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Review of the biological properties andtoxicity of usnic acidA. A. S. Araújoa, M. G. D. de Meloa, T. K. Rabelob, P. S. Nunesa, S.L. Santosa, M. R. Serafinic, M. R. V. Santosa, L. J. Quintans-Júniora

& D. P. Gelainc

a Federal University of Sergipe, São Cristóvão, Brazilb Federal University of Rio Grande do Sul, Porto Alegre, Brazilc Federal University of Sergipe, Lagarto, BrazilPublished online: 24 Feb 2015.

To cite this article: A. A. S. Araújo, M. G. D. de Melo, T. K. Rabelo, P. S. Nunes, S. L. Santos, M.R. Serafini, M. R. V. Santos, L. J. Quintans-Júnior & D. P. Gelain (2015): Review of the biologicalproperties and toxicity of usnic acid, Natural Product Research: Formerly Natural Product Letters,DOI: 10.1080/14786419.2015.1007455

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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

aFederal University of Sergipe, Sao Cristovao, Brazil; bFederal University of Rio Grande do Sul, PortoAlegre, Brazil; cFederal University of Sergipe, Lagarto, Brazil

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

EXTRACTION

LICHEN

REVIEW

Anti-inflammatoryory

healing

Antioxidant

Live injury

UV protectionp

Larvicidal

Antiviral

antiprotozoal

Antimicrobial

inflanalgesic

Since its first isolation in 1844, usnic acid [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione] has become the most extensively studied lichenmetabolite and one of the few that are commercially available. Lichens belonging tousnic acid-containing genera have been used as crude drugs throughout the world.There are indications of usnic acid being a potentially interesting candidate for suchactivities as anti-inflammatory, analgesic, healing, antioxidant, antimicrobial,antiprotozoal, antiviral, larvicidal and UV protection. However, some studies reportedthe liver toxicity and contact allergy. Thus, further studies are needed to establish theefficacy 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 [2,6-

diacetyl-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).

q 2015 Taylor & Francis

*Corresponding author. Email: maiserafini@hotmail.com

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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 (þ ) isomers of UA showed moderate

to strong cytotoxicity against a wide variety of murine and human cancer cell lines (Takai et al.

Figure 1. UA structure (Ingolfsdottir 2002).

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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 adenocarci-

noma), SK-BR-3 (breast adenocarcinoma) HCT-116 p53 þ /þ (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 25mM 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 80mg/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 1.95mg/mL and IC80s of 7.8 and 15.6mg/mL (Pires et al. 2012).

The effects of treatments with (þ ) – UA by oral, subcutaneous or intralesional routes during

the course of infection of BALB/c mice infected with Leishmania amazonensiswas 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

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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).

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 non-

cytotoxic 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 (þ ) 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 anti-

viral activity. With minor exceptions, in the pair of enantiomers, (2 )-UA was more active

comparing with (þ )-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 LPS-

induced 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 (NF-

kB). 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 £ 1026M 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 £ 1024M) 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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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, 3-5-

diiodothyronine 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) 500mg/day for 2 weeks (Durazo et al. 2004).

Han et al. (2004) evaluated in primary cultured murine hepatocytes that the UA treatment

(5mM) resulted in 98% necrosis within 16 h (no apoptosis was detected). UA treatment was

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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 UA-

induced hepatotoxicity based on the following findings: (1) pre-treatment with antioxidants

(butylated hydroxytoluene þ 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 (þ )-UA (200mg/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. (þ )-UA at a high dose (1mM) 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 Ca2þ 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 (þ )-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 PLGA-

nanocapsules 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 multi-

ingredient health supplement UCP-1 (BDC Nutrition, USA) for 3 months. UCP-1 contains UA

(150mg), L-carnitine (525mg) and calcium pyruvate (1050mg) 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 5M 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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labeling patterns. The isotopomer distribution results show that oxidative phosphorylation and

gluconeogenesis were significantly reduced at the 10M 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 þ 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 (1–800mM) produces a negative inotropic effect for concentrations greater than

100mM. This effect was accompanied by an intense diastolic contracture for concentrations

above 500mM. Both these effects were irreversible. The UA addition in concentrations above

100mM also produced changes in the speed of the cardiac muscle contraction, increasing the

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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

(30–300mM) (Mendonc�a 2009). Corroborating these results, Fernandes (2013) demonstrated

that human endothelial cells (Eary926) incubated 24 h with different concentrations of UA

(1 nM–100mM) 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 (O22) productions (Fernandes 2013).

The oral treatment for 7 days with UA (50mg/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 co-

glycolic 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 29mM 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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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 (O’Neill 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 þ /þ , 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, Bcl-

2/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 (2.5–100mM) 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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Funding

The authors would like to thank Conselho Nacional de Desenvolvimento Cientıfico 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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