Hepatotoxicity of usnic acid and underlying mechanisms

Abstract

Since usnic acid was first isolated in 1844 as a prominent secondary lichen metabolite, it has been used for various purposes worldwide. Usnic acid has been claimed to possess numerous therapeutic properties, including antimicrobial, anti-inflammatory, antiviral, anti-proliferative, and antipyretic activities. Approximately two decades ago, crude extracts of usnic acid or pure usnic acid were marketed in the United States as dietary supplements for aiding in weight loss as a “fat-burner” and gained popularity in the bodybuilding community; however, hepatotoxicity was documented for some usnic acid containing products. The US Food and Drug Administration (FDA) received numerous reports of liver toxicity associated with the use of dietary supplements containing usnic acid, leading the FDA to issue a warning letter in 2001 on a product, LipoKinetix. The FDA also sent a recommendation letter to the manufacturer of LipoKinetix, resulting in the withdrawal of LipoKinetix from the market. These events triggered investigations into the hepatotoxicity of usnic acid and its mechanisms.

In 2008, we published a review article titled “Usnic Acid and Usnea Barbata Toxicity”.¹ This review is an updated version of our previous review article and incorporates additional data published since 2008. The purpose of this review is to provide a comprehensive summary of the understanding of the liver toxicity associated with usnic acid, with a particular focus on the current understanding of the putative mechanisms of usnic acid-related hepatotoxicity.

1. Introduction

Dietary and herbal supplements enjoy widespread use in the United States. Unlike pharmaceutical drugs, dietary supplements are not required to undergo pre-market approval by the U.S. Food and Drug Administration (FDA) for safety and effectiveness, according to the Dietary Supplement Health and Education Act (DSHEA) enacted in 1994; thus, rigorous safety evaluations may be lacking. The general perception of herbal supplements is that they are safe, owing to their long history of use in traditional medicine and natural sources. However, toxic effects can arise, particularly when herbs are employed in unconventional ways or when they are used in combinations with drugs or other herbs.^2–6

Usnic acid is present in numerous lichen species and usnic acid content is about 1–3% of dry weight of Usnea lichens.^7,8 Lichens are complex organisms based on a symbiotic co-existence between algae or cyanobacteria and fungi. Lichens can be found on various types of surfaces, and they cover approximately 8% of the earth’s surface.⁹ Because of their abundance, the crude extracts from lichens have been applied for medicinal uses in many countries, particularly for certain types of lichens.

Lichens are known to produce secondary metabolites for defense and adaptation to the environment, to protect them against harsh environment such as ultraviolet rays, bacteria, and viral spectrum. These secondary metabolites are believed to give lichens therapeutic properties. Hundreds of these compounds have been identified, and usnic acid [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzofurandione] (C₁₈H₁₆O₇; molecular weight of 344.32), a secondary metabolite particularly abundant in Usnea species, is one of the most studied and widely used. First isolated in 1844, usnic acid has been found in genera such as Alectoria, Cladonia, Evernia, Flavocetraria, Lecanora, Ramalina, Usnea, and Vulpicida.^8,10

Usnic acid, a yellow pigment, is present in two enantiomeric forms: (+) R-usnic acid (CAS # 7562-61-0) and (−) S-usnic acid (CAS # 6159-66-6). These two usnic acid enantiomers have been observed to exhibit different distributions in lichen species. A single enantiomer form dominates in most species, as exemplified by the prevalence of (+)-usnic acid over (−)-usnic acid in Usnea species.¹¹ In certain genera, such as Flavocetraria and Vulpicida, however; both isomers have been detected.^11,12 Currently there is still no consensus on the differences in the biological and pharmaceutical effects between the two usnic acid enantiomers, although the majority of the discoveries so far are based on (+)-usnic acid. While comparison between the two enantiomers is beyond the scope of this review, a comprehensive review on the enantiospecificity of usnic acid by Galanty et al.¹³ can be read to gain a more insight into the topic. In this review, we generally do not differentiate between the forms of (+)-usnic acid and (−)-usnic acid unless it is necessary.

Because of the long history of medicinal use, Usnea species and usnic acid have received considerable research interest. Earlier studies during the first half of the 20^th century mostly focus on the antibiotic properties of usnic acid.⁹ This interest was renewed in the 1980s due to the discovery that usnic acid is effective against certain drug-resistant bacteria.^1,9 Usnic acid has been used commercially in the perfumery and cosmetic industry, as an ingredient in mouthwashes, toothpastes, deodorants, and creams because of its antibacterial properties. In the recent decades, various properties of usnic acid, such as its anti-cancer potentials, have also been explored. Although not supported by scientific evidence, usnic acid has been also advertised and promoted as a dietary supplement ingredient for weight loss because of its ability to increase fat metabolism, and to raise the basal metabolic rate. However, liver injury, linked to the consumption of products containing usnic acid, was reported in the early 2000s.^14–16 The severity of the liver injury ranged from mild liver enzyme elevation to more serious cases of hepatitis and/or acute liver failure that required liver transplantation.¹ The U.S. FDA received more than 20 reports of liver toxicity associated with the use of dietary supplements containing usnic acid, leading the FDA to issue a warning in 2001 on one product, namely, LipoKinetix. The FDA also sent a letter to Syntrax Innovation Inc., the manufacturer of LipoKinetix, requesting the voluntarily withdrawal of LipoKinetix from the market. The documented liver injury caused by usnic acid, along with the purported novel uses of usnic acid, has sparked interest in investigating its mode of action and exploring potential mechanisms underlying its hepatotoxicity.

In this review, we summarize the current understanding of some of the biological activities of usnic acid and provide an overview of the available data on its hepatotoxicity. We also discuss the mechanisms of action responsible for usnic acid’s toxicity. It’s important to note that this literature review does not serve as a risk assessment.

2. Biological activities of usnic acid

A variety of biological properties of usnic acid have been reported and the majority related to its antibiotic roles. Research interest on usnic acid experienced a significant increase in recent years, based on the number of related publications.¹⁷ This is partially due to the advance of testing methods. Biological and pharmacological activities, such as antibacterial and antibiofilm property, antifungal property, antiviral property, antiprotozoal, antiinflammation, and anti-oxidative properties, have been reviewed extensively.^8,18–21 Here, we mainly summarize the potentials of antitumor properties of usnic acid due to the recent increasing interest in this topic.

The potential biological activity of usnic acid against cancer was first reported in 1975.²² Since then, extensive work has been conducted in various cancer cell lines to explore the antitumor properties of usnic acid, and the interest has further increased in recent years. Most in vivo studies investigating the antitumor properties of usnic acid have used mouse xenograft models. Nude mice, often from a Balb/c background, were injected with various type of cancer cells. When a solid tumor formed, the mice were treated intra-lesionally with usnic acid for a designated period, and the growth of the tumor was examined. In certain cases, the researchers injected cancer cells pretreated with usnic acid and then examined the tumor formation.²³ A list of relevant studies is provided in Table 1. In all these studies, the researchers found that the application of usnic acid suppressed tumor growth. One study used different concentrations of usnic acid to treat the tumors, and reported a dose-dependent inhibition of tumor growth.²⁴ Usnic acid also inhibited tumor angiogenesis²⁵ and induced apoptosis in the tumor tissues.^24,26 Other animal models have also been employed to explore the angiogenesis effect of usnic acid. Song et al. reported that usnic acid effectively blocked the formation of new blood vessels in a chick embryo chorioallantoic membrane assay. Usnic acid also exhibited angiogenesis activity in zebrafish embryos.²⁷

Table 1.

Mouse xenograph studies for antitumor property of usnic acid

Mouse strain	Cancer cells used	Usnic acid treatment	Treatment length	Major finding	Reference
C57BL/6 mice	Bcap-37 Human breast adenocarcinoma cell line	60 mg/kg/day	22 days	Inhibition of tumor growth and tumor angiogenesis	25
Athymic nude mice	H520 Human lung squamous carcinoma cell line	50 mg/kg every 2 days	28 days	Inhibition of tumor growth, which could be reversed by co-application of oxidative scavenger N-acetyl-l-cysteine	63
Balb/c nude mice	BGC823 Human gastric adenocarcinoma cell line	100mg/kg every 2 days	11 days	Inhibition of tumor growth, increased Bax positive cancer cells and reduced Bcl-2 positive cancer cells in tumor tissues	26
Balb/c nude mice	A549 Human lung carcinoma epithelial cell line	A549 cancer cells were pre-treated with 10 μM UA before injection	14 days	Inhibition of tumorgenicity	23
Balb/c nude mice	MCF-7 Human breast adenocarcinoma cell line	25, 50 or 100 mg/kg every 2 days	18 days	Reduced tumor growth in a dosage dependent manner	24

Many studies on the anticancer effect of usnic acid have been conducted in vitro and have indicated cytotoxic effects on a diverse range of cancer cell lines (Table 2). The reported IC₅₀/LC₅₀ concentrations of usnic acid in particular cell lines can vary considerably across studies conducted by different research groups. For instance, in the human breast cancer cell line MCF-7, the IC₅₀/LC₅₀ concentration of usnic acid after 72 h ranged from 11.2 μM to 105.4 μM,^28,29 (The IC₅₀ is the concentration of a substance which inhibits biological function by 50%, while the LC₅₀ is the concentration of a substance which causes 50% lethality.) Such variation may stem from differences in testing methods used to determine IC₅₀/LC₅₀, variations in cell density during treatment, or other technical factors. Also, most of the studies either used (+)-usnic acid or did not specify the stereochemistry, with only a limited number exploring the antitumor effects of (−)-usnic acid. In those few studies, (−)-usnic acid sometimes exhibited considerable difference in its antitumor efficacy compared to (+)-usnic acid.^29,30

Table 2.

Cytotoxicity of usnic acid in cancer cell lines

Cancer type	Cell line	Enantiomer of usnic acid	Treatment length	IC50/LC501 (μM)	Test method	Reference
Human cells
Breast cancer	MCF-7	NA2	24 h	34.12±1.25	MTT assay	24
		NA	48 h	13.11 ± 0.01	MTT assay	66
		(+) usnic acid	48 h	71.4	WST-1 assay	33
		(+) usnic acid	48 h	75.7±3.4	MTT assay	67
		(+) usnic acid	72 h	11.2	MTT assay	28
		NA	72 h	18.9	MTT assay	64
		(−) usnic acid	72 h	51.7±7.3	MTT assay	29
		(+) usnic acid	72 h	94.6±7.9	MTT assay	37
		(+) usnic acid	72 h	105.4±16	MTT assay	29
	MCF-7/Topo	NA	72 h	16.4±2.5	MTT assay	27
	MDA-MB-231	NA	24 h	38.41 ± 1.64	MTT assay	24
		NA	48 h	12.84 ± 0.01	MTT assay	66
		(+) usnic acid	72 h	13.1	MTT assay	28
		NA	72 h	22.3	MTT assay	64
	SK-BR-3	NA	24 h	48.07±1.52	MTT assay	24
		(+) usnic acid	72 h	14.4	MTT assay	28
		(+) usnic acid	72 h	199.2±18.8	MTT assay	37
	T-47D	(+) usnic acid	24 h	8.4	Thymidine uptake assay	68
		(−) usnic acid	24 h	11.6	Thymidine uptake assay	61
		(+) usnic acid	24 h	12.2	Thymidine uptake assay
		(+) usnic acid	72 h	15.9	MTT assay	28
	BT-474	NA	48 h	12.65 ± 0.04	MTT assay	66
		(+) usnic acid	72 h	15.1	MTT assay	28
Cervical cancer	HeLa	(+) usnic acid	24 h	>10	WST-1 assay	69
		(+) usnic acid	48 h	>10	Resazurin assay
		NA	48 h	14.9	MTT assay	70
		(+) usnic acid	48 h	23.7±2.5	MTT assay	67
		(+) usnic acid	72 h	178.3±9.7	MTT assay	37
	Hep2C	NA	48 h	21.8	MTT assay	60
	KB-V1/Vbl	NA	72 h	9.6±0.1	MTT assay	27
Colon cancer	HT29	NA	48 h	95.2±0.85 (MTT)	MTT assay	23
		(+) usnic acid	72 h	99.7±8.4	MTT assay	37
	HCT116	(+) usnic acid	48 h	17.7±5.4	MTT assay	67
	HCT116 p53+/+	(+) usnic acid	72 h	157.2±4.0	MTT assay	37
	HCT116 p53−/−	(+) usnic acid	72 h	143.1±11.3	MTT assay
	Caco2	NA	48 h	7.05	MTT assay	60
	DLD-1	NA	72 h	19.3±5.9	MTT assay	27
Gastric cancer	BCG823	(+) usnic acid	24 h	236.55±11.12	Cell Counting Kit-8 assay	26
	SGC7901	(+) usnic acid	24 h	618.82±1.77	Cell Counting Kit-8 assay
	AGS	NA	48 h	15.01±0.52	MTT assay	23
Glioblastoma	U251	(+) usnic acid	72 h	19.5±1.6	MTT assay	29
		(−) usnic acid	72 h	19.7±4.6	MTT assay
	U87MG	NA	48 h	120.7	MTT assay	71
	U-87	NA	72 h	>50	MTT assay	27
Leukemia	HL60	(+) usnic acid	24 h	5.4	WST-1 assay	69
		(+) usnic acid	48 h	1.7	Resazurin assay
		(+) usnic acid	72 h	10.32±1.03	Trypan blue exclusion assay	72
		(+) usnic acid	72 h	14.3±0.1	MTT assay	73
		(+) usnic acid	72 h	48.5±9.1	MTT assay	37
	K562	(+) usnic acid	72 h	10.49±0.60	Trypan blue exclusion assay	72
		(−) usnic acid	72 h	21.8±3.8	MTT assay	29
		(+) usnic acid	72 h	52.8±8.7	MTT assay
	U937	(+) usnic acid	72 h	14.3 ± 0.1	MTT assay	73
	Jurkat	(+) usnic acid	72 h	76.3±8.2	MTT assay	37
Liver cancer	HepG2	(+) usnic acid	24 h	30	Resazurin assay	74
		(−) usnic acid	24 h	160.6±4.38	MTT assay	75
		NA	48 h	15.4	MTT assay	60
		(−) usnic acid	48 h	50.24±1.23	MTT assay	75
Lung cancer	H520	(+) usnic acid	48 h	32.51±0.44	MTT assay	63
	Calu-1	(+) usnic acid	48 h	34.25±0.05	MTT assay
	A549	NA	48 h	65.3±0.65	MTT assay	23
	H1299	NA	72 h	22.3	MTT assay	64
Melanoma	HTB140	(+) usnic acid	24 h	49.3	LDH assay	30
		(−) usnic acid	24 h	76.2	LDH assay
		(+) usnic acid	48 h	42.8	LDH assay
		(−) usnic acid	48 h	59.9	LDH assay
	A375	(+) usnic acid	24 h	43.9	LDH assay
		(−) usnic acid	24 h	86.8	LDH assay
		(+) usnic acid	48 h	34.4	LDH assay
		(−) usnic acid	48 h	64.3	LDH assay
	WM793	(+) usnic acid	24 h	132.7	LDH assay
		(−) usnic acid	24 h	255.4	LDH assay
		(+) usnic acid	48 h	87.3	LDH assay
		(−) usnic acid	48 h	151.3	LDH assay
518A2	NA	72 h	5.4±0.3	MTT assay	27
Mesothelioma	MM98 cells	(+) usnic acid	24 h	23	Crystal violet assay	76
		(+) usnic acid	24 h	64	Neutral red uptake assay
Neuroblastoma	NS20Y	(−) usnic acid	24 h	217.31±3.51	MTT assay	75
		(−) usnic acid	48 h	52.18±1.71	MTT assay
Pancreatic cancer	Capan-2	(+) usnic acid	24 h	15.4	Thymidine uptake assay	61
		(−) usnic acid	24 h	14.5	Thymidine uptake assay
	Panc-1	(+) usnic acid	24 h	12.5	Thymidine uptake assay	68
		NA	72 h	10.8±1.2	MTT assay	27
Prostate cancer	PC3	(+) usnic acid	24 h	23.8	Thymidine uptake assay	68
	CWR22Rv-1	NA	48 h	24.1±0.63	MTT assay	23
	LNCaP	(+) usnic acid	48 h	77.5	WST-1 assay	33
	DU145	(−) usnic acid	72 h	45.9±7.0	MTT assay	29
		(+) usnic acid	72 h	57.4±2.1	MTT assay
Rhabdomyosarcoma	RD	NA	48 h	22.9	MTT assay	60
Ovarian cancer	A2780	(+) usnic acid	72 h	75.9±2.0	MTT assay	37
Vulvar cancer	A431	(+) usnic acid	24 h	39	Crystal violet assay	76
		(+) usnic acid	24 h	72	Neutral red uptake assay
Murine cells
Mouse fibrosarcoma	Wehi	NA	48 h	15.8	MTT assay	60
Murine lymphocytic leukemia	L1210	(+) usnic acid	48 h	26.4±8.5	MTT assay	29
		(−) usnic acid	48 h	17.4±1.4	MTT assay
Murine Lewis lung carcinoma	3LL	(+) usnic acid	48 h	23±6.6	MTT assay
		(−) usnic acid	48 h	35.1±10.7	MTT assay

¹Some researchers reported the IC50/LC50 in μg/ml or mg/L in their paper. We converted them into μM here for easy comparison.

²NA: The authors did not specify the enantiomer of usnic acid used in the study.

Usnic acid exhibits toxicity towards both cancer cells and normal cells. Some studies showed a comparable toxicity with normal cells and cancer cells. For instance, in normal human L02 liver cell lines, the reported IC₅₀ was 24.4 μM,³¹ compared to hepatic hepatoma-derived HepG2 cells that showed an IC₅₀ of 30 μM.³² However, numerous studies suggest that cancer cells tend to be more susceptible to the cytotoxic effects of usnic acid compared to normal cells. Eryilmaz et al. treated the human breast cancer cell line MCF-7 and human prostate cancer cell line LNCaP with usnic acid and reported IC₅₀ values of 71.4 μM and 77.5 μM, respectively. In contrast, the IC₅₀ values for human breast normal cell line MCF-10A and human prostate normal cell line RWPE-1 surpassed 150 μM, the highest concentration tested by the authors.³³ Similar differences in responses between cancer and normal cells have been reported by several research groups. Kumar et al. found that usnic acid caused severe cytotoxicity in human gastric carcinoma AGS and SNU-1 at concentrations up to 50 μM for 48 h, while no significant toxic effect was observed in normal human embryonic kidney HEK293 cells and normal human keratinocyte HaCaT cells.³⁴ Galanty et al. simultaneously investigated the cancer cell lines melanoma HTB140, prostate DU145 and PC3 along with normal human skin fibroblasts and prostate epithelial PNT2 cells, and found significant cytotoxicity of usnic acid in HTB140 and DU145 cancer cells, with no observable effect on the normal cells.³⁵ Additionally, usnic acid induced cytotoxicity in human hepatocellular carcinoma HepG2 cells and SNU-449 cells but not in normal human endothelial HUVEC cells.³⁶ The variation in sensitivity among the cells is unclear; conducting a comprehensive analysis of these cell lines, including an examination of their genetic profiles, would be intriguing to gain a deeper understanding of the varying sensitivity and molecular targets associated with usnic acid.

In addition to assessing the cytotoxicity of usnic acid in cancer cell lines, researchers have explored its effect on the clonogenesis and migration of cancer cells. Sub-lethal concentrations of usnic acid have demonstrated an inhibitory effect in the soft agar colony formation assay, using lung cancer A549 cells and gastric cancer AGS cells.²³ Numerous research groups have also reported that usnic acid efficiently inhibits the clonogenic ability of various tumor cells, including breast cancer MCF7 cells, cervical cancer HeLa cells, and lung cancer A549 cells.^23,37,38 The antimigratory effect of usnic acid has also been tested using the wound healing scratch assay. Usnic acid treatment resulted in a dose-dependent inhibition of cell migration.^{23,28,39–41} Other tumor invasion assays have confirmed the effect of usnic acid in reducing the mobility and invasion of cancer cells.^23,28 Treatment with usnic acid induced cytoskeletal rearrangement in cancer cells, suggesting that this may be one of the underlying mechanisms for the inhibitory effect of usnic acid on the mobility and invasion of tumor cells.³⁵

3. Toxicity of usnic acid

In the United States, it is estimated that approximately 2000 individuals experience acute liver failure each year, and drug-induced liver injury (DILI) is a significant contributor to this condition. Approximately half of the DILI cases are linked to acetaminophen.⁴² The occurrence of acute liver failure resulting from DILI has raised concerns among both healthcare providers and patients.

In a prospective study conducted in the United States between 1998 and 2007, approximately 11% (133 cases) of the 1198 acute liver failure patients were associated with DILI. Among the DILI cases, 12 were specifically linked to herbal products, including two instances involving the use of usnic acid.⁴³ Another prospective study, covering the period from 2003 to 2015 and involving 2048 adult liver transplant recipients registered in the Scientific Registry of Transplant Recipients, identified 625 cases of DILI. Approximately half of these cases (300) were attributed to acetaminophen, making it the most prevalent cause. Herbal use ranked as the fourth most common cause, contributing to 21 cases of drug-induced liver failure requiring a liver transplant.⁴⁴

3.1. Case reports of hepatotoxicity linked to the use of s usnic acid containing products

With obesity emerging as a major global public health concern, there has been considerable interest in discovering natural or synthetic compounds to aid in weight management. In the early 1930s, an industrial chemical known as 2,4-dinitrophenol, an artificial mitochondrial respiratory chain uncoupler, was formulated into an anti-obesity drug. It gained popularity when it was observed that munition workers exposed to 2,4-dinitrophenol experienced weight loss.⁴⁵ Tainer et al. reported that 2,4-dinitrophenol increased the metabolic rate by approximately 50%, leading to the burning of carbohydrates and fats and subsequent reduction in body weight.⁴⁶ However, the enthusiasm did not last long, due to severe side effects, including cases of death associated with the use of this chemical. Consequently, the U.S. FDA declared 2,4-dinitrophenol too toxic for use in 1938 under the Food, Drug, and Cosmetic Act.^45,47

Despite the known toxicity of 2,4-dinitrophenol, compounds with similar mitochondrial uncoupling properties have been explored, tested, or even directly applied for weight loss purposes, with usnic acid being among them. Although lacking support from clinical evidence, usnic acid was promoted and marketed as a natural dietary supplement that could facilitate fat burning and weight loss. About two decades ago, the use of usnic acid-containing dietary supplements and pure usnic acid resurfaced primarily in the body-building community. Its salt form, sodium usneate, was one of the active ingredients in the product LipoKinetix, that was advertised as a bodybuilding and weight loss supplement. Usnic acid was also promoted for use as a brewed tea or in other forms to stimulate metabolism naturally.

Since 2000, documented cases of mild to severe liver injuries associated with the use of usnic acid have been reported. Many of these cases were linked to the consumption of LipoKinetix. LipoKinetix contained 100 mg usnic acid in each of its capsules, along with caffeine, norephedrine, 3,5-diiodothyronine, and yohimbine. The manufacturer (Syntrax) recommended a dosage of 1–2 capsules, three times per day. In 2002, seven cases of liver injuries related to the use of LipoKinetix were reported.¹⁶ In each instance, the patients were previously healthy, followed the manufacturer’s instruction for LipoKinetix use, and were not taking any other prescriptions or medications at the time. Acute hepatotoxicity developed within three months of taking LipoKinetix for 10 days to 84 days. The symptoms included abdominal pain and fatigue, and there were significantly elevated levels of alanine aminotransferase, aspartate aminotransferase, and bilirubin. Jaundice was also observed in five out of the seven cases.¹⁶ Following a FDA warning letter titled “FDA Warns Consumers Not to Use the Dietary Supplement LipoKinetix”, and a letter to the manufacturer titled “Letter to Distributor on Hazardous Dietary Supplement LipoKinetix”, LipoKinetix was voluntarily removed from the market.

In the subsequent years, a dozen additional cases of liver injury were reported related to usnic acid.^15,48–50 Sanchez et al. documented two cases where previously healthy individuals experienced severe liver failure after taking a recommended dosage of UCP-1 (BDC Nutrition) as a body building aid,¹⁴ with one case resulting in a liver transplant. UCP-1 capsules contained 150 mg of usnic acid, 525 mg of L-carnitine, and 1050 mg of calcium pyruvate, and the recommended dose was three capsules three times daily. Instances of liver failure directly attributed to the use of pure usnic acid were documented. A healthy woman developed liver failure that even required a transplant after taking usnic acid (Industrial Strength AAA Services) for weight loss.⁴⁹ The manufacturer’s recommended dose of pure usnic acid was 500 mg per day.

By 2003, the FDA received more than 20 adverse event reports linked to the consumption of usnic acid containing weight-loss supplements, including LipoKinetix, UCP-1, and pure usnic acid. A review of the literature by Guo et al. in 2008 summarized twelve cases associated with hepatotoxicity.¹ Among them, two cases were linked to the use of UCP-1, one case to pure usnic acid, and nine cases to LipoKinetix. The affected individuals comprised eight females and four males, with a median age of 31 years. Two patients required liver transplantation, while the others recovered.

A decade after the FDA’s warning on usnic acid containing supplements, Yellapu et al. presented a case report of a 28-year-old female professional bodybuilder who suffered severe liver damage after consuming a dietary supplement (Somalyz and Lipolyz, Species Nutrition) for bodybuilding. The patient consumed two “fat burners”, Somalyz and Lipolyz, both of which contained usnic acid. Following the manufacturer’s instructions, she took one to two capsules of Somalyz at bedtime and one capsule of Lipolyz three times a day for a month before the onset of acute hepatitis. Histopathological examination revealed massive hepatic necrosis, and she ultimately underwent liver transplantation.⁵¹ Somalyz contained 4 mg of usnic acid, and Lipolyz contained 12 mg of usnic acid per capsule. This incident highlights that consumers may still easily access usnic acid-containing supplements and may not be fully aware of the associated risks.

3.2. General toxicity and liver toxicity studies in animals

In vivo studies by the National Toxicology Program (NTP) evaluated the toxicity of usnic acid in both male and female F344/N rats and B6C3F1 mice. Rats were administered with usnic acid in the diet at doses of 0, 30, 60, 120, 360, or 720 ppm, while mice were fed doses of 0, 15, 30, 60, 180, or 360 ppm for a duration of 3 months. These exposure levels corresponded to doses of 5, 10, 30, 100, and 200 mg/kg body weight per day (mg/kg/day) for both rats and mice.

In male rats, oral administration of usnic acid at doses of 360 and 720 ppm (100 and 200 mg/kg/day) for 3 months resulted in hepatotoxicity, characterized by severe hepatocellular degeneration and hepatic inflammation. Usnic acid appeared to be relatively non-toxic to female rats and in both sexes of mice, although clinical chemistry results indicated that exposure to 720 ppm (200 mg/kg/day) of usnic acid in female rats led to a moderate increase in alanine aminotransferase (ALT) activity. Additionally, male mice administered with 360 ppm (100 mg/kg/day) of usnic acid showed moderate increases in ALT and alkaline phosphatase levels.¹⁹

The NTP also evaluated the toxicity of ground Usnea Lichens containing usnic acid in both male and female F344/N rats and B6C3F1 mice.⁵² Usnea lichens preparation used in this study contained 2.78% usnic acid by weight. Rats were exposed to Usnea Lichens at 0, 30, 60, 120, 360, or 720 ppm in feed, while B6C3F1 mice were exposed to 0, 15, 30, 60, 180, or 360 ppm in feed for a duration of 3 months.

Exposing rats orally to Usnea lichens containing usnic acid for 3 months led to severe toxicity and morbidity at exposure levels reaching 720 ppm. Notably, significant hepatotoxicity was observed in male rats at exposure levels of 120, 360, and 720 ppm, while female rats exhibited hepatotoxicity at 720 ppm as indicated by an increased incidence of hepatocellular degeneration. An increase in ALT and/or alkaline phosphatase activities was also observed in rats.

In B6C3F1 mice, three months of dietary exposure to Usnea lichens containing usnic acid resulted in hepatotoxicity at 360 ppm for both sexes as indicated by an increased incidence of hepatocellular degeneration. The authors stated that it is not possible to attribute all the observed toxicity to usnic acid, since other potentially toxic lichen secondary metabolites were also present in the Usnea lichens preparation used in these studies.

4. Mechanisms of action

Earlier studies investigated the effects of usnic acid on mitochondrial function, particularly on the uncoupling of oxidative phosphorylation. Johnson et al. demonstrated that 1 μM usnic acid induced an increase in oxygen consumption in the presence of various substrates using rat kidney and liver homogenates.⁵³ However, at higher concentrations of usnic acid (8–30 μM), a reduction in phosphate uptake occurred without a concurrent decrease in oxygen consumption. This observation implies the uncoupling of oxidative phosphorylation. Another study demonstrated that usnic acid reduced the phosphate/oxygen ratio without inhibiting oxygen consumption.⁵⁴ The augmentation of oxygen consumption by usnic acid was evident in the presence of the ATP synthase inhibitor oligomycin, supporting that usnic acid actively uncouples oxidative phosphorylation. Pramyothin et al. evaluated respiration stimulation in isolated rat liver mitochondria, employing different substrates, such as glutamate plus malate or succinate.⁵⁵ They found that usnic acid induced a considerable increase in oxygen consumption, reaching maximal stimulation of respiration (3–7 fold, depending on the substrate) in the absence of ADP. In addition to its uncoupling effect, a study by Han et al. demonstrated that usnic acid also acts as a mitochondria inhibitor.⁵⁶ When bovine serum albumin (BSA) was present in the buffer, an uncoupling effect of usnic acid was observed in mitochondria isolated from rat liver; however, in the absence of BSA, usnic acid acted both as an uncoupler and an inhibitor of mitochondria in primary mouse hepatocytes and rat liver mitochondria. The inhibitory effect of usnic acid on mitochondria led to oxidative stress, resulting in an increase in the generation of hydrogen peroxide. Usnic acids action as an inhibitor of mitochondrial oxidative phosphorylation was future supported by a study using freshly isolated rat hepatocytes.⁵⁷ These findings suggest that the adverse hepatic effects of usnic acid on mitochondrial function may extend beyond the uncoupling of oxidative phosphorylation.

Extensive research has been directed toward exploring the therapeutic potential of usnic acid, with a particular emphasis on its antitumor properties, as outlined in Section 2 “Biological activities of usnic acid”. These studies have also provided insights into the underlying cellular mechanisms and have indicated that the mechanisms and cellular events implicated in the hepatotoxicity of usnic acid may overlap with those identified in its antitumor effects.

One of the most well-studied mechanisms of usnic acid effect is apoptosis. Multiple research groups have demonstrated that exposure to usnic acid induces apoptosis in a concentration-dependent manner in various cell lines (Table 3). Elevated levels of the cleaved PARP and caspase 3, recognized as hallmarks of apoptosis, were detected in these cells, accompanied by increased caspase 3 activity.^{25,26,35,58,59} Usnic acid also activates the intrinsic apoptotic pathway, leading to depolarization of mitochondrial membrane, an increase in the expression of the pro-apoptotic factor Bax, and a reduction in the expression of the anti-apoptotic factor Bcl-2.^23,26,34,60

Table 3.

Cellular mechanism of action of usnic acid

Mechanism	Human cell model	Treatment conditions	Major findings	Reference
Apoptosis	Leukemia cells L1210	17–35 μM, 24 h and 48 h	Induction of apoptosis and increased caspase-3 activity in a dosage dependent manner.	29
	Breast cancer T-47D cells Pancreatic cancer Capan-2 cells	8.7–29.0 μM, 24 h	Depolarization of mitochondrial membrane	61
	Lung cancer cells Calu-1, Ovarian carcinoma A2780, Leukemia cells HL60, Colon cancer cells HCT-116 p53+/+ and HCT-116 p53−/−	50 and 100 μM, 48 h and 72 h	Induction of apoptosis	37
	Liver cancer HepG2 cells	5–100 μM, 24 h	Mitochondrial membrane depolarization	74
	Normal human epithelial HUVEC cells	20 μM, 24 h	Induction of apoptosis, decreased Bcl-xl and survivin protein expression, increased the expression of PARP and cleaved caspase 3	25
	Lung cancer cells A549	25–100 μM, 24 h and 48 h	Induction of apoptosis, dose-dependent increase in the level of cleaved-PARP, depolarization of mitochondrial membrane	58
	Liver cancer HepG2 cells	1.56–50 μM, 24 h and 48 h	Increased caspase 3/7 activity and increased subdiploid nucleus formation	59
	Colon cancer cell HT29, Gastric cancer cell AGS Lung cancer cell A549, Prostate cancer cell CWR22RV-1	25–100 μM, 24 h or 48 h	Induction of apoptosis in a dose-dependent manner, increased levels of cleaved PARP and cleaved caspase-3, increase Bax, but decreased Bcl-xL.	23
	Breast cancer MCF-7 cells	12.5–50 μM, 24 h	Depolarization of mitochondrial membrane potential, induced the activation of intrinsic apoptotic pathway, increased caspase 3 activity	24
	Melanoma HTB140 cells, prostate cancer PC-3 cells	29 μM, 24 h	Induction of apoptosis, increased expression of PARP, increased the levels of cleaved PARP and caspase 3	35
	Cervical cancer Hep2C cells, rhabdomyosarcoma cell line RD	Respective IC50 concentrations for 48 h	Increase in the mRNA level of Bax, and decrease in mRNA levels of Bcl-2	60
	Gastric cancer BGC823 cells	100– 400 μM for 24 h	Induction of apoptosis, increased the protein levels of Bax, cleaved caspase-3, cleaved PARP, decreased Bcl-2	26
	Gastric cancer SGC7901 cells	300–1200 μM, 24 h
	Liver cancer HepG2 cells, liver cancer SNU-449 cells, normal human epithelial HUVEC cells	6.25–100 μM, 48 h	Induction of apoptosis in a dose-dependent manner	36
	Gastric cancer AGS cells, gastric cancer SNU-1 cells	10–50 μM, up to 48 h	Induction of apoptosis, increased the levels of cleaved PARP in a dose-dependent manner, decreased expression of Bcl-2, increased expression of Bax, depolarization of mitochondrial membrane	34
	Lung cancer cells H520	10–40 μM, 12 h, 24 h, 48 h	Induction of apoptosis in a dose-dependent manner	63
	Blood cell culture from healthy donors	72.6–363 μM, 24 h	Induction of apoptosis, increased caspase 3/7 activity in a dose-dependent manner	77
Cell cycle arrest	Breast cancer T-47D cells, pancreatic cancer Capan-2 cells	8.7–29.0 μM, 24 h	Induction of cell cycle arrest at G0/G1	61
	Lung cancer cells Calu-1, ovarian carcinoma A2780, leukemia cells HL60, colon cancer cells HCT-116 p53+/+ and HCT-116 p53−/−	50 and 100 μM, 48 h, 72 h	Induction of cell cycle arrest.	37
	Lung cancer cells A549	25–100 μM, 24 h and 48 h	Induction of cell cycle arrest at G0/G1 stage, dose-dependent decrease in CDK4 and cyclin D1, strong increase of p21/Cip1 protein	58
	Liver cancer HepG2 cells	3.1–50 μM, 2 h, 6 h and 24 h	Induction of S-phase cell cycle arrest, decrease in cell cycle proteins CDK2, CDK4, CDK6, cyclin D1, and cyclin D3 in a dose-dependent manner.	62
	Gastric cancer BGC823 cells	100– 400 μM, 24 h	Induction of cell cycle arrest at G0/G1 and G2/M stages	26
	Gastric cancer SGC7901 cells	300–1200 μM, 24 h
	Liver cancer HepG2 cells, liver cancer SNU-449 cells, normal human epithelial HUVEC cells	6.25–100 μM, 48 h	Induction of cell cycle at G0/G1 and G2/M phase in a concentration-dependent manner.	36
	Breast cancer MCF-7 cells	2.9 and 8.7 μM, 24 h	Induction of cell cycle arrest at G0/G1 phase	38
Autophagy	Liver cancer HepG2 cells	1.56–50 μM, 24 h and 48 h	Induced conversion of LC3B-I to LC3B-II and degradation of P62, inhibition of autophagy that reduced usnic acid-induced apoptosis	59
	Breast cancer cell MCF-7, breast cancer cell MDA-MB-231	15 and 25 uM up to 72 h	Induction of autophagy in a dose-dependent manner, increase in autophagic markers including LC3 and Beclin-1	28
	Gastric cancer BGC823 cells	100– 400 μM, 24 h	Increased the expression of LC3B and decreased p62	26
	Gastric cancer SGC7901 cells	300–1200 μM, 24 h
	Colorectal cancer HCT 116 cells	8 μM, 24 h and 48 h.	Induction of autophagy, transformation of LC3B, decreased phosphorylation of S6K1 and 4E0BP1	40
	Liver cancer HepG2 cells, liver cancer SNU-449 cells, normal human epithelial HUVEC cells	6.25–100 μM, 48 h	Induction of autophagy, increased levels of LC3-II	36
	Blood cell culture from healthy donors	72.6–363 μM, 24 h	Induction of cell cycle arrest at G0/G1 stage.	77
Oxidative stress	Neuroblastoma SH-SY5Y cells	0.006– 58 μM, 24 h	Increased ORS production	78
	Liver cancer HepG2 cells	5–100 μM, 24 h	Induction of oxidative stress	74
	Breast cancer MCF-7 cells	12.5 – 50 μM, 24 h	Induction of ROS generation in a dose-dependent manner, inhibition of ROS by NAC reduced usnic acid-induced apoptosis.	24
	Liver cancer HepG2 cells	3.1–50 μM, 2 h, 6 h and 24 h	ROS accumulation in cells, depletion of glutathione, activation of Nrf2 signaling pathway.	62
	Gastric cancer AGS cells, gastric cancer SNU-1 cells	10–50 μM, up to 48 h	ROS generation in a concentration- and time-dependent manner.	34
	Lung cancer cells H520, lung cancer cells Calu-1	10–40 μM, 12 h, 24 h and 48 h	ROS generation in a dose-dependent manner, damage 0f mitochondrial respiratory complex enzyme I and III, reduced Nrf2 protein levels.	63
	Blood cell culture from healthy donors	72.6–363 μM, 24 h	Induction of autophagy, nuclear shrinkage.	77
DNA damage	Liver cancer HepG2 cells	3.1–50 μM, 2 h, 6 h and 24 h	Induction of DNA damage, increased γ-H2AX in a dosage-dependent manner.	62
	Gastric cancer AGS cells, gastric cancer SNU-1 cells	10–50 μM, up to 48 h	Induction of DNA damage, increased γ-H2AX, DNA-PKcs, Chk-2 and p53.	34
	Blood cell culture from healthy donors	72.6–363 μM, 24 h	Induction of DNA damage in a dose-dependent manner.	77
ER stress	Liver cancer HepG2 cells	3.13–50 μM, 2 h, 6 h and 24 h	Increased expression of ER stress markers, including CHOP, ATF-4, p-eIF2α, and spliced XBP1, impaired ER function, increased free cytosolic Ca2+ concentration.	79
mTOR/pI3K/Akt	Liver cancer cells HepG2	1.56–25 μM 24 h and 48 h	Suppression of mTOR pathway in a concentration dependent manner, reduced overall expression of Akt and mTOR, reduced phosphorylation of PDK1, Akt, mTOR, p70S6K, and 4E-BP1	59
	Breast cancer cell MCF-7 Breast cancer cell MDA-MB-231	15 and 25 μM, up to 72 h	Reduced phosphorylation of Akt, 4EBP1, and p70S6, optimal binding at mTOR kinase pocket.	28
	Lung cancer cells H520 Lung cancer cells Calu-1	10–40 μM, 12h, 24 h and 48 h	Dose-dependent reduction in Akt phosphorylation	63
	Cervical cancer cell HeLa	10–100 μM, 24 h	Suppression in phosphorylation of mTOR and p70S6k in a concentration dependent manner	39
p53	Breast cancer	3.6–29 μM, 24 h	Increased levels of p53 and p21	64
	Cervical cancer Hep2C cells, rhabdomyosarcoma cell line RD	Respective IC50 concentrations for 48 h	Decrease in p53 mRNA levels	60

Another frequently reported finding is the disruption of cell cycle by usnic acid in various cell lines. Several research groups reported that usnic acid caused cell cycle arrest at G0/G1 stage,^38,58,61 although there were also instances where usnic acid treatment led to cell cycle arrest at S phase or G2/M stage.^26,36,62 Corresponding with these findings, usnic acid was observed to modulate the expression levels of crucial cell cycle-regulating proteins, including CDK2, CDK4, CDK6, cyclin D1, and cyclin D3.^58,62

Usnic acid treatment also affects multiple cellular signaling pathways. One such pathway is the MAPK cascades. Song et al. reported that usnic acid inhibited ERK1/2 in MAPK signaling pathway by blocking vascular endothelial growth factor receptor (VEGFR).²⁵ Chen et al. demonstrated that usnic acid treatment increased the phosphorylation of JNK, ERK1/2, and p38 in a concentration-dependent manner, with JNK activation negatively regulating usnic acid-induced apoptosis.⁵⁹ The involvement of JNK in usnic acid-induced apoptosis was also confirmed by Zuo et al.²⁴

Other important pathways that have been shown to be affected by usnic acid treatment are the mTOR/pI3K/Akt and p53, which play critical roles in cell proliferation, DNA replication, and various cellular functions. Usnic acid treatment leads to a reduction in the expression levels and the phosphorylation of the components of the mTOR/pI3K/Akt pathway, such as mTOR, Akt, 4EBP1, and p70S6k.^28,39,59,63 Reduction of mTOR was reported to be associated with autophagy induced by usnic acid.⁵⁹ Findings regarding the effects of usnic acid on the p53 signaling pathway are somewhat conflicting. Mayer et al. reported an upregulation in the expression levels and phosphorylation of p53 upon exposure to usnic acid,⁶⁴ while another research group found a decrease in the mRNA levels of p53 in response to usnic acid treatment.⁶⁰ The discrepancy may arise from variations in the cell lines used and different treatment conditions.

In recent years, advancements in next-generation sequencing and other techniques have enabled scientists to explore additional cellular molecules crucial for cellular functions, such as microRNAs (miRNAs). One research group published a series of work exploring the changes in the levels of miRNA expression levels induced by usnic acid in breast cancer cell lines. Their findings demonstrated that exposure to usnic acid induced alterations in miRNAs associated with apoptosis, MAPK, TGFβ, and hedgehog signaling pathways. Furthermore, they found that the miRNA signature of breast cancer cells exposed to usnic acid shares similarities with those generated from treatment with established anti-breast cancer drugs, such as 5-fluorouracil and sulindac sulfide.⁶⁵

Table 3 provides a summary of various studies revealing additional mechanisms contributing to the cytotoxicity of usnic acid. These mechanisms include autophagy, oxidative stress, DNA damage, and endoplasmic reticulum (ER) stress, each of these mechanisms illuminates unique aspects of usnic acid’s cytotoxic effects.

5. Conclusion and perspectives

Dietary supplement sales have experienced a significant surge following the implementation of the Dietary Supplement Health and Education Act (DSHEA) in 1994, aimed at defining and overseeing these products. Although DSHEA has established a structure for the regulation of dietary supplements, it has faced criticism for certain aspects, particularly the absence of thorough safety evaluations prior to marketing, as compared to the stringent evaluations required for pharmaceutical drugs. As an herbal dietary supplement, usnic acid has been advocated for numerous therapeutic purposes for many years and it has been gaining attention for its antitumor potential. Despite usnic acid’s historical applications and proposed therapeutic potentials, the documented cases of hepatotoxicity highlight the need for more comprehensive examinations of its safety profile. This becomes especially pertinent given the ease of online accessibility for usnic acid and usnic acid-containing products.

In response to these concerns, studies have been performed to address the safety of usnic acid in recent years. Toxicity evaluations have been performed in animals, and mechanistic studies have been initiated to understand the underlying factors contributing to its adverse effects at cellular and molecular levels. However, critical gaps persist, such as the need for long-term and low-dose studies in animals, and investigations into potential susceptibilities in humans. Advanced techniques, such as omics approaches, could be employed to identify key pathways, signaling molecules, and genetic factors involved in the toxic response.

Investigations of the impact of different formulations have been conducted in recent years, especially with the integration of nano technologies to enhance therapeutic benefits and reduce toxicity. In addition, the development of usnic acid analogs or derivatives has sparked interest among researchers. The need for extensive assessment of the toxicity of the nano-formulations and derivatives is growing. Continued research efforts will be crucial in addressing the knowledge gaps and raising consumer awareness regarding the potential risks associated with usnic acid.