Absorption and Metabolism of Urolithin A and Ellagic Acid in Mice and Their Cytotoxicity in Human Colorectal Cancer Cells

Background Ellagic acid is a natural polyphenol compound found in pomegranates, walnuts, and many berries. It is not easily absorbed, but it could be metabolized to urolithins by the gut microbiota. Urolithin A, one of the ellagic acid metabolites, has been proved to prolong the lifespan of C. elegans and increases muscle function of mice. The purpose of this current study was to analyze the absorption and metabolites of urolithin A and ellagic acid in mice and the anticancer effects of urolithin A, urolithin B, and ellagic acid in colorectal cancer cells. Methods Urolithin A and urolithin B were synthesized and analyzed by HPLC and NMR. A pharmacokinetic study of urolithin A was performed in mice by analyzing urolithin A and its metabolites in urines. Absorption and biotransformation of ellagic acid were also studied in mice by analyzing the plasma, liver, and feces. The cytotoxicity of urolithin A, urolithin B, and ellagic acid was assayed in SW480, SW620, HCT 116, and HT-29 cells. Results Urolithin A and urolithin B were synthesized and purified to reach 98.1% and 99% purity, respectively, and the structures were identified by NMR. In urolithin A intake analysis, urolithin A was only detectable at 3 h, not at 6–24 h; it suggested that urolithin A was rapidly metabolized to some unknown metabolites. Using UPLC-MS/MS analysis, the metabolites might be urolithin A 3-O-glucuronide, urolithin A 3-sulfate, and urolithin A-sulfate glucuronide. After feeding mice with ellagic acid for consecutive 14 days, ellagic acid contents could be detected in the fecal samples, but not in plasma and liver, and urolithin A was not detected in all samples. It suggests that ellagic acid is not easily absorbed and that the biotransformation of ellagic acid to urolithin A by intestinal flora might be very low. From the cytotoxicity assay, it was found that there was anticancer effect in urolithin A and urolithin B but not in ellagic acid. In contrast, ellagic acid promoted the proliferation of SW480 and SW620 cells.


Introduction
Colorectal cancer (CRC) is one of the most common malignant tumors and the second leading cause of cancerrelated deaths worldwide [1].Early diagnosis and promotion of healthy eating habits have been established as basic preventive measures for CRC prevention [2].Both hereditary and environmental risk factors play a role in the development of colorectal cancer [3].In terms of environmental risk factors, an aging population and dietary habits are the most common factors [3].Epidemiological studies have shown that the incidence of CRC is inversely related to the large intake of fruits and vegetables rich in phytochemicals, including phenolic compounds [4].However, although it has been reported that diferent single phenols and phenol-rich plant foods have rich preclinical (in vitro and animal model) anticancer activity, so far, clinical evidence is still elusive [5].Ellagic acid, which can be released from ellagitannins, is a polyphenol found in pomegranates, walnuts, and various berries.Ellagitannins, also known as hydrolysable tannins, are not absorbed in the gastrointestinal tract because they adhere to the Lipinskin rule of fve [5].Ellagic acid exhibits low bioavailability, with a maximum concentration of 120 ng/ml in human plasma [6].It is known that ellagic acid is metabolized by the colonic fora and colorectal cells to produce a family of bioavailable metabolites called urolithins, which are 25∼80-fold more bioavailable than ellagic acid [6].Remarkably, urolithins reach relevant concentrations in the lumen and colonic tissues (malignant and normal) of CRC patients after the intake of an ellagic acid-rich pomegranate extract [7].Urolithin D is produced from ellagic acid through the opening of a lactone ring and the removal of a carboxyl group.Urolithin D loses 1, 2, or 3 hydroxyl groups through dehydroxylation to form urolithin C, urolithin A/isourolithin A, and urolithin B, respectively [6] (Figure 1).According to the constitution of urolithins in urine, the gut microbiota composition in humans diferentially determines the 3 urolithin metabotypes including A, B, and 0 [8].Tis dis-similar metabolism is linked to the interindividual variability of the human gut microbiota, which has led to identify three major fnal urolithins, including urolithin A, urolithin B, and isourolithin A [9].
Gut microbiota plays an important role for metabolizing plant phenolic acids to urolithins [10]; therefore, how to monitor the structure, concentration, and distribution of urolithins after catabolization by the gut microbial communities has become an attractive research topic for scientists.Metabolomics is the study of metabolites in a given biological system that provides correlation between specifc metabolites and certain physiological conditions.For example, metabolomics has been employed for the screening of biomarkers for diagnosis, disease pathogenesis, and drug discovery [11][12][13].Generally, the frst step in a metabolomic analysis is to extract metabolites from the samples of interest.Tose metabolites are often separated and analyzed using gas chromatography MS (GC/MS) and liquid chromatography MS (LC/MS) [6,9].Furthermore, the metabolites of interest could be identifed through comparison of exact mass, retention time, and fragmentation information with genuine standards and spectral databases.LC-tandem MS spectrometry is a powerful tool for comparative metabolomic analysis.Using comparative metabolomic analysis would help us to understand the structural relationship of the target compounds and their derivatives.Terefore, using comparative metabolomics is an appropriate strategy for new urolithin derivatives discovery.
Urolithin A is a bioactive metabolite produced by the gut microbiota from ellagitannins and ellagic acid [7] (Figure 1).Recently, it has been proven that urolithin A prolongs lifespan of C. elegans and increases muscle function of mice [14].In a male C57BL/6 mice study, it indicates that urolithin A presents in colon, intestine, liver, and prostate after receiving urolithin A by oral route [15].Because urolithin A was detectable in mice prostate tissue, a human trial about patients with prostate cancer was reported.A double-blind and randomized trial was conducted in men with prostate cancer before radical prostatectomy [16].Te results suggest that urolithin A levels were inversely correlated with 8-OHdG expression in cancer tissues (r = −0.299,P � 0.017).Urolithin A and its related metabolites, urolithin B, and urolithin C have been found to possess the ability to inhibit the proliferation of human prostate cancer cells.However, it is not recommended to coadminister these compounds with the nonsteroidal antiandrogen drug bicalutamide [17].In addition to prostate cancer, urolithin A also induces autophagy and inhibits metastasis in the human colorectal adenoma cell SW620 [18].Other studies about colorectal cancer cells have been reviewed in the article [19], which suggests that some pharmacological mechanisms (apoptosis, autophagy, p21 expression, and G2/M arrest) are induced by urolithins.
In this study, we synthesized urolithin A and urolithin B with high purity by ourselves using the HPLC analysis.Ten, we used the C57BL/6 mice model to identify the metabolites of urolithin A in urine by a single-dose urolithin A regimen.Furthermore, the mice were fed with ellagic acid, the urolithins' precursor, for continuously 14 days, and the plasma, liver, and feces samples were analyzed for ellagic acid and its urolithin metabolites.In addition to in vivo pharmacokinetic study, the cytotoxicity of urolithin A, urolithin B, and ellagic acid was also tested in four diferent human colorectal cancer cell lines.

Purifcation and Identifcation of Urolithin A and Urolithin B.
Te purity of the products was determined by HPLC, 1 H-, and 13 C NMR spectra.All chemical reagents in commercial quality were used as received (Sigma-Aldrich, St. Louis, MO, USA) and were used without further purifcation.Solvents were dried, and the synthesized compounds were purifed using standard techniques.Reactions progression was monitored by TLC on aluminum plates coated with silica gel with a fuorescent indicator (Merck 60 F 254 ).Unless otherwise stated.Melting points were determined in open capillaries using the Fargo MP-2D apparatus and are uncorrected.NMR spectra were recorded using TMS as an internal standard in DMSO-d 6 at 500 MHz for 1 H and at 125 MHz for 13 C (Bruker Biospin GmbH AVANCE III 500 MHz, Rheinstetten, Germany).Chemical shift (δ) was reported in parts per million (ppm) measured relative to the internal standards tetramethylsilane (TMS), and the coupling constant (J) was expressed in Hertz (Hz).Column chromatography was performed with silica gel SiliaFlash ® G60 (60-200 μm) purchased from SiliCycle Inc.
(Quebec City, Canada).In general, the reactions were carried out under anhydrous conditions in a dry solvent and nitrogen atmosphere.Te purity of these compounds was based on the analysis of HPLC (Hitachi High-Technologies, Tokyo, Japan) equipped with a 280 nm detector and a LiChroCART RP-C 18 column (4.6 mm i.d.× 250 mm, 5 μm, Merck, Darmstadt, Germany).Te mobile phase was composed of MeOH-H 2 O (0.05% TFA) (90 : 10), and the fow rate was 1.0 mL/min.Te purity of all compounds was more than 98.1%.Te mass spectra were acquired using a Termo Finnigan model LXQ (Termo Electron Co., Waltham, MA, USA) ion trap mass spectrometer equipped with ESI source interference and controlled by Xcalibur 2.06.Te mass spectra were acquired in a positive ion mode or a negative ion mode [20].

Short-Term Urolithin A Metabolomics Experiment.
Female C57BL/6 mice (n � 20, 8 weeks old, 16-19 g) were purchased from the National Laboratory Animal Center (Tainan, Taiwan).Te diets and water were feed ad libitum and fasted 6 h prior to dosing.Mice were orally administered with urolithin A (0.6 mg/mouse, n � 10) or vehicle control (1.6% DMSO and 6.25% Tween-20 in 160 µL water/mouse, n � 10).Urine samples were collected at 3, 6, 9, 12, and 24 h after oral administration.Te samples were stored at −80 °C until analysis.For the metabolomic analysis, the urine samples were thawed and 15 μL was taken for extraction.Evidence-Based Complementary and Alternative Medicine vortexed and centrifuged at 13,200 g for 10 min at 4 °C.Te supernatants were collected and fltered by a 0.22 μm flter for HPLC analysis.For the fecal metabolite analysis, the cecum contents of mice were collected and then extracted using the method as used in liver tissue extraction.Te supernatants were collected and fltered by a 0.22 μm flter for HPLC analysis.

HPLC Analysis.
All test samples were dissolved in methanol (HPLC grade) at a concentration of 10 mg/mL.Te metabolite profle of each sample was analyzed using an HPLC system (SPD-M20A diode array detector, Shimadzu, Kyoto, Japan) coupled with a C18HQ column (4.6 mm i.d.× 250 mm, 5 μm, Interchim, Montluçon, France).Te mobile phase consisted of 50% of methanol and 50% of ddH 2 O (0.05% of phosphoric acid), and the fow rate was 1 mL/min.In addition, urolithin A, urolithin B, and ellagic acid were quantifed by HPLC with the same condition as aforementioned.For the calibration curve analysis, the stock solution was dissolved in methanol and then diluted to obtain the desired concentrations for the quantifcation (urolithin A and urolithin B: 200, 100, 50, 25, 12.5, and 6.25 μM; ellagic acid: 4, 2, 1, 0.5, 0.25, and 0.125 μM).Te calibration curve was plotted using the linear regression method (peak areas versus compound concentrations).

UPLC-MS/MS Analysis.
All test samples will be dissolved in methanol at a concentration of 10 mg/mL.Te experimental procedure was conducted as previously described [7].Te mass data were acquired using UPLC-ESIMS (Termo Scientifc Orbitrap Elite Mass Spectrometer), and the mass range was set up as m/z 100-1500.

Human Colorectal Adenocarcinoma Cell Lines.
Tere were 4 cell lines used in this study including SW480, SW620, HCT 116, and HT-29 cells (Bioresource Collection and Research Center, Hsinchu, Taiwan).SW480 and SW620 cells were established from a same Caucasian male patient with colon adenocarcinoma, and SW480 was isolated from the primary colon tumor site, SW620 was isolated from a lymph node metastasis site.SW480, but not HT-29, secrets granulocyte-macrophage colony-stimulating factor (GM-CSF) that stimulates the proliferation of acute megakaryoblastic leukemia M-07e [21].SW480 and SW620 synthesize small quantities of carcinoembryonic antigen (CEA) and belong to Broders' grade 4 cancer [22].By Northern blot analysis, the oncogenes c-myc, H-ras, K-ras, N-ras, myb, fos, and p53 were all expressed in SW480 and SW620 cells [23].HT-29 cells were isolated from a Caucasian female patient with colorectal adenocarcinoma (ATCC data).HT-29 and SW480, but not SW620, express vitamin D receptor whose activation by 1,25-dihydroxyvitamin D inhibits cell proliferation and clonogenic growth in soft agar [24].HT 116 has a mutation in codon 13 of the ras protooncogene and expresses transforming growth factor beta 1 and beta 2. SW480 and SW620 cells are cultured in Leibovitz's L-15 medium with 10% FBS, and HT-29 and HCT 116 cells are cultured in McCoy's 5a medium with 10% FBS.

Cytotoxic Analysis of Urolithin A, Urolithin B, and Ellagic
Acid in Cells.Te cytotoxicity of urolithin A, urolithin B, and ellagic acid was analyzed in these 4 cell lines by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, following the method outlined in a previous study [25].Cells (6.5 × 10 4 for SW480, 1.5 × 10 5 for SW620, 2.5 × 10 5 for HCT 116, and 1 × 10 5 for HT-29) were cultured in 24-well plates for 24 h and then were incubated with various concentrations of chemicals for another 24 h.After adding MTT to the medium and incubating the cells for 1.5 h (SW480 and SW620) and 45 min (HCT 116 and HT-29), the cells were dissolved in DMSO and the suspension was collected for OD570 detection by a spectrophotometer.Te result was expressed as a percentage, relative to control group.

Statistical Analysis.
Te values shown are the mean ± SE.Data were statistically evaluated by GraphPad Prism software (version 9.4) using student's t-test for comparison of the two groups, and for more than two groups, we conducted by ANOVA with Dunnett's multiple comparisons test; signifcant diferences are shown as * p < 0.05, * * p < 0.01, and * * * p < 0.001.

Purifcation and Identifcation of Urolithin A and Urolithin B.
Urolithin A and urolithin B have been synthesized from 2-bromo-5-hydroxy benzoic acid or 2-bromo-benzoic acid in two steps as described in Section 1.1.Te purifed urolithin A and urolithin B were obtained as a slight yellow color solid, which was characterized using NMR spectroscopy and HPLC analysis.Te purity of these compounds was based on the analysis of HPLC (Hitachi High-Technologies, Tokyo, Japan) equipped with a 305 nm detector and LiChroCART RP-C 18 column (4.6 mm i.d.× 250 mm, 5 μm, Merck, Darmstadt, Germany).Te mobile phase was composed of MeOH-H 2 O (0.05% phosphoric acid) (50 : 50), and the fow rate was 1.0 mL/min.Te purity of all compounds was greater than 98.1%.Urolithin A and urolithin B showed retention time of 12.3 min and 22.3 min, respectively.

Short-Term Metabolomics Analysis of Urolithin A Intake in Mice.
Te short-term metabolomics analysis using the urine sample collected from C57BL/6 mice fed with urolithin A (0.6 mg/mouse) or vehicle control (1.6% DMSO and 6.25% Tween-20 in 160 µL water/mouse).Te urine samples were collected after gavage with vehicle/urolithin A for 0, 3, 6, 9, 12, and 24 h and then subjected to the HPLC analysis.Te results showed that urolithin A (RT � 7.6 min) was only detectable at 3 h, it suggested that urolithin A was rapidly metabolized before 3 h, and some unknown materials were detected by HPLC analysis as well (Figure 2).Te unknown materials included the signal A (RT � 3.0 min) which was detected in urine samples of mice after feeding with urolithin A for 6 h to 24 h and another signal B (RT � 3.6 min) which was detected in urine samples of mice after feeding with urolithin A for 3 h and 6 h.Because the 2 unknown materials were more hydrophilic than urolithin A, it is suggested that they might be urolithin A-related metabolites by mice phase II metabolic enzymes.However, the exact structure of those metabolites remains unknown, and further UPLC-MS/MS analysis for structural elucidation was applied.

UPLC-MS/MS Analysis.
To check the possible urolithin A-related metabolites in the urine samples, the UPLC-MS/ MS analysis was performed in this study.Because the urine samples were limited, we used pooling sample for this assay.Here, we detected and proposed three possible urolithin Arelated metabolites, including urolithin A 3-O-glucuronide ( ) was detected at m/z � 403.07 and the MS 2 ion at 227.03 (characteristic for neutral loss of 176 amu); urolithin A 3sulfate ( 2) was detected at m/z � 306.99 and the MS 2 ion at 227.03 (characteristic for neutral sulfate loss of 80 amu), and urolithin A-sulfate glucuronide was detected at m/z � 483.02 and the MS 2 ion at 306.99 (neutral loss of 176 amu), which fragmented to 227.033 (characteristic for neutral sulfate loss of 80 amu) (3) in the urine samples (Figure 3).Based on aforementioned, we speculated that the unknown signals A and B of urine samples in Figure 2 may be phase II conjugates of urolithin A.

Long-Term Metabolomics Analysis of Ellagic
Intake in Mice.After feeding mice with ellagic acid continuously for 2 weeks, the mice were euthanized and the feces, liver, and plasma of mice were collected and stored in −80 °C freezer for further analysis.Te mice body weight change is shown in Figure 4(a), and it showed that high-dose ellagic acid intake (1.2 mg/mouse/day) reduced the mice body weight after continuous intake for 14 days.For the plasma and liver samples, no ellagic acid and urolithin A-related signals were detected in HPLC analysis.We also conducted the fecal metabolites extraction and the HPLC analysis.It showed that the ellagic acid (RT � 5 min) contents could be detected in the fecal samples in both of the low-and highdosage groups, while the control group showed a very low ellagic acid concentration compared to the other two groups (Figure 4(b)).In addition, the ellagic acid contents showed a dose-dependent manner in low (2.8 μM) and high-dosage groups (3.7 μM).However, urolithin A was unable to detect in fecal samples in both of low-and high-dosage groups (data not shown).Evidence-Based Complementary and Alternative Medicine

Cytotoxic Analysis of Urolithin A in Cells.
Urolithin A was dissolved in DMSO, and the stock concentration was 100 mM.In a dose-dependent cytotoxic assay, the fnal concentration of DMSO in the culture medium was 0.1%.As shown in Figure 5, IC 50 of urolithin A in SW480 and SW620 was about 50 μM which had reached the plateau with 50% cell viability.In HCT 116, the cytotoxicity reached the plateau at 50 μM with about 75% cell viability.In HT-29 cells, the cell viability was more than 80% by 20-100 μM urolithin A treatment for 24 h, and it means urolithin A had minor cytotoxicity in HT-29 cells.In summary, urolithin A has diferent cytotoxicity in SW480, SW620, HCT 116, and HT-29 cells, but all reached the plateau at 50 μM.

Cytotoxic Analysis of Urolithin B in Cells.
We also analyzed the cytotoxicity of urolithin B in cells.As shown in Figure 6, IC 50 of urolithin B in SW480 was about 75 μM, which was also the highest cytotoxicity with 50% cell viability.In SW620 cells, the cytotoxicity reached the plateau at 75 μM with about 60% cell viability.Tere was no obvious cytotoxicity of urolithin B in HT-29 and HCT 116 cells.In conclusion, the cytotoxicity of urolithin B was lower than that of urolithin A in these 4 cell lines.

Cytotoxic Analysis of Ellagic Acid in Cells.
In 1-4 μM of ellagic acid, the cell viability was not below 80%, and 10 μM ellagic acid increased cell proliferation in HT-29, SW620, and SW480 cells (Figure 7).It suggests no obvious cytotoxicity of ellagic acid in these 4 colorectal cancer cell lines, and it instead to increase cell proliferation at 10 μM.In addition, we used transformed normal epithelial cells SV-HUC1 for the cytotoxic assay, and they also showed no cytotoxicity of ellagic acid.For the cytotoxicity of urolithin A and urolithin B, they exhibit a similar efect to SW620 cells in SV-HUC1 (data not shown).

Discussion
In 2003, urolithins were frst revealed as bioavailable metabolites from the ellagitannins of pomegranate in rat [26,27].Te ellagitannins are hydrolysable tannins that are found in many medicinal plants, herbal teas, and foods [28].However, the low bioavailability of ellagitannins limited their health benefts.Te gut microbiota metabolizes ellagitannins and ellagic acid to several urolithins that were reported to have various health efects in animal models and humans [28].Te characteristic chemical structure of  Evidence-Based Complementary and Alternative Medicine tissues and excreted in urine and feces are urolithin A, urolithin B, and isourolithin A [8]. Te corresponding phase II conjugates (glucuronides or sulfates) of urolithin A have also been found in the aforementioned urine samples as well [10].
Te high variation of urolithins concentration in biological fuids was reported.For example, the concentration of urolithin A reported in human (consuming nectar 20 g/ day for 3 weeks) urine is about 4807 nM, while the concentration of urolithin B in the same sample is about 312 nM [29].Wistar rats were fed a diet containing strawberries that were rich in monomeric ellagitannins (ME), dimeric ellagitannins (DE), or free ellagic acid (FE) at a concentration of 0.10-0.15%for 24 h.Te urine concentrations of urolithin A in the ME, DE, and FE groups were found to be 5.6, 3.9, and 99.5 μM, respectively [30].In the same model, the concentration of urolithin A in the ME, DE, and FE groups is 8.3, 0.2, and 224.3 μg/g of cecal digesta, respectively.However, urolithin A was only detected in the feces sample of the FE group (89.3 μg/g feces) [30].Te variation may be due to the sample dosage, the source of ellagitannins, and the methods used for quantifcation.Our results revealed that 3 possible phase II conjugates (glucuronides or sulfates) of urolithin A were detected in the urine samples after feeding mice with 0.6 mg urolithin A. Te results are consistent with previous studies.After absorption by animals, only traces of nonconjugated urolithins are able to detect in bodily fuids, indicating that urolithins undergo phase II metabolism in a very short time.Tis also afects the presence of urolithins in plasma and tissues.Te presented urolithins predominant in conjugated form included glucuronides (major) and sulfates (minor) [31].In a cytotoxicity assay of SW480, SW620, HCT 116, and HT-29 cells, it shows that there is no obvious cytotoxicity in ellagic acid-treated cells; in contrast, a high-dose (10 μM) ellagic acid induced cell proliferation (Figure 7).In split of the antiproliferation efect of ellagic acid in the breast cancer cell line MCF-7 [32], we still suggest that foods with high ellagic acid content should be consumed with caution by CRC patients.In contrast, the metabolites of ellagic acid, urolithin A and urolithin B, have cytotoxicity to SW480, SW620, and HCT 116 (Figures 5 and 6), but it is necessary to convert ellagic acid to urolithins by intestinal microbiota.In a human study using oral pomegranate extracts, the urolithin A and urolithin B are presented in normal and malignant tissue of CRC patients [7], it suggests that pomegranate extracts could be metabolized to urolithins in the human intestine.In this study, urolithin A-related compounds could not be detected in mice plasma, liver, and feces after ellagic acid intake for 14 days, but ellagic acid was measured in the feces.Tis mice study shows diferent results with the above human study.Some possible explanations for the diferences between the two studies: one is the intake material; the human study used pomegranate extracts, and we used ellagic acid.Second one is a human study, and we did a mouse experiment.Tird, the assay tissues are different.Te human study analyzed colon tissues, and we investigated plasma, liver, and feces.In addition, the diferences in gut microbial ecology infuence the variability in microbial metabolism of ellagic acid; for example, phenotype 0 does not produce any urolithin [6].Neither urolithin A nor urolithin B reduced cell viability to less than 40% (Figures 5 and 6).Especially in HT-29 cells, urolithin A produced lower cytotoxic activity than in SW480, SW620, and HCT 116 cells.One report provides that the resistance mechanism of HT-29 to urolithin A is phase II metabolism which converts urolithin A to urolithin A-glucuronide which has lower cytotoxic activity [33,34].In this study, we found that urolithin A was rapidly metabolized and excreted into urine in mice (Figure 2); it suggests that the single dose of urolithin A might exert a limited biological efect in vivo.In a previous study in mice [15], it shows that after urolithin A intake, little urolithin A was detected in mice plasma and high concentration of urolithin A presented in the prostate, intestine, and colon, and high concentrations of urolithin A-sulfate and urolithin A-glucuronide presented in the liver and kidney.Terefore, our data support the abovementioned report because minor amounts of urolithin A presented in mice urine that refect its low concentration in mice plasma.Te urolithin A-related metabolites were detected after intake for 3 h to 24 h (Figure 2).Te phase II metabolites of urolithin A, including urolithin Aglucuronide and urolithin A-sulfate, also show no efect on CRC cell proliferation and senescence [35].Terefore, if urolithin A could directly enter colon epithelial cells without phase II metabolism, it produces its own biological activity.If urolithin A is absorbed into blood, it might be rapidly metabolized by liver phase II enzymes and produce the metabolites with lower biological activity.
Among the urolithins, urolithin A has been extensively studied for its various biological activities, which include anti-infammatory properties, neuroprotective efects, cognitive improvement, and cardioprotective efects [9].In this study, we also found that urolithin A was better than urolithin B and ellagic acid in anti-CRC cell proliferation.However, urolithin A is a compound derived from the gut bacteria that can be naturally converted from dietary precursors to meaningful levels in only 40% of people.In several recent studies, the positive efects of direct urolithin A administration on health, aging, and age-related conditions have been identifed [36][37][38].Urolithin A is obtained through pharmaceutical synthesis as a modern dietary supplement.Terefore, using urolithin A directly might be a more efective route than ellagitannins or ellagic acid intake in cancer inhibition of CRC patients.A human study also suggests that direct urolithin A supplementation could overcome the limitation of gut microbiome variability to produce a consistent efect [39].

Conclusions
In this study, we developed the method for the synthesis, purifcation, and identifcation of urolithin A and urolithin B. In mice study, it suggests that urolithin A could be absorbed into blood and might be metabolized rapidly by phase II enzymes.In cell cytotoxicity assay, urolithin A has higher cytotoxicity than urolithin B and ellagic acid, and HT-29 cells have resistance to urolithin A compared to HCT 116, SW480, and SW620 cells.Due to the enhanced proliferation of CRC cells by ellagic acid, it is preferable to directly use urolithin A instead of natural extracts containing ellagitannins and ellagic acid in patients with colorectal cancer.

Figure 1 :
Figure 1: Te metabolic pathway of ellagitannins and ellagic acid to urolithins.

Figure 5 :
Figure 5: Cytotoxicity of urolithin A in HT-29, HCT 116, SW480, and SW620 cells.(a) Each cell viability was compared with its own control.* p < 0.05 and * * * p < 0.001.(b) Te photographs show cells that were treated with or without 75 µM urolithin A for 24 h.Te yellow scale bar is 100 µm.