Journal of Chromatography B, 990 (2015) 111–117

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Short Communication

Preparative isolation and purification of urolithins from the intestinal metabolites of pomegranate ellagitannins by high-speed counter-current chromatography Wenhua Zhao a , Yuji Wang a , Weijia Hao a , Hua Yang a , Xueying Song a , Ming Zhao a,b,∗ , Shiqi Peng a,∗ a Beijing area major laboratory of peptide and small molecular drugs; Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China; Beijing Laboratory of Biomedical Materials; College of Pharmaceutical Sciences, of Capital Medical University, Beijing 100069, China b Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan

a r t i c l e

i n f o

Article history: Received 11 December 2014 Accepted 27 March 2015 Available online 3 April 2015 Keywords: Pomegranate husk Urolithins High-speed counter-current chromatography (HSCCC) Intestinal metabolites Anti-oxidant

a b s t r a c t Urolithins were separated from the intestinal metabolites of pomegranate ellagitannins by high-speed counter current chromatography in two steps using two solvent systems composed of n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5, v/v/v/v/v) and n-hexane-ethyl acetate-methanolacetic acid-water (2.5:0. 8:0.25:5, v/v/v/v/v) for the first time. Each injection of 100 mg extract yielded 21 mg of pure urolithin A and 10 mg of pure urolithin B. High-performance liquid chromatography analyses revealed that the purity of urolithin A and urolihtin B was over 98.5%. The structures of urolithin A and urolitihn B were identified by high resolution-MS, NMR and single crystal x-ray analysis. Urolithins reduced the oxidative stress status in colon cancer by decreasing the intracellular ROS and malondialdehyde levels, and increasing SOD activity in H2 O2 treated Caco-2 cells. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Pomegranate (Punica granatum L.) husk possesses anticarcinogenic [1], anti-inflammatory [2], antimicrobial [3], antidiabetic [4], antioxidant [5] and anti-plasmodial activities [6]. These pharmacology actions were attributed to the ellagitannins [7,8]. Of note, it was reported that pomegranate ellagitannins absorption is very few, and the metabolites analysis is well established that pomegranate ellagitannins are mainly metabolized to urolithins in the colon. The major metabolites in the gut are urolithin A and urolithin B by sequential removal of hydroxyls [9]. It was reported that urolithins can be absorbed, and, afterwards, reach different tissues in the body and offer anti-oxidation [10,11], anti-inflammatory [12], anti-carcinogenic [13] and anti-microbial actions[14,15] in vitro. For now, the biological activity in vivo becomes the focus of research. So, to providing abundant urolithins

∗ Corresponding author. Tel./fax: +86 10 8391 1528. E-mail addresses: [email protected] (M. Zhao), [email protected] (S. Peng). http://dx.doi.org/10.1016/j.jchromb.2015.03.024 1570-0232/© 2015 Elsevier B.V. All rights reserved.

for animal experiment is of great importance. However, the commercial products are too expensive, and more notably, the synthetic urolithins could contain trace amount of metals [16], the copper may present in the final formulation, and as a consequence, dramatically influence the results. So, the preparation of high-purity and abundant urolithins from the intestinal extracts of pomegranate ellagitannins is of great interest. High-speed counter-current chromatography (HSCCC) has been successfully used in separation and purification of bioactive compounds from natural products such as, food-related polyphenols [17,18], flavonoids [19,20], cyanidin 3-glucoside [21], phthalide [22], tea catechins [23,24], oils [25] and fatty acids[26]. Being a continuous liquid–liquid partition chromatography and the excellent sample recovery, HSCCC provides an optimal choice for the preparation of the bioactive compounds from the physiological metabolites such as intestinal metabolites. The present study described the successful preparative separation and purification of urolithins from the intestinal extracts of pomegranate ellagitannins by HSCCC. And the antioxidant activity of urolithins in Caco-2 cells was also detected.

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2. Experimental 2.1. Apparatus The preparative HSCCC instrument employed in the study was TBE-300 high-speed counter-current chromatograph (Tauto Bio technique, Shanghai, China) with multilayer coil and separation columns connected in series (i.d. of the tubing = 1.5 mm), and a total volume of 300 mL. The ␤ values varied from 0.5 to 0.8. The speed of the apparatus was 800 rpm. The solvent was pumped into the column with a Tauto TBP-5A constant-flow pump (Tauto Biotech, Shanghai, China). Continuous monitoring of the effluent was achieved with a Waters 2487 (USA) Dual ␭ Absorbance Detector (Waters, USA). A manual sample injection valve with a 20 mL loop (Tianjin High New Science Technology Co, China) was used to introduce the sample into the column. An Agilent Technology 1200 Series HPLC system (Agilent, USA) equipped with an auto injector, a quaternary pump, a degasser, a thermostatic auto-sampler, and a photodiode array detector (DAD), and an Agilent 1200 Chem Station software was used for the analysis of each compound in the intestinal extracts of pomegranate ellagitannins and fractions collected from the HSCCC separation. Xray diffraction (Gemini A ULTRA Single Crystal Diffractometer, USA) was analyzed using the Bruker AXS D8 Advance, X-ray diffractometer with Cu K␣— targets at a scanning rate of 0.010 2/s, applying 40 kV, 40 mA. 2.2. Reagents All solvents used for HSCCC were of analytical grade and were purchased from Beijing Chemical Factory, China. Acetonitrile used for HPLC analysis was of chromatographic grade (Fisher). The dried pomegranate husk was purchased from Tongrentang drugstore, Beijing, China. 2.3. Preparation of two-phase solvent system The two-phase solvent system composed of n-hexaneethyl acetate-methanol-acetic acid-water at volume ratios of 2.5:2:0.25:5(v/v/v/v) and 2.5:0. 8:0.25:5 (v/v/v/v/v) were used for HSCCC separation. The upper phase and the lower phase were separated to be degassed by sonication for 30 min before use. 2.4. Preparation of intestinal metabolites of pomegranate ellagitannins 2.4.1. Preparation of pomegranate ellagitannins extracts An amount of 200 g raw husk of pomegranate was extracted three times by 50% ethanol (1000 mL for each time) with ultrasonic treatment at room temperature and yielded 63 g of crude extract. Then, the extract was evaporated to dryness under reduced pressure. The obtained residue was dissolved in water. After filtration, the aqueous solution was extracted with water-saturated ethyl acetate for three times. The incorporate ethyl acetate extract (12 g) was further evaporated to dryness under reduced pressure to give the crude sample of pomegranate ellagitannins extract for further use. 2.4.2. Preparation of intestinal bacteria culture solution The anaerobic medium broth for intestinal bacteria culture was prepared as follows: The anaerobic medium mixed with fresh feces of rats were cultured under an anaerobic condition at 37 ◦ C for 30 min, and then the intestinal bacteria culture solution was prepared for further use.

2.4.3. Preparation of intestinal bacteria culture solution of pomegranate Pomegranate husk extract (5 g) was weighed and dissolved in 50% methanol (50 mL). Pomegranate husk extract (10 mL) was added into the intestinal bacteria solution (100 mL), and then incubated at 37 ◦ C for 72 h. After the addition of methanol (100 mL) and 2% formic acid, the incubation samples were centrifuged at 10,000 × g for 15 min. The supernatant was dried under nitrogen flow before subjected to HSCCC. The sample solutions were prepared by dissolving the intestinal metabolites extract in the lower phase of the solvent system used for HSCCC separation. 2.5. Measurement of partition coefficient (K) Successful separation by HSCCC largely depends on the selection of suitable two-phase solvent system. Approximately 10 mg of each sample was weighed in a test tube into which 10 mL of each phase of the pre-equilibrated two-phase solvent system was added. The test tube was shaken for 1 min and placed stand until it separated completely. The aliquot of 4 mL of each layer was taken out and evaporated separately to dryness in vacuum at 35 ◦ C. The residue was dissolved in methanol and filtered through a 0.45 ␮m filter. The K value was calculated as the peak area of target compound in the upper phase divided by that in the lower phase by LC. 2.6. HSCCC separation procedure The preparative HSCCC separation was performed as follows: The separation multilayer coiled column was first entirely filled with the upper organic phase (stationary phase). Then the apparatus was rotated at a revolution speed of 800 rpm, while the lower phase (mobile phase) was pumped into the head end of the column at a flow rate of 2.0 mL/min. After hydrodynamic equilibrium was achieved, as indicated by the clear mobile phase front emerged, the sample was injected into the separation column through the injection valve. The effluent from the outlet of the column was continuously monitored with the UV detector at 230 nm. Each peak fraction was manually collected according to the chromatogram and evaporated under reduced pressure. 2.7. HPLC analysis and structural assign of the fractions The HPLC analysis of each peak fraction obtained by HSCCC were performed by Agilent Technology 1200 Series HPLC system equipped with a quaternary pump, a degasser, a thermostatic autosampler, an auto injector, a photodiode array detector (DAD), and an Agilent 1200 Chem Station software. The analysis was carried out with a Waters SymmetryR C18 column (4.6mm × 250 mm, 5 ␮m) at room temperature. The binary mobile phase consisted of acetonitrile (solvent A) and water containing 0.4% acetic acid (solvent B). All solvents were filtered through a 0.45 ␮m filter prior to use. The system was run with a solvent A–solvent B (0 min,5:95,v/v; 10 min, 8:92,v/v; 15 min, 15:85,v/v; 30 min, 20:80,v/v; 70 min, 50:50,v/v; 71 min, 95:5,v/v; 80 min, 95:5,v/v; 81 min, 5:95,v/v; stop time: 120 min) and the effluent was monitored at 230 nm. Identification of HSCCC peak fractions was carried out by high resolution MS, 1 HNMR, 13 CNMR and X-ray spectra. 2.8. Measurement of intracellular levels of ROS, MDA and SOD The antioxidant activity was evaluated by measuring the intracellular ROS, SOD and MDA level in Caco-2 cells. ROS content was determined by the DCFH-DA method. Briefly, Caco-2 cells were seeded onto a 96-well culture plate. After

W. Zhao et al. / J. Chromatogr. B 990 (2015) 111–117 Table 1 The K (partition coefficient) values of urolithin A (K1) and urolithin B (K2) in different two-phase solvent systems used in HSCCC. Solvent system

K1

K2

n-Butanol-n-propyl alcohol-water (4:1:5, v/v/v) n-Butanol-n-propyl alcohol-water (2:1:3, v/v/v) n-Butanol-acetic acid-water (4:1:5, v/v/v) Ethyl acetate-methanol-water (5:1:5, v/v/v) n-Hexane-ethyl acetate-methanol-water (5:5:5:5, v/v/v/v) n-Hexane-ethyl acetate-methanol-water (4:5:4:5, v/v/v/v) n-Hexane-ethyl acetate-methanol-water (2:5:2:5, v/v/v/v) n-Hexane-ethyl acetate-methanol- acetic acid-water (2.5:2:0.05:5, v/v/v/v/v) n-Hexane-ethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5, v/v/v/v/v)

0.10 0.23 0.13 6.98 0.05 0.11 0.38 0.59

0.04 0.16 0.08 4.77 0.28 0.35 0.89 1.37

0.86

1.58

the urolithins treatment (10 ␮M urolithin A; 10 ␮M urolithin B; 10 ␮M urolithin A and 10 ␮M urolithin B; 0.10 mg/mL pomegranate ellagitannins (PE); 0.01 mg/mL intestinal extracts of pomegranate ellagitannins (PE-I); 10 ␮M vitamin E) was finished, cells were washed 3 times in PBS, and incubated with 100 mM H2 O2 . After 3 h incubation, the cells were washed with PBS and incubated with DCFH-DA at the concentration of 10 ␮M for 30 min at 37 ◦ C. Then the cells were washed with PBS and the DCFH-DA reactivity was measured with micro plate reader (Thermo scientific) at an excitation and emission wavelength of 485 nm and 538 nm, respectively. The SOD activity and MDA content were measured using a SOD activity assay kit and an MDA activity assay kit (Biyuntian, China) according to the manufacturer’s protocol, respectively.

3. Results and discussion 3.1. Selection of solvent system and other conditions of HSCCC The sample of intestinal bacteria culture solution of pomegranate ellagitannins was analyzed by HPLC. The result indicates that the sample, but not the control, contains several compounds including urolithins and some unknown compounds (Fig. 1). Successful selection of the two-phase solvent system is the critical step in an HSCCC experiment; the good solvent system can provide an ideal partition coefficient (K) for the target compounds. Small K-values and large K-values result in poor peak resolution or excessive sample band broadening. In our experiment, the measured K-values for urolithins in different solvent systems are summarized in Table 1. Several kinds of solvent systems including n-butanol-n-propanol-water (2:1:3,v/v/v), n-butanol-acetic acidwater (4:1:5,v/v/v) and n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5,v/v/v/v/v) were tested. The results indicated that the urolithin A had appropriate K values (0.5-2.0) basically in the two systems; the urolithin B has appropriate K values in three systems. The system composed of n-hexaneethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5, v/v/v/v/v) was able to achieve two-phase equilibrium very quickly and gave the best results (Table 1). Consequently, the pomegranate ellagitannins samples from the intestinal bacteria culture of pomegranate husk were separated by HSCCC using a solvent system composed of n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5,v/v/v/v/v), yielding three peak fractions (I–III, and the residual in the column) (Fig. 1A) among which fraction II and III contained urolithin A and urolithin B. This partially purified fraction was further subjected to HSCCC using the second solvent system composed of n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:0.8:0.25:5, v/v/v/v/v) (Fig. 1A insert). Under this

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condition 3.5 mL of the stationary phase was carried-over during every 200 mL of the mobile phase eluted and lost from the column. 3.2. Purity analyses by HPLC The HPLC chromatograms of these compounds are shown in Fig. 1. Three HSCCC fractions were obtained and the yields of fraction I, II, and III were 30, 5.0, and 2.0 mg, respectively. The analysis of these fractions indicated that peak I was impure and the purity of peak I was 47% of ellagic acid at 254 nm (Fig. 1B and C). It should be noted that the peak II, which was collected according to the shaded part in Fig. 1A, contained urolithin A at over 98.5% purity as measured from HPLC peak areas at 230 nm (Fig. 1C). The peak III was urolithin B at over 99.0% purity (Fig. 1C). Each injection of 100 mg crude extract yielded 21 mg of pure urolithin A and 10 mg of pure urolithin B. 3.3. Confirmation of chemical structure The structural identification of urolithin A was carried out by high resolution MS, 1 H NMR and 13 CNMR spectra as follows: High resolution-MS m/z: 227.03511 (M − 1) (Fig. 2A); 1 HNMR (300 MHz, DMSO-d6 ): ␦ppm 10.23 (br, s, 2H), 8.13(d, J = 8.7 Hz, 1H), 8.03(d, J = 8.7 Hz, 1H), 7.51(d, J = 2.7 Hz, 1H), 7.33(dd, J = 8.7, 2.7 Hz, 1H), 6.81(dd, J = 8.7, 2.4 Hz, 1H), 6.73(d, J = 2.4 Hz, 1H). 13 CNMR (125 MHz, DMSO-d6 ): ␦ppm 160.04, 158.86, 157.27, 151.39, 127.48, 124.54, 124.26, 124.03, 120.67, 113.97, 113.43, 110.36, 103.27. Comparing with the reported data, the high resolution MS, 1 HNMR and 13 CNMR data are in agreement with those of urolithin A (Fig. 2C)[27]. The structural identification of urolithin B was carried out by high resolution MS, 1 H NMR and 13 CNMR spectra as follows: High resolution-MS m/z: 211.03976 (M − 1) (Fig. 2B); 1 HNMR(300 MHz, DMSO-d6 ): ␦ppm 10.36 (s, 1H), 8.27(d, J = 8.1 Hz, 1H), 8.20(dt, J = 8.7, 2.4 Hz, 1H), 8.17(dd, J = 7.8, 2.4 Hz, 1H),7.99(dt, J = 8.1, 1.5 Hz, 1H), 7.57(dt, J = 7.8, 1.5 Hz, 1H), 6.85(dd, J = 8.1, 2.4 Hz, 1H), 6.76(d, J = 2.4 Hz, 1H). 13 CNMR (125 MHz, DMSO-d6 ): ␦ppm 161.08, 160.35, 152.61, 135.77, 135.59, 130.16, 128.14, 125.32, 122.13, 119.44, 113.65, 109.87, 103.43. Comparing with the reported data, the high resolution MS, 1 HNMR and 13 CNMR data are in agreement with those of urolithin B (Fig. 2D)[27]. 3.4. X-ray diffraction of urolithin A and urolithin B The XRD data of urolithin A and urolithin B are shown in Fig. 3. The crystal color of urolithin A and urolithin B is canary yellow and white, respectively. The diffractogram shows the diffraction pattern of urolithin B (Fig. 3A). However in the urolithin A (Fig. 3B), many additional peaks are observed at 2 values between 18 and 20◦ . Crystal structure determination of urolithin B as follows: Crystal data for C13 H8 O3 (M = 212.21): monoclinic, space group ˚ b = 13.3459(3) A, ˚ c = 7.26574(19) A, ˚ P21 /c (no. 14), a = 10.3193(3) A, ˛ = 90◦ , ˇ = 101.552(3)◦ ,  = 90◦ ,Volume= 980.37(4) Å3 , Z = 4, ␮(Cu K␣) = 0.851 mm−1 , Dcalc = 1.4376 g/mm3 , 5292 reflections measured (8.74 ≤ 2␪ ≤ 132.94), 1715 unique (Rint = 0.0227) which were used in all calculations. The final R1 was 0.0378 (I > = 2 (I)) and wR2 was 0.1055 (all data). The structure of urolithin B is given with Olex 2 software in Insret (Fig. 3A). 3.5. The levels of ROS, SOD and MDA in Caco-2 cells The level of H2 O2 -induced oxidative stress was reduced by urolithins and pomegranate ellagitannins to confirm the antioxidant activity of urolithins and pomegranate ellagitannins (Fig. 4).

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Fig. 1. HSCCC and HPLC analysis spectra. (A) Preparative HSCCC separation of urolithin A and urolithin B from the intestinal metabolites of pomegranate ellagitannins with two steps. Solvent system, n-Hexane-ethyl acetate-methanol- acetic acid-water (2.5:2:0.25:5, v/v/v/v/v) and n-Hexane-ethyl acetate-methanol-acetic acid-water (2.5:0.8:0.25:5, v/v/v/v/v); Solvent system, n-Hexane-ethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5, v/v/v/v/v); stationary phase, upper phase; mobile phase, lower phase; flowrate, 2.0 ml/min; sample, 100 mg dissolved in 20 ml of lower phase; revolution speed, 800 rpm; retention of stationary phase, 64%; detection wavelength, 230 nm; Peak I, unknown compound content ellagic acid; peak II (shaded portion), urolithin A; peak III (shaded portion), urolithin B; peak IIA in the insert illustration (shaded portion), urolithin A from the second step; peak IIIB in the insert illustration (shaded portion), urolithin B from the second step;. (B) HPLC analysis of the HSCCC separation fractions of peak 1(contain ellagic acid) and standard sample peak of ellagic acid at 254 nm; (C) HPLC analysis of HSCCC fraction of peak IIA (urolithin A), peak IIIB (urolithin B), control sample – the intestinal metabolites and original sample – the intestinal metabolites of pomegranate husk at 230 nm. Experimental conditions: HPLC column, Waters SymmetryR C18 column (4.6 mm × 250 mm, 5 ␮m); sample inject, 10 ␮L; column temperature, 25 ◦ C; mobile phase, acetonitrile-0.4% acetic acid (0 min,5:95,v/v; 10 min, 8:92,v/v; 15 min, 15:85,v/v; 30 min, 20:80,v/v; 70 min, 50:50,v/v; 71 min, 95:5,v/v; 80 min, 95:5,v/v; 81 min, 5:95,v/v; stop time: 120 min); flow-rate,1.0 ml/min; detection wavelength, 230 nm.

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Fig. 2. High resolution-MS of urolithins and Chemical structure of Urolithins. (A) High resolution-MS of urolithin A (negative mode); (B) High resolution-MS of urolithin B (negative mode). MS conditions, instrument model, Bruker FT-ICR-MS solarix Maldi/ESI 9.4T; data acquisition software, ftmsControl 2.0; data process software, DataAnalysis 4.1; source type, ESI; gas, 4 L/min; gas temperature, 180 ◦ C; Nebulize, 1.0 bar; polarity, negative; capillary, 4 KV; end plate offset, −500 V. (C) Chemical structure of Urolithin A. (D) Chemical structure of Urolithin A.

Both the intracellular ROS level (Fig. 4A) and the MDA level (Fig. 4B) significantly decreased, whereas the SOD activity (Fig. 4C) is significantly higher than that of negative control. The components of pomegranate ellagitannins might appear to synergistically resist oxidation. The MDA level of the combination of urolithin

A and urolithin B groups were decreased more significantly than those of the single compound treatment groups (Fig. 4B). The combination of urolithin A and urolithin B might be beneficial to human health and useful for the further pharmacology study.

Fig. 3. X-ray diffraction of urolithins. (A) Single crystal X-ray diffraction of urolithin B; (B) X-ray diffraction of urolithin A. Diffraction conditions, the Bruker AXS D8 Advance, Cu K␣-targets at a scanning rate of 0.010 2␪/s, 40 kV, 40 mA.

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Fig. 4. Effects of Urolithin A, Urolithin B, pomegranate ellagitannins (PE), the intestinal extracts of pomegranate ellagitannins (PE-I);Urolithin A + Urolithin B, and Vitamin E on H2 O2 -induced oxidative stress in Caco-2 cells. Oxidative stress was assessed by measuring the intracellular ROS level (A) SOD level (B), MDA level (C). The values given are the mean ± SD of six independent experiments. n = 6 # p < 0.05 (compared with the Control group).

4. Conclusion

References

Some of the chemical constituents of intestinal bacteria culture solution of pomegranate husk were isolated and purified systematically by HSCCC. The above results of our studies clearly demonstrated that two steps chromatographic separation by preparative HSCCC is able to yield pure urolithins from the intestinal bacteria culture solution of pomegranate. But for HPLC, the intestinal metabolites of pomegranate ellagitannins has to be prepurified with sephadex LH-20 before separation using preparative HPLC as we found that the RP-C 18 column might be polluted with injection of crude sample (1.0 mg/ml × 10 ␮l) for three times. Compared to preparative HPLC, the method of separating urolithins by HSCCC has no irreversible adsorption effects of analytes to solidphase column material, high sample loading capacity, complete sample recovery and saves time. The results provided a successful pattern for isolation of bioactive compounds from the mechanism by HSCCC. We believe that the method may be successfully applied to the separation of other polyphenols mechanism by selecting a suitable two-phase solvent system.

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Acknowledgments This work is supported by National Science Foundation of China (21201124), TJSHG (201310025008), the Scientific Research Common Program of Beijing Municipal Commission of Education (KM201310025007), the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (CIT&TCD201304176) and the Beijing Municipal Science & Technology Commission (Z141100002114049).

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