Poliumoside, one of the major phenylethanoid glycosides (PhGs) abundantly found in Callicarpa kwangtungensis Chun (Verbenaceae, CK), is a key ingredient in a Chinese patented medicine named "Kang-Gong-Yan Tablet" or "Kang Gong Yan Capsule", which has been widely used for treating gynecological diseases for nearly 20 years . Poliumoside also exists in many other plants, such as Cistanche sinensis G. Beck , Buddleja officinalis Maxim , and Brandisia hancei Hook. F . A broad range of biological activities have been reported for poliumoside, including antioxidation , hemostasis , neuroprotection , and cell aggregation inhibition .
The chemical structure of poliumoside consists of three chemical moieties: caffeic acid (CA), hydroxytyrosol (HT, 3, 4-dihydroxyphenethyl alcohol, phenylethanoid aglycone), and two rhamnose (Rha) groups. The chemical bonds linking these moieties are easily hydrolysed in biological environments . Recently, we reported that the bioavailability of poliumoside is 0.69% in rats . The complex microbial ecosystem in the gastrointestinal tract (GIT) is generally considered as a separated organ with a powerful metabolic capacity [11-12]. The metabolism in the GIT may directly decrease the absorption of poliumoside, ultimately resulting in its low bioavailability. Moreover, the metabolites in plasma, urine, and bile have not been determined. Therefore, it is of great value to explore the metabolism of poliumoside, especially by the intestinal bacteria.
High resolution mass spectrometry, such as quadrupole time-of-flight mass spectrometry (Q-TOF-MS), linear ion trap quadrupole Orbitrap mass spectrometry (LTQ-Oribitrap-MS), and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), can provide information pertaining to the molecular weight, elemental composition, and molecular structures of targeted metabolites, and has been broadly used in biological sample analysis, such as chemical profiling , metabolomics studies , and metabolite identification [15-17].
In the present study, we established an UPLC/Q-TOF-MS method for identification of metabolites of poliumoside in rat plasma, urine, and bile after oral administration. The metabolites, metabolic pathways, and metabolite fragmentation patterns were proposed. We also investigated the metabolic process and metabolites of poliumoside after incubated with rat intestinal bacteria.Materials and Methods Chemicals and reagents
Acteside, hydroxytyrosol (HT), CA, and 3-hydroxyphenylpropionic acid (3-HPP), all with purity > 98%, were purchased from Aladdin Industrial Inc. (Shanghai, China). Tryptone soya broth (TSB) was purchased from Beijng Aoboxing Bio-Tech Co. Ltd. (Beijing, China). Analytical grade trichloroacetic acid (TCA) was purchased from Tianjin Chemical Corp. (Tianjin, China). Isoacteoside and poliumoside (Fig. 1, purity > 98%, as determined by HPLC-UV) used for oral administration were isolated from Cistanche sinensis in our laboratory and their chemical structures were confirmed by MS, NMR, and HPLC. Acetonitrile, methanol, and formic acid used in the present study were all purchased from Thermo Fisher Scientific (USA).
Chromatographic separation was performed on an Acquity UPLC system with a BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, MA, USA). An aliquot of the sample (5 μL) was injected onto the UPLC system for analysis. A gradient elution was performed with formic acid (0.1%) in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.4 mL·min-1. The gradient elution process was as follows: 5% B from 0 to 1 min, 5% to 20% B from 1 to 2 min, 20% to 80% from 2 to 8 min, 80% to 95% from 8 to 9 min, then back to 5% for 1 min before the next run. Before the next injection, a needle wash program was performed to minimize pollution. The solvent for the needle wash contained methanol and water (50 : 50, V/V). The column temperature was maintained at 45 ℃.
Mass spectrometry was performed on a Xevo G2 Q-TOF MS (Waters), equipped with the electronic spray ion (ESI) under the negative ion mode. The extraction cone, sampling cone, and capillary voltages were 4, 40, and 3000 V, respectively. Nitrogen gas, used as the cone and desolvation gas, was set at 30 and 800 L·h-1, respectively. The temperature of the source and desolvation was set at 100 and 250 ℃, respectively. Mass spectra was recorded in the range of m/z 50-1000. An external reference (2 μg·mL-1 of leucine enkephalin, m/z 556.277) was infused at 5 μL·min-1 to guarantee the mass accuracy of the entire assay. The mass-to-charge ratio of the poliumoside was m/z 769.2537. The data for the metabolite identification was acquired in the MS/MS mode, and the collision energy was set at 35 eV. The MS identification for the metabolites was performed on full scan analysis and extracted ion chromatograms using MassLynx, version 4.1.Animals
Male Sprague-Dawley (SD) rats, weighing 220-250 g, were obtained from the Laboratory Animal Center of Jinan University (Guangdong, China). The rats were housed in a humidity-controlled room maintained on 12 h/12 h light/dark cycle for 5 days before the experiment. Standard animal food and water were provided ad libitum. Six rats were anesthetized using 10% chloral hydrate and the jugular veins were cannulated for blood collection 24 h before the experiment. Diet was prohibited for 12 h before the experiment, while water was provided ad libitum. All the procedures were approved by the Animal Care and Ethics Committee of Jinan University (No. 20160423-01; Guangzhou, China).Sample collection and pretreatment
Poliumoside, dissolved in saline, was orally administrated to rats at a dose of 1.5 g·kg-1. Approximately 0.3 mL of blood samples were collected from the jugular vein at 0, 30, 60, 120, and 240 min after administration and transferred into 1.5-mL heparinized centrifuge tubes. The blood samples were immediately centrifuged (5000 g, 4 ℃, 8 min), and the supernatant (plasma) was collected. Urine samples were collected for 0-36 h after administration. Bile samples were collected for 0-8 h through a plastic cannula that had been surgically inserted into the bile duct as described previously . All the biological samples were immediately stored at -80 ℃ after collection.Sample analysis
The plasma samples were thawed at room temperature before use and put on ice . Extracts were obtained with the protein precipitation method with 10% TCA. In brief, an aliquot of 100 μL of 10% TCA was added to the 100 μL of plasma samples in 1.5-mL polypropylenes tubes, followed by vortexing for 30 s. The mixture was centrifuged (13 000 g, 4 ℃, 15 min) and the supernatant was collected and transferred into a new tube. The supernatant (5 μL) was injected into the UPLC/Q-TOF-MS system for analysis.
Aliquots of 5 mL of urine were loaded onto a solid phase extraction (SPE) column with a SupelcleanTM C18 cartridge (500 mg, 3 mL), which was activated well with 5 mL of methanol and 5 mL of distilled water. The SPE column was washed with 2 mL of water and 1 mL of methanol was added to elute the metabolite. The eluent (5 μL) was injected into the UPLC/Q-TOF-MS system for analysis. The processing method for the bile was consistent with that of urine samples. Aliquots of 2 mL of bile were loaded onto a activated SPE column, then The SPE column was washed with 2 mL of water and 1 mL of methanol was added to elute the metabolite.Metabolism study of poliumoside by rat intestinal bacteria in vitro
Fresh fecal samples were obtained from male Sprague-Dawley rats. The feces were mixed and immediately homogenized with 5-time volume of TSB. The mixed solutions were centrifuged (3000 g, 4 ℃, 8 min) and the supernatant was transferred onto a culture dish. The dish was then placed into an anaerobic culture bag with a gas bag (AnaeroPack, Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) that could convert oxygen to carbon dioxide. The culture bag was placed into an incubator (70 r·min-1, 37 ℃, 24 h) for the breeding of intestinal bacteria. Fecal lysate was incubated with poliumoside at an initial concentration of 50 mmol·L-1. At 0, 0.5, 1, 2, 4, 6, and 8 h, aliquots of 500 μL of the solutions were removed and mixed with a 500 μL of methanol. The mixture was centrifuged (13 000 g, 15 min). An aliquot of 5 μL of the supernatant was injected into the UPLC/Q-TOF-MS for further analysis.Results and Discussion Identification of metabolites in vivo
In the present study, the contents of poliumoside, acteoside, isoacteoside, CA, HT, and 3-HPP were analyzed by the UPLC/Q-TOF-MS method (Table 1). The proposed fragmentation pathways of poliumoside are shown in Fig. 2. Full scan mass spectra of biological samples from rats after oral administration were compared with blank samples to screen for the parent compound and its potential metabolites (Fig. 3). A total of 34 metabolites were detected in rat plasma, urine, and bile samples. The detailed mass spectral data are shown in Table 2. Moreover, the metabolites, acteoside, and M25 (m/z 877.2558), were found to be the major metabolites in urine and bile samples, respectively. Fig. 4 shows the proposed metabolic pathways of poliumoside, including rearrangement, reduction, hydration, hydrolyzation, dehydration, methylation, hydroxylation, acetylation, and sulfation. The main metabolites are described in detail in the following section.
The fragmentation pattern analysis of the parent compound is of great value for identifying the metabolites. 2 μg·mL-1 of poliumoside authentic samples prepared in 50% methanol were used for the fragmentation pattern study. Poliumoside (m/z 769.2537, C35H45O19, 4.62 min) produced a fragment ion at m/z 607.2238 by loss of a CA moiety, and it then generated an ion at m/z 461.1647 by the further loss of a Rha moiety. The CA moiety was found at m/z 179.0328 with its fragment ions appearing at m/z 161.0237 and m/z 135.0448, by the loss of one H2O and one CO2, respectively. M0a (m/z 769.2537, C35H45O19, 4.62 min) was identified by comparing the UPLC retention time, the accurate MS, and MS/MS spectra to those of the authentic standard of poliumoside. M0b (m/z 769.2546, C35H45O19, 5.14 min) was tentatively identified as isopoliumoside.Degradation products of poliumoside
M6a (m/z 623.1998, C29H35O15, 4.25 min) and M6b (m/z 623.2003, C29H35O15, 4.63 min) were 146 Da (C6H10O4) less than M0. The UPLC retention time, accurate MS, and MS/MS spectra were the same as the reference standards. Thus, M6a and M6b were confirmed as acteoside and isoacteoside, respectively. M6a was the main metabolite found in urine, which was in accordance with the metabolite found in feces . This also provided further evidence that the acteoside transformed from poliumoside was likely performing a pro-drug function.
M1 (m/z 179.0328, C9H7O4, 1.99 min) was clearly assigned as CA since it shared the same UPLC retention time, accurate MS, and MS/MS spectra with the authentic standards. M7 (m/z 161.0228, C9H6O3, 2.79 min) was 18 Da (H2O) less than M1 by the loss of H2O. The MS/MS spectrum showed the product ion at m/z 117.0526 by the loss of CO2. Ultimately, M7 was identified as dehydrated caffeic acid.
M13 (m/z 165.0542, C9H9O3, 3.38 min) showed the same characteristics as 3-HPP. The MS/MS spectrum displayed a fragment ion at m/z 121.0545, formed by the elimination of CO2. Therefore, M13 was assigned as 3-HPP.
M5 (m/z 607.2197, C26H39O16, 2.41 min) was 162 Da (C9H6O3) less than poliumoside. The fragment ion of M5 at m/z 461.1573 and m/z 135.0447 further suggested that M5 was decaffeoyl poliumoside. According to the MS and MS/MS characteristics of M14 (m/z, 181.0519), it was tentatively identified as cis-Kankanoside G .
M4 (m/z 461.1673, C20H29O12, 3.25 min) was 146 Da (C6H10O4) less than M5. From the MS/MS spectrum of M4, the fragment ion at m/z 315.1097 could be formed by the loss of a Rha moiety from the deprotonated molecular ion of M4. Consequently, M4 was tentatively identified as the de-CA and de-Rha product of M0a. M3 (m/z 315.1107, C14H19O8, 4.90 min) was 146 Da (C6H10O4) less than M4, by the loss of Rha. The fragment ion at m/z 153.0502 was attributed to the HT group, with the additional peak at m/z 135.0447 generating by the loss of an H2O. Thus, M3 appeared to be the de-CA and de-2 Rha product of M0.
M10 (m/z 459.1474, C20H27O12, 3.40 min) was 2 Da (2H) less than M4, and it was considered as the dehydrogenated metabolite of M4. As part of the same metabolic pathway, M11 (m/z 313.0939, C14H17O8, 5.07 min) and M8 (m/z 767.2488, C37H49O19, 4.84 min) were considered to be the dehydrogenated metabolites of M3 and M0a, respectively. The MS/MS spectra of these metabolites showed the same fragment ion of m/z 151.0428, suggesting that the elimination occurred on part of the HT moiety.
M12 (m/z 625.2107, C29H37O15, 4.39 min) was 2 Da more than M6a, indicating that M12 was a reduced product of M6a. In the MS/MS spectra, the fragment ion at m/z 181.0503 implied that reduction occurred on part of the CA moiety. And the bacteria could reduce CA to dihydro-CA  in vitro. In addition, other fragment ions were consistent with the product ions of M6a.Methylated, sulfated and glucuronide metabolites
M2 (m/z 193.0507, C10H10O4, 4.85 min) was 14 Da more than CA. The MS/MS spectrum showed the fragment ion at m/z 149.0614 by the loss of CO2. Thus, M2 was considered to be methylated CA. M16 (m/z 653.2108, C30H37O16, 7.52 min) was 30 Da more than M6a, suggesting that M16 was the metabolite from methoxylation, or separate methylation and hydroxylation. M16 produced a fragment ion at m/z 151.0427, indicating that the conjugation occurred at the HT moieties. M17 (m/z 703.1484, C29H35O18S, 4.13 min) was 80 Da (SO3) more than M6. In the MS/MS spectrum of M17, the fragment ions at m/z 623.2047 and m/z 541.1314 were formed by the loss of SO3 and CA, respectively. Thus, M17 was proposed to be the sulfated metabolite of M3 and the conjugation site was proposed to be at the HT moiety. The proposed fragmentation mechanism of M17 is shown in Fig. 5. M18 (m/z 329.0867, C14H17O9, 5.33 min) was 176 Da more than HT, suggesting that it was a glucuronide conjugate product of HT. In the MS/MS spectrum of M18, the fragment ions at m/z 175.0438 and m/z 153.0507 lend further support to our proposed structures.
M21 (m/z 797.3002, C37H49O19, 6.00 min) was 28 Da more than M0a, indicating that M21 was a dimethyl product of poliumoside. Based on the fragment ions at m/z 193.0502 and m/z 475.1824 of M21, it was proposed that the dimethylation occurred separately at the hydroxyls of the CA and HT moieties. Meanwhile, according to literature , methylation in the two contiguous catechol hydroxyls can't happen, which supports our speculation. Nevertheless, because of the limited information, the accuracy of the conjugation site cannot be confirmed.
M22 (m/z 799.2716, C36H47O20, 4.13 min) was 30 Da more than M0a, suggesting that it was the metabolite of M0a conjugated with methyl and hydroxyl at different positions or it was the methoxylated metabolite. In the MS/MS spectrum of M22, the fragment ion at m/z 637.23 was derived from the loss of a CA moiety, suggesting that the conjugation mostly occurred at the HT moiety, and the fragment ion of m/z 491.1717 was formed by the subsequent loss of a Rha. Because of the limited information, we could not confirm the accurate position for the methyl and hydroxyl groups. The proposed fragmentation pattern of M22 is shown in Fig. 5.
M24 (m/z 785.2547, C35H45O20, 3.84 min) was 16 Da higher than M0, and it was considered to be a hydroxylated metabolite. In the MS/MS spectra of M24, the fragment ion at m/z 151.0427 was ascribed as the dehydrogenated HT group, suggesting that the hydroxylation site of M24 was at the position of the HT moiety. The other fragments of M24 were consistent with poliumoside and in accordance with β-OH poliumoside , which supported the structural assignment.
M25 (m/z 877.2558, C37H49O19, 6.88 min) was 80 Da (SO3) more than M21 and thus considered as a sulfated conjugate of M21. In the MS/MS spectra of M25, the fragment ions at m/z 475.1823 and m/z 193.0502 suggested that the dimethylation occurred separately at the hydroxyl groups of the CA and HT moieties. Moreover, the fragment ion at m/z 683.1824 indicated that the sulfated conjugation site was at the HT moiety. Sulfated metabolites could improve the water solubility of poliumoside and accelerate its elimination. The other fragments at m/z 651.2122, m/z 621.2439, and m/z 603.2346 all supported our proposed structure. Moreover, M25 was largely found in bile samples. The proposed fragmentation pattern of M25 is shown in Fig. 6.
M26 (m/z 827.2632, C37H47O21, 4.43 min) was 42 Da (C2H2O) more than M24. Thus, M26 was assigned as the hydroxylated and acetylated metabolite of M0a. In the MS/MS spectrum of M26, the fragment at m/z 809.2585 originated from m/z 827.2632 by the loss of H2O, and the fragment ion at m/z 647.2213 was formed by the continuous loss of CA. Thus, the hydroxyl group was likely conjugated at the HT moiety. The other fragments at m/z 605.2117 and m/z 151.0428 suggested that the acetyl group was also positioned at the HT moiety. The proposed fragmentation is shown in Fig. 6.
M28 (m/z 787.2617, C35H47O20, 3.98 min) was 18 Da (H2O) more than M0a. In the MS/MS spectrum of M28, a series of fragment ions was seen at m/z 769.2323, m/z 607.2237, m/z 461.1764, m/z 179.0345, and m/z 161.0239, which were consistent with the fragment ions of M0a. Therefore, M28 was proposed as the hydrated metabolite of M0a, and the hydration site was mostly at the CA moiety.
M29 (m/z 811.2745, C37H47O20, 5.59 min) was 42 Da (C2H2O) higher than M0a, suggesting that it was the acetylated metabolite of poliumoside. The MS/MS spectrum showed the product ion at m/z 649.2403 formed from m/z 811.2745 by the loss of CA. The other fragment ions of M29 were in accordance with poliumoside. Therefore, the site of the acetyl group was at the HT moiety. Fig. 6 shows the proposed chemical structure and the fragmentation mechanism of M30.
M30 (m/z 849.2154, C35H45O22S, 4.39 min) was 80 Da (SO3) more than M0a. In the MS/MS spectrum, the fragment ions at m/z 769.2331 and m/z 687.1947 were formed by the loss of SO3 and Rha, respectively. M30 was assigned as a sulfated conjugated product, and the conjugation site was mostly at the HT moiety based on these fragmentations.Metabolism of poliumoside by gut bacteria in vitro
From the UPLC-MS analysis, we clearly found one metabolite and its concentration changed with incubation time. Based on the idea that the m/z 623.1998 fragment ion was derived from the metabolite, we suspected that the metabolite was acteoside. In the subsequent analysis, the standard solution of acteoside was used for comparison and the metabolite demonstrated the same characteristics as the standard, in terms of retention time, accurate MS, and MS/MS spectra. Therefore, we concluded that the metabolite was acteoside. We also measured the concentrations of poliumoside and the metabolite in the fecal lysate at different times of incubation. After 8 h of in vitro incubation and the UPLC-MS analysis, no other obvious metabolites were found in the samples. Fig. 7 shows the time-course for the bacterial metabolism of poliumoside. Acteoside (m/z 623.1998, C29H35O15, 4.25 min) produced an ion that was similar to that produced from poliumoside at m/z 461.1647, m/z 179.0345, m/z 161.0246, and m/z 135.0447. The proposed fragmentation pathway of acteoside is shown in Fig. 2. As the main metabolite, acteoside exhibits various activities including antibacterial , neuroprotective , antioxidant , and anti-inflammatory effects .
In the present study, the in vivo and in vitro metabolic profiles of poliumoside were thoroughly investigated using a UPLC/Q-TOF-MS method. In vitro, poliumoside was incubated with fecal lysate under anaerobic conditions, and acteoside was identified as the major metabolite. As an active compound, acteoside may function as an intermediate for poliumoside in performing its pharmacological activities. In vivo metabolites in plasma, urine, and bile samples were identified by UPLC/Q-TOF-MS and the MS/MS method after oral administration of poliumoside. A total of 34 metabolites and 9 metabolic pathways (rearrangment, reduction, hydration, hydrolyzation, dehydration, methylation, hydroxylation, acetylation, and sulfation) were proposed. The amounts of metabolites found in plasma were very low, and the metabolites of acteoside and M25 mainly existed in urine and bile, respectively. In summary, the present study presented a fast and accurate method for systematically identifying metabolites in vitro and in vivo. The findings presented here lay the foundation for further studies of poliumoside metabolism.
Jia A, Yang YF, Kong DY, et al. GC-MS analysis of chemical constituents of essential oil from Callicarpa kwangtungensis and their antimicrobial activity[J]. J Chin Med Mater, 2012, 35(3): 415-418.
Tu PF, Shi HM, Song ZH, et al. Chemical constituents of Cistanche sinensis[J]. J Asian Nat Prod Res, 2007, 9(1): 79-84. DOI:10.1080/10286020500384450
Tai BH, Jung BY, Cuong NM, et al. Total peroxynitrite scavenging capacity of phenylethanoid and flavonoid glycosides from the flowers of Buddleja officinalis[J]. Biol Pharm Bull, 2009, 32(12): 1952-1956. DOI:10.1248/bpb.32.1952
Yu SY, Lee IS, Jung SH, et al. Caffeoylated phenylpropanoid glycosides from Brandisia hancei inhibit advanced glycation end product formation and aldose reductase in vitro and vessel dilation in larval zebrafish in vivo[J]. Planta Med, 2013, 79(18): 1705-1709. DOI:10.1055/s-00000058
De Marino S, Festa C, Zollo F, et al. Antioxidant activity of phenolic and phenylethanoid glycosides from Teucrium polium L.[J]. Food Chem, 2012, 133(1): 21-28. DOI:10.1016/j.foodchem.2011.12.054
He ZD, Lau KM, Xu HX, et al. Antioxidant activity of phenylethanoid glycosides from Brandisia hancei[J]. J Ethnopharmacol, 2000, 71(3): 483-486. DOI:10.1016/S0378-8741(00)00189-6
Koo KA, Sung SH, Park JH, et al. In vitro neuroprotective activities of phenylethanoid glycosides from Callicarpa dichotoma[J]. Planta Med, 2005, 71(8): 778-780. DOI:10.1055/s-2005-871213
Tatli II, Takamatsu S, Khan I, et al. Screening for free radical scavenging and cell aggregation inhibitory activities by secondary metabolites from Turkish Verbascum species[J]. Z Naturforsch C, 2007, 62(9-10): 673-678. DOI:10.1515/znc-2007-9-1008
Li Y, Zhou GS, Peng Y, et al. Screening and identification of three typical phenylethanoid glycosides metabolites from Cistanches Herba by human intestinal bacteria using UPLC/ Q-TOF-MS[J]. J Pharm Biomed Anal, 2016, 118: 167-176. DOI:10.1016/j.jpba.2015.10.038
Qian H, Lu DY, Li W, et al. Validated UPLC/Q-TOF-MS method for determination of poliumoside in rat plasma and its application to pharmacokinetic study[J]. Am J Anal Chem, 2016, 7(3): 266-274. DOI:10.4236/ajac.2016.73024
Payne AN, Zihler A, Chassard C, et al. Advances and perspectives in in vitro human gut fermentation modeling[J]. Trends Biotechnol, 2012, 30(1): 17-25. DOI:10.1016/j.tibtech.2011.06.011
Wang P, Chen HD, Zhu YD, et al. Oat avenanthramide-C (2c) is biotransformed by mice and the human microbiota into bioactive metabolites[J]. J Nutr, 2015, 145(2): 239-245. DOI:10.3945/jn.114.206508
Wang P, Wang B, Xu JY, et al. Detection and chemical profiling of Ling-Gui-Zhu-Gan decoction by ultra performance liquid chromatography-hybrid linear ion trap-Orbitrap mass spectrometry[J]. J Chromatogr Sci, 2015, 53(2): 263-273. DOI:10.1093/chromsci/bmu051
Zhu YD, Wang P, Sha W, et al. Urinary biomarkers of whole grain wheat intake identified by non-targeted and targeted metabolomics approaches[J]. Sci Rep, 2016, 6: 36278. DOI:10.1038/srep36278
Wang P, Zhao YL, Zhu YD, et al. Metabolism of dictamnine in liver microsomes from mouse, rat, dog, monkey, and human[J]. J Pharm Biomed Anal, 2016, 119: 166-174. DOI:10.1016/j.jpba.2015.11.016
Wang P, Chen HD, Sang SM. Trapping methylglyoxal by genistein and its metabolites in mice[J]. Chem Res Toxicol, 2016, 29(3): 406-414. DOI:10.1021/acs.chemrestox.5b00516
Men L, Lin HL, Zhao YL, et al. Metabolism of TM-2, a potential antitumor drug, in rats by using LC-MS[J]. J Sep Sci, 2014, 37(6): 625-629. DOI:10.1002/jssc.201301251
Liu HM, Sun H, Lu DY, et al. Identification of glucuronidation and biliary excretion as the main mechanisms for gossypol clearance: in vivo and in vitro evidence[J]. Xenobiotica, 2014, 44(8): 696-707. DOI:10.3109/00498254.2014.891780
Sun XM, Liao QF, Liu GH, et al. Simultaneous determination of three phenylethanoid glycosides from Callicarpae Caulis et Folium in rat plasma by LC-MS/MS and its application to PK study[J]. Bioanalysis, 2013, 5(15): 1883-1895. DOI:10.4155/bio.13.124
Deng R, Xu YH, Feng F, et al. Identification of poliumoside metabolites in rat feces by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry[J]. J chromatogr B, 2014, 965: 285-296.
Zhang JY, Li C, Che Y, et al. LTQ-Orbitrap-based strategy for traditional Chinese medicine targeted class discovery, identification and herbomics research: a case study on phenylethanoid glycosides in three different species of Herba Cistanches[J]. RSC Adv, 2015, 5(98): 80816-80828. DOI:10.1039/C5RA13276B
Peppercorn MA, Goldman P. Caffeic acid metabolism by bacteria of the human gastrointestinal tract[J]. J Bacteriol, 1971, 108(3): 996-1000.
Mannisto PT, Kaakkola S. Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[J]. Pharmacol Rev, 1999, 51(4): 593-628.
Cai H, Xie ZY, Liu GH, et al. Isolation, identification and activities of natural antioxidants from Callicarpa kwangtungensis Chun[J]. PLoS One, 2014, 9(3): e93000. DOI:10.1371/journal.pone.0093000
Rao YK, Lien HM, Lin YH, et al. Antibacterial activities of Anisomeles indica constituents and their inhibition effect on Helicobacter pylori-induced inflammation in human gastric epithelial cells[J]. Food Chem, 2012, 132(2): 780-787. DOI:10.1016/j.foodchem.2011.11.037
Owen RW, Haubner R, Mier W, et al. Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes[J]. Food Chem Toxicol, 2003, 41(5): 703-717. DOI:10.1016/S0278-6915(03)00011-5
Diaz AM, Abad MJ, Fernandez L, et al. Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro anti-inflammatory activity[J]. Life Sci, 2004, 74(20): 2515-2526. DOI:10.1016/j.lfs.2003.10.008