Multi-Dimensional Analysis of Key Points in the Biological Activity, Chemical Synthesis and Biotransformation of Urolithin A

Abstract

Urolithin A (Uro-A) is a natural metabolite generated via the gut microbiota-mediated transformation of plant polyphenols. This review systematically summarizes the notable biological activities, preparation methods, metabolic characteristics, and prospects for product development and application of Uro-A. In the Conclusions and Perspectives section, it not only analyzes the technical and economic feasibility related to Uro-A, but also focuses on the critical analysis of its clinical research limitations, safety controversies, and industrial large-scale production challenges. Finally, specific suggestions and prospects are put forward for the future research directions of Uro-A. In summary, this review systematically organizes the current research progress on Uro-A, clearly identifies future development directions, and provides strong support for in-depth research in this field.

1. Introduction

Urolithins were first identified in the liver and kidney tissues of experimental animals in 2003, with their chemical name designated as 3,8-dihydroxy-6H-dibenzo[b, d]pyran-6-one [1]. Subsequently, in 2005, urolithin B (Uro-B) was further identified in urine [2]. Urolithins are synthesized via microbial transformation of plant-derived substrates, such as ellagitannins (ETs) and ellagic acid (EA) from pomegranates and other plants [3]. To date, 13 urolithin derivatives have been identified, which are classified into five categories based on their hydroxylation patterns: pentahydroxylated (urolithin M5/Uro-M5), tetrahydroxylated (urolithin M6R, E, M6, D), trihydroxylated (urolithin CR, M7R, M7, C), dihydroxylated (urolithin AR, A, isourolithin A/Iso-uro-A), and monohydroxylated Uro-B variants [4]. Notably, the dihydroxy metabolite Uro-A exhibits significantly more potent biological activities than other urolithin analogs [5].

2. Biological Activities of Uro-A

Uro-A, a versatile natural metabolite, exhibits broad therapeutic potential in healthcare, functional foods, pharmaceutical research, and beauty and cosmeceuticals [6,7,8,9]. Notably, accumulating evidence from recent studies has substantiated its multimodal bioactivities, including antioxidant, anti-inflammatory, anticancer, and anti-aging effects [10]. The mechanistic underpinnings of these bioactivities are delineated below.

2.1. Antioxidant

Studies demonstrated that Uro-A exhibits potent antioxidant activity, with its efficacy ranking second only to that of proanthocyanidin oligomers, catechins, epicatechins, and 3,4-dihydroxyphenylacetic acid [11,12]. From a molecular docking perspective, among oxidation-related receptors, human topoisomerase II ATPase/AMP-PNP (PDB ID: 1ZXM) showed a binding energy of −7.4 kcal/mol. In comparison, human cytochrome P450 CYP2C9 (PDB ID: 1OG5) displayed stronger binding affinity, with a binding energy of −7.9 kcal/mol. These findings highlight Uro-A’s binding capacity and diverse interaction patterns under oxidative stress conditions [13]. The phenolic hydroxyl groups in Uro-A’s molecular structure donate hydrogen atoms to quench reactive oxygen species (ROS), such as hydroxyl radicals and superoxide anion radicals, thereby stabilizing free radicals and interrupting oxidative chain reactions. This mechanism was experimentally validated in Neuro-2a cells, where a significant reduction in intracellular ROS levels was observed [14]. Furthermore, Haddad et al. [11] conducted oxygen radical absorbance capacity assays on plasma samples from 16 healthy adults (6 males and 10 females, aged 23–44 years) and found that urinary Uro-A concentrations increased markedly within hours following consumption of 90 g raw walnut kernels. This increase correlated with a 32% enhancement in antioxidant capacity and attenuated oxidative stress responses. Notably, a study that established a myocardial ischemia–reperfusion (I/R) model in 8-week-old male C57BL mice [15] showed that the Uro-A treatment group activated the Nrf2 signaling pathway, inhibited oxidative stress and ferroptosis, and thus protected the heart against ischemia–reperfusion injury. This suggests that Uro-A holds promise as a therapeutic agent for acute myocardial infarction. Collectively, these studies indicate that Uro-A exhibits strong antioxidant activity not only in vitro but also exerts significant antioxidant protection in vivo, providing robust experimental evidence for its potential application in preventing and treating oxidative stress-related diseases.

2.2. Anti-Inflammatory

To date, investigations into the anti-inflammatory properties of urolithin derivatives have predominantly focused on colonic environments [16]. Research [17,18] demonstrated that Uro-A potently inhibited the migration of normal human colonic fibroblast cells (CCD18-Co line) at specific concentrations, suggesting its potential to ameliorate colonic inflammation. Furthermore, this compound significantly downregulated key inflammatory mediators, including prostaglandin E₂ (PGE₂), plasminogen activator inhibitor-1, and interleukin-8, while exerting modulatory effects on other critical regulators of cellular migration and adhesion. Notably, beyond suppressing PGE₂ production, Uro-A suppressed the expression of mPGES-1 and COX-2—two pivotal enzymes in prostaglandin biosynthesis under inflammatory conditions. Uro-A affected the expression rhythms of clock genes and tight junction proteins in intestinal epithelial cells (Caco-2 cells, HT-29 cells), and the abnormalities of these genes were associated with inflammation (a well-established scientific fact). In a dextran sulfate sodium (DSS)-induced colitis mouse model (7-week-old female C57BL/6J mice, n = 50), oral administration of Uro-A [19] improved fecal IgA concentrations and the expression of related tight junction proteins and clock genes. It had been confirmed that the Nrf2-SIRT1 signaling pathway was involved in the regulation of intestinal epithelial cell circadian rhythm by Uro-A (a validated mechanistic conclusion). In addition, Uro-A can also affect the central circadian clock (an inherent biological activity of Uro-A). In conclusion, Uro-A was expected to be used for the treatment of inflammatory bowel disease accompanied by sleep disorders. These findings indicate that Uro-A may exert its anti-inflammatory functions by regulating nuclear transcription factors, providing a scientific basis for researchers to further understand the anti-inflammatory mechanisms of Uro-A and develop novel therapies.

2.3. Anticancer

Since 2009, studies have revealed the therapeutic effects of Uro-A against various types of cancer cells—via molecular docking, as well as in vitro and in vivo experiments—including Caco-2/HT-29/SW480 colorectal cancer cells [20], 22Rv1/LNCaP/PC-3 prostate cancer cells [21], MCF-7/MCF-7aro/MDCKII breast cancer cells [22,23,24], U251/U118 MG/U87 MG glioblastoma cells [25], PDAC (pancreatic ductal adenocarcinoma) cells and PKT mouse models [26], T24/UMUC3 bladder cancer cells [27,28], HEC1A/Ishikawa/RPE-1/Hec1A/HESCs endometrial cancer cells [29,30], SAS/RCB1015 oral squamous cell carcinoma cells [31], HepG2/DU145/T2 liver cancer cells [32], A549/H460/H1299 lung cancer cells [33,34], HGC-27/MKN-45/MFC/AGS gastric cancer cells [35,36], and Jurkat/K562 leukemia cells [37]. Uro-A inhibits cancer cell proliferation by inducing cell cycle arrest and blocking pro-proliferative signaling pathways (e.g., PI3K/AKT/mTOR and MAPK/ERK). It induces cancer cell apoptosis by enhancing mitophagy, activating death receptor pathways, and promoting the expression of apoptotic genes. Additionally, since tumor growth and metastasis rely on nutrients supplied by newly formed blood vessels, Uro-A exerts anti-tumor effects by inhibiting key angiogenic factors such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). Furthermore, Uro-A can reshape the tumor microenvironment: it downregulates the NF-κB signaling pathway to reduce the release of pro-inflammatory factors (e.g., interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) and enhances immune surveillance by activating immune cells, thereby suppressing cancer progression. Uro-A also prevents DNA damage and malignant transformation of cancer cells by activating the Nrf2 pathway and selectively inducing reactive oxygen species (ROS) accumulation in cancer cells. These anticancer mechanisms of Uro-A provide a theoretical foundation for the development of anticancer agents. Future studies need to further clarify three key aspects: the dose–response relationship of Uro-A in different cancer types, its synergistic effects with chemotherapy or immunotherapy, and the impact of gut microbiota metabolism on its bioactivity—thereby providing a basis for its clinical translation.

2.4. Anti-Aging

The mechanisms of aging are closely associated with mitochondrial dysfunction and impaired autophagic capacity. The senescence process involved extracellular ROS-mediated signaling that downregulated AMP-activated protein kinase pathways while upregulating PI3K-Akt signaling, thereby leading to hyperactivation of mammalian target of rapamycin cascades [38]. This aberrant signaling suppressed autophagic flux, ultimately driving alterations in cellular lifespan. Uro-A functioned as a potent autophagic inducer, activating selective mitophagy through either the PINK1/Parkin ubiquitin-dependent pathway or BNIP3 receptor-mediated mechanisms—thus attenuating age-related decline in mitochondrial function [39,40]. As a natural compound capable of inducing mitophagy, Uro-A significantly extended the lifespan of Caenorhabditis elegans while increasing their pharyngeal pumping frequency and locomotor ability. In mammalian cells (C2C12 myoblasts and Mode-K intestinal cells) and rodent models (aged C57BL/6J male mice and young Wistar Han male rats), Uro-A similarly exerted effects of promoting mitophagy and enhancing muscle function [41]. Uro-A treatment significantly improved nuclear maturation of Cumulus–Oocyte Complexes (COCs), mitochondrial membrane potential, and embryonic developmental capacity, with particularly more pronounced effects observed in immature heifers. By inducing mitophagy and extending cell lifespan, Uro-A prevented age-related mitochondrial dysfunction, thereby improving COCs quality [42]. To date cellular experiments and animal models have laid a solid foundation for the clinical translation of Uro-A in anti-aging research. Further exploration of Uro-A’s potential anti-aging mechanisms in humans and its potential in clinical application is of great significance.

3. Synthesis of Uro-A

The synthesis of Uro-A is strategically categorized into two principal approaches: chemical synthesis and biosynthesis. Current research has identified eight distinct chemical synthetic methodologies, while biosynthesis relies on microbial fermentation systems for efficient and green production.

3.1. Chemical Synthesis Pathway

In the Uro-A chemical synthesis pathway, researchers widely use a specific chemical reaction to realize the connection of two benzene rings (i.e., Ullmann coupling reaction), which needs to be catalyzed by substances such as copper sulfate under alkaline conditions. The specific synthesis methods are shown in

Table 1. Methods for chemical synthesis of Uro-A.

Synthesis Method	Synthetic Route	Uro-A Yield	Cost- Effectiveness	Environmental Friendliness	Reference
Copper catalytic two-step method		42%	Moderate cost, moderate yield.	Moderate (AlCl3 corrosive, Cu2+ heavy metal)	[43]
Copper catalytic four-step method		—	High cost, low efficiency.	Poor (hazardous reagents, large waste)	[44]
Subcritical water-copper method		15%	High cost, low yield.	Poor (IBr/DCM pollution, low atom economy)	[45]
Copper catalytic three-step method		25%	Moderate cost, low-moderate yield.	Moderate (Br2, AlCl3, Cu2+ impact)	[46]
Palladium-catalyzed three-step method		—	High cost, yield unreported.	Moderate (precious metals, HFIO solvent)	[47]
Copper-catalyzed acid reduction method		—	Moderate cost, yield unreported.	Moderate (50%NaOH/Cu2+ disposal)	[48]
Palladium-catalyzed five-step method		(1) 63% (2) 98% (3) 99%	Very high cost, high step yields → total cost remains high.	Moderate (precious metals, multi-step waste; high step yields partly offset)	[49]
NBS-copper catalyzed method		86%	Low to moderate cost, high yield → high efficiency.	Relatively good (NBS safer than Br2, high atom economy)	[50]

“—” denotes data not provided; (1) 63%, (2) 98%, and (3) 99% represent the Uro-A (Urolithin A) yields of the three synthesis methods, respectively.

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For the copper catalytic two-step method [43], the yield of Uro-A reached 66%, while the overall yield of the two-step reaction was only 42%, indicating notably low efficiency. Aluminum trichloride, used in the first step, was highly hygroscopic and prone to deterioration, and the hydrogen chloride gas generated posed environmental risks. Furthermore, the raw material 2-bromo-5-methoxybenzoic acid was relatively expensive. These limitations rendered this method unsuitable for industrial-scale production. The copper catalytic four-step method [44] optimized the copper catalytic two-step method by using cheaper m-methoxybenzoic acid as a raw material to synthesize 2-bromo-5-methoxybenzoic acid. However, this report failed to provide corresponding yield data or spectral characterization results, precluding accurate assessment of its yield. The initial step in the original Uro-A synthesis involved bromination at the ortho-position of the carboxyl group in m-methoxybenzoic acid to prepare 2-bromo-5-methoxybenzoic acid. Poor regioselectivity of this bromination reaction led to concurrent meta-substitution, generating multiple isomers and by-products. This resulted in low purity of the target product, which necessitated repeated purification; additionally, the environmental harm associated with bromine usage contradicted the principles of green chemistry. The subcritical water-copper method [45] exhibited an overall yield of 15%, characterized by low efficiency, lengthy procedures, and complex reaction systems, rendering it industrially impractical. Copper catalytic three-step method [46] achieved a 25% total yield but suffered from similar drawbacks including low efficiency and environmental pollution from bromine utilization. Palladium-catalyzed three-step method [47] demonstrated merely 36% combined yield for its first two steps, with unreported yield for the final step. Although its electrochemical technology showed environmental advantages, the prohibitive costs precluded industrial application. The copper-catalyzed acid reduction method [48] was described as versatile and concise, with time-saving benefits that made it potentially suitable for industrial scale-up, albeit without reported yield data. For the palladium-catalyzed five-step method [49], three synthetic approaches were reported: When R₁ = Me (methyl) and R₂ = Bn (benzyl), the coupling reagents and conditions were Pd₂dba₃ (tris(dibenzylideneacetone)dipalladium), Na₂CO₃, DME (dimethoxyethane), and 85 °C; R₂ deprotection used 10% Pd/C, H₂, EtOH (ethanol), and room temperature (RT); R₁ deprotection used BBr₃, CH₂Cl₂ (dichloromethane), and −78 °C to RT. The yield of Uro-A was 63%. When R₁ = TBS (tert-butyldimethylsilyl) and R₂ = Me, the coupling reagents and conditions were Pd(PPh₃)₄ (tetrakis(triphenylphosphine)palladium), K₂CO₃, THF (tetrahydrofuran), and reflux; R₁ deprotection used BBr₃, CH₂Cl₂, and −78 °C to RT; R₁ deprotection used TBAF (tetrabutylammonium fluoride), THF, and RT. The yield of Uro-A was 98%. When R₁ = Bn and R₂ = MOM (methoxymethyl), the coupling reagents and conditions were Pd₂dba₃, Na₂CO₃, DME, and 85 °C; R₂ deprotection used HCl, MeOH (methanol), and reflux; R₁ deprotection used 10% Pd/C, H₂, EtOAc (ethyl acetate), and RT. The yield of Uro-A was 99%. Although these three methods had high yield, they involved too many protecting and deprotection reactions and expensive reagents, which were not suitable for industrial production. NBS-copper catalyzed method [50] achieved 86% yield for Uro-A with 74% overall process yield, enabling potential industrialization by eliminating column chromatography. However, the concentrated sulfuric acid used in bromination generated environmentally hazardous waste acid, whose treatment incurs additional costs that may impact process economics. Overall, these methods have been used for the synthesis of Uro-A, and similarities such as raw materials, reagents, catalysts, intermediates, etc. However, they exhibited significant differences in yield, cost, environmental impact, and industrial applicability. Currently, no single method can simultaneously meet the requirements of high yield, low cost, environmental friendliness, and industrial applicability. Future research needs to achieve breakthroughs in improving yield, reducing costs, minimizing environmental impact, and simplifying the process flow to identify the optimal synthetic route suitable for industrial production.

3.2. Biotransformation Pathways

The biosynthesis of Uro-A is carried out using tannins such as ETs and EA from plant components as substrates. It is gradually transformed into Uro-A through a series of enzymatic reactions catalyzed by appropriate strains.

3.2.1. The Biological Metabolism Process of Uro-A

Punicalagin, a type of ET, is converted to Uro-A through a gut microbiota-mediated multi-step enzymatic cascade as shown in Figure 1 [4,51,52,53,54,55].

ETs are first hydrolyzed to produce the intermediate hexahydroxydiphenyl-glucose (HHDP), which forms EA through lactonization. The lactone ring of EA is cleaved to form luteic acid, which undergoes deoxygenation to form pentahydroxy urolithin (Uro-M5). Uro-M5 is dehydroxylated at different positions by dehydroxylases to produce tetrahydroxy urolithins (Uro-D, Uro-M6, Uro-E and Uro-M6R). Uro-D, Uro-M6, Uro-E and Uro-M6R are further dehydroxylated at different positions by dehydroxylases to produce trihydroxy urolithins (Uro-G and Uro-C, Uro-C and Uro-M7, Uro-M7, Uro-M7R and Uro-CR). Uro-G, Uro-C, Uro-M7, Uro-M7R and Uro-CR are dehydroxylated at different positions by dehydroxylases to produce dihydroxy urolithins (Uro-A, Uro-AR and Iso-uro-A). Uro-A and Iso-uro-A are dehydroxylated at different positions by dehydroxylases to produce dihydroxy Uro-B, and Uro-A and Uro-B are interconvertible. Additionally, Uro-A undergoes phase II metabolism (glucuronidation and sulfation) by human cellular enzymes to form phase II conjugates Uro-A glucuronide and Uro-A sulfate, respectively. The last two compounds are phase II conjugates of Uro-A.

3.2.2. Urolithin A-Producing Microbial Strains

Currently, 11 strains have been confirmed to possess the ability to produce urolithins, as shown in

Table 2. Strains capable of converting to produce Uro-A.

Strain Name	Colony Characteristics	Source	Screening Methods	Identification Techniques	Fermentation Conditions	Reference
Bifidobacterium pseudocatenulatum INIA P815	Round, convex, with regular edges, smooth and moist surface, creamy white to pale grayish white, and a diameter of approximately 1.5–2.0 mm.	Intestinal contents	Fecal samples from humans producing Uro-A and B → after dilution, inoculated separately onto RCM, BHI, WC, MRS, and GM17 agar plates → strains + ellagic acid → the strain that produce Uro-A and B	HPLC, HPLC -MS/MS, 16S rRNA.	BHI Anaerobic Basal Broth Medium, 0.1% seed culture inoculum size, pH 7.4, 37 °C, anaerobic culture for 5 d.	[56]
Lactococcus garvieae FUA009	Circular, milky white and translucent, with a moist surface, regular edges, no halo, a central protrusion, a diameter of 0.5–1.0 mm, and easy to pick.	Intestinal contents	Fecal samples from volunteers verified to produce UroA → dilution plating → strains + ellagic acid → the Uro-A- producing strain.	HPLC, HPLC -MS/MS, morphological identification, physiological and biochemical Identification, 16S rRNA.	ABB Anaerobic Basal Broth Medium, 2% seed culture inoculum size, pH 7.0, 37 °C, anaerobic culture for 48 h.	[57,58]
Streptococcus thermophilus FUA329	The colonies are circular, milky white and translucent, with a moist surface, regular edges, no halo, a central protrusion, and a diameter of 0.5–1.0 mm, and easy to pick.	Human breast milk	Breast milk + ellagic acid → Uro-A-producing breast milk → dilution plating → strains + ellagic acid → the Uro-A-producing strain.	HPLC, HPLC -MS/MS, morphological identification, physiological and biochemical Identification, 16S rRNA.	ABB Anaerobic Basal Broth Medium, 2% seed culture inoculum size, pH 7.0, 37 °C, anaerobic culture for 48 h.	[59,60]
Lactobacillus plantarum CCFM1286	Convex, white, smooth, and circular, with a diameter of approximately 2–3 mm.	The Feces of healthy people	Obtain enzyme sequences that may catalyze the conversion of ETs to Uro-A from NCBI → Perform NCBI-Blast alignment of these enzyme sequences against a local genomic database, and screen out strains with similarity ≥ 70% and coverage ≥ 30% → In vitro fermentation of strains with ETs → the Uro-A-producing strain.	HPLC, HPLC -MS/MS, morphological identification, 16S rRNA.	BHI Liquid Medium, 2% seed culture inoculum size, pH 7.0, 37 °C, aerobic culture for 48 h.	[61]
Lactobacillus plantarum CCFM1290	Convex, white, smooth, and circular, with a diameter of approximately 3 mm.	The Feces of healthy people	Obtain enzyme sequences that may catalyze the conversion of ETs to Uro-A from NCBI → Perform BLAST alignment of these enzyme sequences against a local genomic database, and screen out strains with similarity ≥ 70% and coverage ≥ 30% → In vitro fermentation of strains with ETs → the Uro-A-producing strain.	HPLC, HPLC -MS/MS, morphological identification, 16S rRNA.	BHI Liquid Medium, 2% seed culture inoculum size, pH 7.0, 37 °C, aerobic culture for 48 h.	[62]
Lactobacillus plantarum CCFM1291	Convex, white, smooth, and circular, with a diameter of approximately 3 mm.	The Feces of healthy people	Strain Bank of the Food Biotechnology Center, Jiangnan University (source: fecal samples from healthy humans)-edible strains + ellagitannins → the Uro-A-producing strain.	HPLC, HPLC -MS/MS, morphological identification, 16S rRNA.	MRS Liquid Medium, 2–4% seed culture inoculum size, pH 6.2, 37 °C, aerobic culture for 48 h.	[63]
Limosilactobacillus fermentum FUA033	Milky white, opaque, and circular (with a diameter of 1–2 mm), featuring regular and neat edges, a convex center, a bright surface, and a moist texture that is easy to pick.	Intestinal contents	20 healthy adult volunteers (age: 22–27 years old) + 0.7 g raw walnuts/kg body weight → fecal + ellagic acid → dilution plating → colonies + ellagic acid → the Uro-A-producing strain.	HPLC, HPLC -MS/MS, morphological identification, physiological and biochemical Identification, 16S rRNA.	Wilkins–Chalgren Anaerobe Broth, 2% seed culture inoculum size, pH 7.1, 37 °C, anaerobic culture for 48 h.	[64]
Enterococcus faecium FUA027	Circular, milky white, with a moist surface, regular edges, no halo, a central protrusion, a diameter of 0.1–0.5 mm, and easy to pick.	Intestinal contents	7 volunteers with no history of gastrointestinal diseases (age: 22–27 years old) + 0.7 g raw walnuts/kg body weight → fecal + ellagic acid → dilution plating → colonies + ellagic acid → the Uro-A-producing strain.	HPLC, HPLC -MS/MS, morphological identification, physiological and biochemical Identification, 16S rRNA.	ABB Anaerobic Basal Broth Medium, 2% seed culture inoculum size, pH 6.8, 37 °C, anaerobic culture for 48 h.	[65]
Lactobacillus plantarum CCTCCAB 2013128	Convex, white, smooth, and circular, with a diameter of approximately 3 mm.	China Center for Type Culture Collection	Various strains from the Culture Collection Center → Strain activation → Seed culture + EA → Strains with a high Uro-A conversion rate.	HPLC, HPLC -MS/MS, morphological identification.	The medium consists of: 4 g rice flour, 1 g yeast extract, 1 g molasses, 1.2 g sodium acetate, 0.02 g copper sulfate, 0.05 g magnesium sulfate, 2 g malt flour, 0.5 g dipotassium hydrogen phosphate; with a 1.5% seed culture inoculum size, pH 7.1, 37 °C, aerobic culture for 80 h.	[66]
Lepista sordida	Radially spreading hyphae, pale purple (hyphae), spherical mycelial pellets.	Institute of Microbiology, Chinese Academy of Sciences	Edible and medicinal fungi existing in the laboratory → fungal activation → seed culture + EA → the Uro-A-producing fungi.	HPLC, HPLC -MS/MS.	CYM Liquid Medium, 10% inoculum size, pH6.0–6.5, 25 °C, shaking, aerobic fermentation for 9–17 days.	[67]

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These strains exhibited certain resistance, acid and bile tolerance, and no hemolytic activity, displaying different product characteristics during the biometabolism of EA. Bifidobacterium pseudocatenulatum INIA P815 [56] was the first reported Bifidobacterium strain capable of converting EA into Uro-A and Uro-B. Lactococcus garvieae FUA009 [57,58] possessed significant safety and probiotic properties, and was expected to be applied in the development of functional foods and health products. Streptococcus thermophilus FUA329 [59,60] showed a high production yield of up to 82%. Postbiotics and synbiotics developed from Lactobacillus plantarum CCFM1286 [61] exerted anti-aging effects by regulating mitochondrial autophagy. Fermented products or complex preparations containing Lactobacillus plantarum CCFM1290 [62] can improve muscle function damage and exhibit anti-aging effects, making them applicable for the development of functional foods. Lactobacillus plantarum CCFM1291 [63] exhibited anti-aging effects through the PINK1-Parkin pathway and BNIP3 receptor-mediated pathway; this strain, its fermented products, and synbiotic preparations promoted the autophagy of dysfunctional mitochondria, thereby exerting significant anti-aging effects. Therefore, Lactobacillus plantarum CCFM1291 can be used to assist in developing anti-aging products [38]. In addition to producing Uro-A, Limosilactobacillus fermentum FUA033 [64] can also produce fermentative proteases and lipases. The enzymatic activities of these strains can improve protein digestibility, regulate fatty acid release, and maintain human health by colonizing the gastrointestinal tract. Enterococcus faecium FUA027 [65] and Lactobacillus plantarum CCTCCAB 2013128 [66] were utilized for the production of Uro-A with a relatively high yield, and these strains were suitable for application in the large-scale production of Uro-A. Lepista sordida [67] was an edible and medicinal fungus that can convert ellagic acid into Uro-A under aerobic conditions.

3.2.3. Metabolic Characteristics of Uro-A in Animals and Humans

The indirect source of Uro-A (food precursor) and the metabolic distribution characteristics in different species. After ingestion of foods rich in ETs (and their precursors), researchers observed tissue-specific distribution characteristics of Uro-A and its derivatives in a variety of experimental animal models and human populations. Among them, the concentration of Uro-A was the highest in the colon, which was consistent with the source characteristics of microbial metabolism. The higher level of Uro-A glucuronide in the liver and circulatory system suggests that Uro-A will bind to the liver after intestinal absorption. In peripheral tissues such as skeletal muscle and adipose tissue, the content of Uro-A was significantly reduced, which was mainly attributed to the systemic dilution effect and potential catabolism. Quantitative distribution data are presented in

Table 3. Distribution of Uro-A and Its Metabolites in Biological Systems.

Test Subject	Food Source	Detection Site	Uro-A Form	Reference
Mouse	Pomegranate	Kidney, liver	Uro-A glucuronide	[68]
Pig	Oak-flavored milk powder	Plasma	Uro-A glucuronide	[69]
		Urine, intestinal contents	Uro-A
	Fresh acorns	Bile, intestinal lumen, gastrointestinal tissue	Uro-A glucuronide	[70]
		Urine, intestinal contents	Uro-A
Bull	Hay and oak leaves	Gastric juice	Uro-A glucuronide	[71]
		Blood	Uro-A glucuronide
		Urine, intestinal contents	Uro-A glucuronide
Adult	Walnut	Intestinal contents	Uro-A	[72]
	Black tea	Urine	Uro-A	[73]
	Pomegranate extract	Blood	Uro-A	[74]
Infant	Pomegranate juice	Urine, intestinal contents	Uro-A glucuronide	[75]

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The variability of metabolism among individuals. Studies have confirmed that there was significant individual variability in the type and quantity of urolithin generated in the human body after ingestion of EA, of which only 40% of the population can endogenously generate Uro-A. This proportion may further decline due to age-related gut microbiota alterations. Based on urolithin production profiles, individuals were classified into three urolithin metabotypes (UM): urolithin metabotype A (UM-A), urolithin metabotype B (UM-B), urolithin metabotype 0 (UM-0) [76]. The end products of these three metabotypes were Uro-A; Uro-A, Uro-B, Iso-uro-A; and Uro-M6, respectively. The difference in the composition of individual intestinal microbial communities was the reason for the difference in the type and quantity of urolithin production in the body, and may also be one of the key reasons for the individual to present different urolithin metabotypes [77]. Further studies indicated that the distribution of urolithin metabolites in humans was influenced by multiple factors, including gut microbiota composition, age, sex, dietary habits, and genetic background. UM-0 individuals exhibited reduced gut microbiota diversity and richness compared to UM-B and UM-A subgroups, characterized by lower abundances of genera such as Bacteroides, Prevotella, and Ruminococcus, as well as Phascolarctobacterium, Bilophila, Alistipes, and Butyricimonas [78]. A study [79] demonstrated that in a non-urolithin-producing rat model, the intake of bacterial consortia A (Gordonibacter + Enterocloster) and B (Ellagibacter + Enterocloster) was sufficient to establish gut colonization by urolithin-producing bacteria. In contrast, continuous ingestion of ETs alone was insufficient to convert the UM-0 into UM-A or UM-B. García-Mantrana et al. [80] demonstrated that walnut consumption modulated gut microbial communities in healthy volunteers: UM-B individuals showed increased levels of Bifidobacterium, Blautia, and Coriobacteriaceae members such as Gordonibacter following walnut intervention, while specific Lachnospiraceae taxa were selectively reduced only in UM-A individuals. These findings highlight the potential of dietary strategies to regulate UM. Among 415 Spanish children and adolescents, the UM-B and UM-0 metabotypes were significantly associated with the risk of obesity, and exhibited a synergistic effect with low adherence to the Mediterranean diet. For the first time, the study integrated genetic polymorphisms (e.g., rs8061518-FTO), gut microbiota metabotypes, and dietary patterns into a predictive model, which accounted for obesity variation [81]. Notably, UM-0 appeared temporally stable, whereas UM-A and UM-B distributions exhibited age-dependent dynamics [82]. A study [83] on 35 healthy young adults of Han Chinese ethnicity aged 21–30 years showed that the distribution of their urolithin metabotypes was as follows: UM-A (54.3%), UM-B (31.4%), and UM-0 (14.3%). Additionally, the abundances of Gordonibacter and Akkermansia in the gut microbiota were significantly associated with the UM-A and UM-B, while the population with the UM-0 exhibited lower gut microbiota diversity. Gender distribution analysis identified a higher prevalence of UM-B in males and UM-0 dominance in females. Pregnant women also showed different characteristics, with the gut microbiome changing significantly during and after pregnancy [84,85].

4. Development and Application of Uro-A-Related Products

4.1. Products Directly Supplemented with Uro-A

The current market is dominated by high-purity monomeric supplements, such as UltraUro™ (purity > 98%) and Mitopure™ postbiotic preparations. These products are typically available in capsule or powder form, with a daily recommended dosage of 250–1000 mg, suitable for applications such as anti-aging and muscle health. Notably, significant quality variations exist among commercially available products: a 2024 study by the National University of Singapore found that the Uro-A content in five tested supplements deviated from label claims by −15.5% to +28.6%, highlighting the importance of high-purity raw materials [86]. Uro-A can significantly inhibit melanin production in B16 melanoma cells, providing a theoretical basis for the development of new skin-whitening products [87]. Uro-A effectively protects dermal fibroblasts against ultraviolet A-induced damage through a dual mechanism, supporting its potential application in sunscreen products [88]. It exerts anti-aging effects on human dermal fibroblasts at the replicative senescence stage. Compared with retinoic acid, Uro-A not only has similar antioxidant capacity but also enhances antioxidant efficacy by activating the Nrf2/ARE pathway, thereby avoiding the cytotoxicity potentially induced by high concentrations of retinoic acid. As a potential anti-aging ingredient, Uro-A is expected to replace retinoic acid as the core anti-aging component in cosmetics [89].

4.2. Products in Which Uro-A Is Generated Through Conversion by Probiotics

Strains that can convert and produce Uro-A with excellent probiotic properties (such as those strains with high safety and good probiotic properties listed in Table 2) can be used to develop a variety of functional products. For example, there are pomegranate lactic acid bacteria beverages produced by fermenting these strains with pomegranate juice as the base [90]. There are also synbiotic preparations [63] formulated by compounding probiotics with freeze-dried pomegranate powder, which enhance the production efficiency of Uro-A through the synergistic effect between the activity of probiotics and ellagic acid precursors in pomegranate powder. In addition, these strains can also be used to develop daily products such as fermented beverages and yogurts, enabling consumers to not only regulate intestinal flora but also naturally obtain Uro-A when consuming these common foods, thus achieving daily supplementation of health benefits.

4.3. Products Added with EA/ETs as Precursors

EA, the precursor of Uro-A, is widely present in plants such as pomegranates, nuts, and berries. This type of product, by adding raw materials rich in EA (e.g., pomegranate juice, pomegranate extract, ellagic acid supplements), relies on intestinal microbiota to metabolize it into Uro-A. It also helps improve the intestinal environment and increase the abundance of beneficial microbiota [91,92,93]. The consumption scenarios of such foods have expanded from single snacks to various forms including pomegranate juice, mixed berry juice, sports nutrition bars, and meal replacement powders, which fully meet the needs of daily intervention for healthy populations.

5. Conclusions and Perspectives

This review highlights the robust research momentum and significant application value/potential of Uro-A. By elucidating the mechanisms and underlying essence of Uro-A-mediated health improvement at the molecular level, this review supports the development of functional foods and pharmaceuticals, ultimately advancing human health and longevity.

Uro-A is a natural metabolite that targets mitochondrial health. In recent years, it has attracted significant attention due to its potential anti-aging, anti-inflammatory, and mitophagy-promoting effects. From a technical perspective, large-scale production of Uro-A can be achieved through synthetic biology approaches or microbial fermentation. Some enterprises (e.g., Amazentis) have developed high-purity and stable formulations (e.g., Mitopure^®) and completed a number of human clinical trials, which have verified its safety and bioavailability. Economically, with the rapid growth of the global anti-aging market, Uro-A has high value-added potential as a functional food, dietary supplement and possible future prescription drug ingredient.

While studies on Uro-A have demonstrated its potential in the field of metabolic health, significant limitations remain. Clinical research primarily relies on animal models, with most being small-sample, short-follow-up designs. Moreover, metabolic heterogeneity caused by inter-individual differences in gut microbiota undermines the generalizability of research conclusions. Regarding safety, while safety data for short-term, low-dose applications are relatively sufficient, potential risks associated with long-term, high-dose exposure (e.g., impacts on liver and kidney function) and safety data for special populations (pregnant women, children, and patients with chronic kidney disease) remain lacking, leading to controversies. Industrial production faces technical bottlenecks such as low efficiency of raw material extraction, difficulties in quality control of isomers, poor water solubility resulting in low bioavailability, low microbial conversion efficiency, and high purification costs. Additionally, differences in product purity and batch stability caused by varied sources of precursor substances (e.g., ETs) further restrict its industrial application.

Key research priorities include: (1) Optimization of the chemical synthesis method for Uro-A can utilize ellagic acid extracted from agroforestry wastes (such as walnut shells, pomegranate peels, and blackberry pomace) as the raw material. Specifically, in a water-ethanol mixed solvent, through “one-pot” cyclization catalyzed by supported solid acids, combined with continuous-flow reactors and crystallization separation, it enables the efficient, low-cost, and environmentally friendly synthesis of Uro-A. (2) Screening for strains with high Uro-A conversion efficiency via modern biotechnological approaches such as genetic engineering and metabolic engineering. (3) Using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) or isotope labeling technology to analyze the metabolic flux and key enzymes involved in UroA conversion, followed by identifying regulatory factors at the transcriptional, post-translational, and global levels. Then, validate their functions through gene editing and in vitro reconstitution, and finally integrate multi-omics data to construct regulatory networks. (4) Treating diseases by targeted delivery of Uro-A, Uro-A is encapsulated in nanocarriers (e.g., liposomes, PLGA nanoparticles, etc.) and modified with specific ligands to achieve precise delivery, followed by responsive release at the target site, thereby enabling targeted therapy. (5) The dietary strategy of “ellagitannin-rich foods providing substrates + targeted prebiotics enriching functional microbiota” can gradually reshape the metabolic potential of the gut microbiota, enabling individuals with UM-0 to acquire the ability to convert ETs into Uro-A (corresponding to UM-A) or Uro-B (corresponding to UM-B). (6) Further exploration of the multi-pathway crosstalk of Urolithin A in different tissues and across various life stages should be conducted, alongside the implementation of long-term safety studies—with particular attention paid to its metabolites and interactions with commonly used drugs. Advancing research technologies will enable broader exploration and clinical translation of urolithins, potentially yielding significant health benefits.