconeogenesis were significantly reduced at the 10 M UA concentration (Sonko et al. 2011).
Sahu et al. (2012) showed that UA is cytotoxic to humam hepatoblastoma HepG2 cells,
and in 2012 evaluated the possible AU interaction, if any, with other food-related products. They
evaluated its interactive toxicity with LPS, a potential contaminant of food, at low non-toxic
concentrations of both UA and LPS, to see whether their simultaneous mixed exposures could
have any effect on the HepG2 cells. They used both the traditional biochemical assays and the
pathway-focused gene expression profiles as the endpoints of toxicity. The results of the
biochemical analysis in this study show that at the lower levels, UA and LPS alone had no
significant effect on HepG2 cells compared with the controls. However, their binary mixture
significantly decreased cell viability and significantly increased cellular oxidative stress and
mitochondrial injury and mitochondrial membrane damage leading to cell death, compared with
the controls suggesting interactions of UA with LPS. The results of our gene expression profiles
of the HepG2 cells exposed to UA, LPS and their binary mixture (UA LPS) show altered
expression of stress and toxicity pathway-focused genes (Sahu et al. 2012).
In animal studies, UA induced extensive liver necrosis. These investigators identified that the
hepatotoxic effect of UA has been shown to uncouple oxidative phosphorylation with resultant
loss of mitochondrial respiratory control and inhibition of ATP synthesis. The effect is
analogous to a mechanism similar to carbon tetrachloride, which involves free radical generation
with resultant cell membrane and mitochondrial injury, lipid peroxidation, disturbed calcium
homeostasis and cell death (Sonko. et al. 2011).
Based on the temporal relationship between the use of the dietary supplements and the onset
of liver failure, and also on literature supporting reports of hepatotoxicity associated with dietary
supplements and exclusion of other causes, it is fair to assume that the patient developed
fulminant hepatic failure due to dietary supplements (Krishna et al. 2011)
The mixture of UA with the exposure to supplements increased the cellular oxidative stress
and mitochondrial membrane damage leading to cell death (Durazo et al. 2004)
Health care professionals should continue vigilant in inquiring about the use of health
supplements and alternative medicines by patients who have liver injury with no obvious cause.
The use of UA must be considered as a potential risk factor for fulminant hepatic failure
(Krishna et al. 2011)
9. Contact allergy
According to Thune and Solberg (1980), UA is chemically related to furocoumarin and exhibits
allergic cross-reactivity, but does not typically cause photosensitivity. Mitchell and Shibata
(1969), found that only the D -isomer is allergenic. However, Salo et al. (1981), showed that both
the D - and L -isomers are allergenic and that individuals may react to one or both enantiomers
(Salo et al. 1981). Sheu et al. (2006), reported that four patients had positive patch test reactions
to lichen acid mix and D -UA. Out of the three patients who were patch-tested for the botanical
deodorant, all had positive reactions (Thune and Solberg 1980).
10. Cardiovascular effects
Mendonc a (2009) carried out a study in guinea pig left atrium which in isolated organ bath.
In this study, it was demonstrated that the addition of increasing and cumulative concentrations
of UA doses (1 800 mM) produces a negative inotropic effect for concentrations greater than
100 mM. This effect was accompanied by an intense diastolic contracture for concentrations
above 500 mM. Both these effects were irreversible. The UA addition in concentrations above
100 mM also produced changes in the speed of the cardiac muscle contraction, increasing the

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Natural Product Research

time for both systole and diastole (lusitropic effects). However, this effect was reversible. The
lusitropic and inotropic responses observed in this study suggest that the UA produces its effects
by blocking the L-type calcium channels. In spite of the above-mentioned effects, no alteration
was identified in the integrity of the sarcolemmal membrane by monitoring markers of cardiac
damage as creatine kinase (MB isoform), when the atria were incubated for 1 h with UA
(30 300 mM) (Mendonc a 2009). Corroborating these results, Fernandes (2013) demonstrated
that human endothelial cells (Eary926) incubated 24 h with different concentrations of UA
(1 nM 100 mM) did not show any change in cell viability (Fernandes 2013).
Fernandes (2013) demonstrated that cardiac cells exposed in vitro to UA (0.1 10 nM) had no
effect on the cellular contractility, as well as on the intracellular calcium transient which is
responsible for triggering the mechanical phenomenon of contraction. However, in these same
cells, 10 nM of UA was able to induce a significant reduction of nitric oxide (NO), hydrogen
peroxide (H2O2) and superoxide anion radical (O2
2 ) productions (Fernandes 2013).
The oral treatment for 7 days with UA (50 mg/kg/day) increased significantly the activity and
protein expression of endogenous antioxidant enzymes such as glutathione peroxidase and
superoxide dismutase. However, this effect was observed only on the Mn-SOD isoform
(Mendonc a 2009).
UA was also able to reduce the activity of catalase, without any effect on its expression.
Enhancing the antioxidant effects, UA induced a decrease in the expression of the pro-oxidant
enzyme NADPH oxidase. In this study, it was proposed that the decrease in NO production is
due to the reduction in the expression of the enzyme endothelial nitric oxide synthase (eNOS)
(Mendonc a 2009).
Behera et al. (2012) demonstrated that the UA in vitro presented ACE inhibition, fibrinolytic
potential and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibition. From these
results, the authors suggested the use of UA as an important therapeutic tool in the prevention
and treatment of cardiovascular diseases (Behera et al. 2012).
11. Antitumor Activity
Mayer et al. (2005) showed that UA has anti-proliferative activity against the wild-type p53
(MCF7) as well as the non-functional p53 (MDA-MB-231) breast cancer cell lines, and the lung
cancer cell line H1299, which is null for p53. The properties of UA as a non-genotoxic
anticancer agent that works in a p53-independent manner support the need for further studies in
order to establish a safe therapeutic range in vivo. Thus, UA has potential as either a systemic
therapy or as a topical agent for the treatment of tumors (Mayer et al. 2005).
The antitumour activity of UA encapsulated into nanocapsules prepared with lactic coglycolic acid polymer was tested by Santos et al. (2006). The antitumour activity was confirmed
on an ascitic tumour (Sarcoma-180) implanted in Swiss mice and estimated by means of the
tumour inhibition. The results of antitumour activity confirmed that the encapsulation of UA into
PLGA-nanocapsules produced a 26.4% increase in tumour inhibition as compared with the
standard free UA treatment. Vacuolisation of hepatocytes and a mild lymphocytic infiltration in
portal spaces were observed in animals treated with free UA. However, this hepatotoxicity was
substantially reduced when animals were treated with UA-loaded nanocapsules. Furthermore, no
histological changes were noticed in the kidneys or spleen of animals treated either with UA or
UA-loaded nanocapsules. These authors suggest that nanoencapsulation may be a way of
enabling UA to be used in chemotherapy (da Silva Santos et al. 2006).
In another study, the breast cancer cell line MCF7 and the lung cancer cell line H1299 were
treated with UA 29 mM for 24 h and two positive controls: vincristine (which prevents the
formation of microtubules) or taxol (which stabilises microtubules) in order to investigate
whether UA affects the formation and/or stabilisation of microtubules by visualising

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10

A. Araujo et al.

microtubules and determining mitotic indices after treatment. The treatment of MCF7 and
H1299 cells with UA did not result in any morphological changes in microtubules or increase in
the mitotic index. These results suggest that the antineoplastic activity of UA is not related to
alterations in the formation and/or stabilisation of microtubules (ONeill et al. 2010).
Backorova et al. (2011) reported on the sensitivity of up to nine human cancer cell lines
(A2780, HeLa, MCF-7, SK-BR-3, HT-29, HCT-116 p53 /, HCT-116 p532/2, HL-60 and
Jurkat) to the anti-proliferative/cytotoxic effects of four typical secondary metabolites of lichens
(parietin, atranorin, UA and gyrophoric acid). In this work, variations in the dynamics of tumour
cell line populations were evaluated through the MTT, clonogenic and viability assays, cell
proliferation and detachment, cell cycle transition and apoptotic nuclear morphology, thereby
confirming their concentration- and time-dependent cytotoxicity. However, in comparison with
parietin and gyrophoric acid, the suppression of viability and cell proliferation by UA or atranorin
was found to be more efficient at equitoxic doses and correlated more strongly with an increased
number of floating cells or a higher apoptotic index. Moreover, the analysis of cell cycle
distribution also revealed an accumulation of cells in the S-phase. This study confirmed a
differential sensitivity of cancer cell lines to lichen secondary metabolites (Backorova et al. 2012).
Backorova et al. (2012) also investigated the mechanisms of cytotoxicity of four lichen
secondary metabolites (parietin, atranorin, UA and gyrophoric acid) on A2780 and HT-29
cancer cell lines. The work shows that UA and atranorin were more effective anti-cancer
compounds when compared with parietin and gyrophoric acid. UA and atranorin were capable of
inducing a massive loss in the mitochondrial membrane potential, along with caspase-3
activation (only in HT-29 cells) and phosphatidylserine externalisation in both cell lines tested.
Induction of both ROS and especially RNS may be responsible, at least in part, for the cytotoxic
effects of the compounds tested. Based on the detection of protein expression (PARP, p53, Bcl2/Bcl-xL, Bax, p38, pp38), the authors found that UA and atranorin are activators of
programmed cell death in A2780 and HT-29, probably through the mitochondrial pathway
(Backorova et al. 2011, 2012).
Brisdelli et al. (2013) investigated the effects of six lichen metabolites (diffractaic acid,
lobaric acid, UA, vicanicin, variolaric acid and protolichesterinic acid) on the proliferation,
viability and ROS level towards three human cancer cell lines, MCF-7 (breast adenocarcinoma),
HeLa (cervix adenocarcinoma) and HCT-116 (colon carcinoma). Cells were treated with
different concentrations (2.5 100 mM) of these compounds for 48 h. In this comparative study,
the lichen metabolites showed various cytotoxic effects in a concentration-dependent manner,
and UA was the most potent cytotoxic agent (Brisdelli et al. 2013).
Song et al. (2012) demonstrated that UA strongly inhibited in vivo angiogenesis in a chick
embryo chorioallantoic membrane assay. In a mouse xenograft tumour model, UA suppressed
Bcap-37 breast tumour growth and angiogenesis without affecting mice body weight. In an
in vitro assay, UA not only significantly inhibited endothelial cell proliferation, migration and
tube formation, but also induced morphological changes and apoptosis in endothelial cells.
In addition, UA inhibited Bcap-37 tumour cell proliferation. Moreover, Western blot analysis of
cell signaling molecules indicated that UA blocked vascular endothelial growth factor receptor
(VEGFR) 2. These results provided the first evidence of the biological function and molecular
mechanism of UA in tumour angiogenesis (Song et al. 2012).
12. Conclusion
In conclusion, with the scientific information available, it is not possible to validate the use and
safety of UA: these are limited to preclinical pharmacological studies. Furthermore, the
toxicological research to support the safety is insufficient. Thus, further studies are needed to
establish the efficacy and safety of UA.

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11

Funding
The authors would like to thank Conselho Nacional de Desenvolvimento Cientfico e Tecnologico/CNPq/
Brazil) and Fundac ao de Amparo a` Pesquisa do Estado de Sergipe/FAPITEC-SE for the financial supports.

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