Abstract
Polygoni Multiflori Radix Praeparata (PMRP), as the processed product of tuberous roots of Polygonum multiflorum Thunb., is one of the most famous traditional Chinese medicines, with a long history. However, in recent years, liver adverse reactions linked to PMRP have been frequently reported. Our work attempted to investigate the chemical constituents of PMRP for clinical research and safe medication. In this study, an effective and rapid method was established to separate and characterize the constituents in PMRP by combining ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). Based on the accurate mass measurements for molecular and characteristic fragment ions, a total of 103 compounds, including 24 anthraquinones, 21 stilbenes, 15 phenolic acids, 14 flavones, and 29 other compounds were identified or tentatively characterized. Forty-eight compounds were tentatively characterized from PMRP for the first time, and their fragmentation behaviors were summarized. There were 101 components in PMRP ethanol extract (PMRPE) and 91 components in PMRP water extract (PMRPW). Simultaneously, the peak areas of several potential xenobiotic components were compared in the detection, which showed that PMRPE has a higher content of anthraquinones and stilbenes. The obtained results can be used in pharmacological and toxicological research and provided useful information for further in vitro and in vivo studies.
Keywords: polygoni multiflori radix praeparata, UHPLC-Q-Exactive Orbitrap-MS, chemical constituents, traditional Chinese medicine
1. Introduction
Polygoni Multiflori Radix Praeparata (Zhiheshouwu in Chinese, PMRP), as a processed root of Polygonum multiflorum Thunb. (heshouwu in Chinese, PMR), has a long history in clinical application. The common processing method of PMRP is steaming or boiling PMR with a black bean decoction, as prescribed by the Chinese Pharmacopoeia. After processing, the concentrations of major components and traditional usage have changed. PMR contains more combined anthraquinones, while fewer stilbenes and more free anthraquinones are found in PMRP [1]. PMRP could enhance immune function, nourish the liver and kidney, prevent premature loss of hair, protect the nervous system, and inhibit atherosclerosis et al. [2,3,4]. Modern research has revealed that anthraquinones, stilbenes, flavonoids, and phenolic acids in PMRP are the major compounds of its pharmacological activities [5,6]. Several polyhydroxy stilbenes such as 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside(THSG) have a similar structure to resveratrol, and they have also been proven to have a strong ability to antioxidize and perform free radical scavenging activities [7]. Besides, THSG show great lipid-regulation and protection against neurotoxicity [8]. Anthraquinones are the major compounds with extensive activity, such as anti-tumor, antibacterial, and neuroprotective effects. Emodin induces neuronal differentiation through PI3K/Akt/GSK-3β pathways in Neuro2a cells [9]. Three anthraquinones, including physcion, emodin, and questin, were regarded as Cdc25B phosphatase inhibitors by strongly inhibiting the growth of human colon cancer cells [10]. Proanthocyanidins, isolated from MPRP, have the potential to be functional ingredients in reducing postprandial hyperglycemia, by inhibiting α-amylase and α-glucosidase [11].
However, with the widespread application of PMRP in the clinic, many adverse events of PMRP, including dyspnea, fever, rash, nephrotoxicity, and hepatotoxicity, have been reported in many countries such as Japan, China, Korea, Italy, Singapore, Spain, Australia, and the USA [12,13,14]. As the main organ of drug metabolism, the liver seems to be more susceptible to xenobiotic components. Therefore, the incidence of liver injury induced by PMRP has increased year by year [15]. Though some compounds of PMRP have positive physiological effects [16,17], there have been many studies reporting that several xenobiotic compounds could induce idiosyncratic hepatotoxicity. Anthraquinones are generally assigned as the major compounds of xenobiotics, because other anthraquinone-containing herbal medicines were also reported to induce liver injury [18,19]. Constituents other than anthraquinones, such as stilbenes and phenolic acids, were also considered to have a major contribution to the idiosyncratic hepatotoxicity of PMRP [20]. To find the potentially xenobiotic components and mechanisms of hepatotoxicity, qualitative and quantitative research has been explored. Zhang et al. [21] reported that the emodin-8-O-β-d-glucoside (EG) could induce hepatotoxicity, and the combination of EG and THSG could cause more severe liver injury. Moreover, in previous literature, the THSG, physcion and emodin showed no, moderate and severe cytotoxicity, respectively [22]. Rhein, which has weaker toxicity than emodin, has been demonstrated to exert concentration- and time-dependent toxic effects on L-02 cells [23].
At present, only a few compounds have been explored in xenobiotic studies. Considering the multi-component and multi-target characteristics of traditional Chinese medicine, the chemical constituents of PMRP should be identified for further studies. Meanwhile, previous studies have suggested that PMRP, extracted with different extraction solvents, showed various degrees of liver injury, and the order of toxicity was described as PMRP ethanol extract (PMRPE) > PMRP water extract (PMRPW) [24,25]. Consequently, to identify and compare the different components between PMRPE and PMRPW, an effective and sensitive ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) method was established for characterization of the constituents of them. The results of this investigation are meaningful, and would provide a material basis for further pharmacological and toxicological studies.
2. Results and Discussion
2.1. Optimization of LC and MS Conditions
LC conditions including mobile phase, flow rate, column, and column temperature were optimized to obtain a good separation and resolution. Compared with methanol, using acetonitrile as the organic phase showed stronger elutive power and detection sensitivity. Due to most compounds in PMRP contain carboxyl and phenolic hydroxyl, the addition of 0.1% formic acid in the phase system can obtain better mass spectrometric responses and improve the shapes of most peaks. Therefore, the mobile phase was acetonitrile (A)-0.1% formic acid in water (B), with optimized gradient elution. The Waters HSS T3 column (2.1 mm × 100 mm, 1.8 μm, UK) is suitable for the high polar compounds and high percentage of the aqueous phase, which have been applied to the characterization of the constituents of other botanical extracts. For the MS conditions, we chose the negative mode by comparing the intensity of compounds in both positive and negative modes. Meanwhile, according to the base peak intensity chromatograms (BPC), more compounds can be detected in the negative mode. Finally, other MS parameters were optimized to obtain high sensitivity for most compounds. The results indicated that the UHPLC-Q-Exactive Orbitrap-MS developed in this study is appropriate to detect the chemical constituents in PMRP.
2.2. Identification of the Chemical Constituents in PMRP
An in-house database that includes chemical names, molecular formulas, accurate molecular mass, chemical structures, and relevant fragments was established by searching Science Direct of Elsevier, Chemspider, PubMed, and CNKI (Chinese National Knowledge Infrastructure). We used Xcalibur™ and TraceFinder to obtain accurate mass, elemental composition, and multiple-stage mass data. By matching the in-house database to compare and characterize the compounds in PMRPE and PMRPW, these formulas which have been reported in the literature can be considered. A total of 103 chemical constituents were tentatively represented, including 24 anthraquinones, 21 stilbenes, 15 phenolic acids, 14 flavones, and 29 other compounds. The base peak intensity chromatogram (BPC) is shown in Figure 1 and Figure 2. The details of the identified compounds are summarized in Table 1 and the chemical structures of major constituents are shown in Figure S1.
Figure 1.
UHPLC-Q-Exactive Orbitrap-MS base peak intensity chromatogram (BPC) of PMRPE.
Figure 2.
UHPLC-Q-Exactive Orbitrap-MS base peak intensity chromatogram (BPC) of PMRPW.
Table 1.
Chemical constituents identified in PMRP by UHPLC-Q-Exactive Orbitrap-MS.
| NO. | RT (min) |
Identification | Molecular Formula |
Measured Mass [M−H]− |
Accuracy Mass[M−H]− |
Error (ppm) |
Characteristic Fragment Ions | Source |
|---|---|---|---|---|---|---|---|---|
| Anthraquinones and derivatives | ||||||||
| 30 | 5.88 | Physcion-8-O-(6’-O- malonyl)-hexose |
C26H26O12 | 529.13391 | 529.13405 | −0.269 | 366.07205[M-H-C6H11O5]− 348.06122[M-H-C6H11O5-H2O]− 320.06482[M-H-C6H11O5-H2O-CO]− |
PMRPW, PMRPE |
| 33 | 6.09 | Rumejaposide D a | C21H22O11 | 449.10751 | 449.10784 | −0.730 | 287.04864[M-H-C6H10O5]− 269.04453[M-H-C6H10O5-H2O]− 259.06021[M-H-C6H10O5-CO]− |
PMRPW, PMRPE |
| 57 | 8.41 | Di-emodin-Di-glucoside a | C42H42O18 | 833.22919 | 833.22874 | 0.539 | 671.17426[M-H-C6H10O5]− 509.12094[M-H-2C6H10O5]− 253.04974[M-H-2C6H10O5-C15H12O4]− |
PMRPW |
| 58 | 8.86 | Isomer emodin-8-O-(6’-O-acetyl)-β-d- glucoside |
C23H22O11 | 473.10638 | 473.10784 | −3.081 | 311.05423[M-H-C6H10O5]− 283.06085[M-H-C6H10O5-CO]− 255.06544[M-H-C6H10O5-2CO]− |
PMRPW, PMRPE |
| 77 | 10.83 | Citreorosein-O-glucoside a | C21H20O11 | 447.09048 | 447.09219 | −3.820 | 300.02811[M-H-C6H10O4]− 268.03757[M-H-C6H10O4-2O]− 240.04250[M-H-C6H10O4-CO]− |
PMRPW, PMRPE |
| 78 | 10.95 | Chrysophanol b | C15H10O4 | 253.05020 | 253.04954 | 2.627 | 225.05373[M-H-CO]− 197.56078[M-H-2CO]− 181.06459[M-H-CO2-CO]− |
PMRPW, PMRPE |
| 84 | 11.97 | 2-Acetylemodin-8-O-β-d-glucoside | C23H22O11 | 473.10583 | 473.10784 | −4.424 | 311.05438[M-H-C6H10O5]− 269.04575[M-H-C6H10O5-C2H2O]− 241.04889[M-H-C6H10O5-C2H2O-CO]− |
PMRPW, PMRPE |
| 85 | 12.12 | Emodin-8-O-β-d-glucoside b | C21H20O10 | 431.09637 | 431.09727 | −2.095 | 269.04520[M-H-C6H10O5]− 241.04926[M-H-C6H10O5-CO]− 225.05426[M-H-C6H10O5-CO2]− |
PMRPW, PMRPE |
| 86 | 12.35 | Emodin-O-glucoside-gallate a | C28H24O14 | 583.10736 | 583.10823 | −0.872 | 269.04562[M-H-C6H10O5-C7H4O4]− 225.05466[M-H-C6H10O5-C7H4O4-CO2]− |
PMRPE |
| 87 | 13.00 | 6-Carboxyl emodin a | C16H10O7 | 313.03448 | 313.03428 | 0.642 | 269.04544[M-H-CO2]− 241.05034[M-H-CO2-CO]− 225.95458[M-H-2CO2]− |
PMRPW, PMRPE |
| 89 | 13.54 | Physcion-8-O-β-d-glucoside | C22H22O10 | 445.11273 | 445.11292 | −0.434 | 283.06104[M-H-C6H10O5]− 255.06458[M-H-C6H10O5-CO]− 239.06963[M-H-C6H10O5-CO2]− |
PMRPW, PMRPE |
| 90 | 13.65 | Physcion b | C16H12O5 | 283.06113 | 283.06010 | 3.639 | 268.03760[M-H-CH3]− 240.04179[M-H-CH3-CO]− 212.04668[M-H-CH3-2CO]− |
PMRPW, PMRPE |
| 91 | 14.09 | Citreorosein | C15H10O6 | 285.04041 | 285.03936 | 3.668 | 257.04422[M-H-CO]− 241.04965[M-H-CO2]− 227[M-H-CO-CH2O]− |
PMRPW, PMRPE |
| 92 | 14.59 | Chrysophanol anthrone a | C15H12O3 | 239.07027 | 239.06989 | −1.593 | 210.89307[M-H-CO]− 182.89648[M-H-2CO]− |
PMRPE |
| 93 | 14.69 | Questinol | C16H12O6 | 299.05493 | 299.05501 | −0.283 | 268.03696[M-H-CH3O]− 253.04982[M-H-CH3O-CH3]− 240.04204[M-H-CH3O-CO]− |
PMRPW, PMRPE |
| 94 | 14.87 | Hydroxyl-rhein a | C15H8O7 | 299.01867 | 299.01863 | 0.070 | 255.02777[M-H-CO2]− 227.03313[M-H-CO2-CO]− 199.03928[M-H-CO2-2CO]− |
PMRPW, PMRPE |
| 95 | 15.94 | Digitolutein a | C16H12O4 | 267.06601 | 267.06519 | 3.088 | 224.04675[M-H-CO-CH3]− 220.53163[M-H-CO-H2O]− 149.02373[M-H-2CO-CH2O-CH2-H2O]− |
PMRPE |
| 96 | 16.13 | Isomer Citreorosein | C15H10O6 | 285.04041 | 285.03936 | 3.668 | 257.04462[M-H-CO]− 241.04955[M-H-CO2]− 211.03883[M-H-CO2-CH2O]− |
PMRPW, PMRPE |
| 98 | 17.02 | Emodin anthrone a | C15H12O4 | 255.06572 | 255.06519 | 2.096 | 240.04176[M-H-CH3]− 225.05334[M-H-CH2O]− 212.04684[M-H-CH3-CO]− |
PMRPW, PMRPE |
| 99 | 17.05 | Isomer physcion | C16H12O5 | 283.06067 | 283.06010 | 2.014 | 240.04173[M-H-CH3-CO]− 268.03635[M-H-CH3]− |
PMRPW, PMRPE |
| 100 | 18.20 | Emodin-3- ethyl ether |
C17H14O5 | 297.07452 | 297.07575 | 0.606 | 282.05472[M-H-CH3]− 269.08282[M-H-CO]− 254.05786[M-H-C2H5-CH2]− |
PMRPW, PMRPE |
| 101 | 18.86 | 2-Acetylemodin | C17H12O6 | 311.05408 | 311.05501 | −3.004 | 296.03165[M-H-CH3]− 283.06100[M-H-CO]− 269.04504[M-H-C2H2O]− |
PMRPW, PMRPE |
| 102 | 19.52 | Isomer 2-acetylemodin | C17H12O6 | 311.05426 | 311.05501 | −2.426 | 283.06119[M-H-CO]− 269.06583[M-H-C2H2O]− 240.04160[M-H-2CO-CH3]− |
PMRPW, PMRPE |
| 103 | 20.37 | Emodin b | C15H10O5 | 269.04514 | 269.04445 | 2.565 | 241.04941[M-H-CO]− 213.05467[M-H-2CO]− 225.05450[M-H-CO2]− |
PMRPW, PMRPE |
| Stilbenes and derivatives | ||||||||
| 29 | 5.24 | Polygonumosides C | C40H44O19 | 827.23828 | 827.23931 | −1.239 | 421.11276[M-H-C20H22O9]− 259.06009[M-H-C20H22O9-C6H10O5]− 241.04927[M-H-C20H22O9- C6H10O5-H2O]− |
PMRPW |
| 37 | 6.69 | Isomer 3,4,5,4’-tetrahydroxystilbene | C14H12O4 | 243.06465 | 243.06519 | −2.202 | 225.05467[M-H-H2O]− 215.06952[M-H-CO]− 197.05991[M-H-H2O-CO]− |
PMRPW, PMRPE |
| 41 | 7.07 | Rhapontin a | C21H24O9 | 419.13242 | 419.13366 | −2.955 | 257.07297[M-H-C6H10O5]− 239.03387[M-H-C6H10O5-H2O]− 227.03279[M-H-C6H10O5-CH2O]− |
PMRPW, PMRPE |
| 44 | 7.31 | Tetrahydroxystilbene-O-di-glucoside | C26H32O14 | 567.17004 | 567.17083 | −1.396 | 243.06516[M-H-2C6H10O5]− 225.05428[M-H-2C6H10O5-H2O]− 197.06007[M-H-2C6H10O5-H2O-CO]− |
PMRPW, PMRPE |
| 45 | 7.38 | β-d-glucoside,4-[2,3-dihydro-3-(hydroxymethyl)-5-(3-hydroxypropyl)-7-methoxy-2-yl]-2-methoxyphenyl a | C26H34O11 | 521.20062 | 521.20174 | −2.145 | 359.147229[M-H-C6H10O5]− 313.10608[M-H-C6H10O5-H2O-CO]− |
PMRPW, PMRPE |
| 47 | 7.76 | Resveratrol a | C14H12O3 | 227.07057 | 227.07027 | 1.318 | 185.05931[M-H-H2O-CH2]− 170.97986[M-H-H2O-2CH2]− 143.04947[M-H-C4H4O2]− |
PMRPW, PMRPE |
| 48 | 7.67 | Isomer Tetrahydroxystilbene-O-di-glucoside | C26H32O14 | 567.17059 | 567.17083 | −0.427 | 243.06509[M-H-2C6H10O5]− 225.05261[M-H-2C6H10O5-H2O]− |
PMRPW, PMRPE |
| 50 | 7.89 | 3,4,5,4’-Tetrahydroxystilbene | C14H12O4 | 243.06517 | 243.06519 | −0.063 | 225.05469[M-H-H2O]− 197.05965[M-H-H2O-CO]− 169.06514[M-H-H2O-2CO]− |
PMRPW, PMRPE |
| 52 | 7.94 | 2, 3, 5, 4′-Tetrahydroxystilbene-2-O-β-d-glucoside b | C20H22O9 | 405.11670 | 405.11801 | −0.885 | 243.06503[M-H-C6H10O5]− 225.05450[M-H-C6H10O5-H2O]− 215.07039[M-H-C6H10O5-CO]− |
PMRPW, PMRPE |
| 56 | 8.32 | Multiflorumisides A a | C40H44O18 | 811.24438 | 811.24439 | −0.013 | 649.19263[M-H-C6H10O5]− 405.11447[M-H-C20H22O9]− 243.06512[M-H-C20H22O9-C6H10O5]− |
PMRPW, PMRPE |
| 61 | 9.17 | Polygonumoside A | C27H24O13 | 555.11487 | 555.11332 | 2.797 | 393.05923[M-H-C6H10O5]− 349.07019[M-H-C6H10O5-CO2]− 300.99774[M-H-C6H10O5-C6H4O]− |
PMRPW, PMRPE |
| 64 | 9.53 | Isomer polygonumoside A | C27H24O13 | 555.11432 | 555.11332 | 1.807 | 393.05942[M-H-C6H10O5]− 349.06873[M-H-C6H10O5-CO2]− 300.99670[M-H-C6H10O5-C6H4O]− |
PMRPW, PMRPE |
| 66 | 9.56 | 2,3,5,4′-Tetrahydroxystilbene-O-(malonyl)- β-d-glucoside |
C23H24O12 | 491.11929 | 491.11840 | 1.807 | 329.09622[M-H-C6H10O5]− 313.03226[M-H-C6H10O5-H2O]− 285.04071[M-H-C6H10O5-H2O-CO]− |
PMRPW, PMRPE |
| 67 | 9.57 | Tetrahydroxystilbene-O-(galloyl)-glucoside | C27H26O13 | 557.12933 | 557.12897 | 0.651 | 405.05499[M-H-C7H4O4]− 243.06503[M-H-C7H4O4-C6H10O5]− 225.05434[M-H-C7H4O4-C6H10O5-H2O]− |
PMRPW, PMRPE |
| 68 | 9.87 | 2,3,5,4’-Tetrahydroxystilbene-2-O-(2”-O-acetyl)-β-d-glucoside | C22H24O10 | 447.12930 | 447.12857 | 1.625 | 243.06511[M-H-C6H10O5-C2H2O]− 225.05455[M-H-C6H10O5-C2H2O-H2O]− 284.08289[M-H-C6H11O5]− |
PMRPE |
| 69 | 9.95 | Piceatannol-3-O-β-d-(6″-O-galloyl)- glucoside |
C27H26O13 | 557.12817 | 557.12897 | −1.431 | 405.11728[M-H-C7H4O4]− 243.06511[M-H-C7H4O4-C6H10O5]− 225.05495[M-H-C7H4O4-C6H10O5-H2O]− |
PMRPW, PMRPE |
| 73 | 10.70 | Tetrahydroxystilbene-O-(caffeoyl)-glucoside a | C29H28O12 | 567.14502 | 567.14970 | −8.256 | 243.06516[M-H-C6H10O5-C9H6O3]− | PMRPE |
| 75 | 10.81 | Polydatin a b | C20H22O8 | 389.12457 | 389.12309 | 0.984 | 227.07018[M-H-C6H10O5]− 209.05940[M-H-C6H10O5-H2O]− 199.07462[M-H-C6H10O5-CO]− |
PMRPW, PMRPE |
| 76 | 10.83 | Isorhapontigenin a | C15H14O4 | 257.08127 | 257.08084 | 1.691 | 242.05462[M-H-CH3]− 187.56930[M-H-CH2-2CO]− 136.18150[M-H-C7H5O2]− |
PMRPW, PMRPE |
| 81 | 11.39 | 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-(2″-O-coumaroyl)-glucoside | C29H28O11 | 551.15112 | 551.15479 | −3.668 | 389.10031[M-H-C6H10O5]− 225.05389[M-H-C6H10O5-C9H6O2-H2O]− |
PMRPE |
| 88 | 13.17 | Tetrahydroxystilbene-2-(feruloyl)- glucoside |
C30H30O12 | 581.16583 | 581.16535 | 0.821 | 419.11136[M-H-C6H10O5]− 405.21970[M-H-C10H8O3]− 295.05981[M-H-C6H10O5-C6H4O3]− |
PMRPW, PMRPE |
| Flavonoids and derivatives | ||||||||
| 18 | 3.63 | Liquiritigenin-glucoside-xyl/ara | C26H30O13 | 549.1604 | 549.16027 | 0.023 | 387.10541[M-H-C6H10O5]− 369.09552[M-H-C6H10O5-H2O]− 279.06604[M-H-C6H10O5-C5H8O4]− |
PMRPW, PMRPE |
| 28 | 4.79 | Catechin | C15H14O6 | 289.07111 | 289.07066 | 1.541 | 151.03955[M-H-C7H5O3]− 137.02376[M-H-C8H7O3]− 123.04458[M-H-C7H5O3-CO]− 109.02898[M-H-C8H7O3-C2H2O]− |
PMRPW, PMRPE |
| 31 | 5.93 | Epicatechin | C15H14O6 | 289.07080 | 289.07066 | 0.468 | 151.03955[M-H-C7H5O3]− 137.02376[M-H-C8H7O3]− 123.04458[M-H-C7H5O3-CO]− 109.02898[M-H-C8H7O3-C2H2O]− |
PMRPW, PMRPE |
| 35 | 6.36 | Acetyl-epicatechin-O-glucoside a | C23H26O12 | 493.13406 | 493.13405 | 0.015 | 330.07205[M-H-C6H11O5]− 255.06543[M-H-C6H10O5-C2H2O2-H2O]− 227.07016[M-H-C6H10O5-C2H2O2-H2O-CO]− |
PMRPW, PMRPE |
| 39 | 7.04 | Hesperetin-7-O-glucoside a | C22H24O11 | 463.12482 | 463.12349 | 2.876 | 419.13446[M-H-CO2]− 256.07315[M-H-CO2-C6H11O5]− |
PMRPW, PMRPE |
| 49 | 7.73 | Trihydroxy-dimethoxychalcone-O-glucoside | C23H26O11 | 477.13867 | 477.13914 | −0.981 | 315.08606[M-H-C6H10O5]− 297.07486[M-H-C6H10O5-H2O]− 243.06522[M-H-C6H10O5-2CO-O]− |
PMRPW, PMRPE |
| 51 | 7.90 | Epicatechin-O-gallate | C22H18O10 | 441.08109 | 441.08162 | −3.706 | 289.07086[M-H-C7H5O4]− 243.06519[M-H-C7H5O4-H2O-CO]−225.05489[M-H-C7H5O4-CO2]− 169.01367[M-H-C15H13O5]− |
PMRPE |
| 55 | 8.19 | Cirsimarin a | C23H24O11 | 475.12415 | 475.12349 | 1.394 | 313.06976[M-H-C6H10O5]− 285.07596[M-H-C6H10O5-CO]− 242.05670[M-H-C6H10O5-CO2-2CH2]− |
PMRPW, PMRPE |
| 59 | 9.03 | Epimedium | C20H20O7 | 371.11102 | 371.11253 | −4.067 | 281.08215[M-H-C4H10O2]− 161.02383[M-H-C4H8O2-C7H4O2]− |
PMRPE |
| 60 | 9.11 | Kaempferol-3-β-d-glucoside | C21H20O11 | 447.09103 | 447.09219 | −3.529 | 285.03925[M-H-C6H10O5]− 257.04456[M-H-C6H10O5-CO]− 229.04724[M-H-C6H10O5-CO]− |
PMRPW, PMRPE |
| 63 | 9.49 | Quercetin | C15H10O7 | 301.03424 | 301.03428 | −0.130 | 283.03264[M-H-H2O]− 273.04050[M-H-CO]− 255.02896[M-H-CO-H2O]− |
PMRPW, PMRPE |
| 65 | 9.55 | Kaempferol a | C15H10O6 | 285.04022 | 285.03936 | 3.001 | 241.04958[M-H-CO2]− 257.67783[M-H-CO]− |
PMRPW, PMRPE |
| 70 | 10.27 | Kaempferol-O-glucoside-rhamnose a | C27H30O15 | 593.14893 | 593.15010 | −1.967 | 269.04529[M-H-2C6H10O5]− 225.05469[M-H-2C6H10O5-CO2]− 241.04984[M-H-2C6H10O5-CO]− |
PMRPW, PMRPE |
| 74 | 10.76 | Dihydroquercetin | C15H12O7 | 303.04868 | 303.04929 | −2.109 | 151.03946[M-H-CO2-C6H4O2]− 153.01883[M-H-C8H7O3]− 125.02396[M-H-C8H7O3-CO]− |
PMRPW, PMRPE |
| Phenolic acids and derivatives | ||||||||
| 9 | 1.99 | Gallic acid b | C7H6O5 | 169.01358 | 169.01315 | 2.546 | 125.02383[M-H-CO2]− 107.01351[M-H-CO2-H2O]− 97.02921[M-H-CO2-CO]− |
PMRPW, PMRPE |
| 10 | 2.06 | Gallic acid-O- glucoside |
C13H16O10 | 331.06595 | 331.06597 | −0.070 | 169.01357[M-H-C6H10O5]− 125.02380[M-H-C6H10O5-CO2]− |
PMRPW, PMRPE |
| 12 | 2.75 | Dihydroxy-benzoic acid a | C7H6O4 | 153.01859 | 153.01824 | 2.319 | 125.02429[M-H-CO]− 109.02917[M-H-CO2]− |
PMRPW, PMRPE |
| 14 | 2.83 | Galloyl-glycerol a | C10H12O7 | 243.04993 | 243.04993 | 0.004 | 169.01321[M-H-C3H6O2]− 125.02386[M-H-C3H6O2-CO2]− 118.96574[M-H-C3H6O2-3O]− |
PMRPW, PMRPE |
| 15 | 2.90 | Vanillic acid a | C8H8O4 | 167.03433 | 167.03389 | −0.331 | 137.02341[M-H-CH2O]− 123.04459[M-H-CO2]− |
PMRPW, PMRPE |
| 16 | 3.30 | Isomer Dihydroxy- benzoic acid |
C7H6O4 | 153.01859 | 153.01824 | −0.230 | 137.45282[M-H-O]− 125.02434[M-H-CO]− 109.02898[M-H-CO2]− |
PMRPW, PMRPE |
| 17 | 3.34 | Protocatechuic acid-O-glucoside | C13H16O9 | 315.06995 | 315.07106 | −3.518 | 153.01865[M-H-C6H10O5]− 109.02901[M-H-C6H10O5-CO2]− |
PMRPW, PMRPE |
| 19 | 3.71 | Caffeic acid a | C9H8O4 | 179.03412 | 179.03389 | 2.093 | 135.04448[M-H-CO2]− 107.04977[M-H-C3H4O2]− |
PMRPW, PMRPE |
| 20 | 3.75 | 1-(5-Methylfuran-2-yl) ethanone a | C7H8O2 | 123.04462 | 123.04406 | 4.584 | 108.02116[M-H-CH3]− 95.01338[M-H-CO]− 79.05503[M-H-CO2]− |
PMRPW, PMRPE |
| 21 | 4.16 | Veratric acid a | C9H10O4 | 181.05014 | 181.04954 | 2.567 | 137.02396[M-H-CO2]− 122.03658[M-H-2CH2O]− 107.04949[M-H-CO-CH2O]− |
PMRPW, PMRPE |
| 25 | 4.56 | 3-Hydroxybenzoic acid | C7H6O3 | 137.02371 | 137.02332 | 2.842 | 119.01297[M-H-H2O]− 93.03405[M-H-CO2]− |
PMRPW, PMRPE |
| 26 | 4.75 | Coumaric acid a | C9H8O3 | 163.03937 | 163.03897 | 2.4500 | 119.04978[M-H-CO2]− 134.91408[M-H-CO]− 107.04973[M-H-C2O2]− |
PMRPW, PMRPE |
| 27 | 4.77 | 2-Methyl gallic acid a | C8H8O5 | 183.02893 | 183.02880 | 0.711 | 168.00560[M-H-CH3]− 139.00296[M-H-CO2]− 111.00824[M-H-CO2-CO]− |
PMRPW, PMRPE |
| 34 | 6.24 | Methyl gallate a | C8H8O4 | 167.03424 | 167.03389 | 2.124 | 151.00310[M-H-CH3]− 125.02368[M-H-C2H2O]−107.01314[M-H-C2H2O2]− |
PMRPW, PMRPE |
| 40 | 7.06 | Syringic acid a | C9H10O5 | 197.04417 | 197.04445 | −1.420 | 169.01370[M-H-CO]− 125.02389[M-H-CO-CO2]− |
PMRPE |
| Others | ||||||||
| 1 | 0.73 | L-Arginine | C6H14N4O2 | 173.10316 | 173.10330 | −0.821 | 131.08197[M-H-CN2H2]− 114.05562[M-H-NH-CO2]− |
PMRPW, PMRPE |
| 2 | 0.79 | Glucose | C6H12O6 | 179.05534 | 179.05501 | 1.818 | 161.06087[M-H-H2O]− 131.03432[M-H-H2O-CH2O]− 85.02903[M-H-CH2O-4O]− |
PMRPW, PMRPE |
| 3 | 0.82 | L-Threonine | C4H9NO3 | 118.05041 | 118.04987 | 4.577 | 74.02446[M-H-CO2]− 59.01369[M-H-CO2-CH3]− |
PMRPW, PMRPE |
| 4 | 0.85 | (2S)-2-Hydroxybutanedioic acid | C4H6O5 | 133.01361 | 133.01315 | 3.460 | 115.00312[M-H-H2O]− 89.02412[M-H-CO2]− 71.01358[M-H-H2O-CO2]− |
PMRPW, PMRPE |
| 5 | 1.29 | Citric acid a | C6H8O7 | 191.01889 | 191.01863 | 1.366 | 129.01921[M-H-CO2-H2O]− 111.00829[M-H-CO2-2H2O]− 87.00838[M-H-2CO2-O]− |
PMRPW, PMRPE |
| 6 | 1.39 | L-Tyrosine a | C9H11NO3 | 180.06575 | 180.06552 | 1.279 | 163.03926[M-H-OH]− 137.02368[M-H-NH-CO]− 119.04951[M-H-CO2-OH]− |
PMRPW, PMRPE |
| 7 | 1.40 | 3-O-feruloylquinic acid a | C17H20O9 | 367.10272 | 367.10236 | 0.985 | 277.07294[M-H-COOH-CH2O-H2O]− 157.03020[M-H-C10H8O3-2OH]− |
PMRPW, PMRPE |
| 8 | 1.43 | Leucine | C6H13NO2 | 130.08676 | 130.08626 | 3.881 | 85.02912[M-H-COOH]− 88.04015[M-H-3CH2]− |
PMRPW, PMRPE |
| 11 | 2.50 | 3,5-Dihydroxy-2- methyl-4hydro-pyran-4-one |
C6H6O4 | 141.01833 | 141.01824 | 0.673 | 112.95596[M-H-CO]− 97.02898[M-H-CO2]− 69.03445[M-H-CO2-H2O]− |
PMRPW, PMRPE |
| 13 | 2.79 | 5-Hydroxymethylfurfural | C6H6O3 | 125.02386 | 125.02332 | 2.795 | 97.02918[M-H-CO]− 81.03435[M-H-CO2]− |
PMRPW, PMRPE |
| 22 | 4.35 | Altechromone A a | C11H10O3 | 189.05476 | 189.05462 | 0.737 | 174.03186[M-H-CH3]− 161.06018[M-H-CO]− 146.03635[M-H-CO-CH3]− |
PMRPW, PMRPE |
| 23 | 4.52 | Acetyl 1-methyl-1- acetoxyethyl peroxide a |
C7H12O5 | 175.06026 | 175.06010 | 0.914 | 160.97757[M-H-CH2]− 146.96054[M-H-2CH2]− 115.03953[M-H-2CH2O]− |
PMRPW, PMRPE |
| 24 | 4.56 | 2-Vinyl-1H-indole-3-carboxylic acid | C11H9NO2 | 186.05539 | 186.05496 | 2.338 | 142.06551[M-H-CO2]− 159.93617[M-H-C2H3]− 116.05013[M-H-C2H2-CO2]− |
PMRPE |
| 32 | 6.01 | P-hydroxybenzal-dehyde a | C7H6O2 | 121.0289 | 121.02841 | 4.082 | 93.03405[M-H-CO]− | PMRPW, PMRPE |
| 36 | 6.54 | Vanillin a | C8H8O3 | 151.03902 | 151.03897 | 0.327 | 136.01607[M-H-CH3]− 123.04456[M-H-CO]− 107.04993[M-H-CO2]− |
PMRPW, PMRPE |
| 38 | 7.03 | 6-Methoxyl-2-Acetyl-3-methyljuglone-8-O-β-d-glucoside | C20H22O10 | 421.11276 | 421.11292 | −0.388 | 259.06027[M-H-C6H10O5]− 241.04961[M-H-C6H10O5-H2O]− 213.05441[M-H-C6H10O5-H2O-CO]− |
PMRPW, PMRPE |
| 42 | 7.13 | Nudiposide a | C27H36O12 | 551.21313 | 551.21230 | 1.501 | 389.15720[M-H-C6H10O5]− 359.11261[M-H-C6H10O5-2CH2O]− 341.09985[M-H-C6H10O5-2CH2O-H2O]− |
PMRPW, PMRPE |
| 43 | 7.21 | (+)-lyoniresinol-2α-O-β-glucoside a | C28H38O13 | 581.22284 | 581.22287 | −0.994 | 419.16949[M-H-C6H10O5]−389.12219[M-H-C6H10O5-CH2O]−359.11096[M-H-C6H10O5-2CH2O]− | PMRPW, PMRPE |
| 46 | 7.40 | Isomer Altechromone A |
C11H10O3 | 189.0547 | 189.05462 | 0.420 | 174.03166[M-H-CH3]− 161.06047[M-H-CO]− 147.04448[M-H-CO-CH2]− |
PMRPW, PMRPE |
| 53 | 8.08 | Cinnamyl-galloyl-O- glucoside a |
C22H22O11 | 461.10611 | 461.10784 | −3.747 | 417.11594[M-H-CO2]− 254.05766[M-H-CO2-C6H11O5]− |
PMRPW, PMRPE |
| 54 | 8.11 | 2-Methyl-5-carboxymethyl-7-hydroxychromone a | C12H10O5 | 233.04443 | 233.04555 | −0.085 | 205.04994[M-H-CO]− 191.03783[M-H-CO-CH2]− 161.02485[M-H-CO-CO2]− |
PMRPE |
| 62 | 9.24 | Trans-N-caffeoyltyramine a | C17H17NO4 | 298.10773 | 298.10738 | 1.159 | 135.04459[M-H-C9H7O3]− 178.04970[M-H-C8H8O]− 148.05200[M-H-C8H6O-OH]− |
PMRPW, PMRPE |
| 71 | 10.30 | Noreugenin a | C10H8O4 | 191.03399 | 191.03389 | 0.549 | 149.02406[M-H-CO-CH2]− 147.04459[M-H-CO2]− |
PMRPW, PMRPE |
| 72 | 10.61 | 1,2-Dihydroxypropane-1-(4-hydroxy-phenyl) a | C9H12O3 | 167.07063 | 167.07053 | 1.552 | 152.04729[M-H-CH3]− 138.92862[M-H-CO]− |
PMRPW, PMRPE |
| 79 | 10.98 | N-trans-Feruloyl tyramine | C18H19NO4 | 312.12286 | 312.12303 | −0.559 | 190.05000[M-H-C7H6O2]− 178.05019[M-H-C8H6O2]− 148.05235[M-H-C9H10NO2]− |
PMRPW, PMRPE |
| 80 | 11.34 | Trans-N-Feruloyl-3-O-methyldopamine | C19H21NO5 | 342.13211 | 342.13360 | −4.353 | 327.10962[M-H-CH3]− 178.05003[M-H-C9H8O3]− |
PMRPW, PMRPE |
| 82 | 11.43 | Thunberginol C-6- O-β-d-glucoside a |
C21H22O10 | 433.11395 | 433.11292 | 2.371 | 271.06082[M-H-C6H10O5]− 253.05016[M-H-C6H10O5-H2O]− 243.06531[M-H-C6H10O5-CO]− |
PMRPW, PMRPE |
| 83 | 11.87 | Torachrysone a | C14H14O4 | 245.08078 | 245.08084 | −0.226 | 230.05690[M-H-CH3]− 215.03368[M-H-CH2O]− 159.04398[M-H-CH2O-CH2-C2H2O]− |
PMRPE |
| 97 | 16.62 | 3,8-Dihydroxy-1- methoxyxanthone a |
C14H10O5 | 257.04535 | 257.04445 | 3.502 | 239.03391[M-H-H2O]− 229.04854[M-H-CO]− 211.03917[M-H-CO-H2O]− |
PMRPW, PMRPE |
Note: PMRPE: Ethanol extract of Polygoni Multiflori Radix Praeparata; PMRPW: Water extract of Polygoni Multiflori Radix Praeparata; a means first reported in PMRP; b means components compared with standards.
2.2.1. Identification of Anthraquinones and Derivatives
Anthraquinones, which have the pharmacological effects of being anti-inflammatory, anti-virus, anti-cancer, lipid-lowering, and anti-diabetes [26], are the primary compounds in PMRP. There has been much literature which has revealed that anthraquinones can attenuate liver damage and demonstrate an anti-cirrhosis effect by reducing lipid peroxidation and inhibiting the proliferation of hepatic stellate cells [27,28,29]. Moreover, emodin and its oxidative metabolites were deemed as the main xenobiotic components, as they can combine with glutathione (GSH) to disturb cellular GSH and fatty acid metabolism in the liver [30,31]. Most anthraquinones in this family produced the characteristic fragment ions at m/z 269 and m/z 240, and the loss of two CO sequentially could be considered as the characteristic fragment behavior of anthraquinones and their derivates. In detail, peak 84, with a retention time of 11.97 min, generated an [M–H]− ion with mass accuracy at m/z 473.10583. The molecular formula was predicted as C23H22O11 using Xcalibur (Thermo Fisher Scientific) within 5 ppm. As shown in Figure 3, the characteristic fragment ions at m/z 311.05438 indicated a loss of glucuronic acid from the precursor ion at m/z 473.10583. Characteristic ions at m/z 269.04575 and 282.05304, which could be identified as losing C2H2O and CO from m/z 311.05438, respectively, were obtained. The [M−H]− ion fragmented into other characteristic ions at m/z 254.05710, m/z 240.04149, and m/z 225.05450, which corresponded to [M-H-glc-C2H2O-CH2]−, [M-H-glc-C2H3O-CO]− and [M-H-glc-C2H3O-CO-CH3]−. It was putatively identified as 2-acetylemodin-8-O-β-d-glucoside, and the proposed fragmentation pathways of 2-acetylemodin-8-O-β-d-glucoside are depicted in Figure 4. Peak 90 was found at 13.65 min and showed a precise molecular weight at m/z 283.06113. The fragment ion at m/z 268.03760 was produced by losing CH3. Other characteristic ions at m/z 240.04179 and 212.04668 were observed by losing two CO successively. According to the in-house database and reference standard, compound 90 was identified as physcion. Peak 93 was found at 14.69 min and generated a [M−H]− ion at m/z 299.05493. MS/MS fragment at m/z 268.03696, 253.04982, and 240.04204 have corresponded to [M-H-CH3O]−, [M-H-CH3O-CH3]− and [M-H-CH3O-CO]−. As a result, the compound was putatively identified as questinol. Peak 103 produced [M−H]− ions at m/z 269.04514, and further characteristic fragment ions were acquired at m/z 241.04941 and 213.05467 by losing two CO successively. By comparing with the reference standard, the compound was identified as emodin.
Figure 3.
Mass spectrum of 2-acetylemodin-8-O-β-d-glucoside in negative mode.
Figure 4.
The proposed fragmentation pathways of 2-acetylemodin-8-O-β-d-glucoside.
2.2.2. Identification of Stilbenes and Derivatives
Stilbenes are the main characteristic components in Polygoni Multiflori Radix Praeparata, showing great lipid-regulating and antioxidant activity [8]. Specifically, THSG as a unique active constituent plays a vital role in hepatoprotective effects, with various abilities as to the improvement of mitochondrial function and the clearance of intracellular reactive oxygen species [32,33]. On the other hand, some studies have reported that THSG was regarded as a contributor to liver injury associated with the transformation of trans-THSG to cis-THSG [34]. Stilbenes and its derivatives displayed characteristic fragment ions at m/z 405 and m/z 243 in negative ion mode. The other two prominent ions at m/z 225 and m/z 215 were obtained as loss CO and H2O in A-ring after rearrangement, respectively. In detail, peak 52 was found at 7.94 min and generated an [M–H]− ion at m/z 405.11670. The characteristic ion at m/z 243.06503 was produced by losing C6H10O5 from the precursor ion. Other characteristic ions at m/z 225.05450 and 215.07039 were obtained by losing H2O and CO from m/z 243.06503, respectively. Compound 52 was identified as THSG by comparing the reference standard. Figure 5 shows the MS/MS mass spectrum of THSG. The details of proposed fragmentation pathways are depicted in Figure 6. Peaks 67 and 69 were observed at 9.57min and 9.95min, respectively. Their molecular formulas were predicted as C27H26O13 within 5 ppm. They all produced fragment ions at m/z 405.11, 243.06 and 225.05, which were indicated as [M–H–gal]−, [M–H–gal–glc]−, [M–H-gal–glc–H2O]−, respectively. Although it was difficult to distinguish them by MS spectra, it was easier to identify them by comparing their retention time. According to the in-house database, the two compounds were Tetrahydroxy-stilbene-O-(galloyl)-glucoside and Piceatannol-3-O-β-d-(6″-O-galloyl)-glucoside. Based on the different positions of hydroxy in the benzene ring, the dehydration ability of them was different. Tetrahydroxystilbene-O-(galloyl)-Glucoside is more polar and can be more quickly eluted than Piceatannol-3-O-β-d-(6″-O-galloyl)-glucoside on reserved phase column. Therefore, peak 67 was putatively identified as Tetrahydroxystilbene-O-(galloyl)-Glucoside, and peak 69 was Piceatannol-3-O-β-d-(6″-O-galloyl)-glucoside. Peak 68 was found at 9.87 min and generated [M–H]− ion at m/z 447.12930. MS/MS fragment at m/z 243.06511 and 225.05455 corresponded to [M–H–glc–acetyl]− and [M–H–glc–acetyl–H2O]−. Compound 68 was putatively identified as 2,3,5,4’-tetrahydroxy-stilbene-2-O-(2”-O-acetyl)-β-d-glucoside.
Figure 5.
Mass spectrum of THSG in negative mode.
Figure 6.
The proposed fragmentation pathways of THSG.
2.2.3. Identification of Flavonoids and Derivatives
As the main antioxidant in the root, flavonoids and their derivatives exhibit antioxidant and free radical scavenging activities [35]. In addition, flavonoids can protect against liver injury through the regulation of NF-κB/IκBα, p38 MAPK, and Bcl-2/Bax signaling [36]. There were 14 compounds tentatively identified as flavonoids and their derivatives. Catechin and epicatechin are isomers and they were used as examples to illustrate the characterization process of flavonoids, which can undergo an RDA reaction by cleavage of C3–C4 and C2–C1 bonds of the C ring rearranging, and produced the characteristic fragment ions of m/z 151 and m/z 137. Figure 7 shows the MS/MS mass spectrum of epicatechin. The proposed fragmentation pathways are depicted in Figure 8. Peak 28 and Peak 31 generated an [M–H]− ion at m/z 289.07111 and 289.07080, respectively. Their molecular formulas were all predicted as C15H14O6 within 5 ppm. The common characteristic ions were observed at m/z 151.03, 37.02, 123.04. The precursor ions undergo an RDA reaction to produce m/z 151.03 and 137.02, corresponding to [M-H-C7H5O3]− and [M-H-C8H7O3]−, respectively. Characteristic ions at m/z 123.04 were obtained by losing CO from m/z 311.05438. Based on their different hydroxyl configuration at the C3 position and combined with the literature, catechin is more polar than epicatechin. Therefore, in this separation condition, the retention time of catechin is shorter. Peak 28 was putatively identified as catechin. Peak 31 was epicatechin.
Figure 7.
Mass spectrum of epicatechin in negative mode.
Figure 8.
The proposed fragmentation pathways of epicatechin.
2.2.4. Identification of Phenolic Acids and Derivatives
Fifteen phenolic acids and their derivatives were tentatively identified in PMRP. It has been reported to exhibit hepatoprotective effects and good inhibitory activity towards α-glucosidase[37]. Phenolic acids were structures containing one or more phenolic hydroxyl moieties. Therefore, the loss of 44 Da (−COO) and 18Da (H2O) could be considered as the characteristic fragment behavior of phenolic acids and its derivatives. Gallic acid was reported as a potentially xenobiotic component with anti-inflammatory activities and hepatotoxicity[38,39]. As shown in Figure 9, peak 9 with a retention time of 1.99 min generated an [M–H]− ion with mass accuracy at m/z 169.01358. The characteristic ion at m/z 125.02383 was produced by losing CO2 from the precursor ion. The consecutive loss of CO2 and H2O leads to characteristic ions at m/z 125.02383 and 107.01351. Compound 9 was identified as gallic acid by comparing with the reference standard. The proposed fragmentation pathways of gallic acid are illustrated in Figure 10. Peak 17 was found at 3.34 min and generated an [M–H]− ion at m/z 315.06995. It produced fragment ion at m/z 153.01865 by losing 162 Da which be considered as a loss of C6H10O5 group, and ion at m/z 109.02901 by losing a CO2 (44 Da). Therefore, compound 17 could be tentatively identified as protocatechuic acid-O-glucoside.
Figure 9.
Mass spectrum of gallic acid in negative mode.
Figure 10.
The proposed fragmentation pathways of gallic acid.
2.2.5. Identification of Other Compounds
Peak 13 generated an [M−H]− ion at m/z 125.02386, and the molecular formula was predicted as C6H6O3 within 5 ppm. Diagnostic ions at m/z 97.02918 and 81.03435 indicated the loss of CO and CO2 groups, respectively. Peak 13 was putatively identified as 5-Hydroxymethylfurfural, matched with the in-house database. Peak 22 was found at 4.35 min and produced an [M–H]− ion at m/z 189.05476. The diagnostic fragment ions such as m/z 174.03186 and 161.06018 corresponded to [M–H–CH3]− and [M–H–CO]−. Compound 22 was tentatively identified as Altechromone A. The [M−H]− ion at m/z 151.03902, which was found at 6.54 min in peak 36, indicated a molecular formula of C8H8O3 within 5 ppm. It produced characteristic fragments at m/z 136.01607, 123.04456, and 107.04993 due to the elimination of [M–H–CH3]−, [M–H–CO]− and [M–H–CO2]−, respectively. Finally, peak 36 was tentatively identified as Vanillin. Aside from the major compounds analyzed above, the remaining constituents were also identified by comparing them with the in-house database.
2.2.6. Comparison of Chemical Constituents Between PMRPE and PMRPW
Based on the identification strategy we have established, 101 components were identified in PMRPE and 91 components were identified in PMRPW. The results showed that there were 12 characteristic components in PMRPE, which were 2-vinyl-1H-indole-3-carboxylic acid, syringic acid, epicatechin-O-gallate, 2-methyl-5-carboxymethyl-7-hydroxychromone, epimedium, 2,3,5,4’-tetrahydroxystilbene-2-O-(2”-O-acetyl)-β-d-glucoside, 2,3,5,4’-tetrahydroxystilbene-2-O-β-d-(2″-O-coumaroyl)-glucoside, tetrahydroxystilbene-O-(caffeoyl)-glucoside, torachrysone, emodin-O-glucoside-gallate, chrysophanol anthrone and digitolutein. Two characteristic constituents were identified in PMRPW, which were polygonumosides C and di-emodin-di-glucoside.
Meanwhile, the peak area of potentially xenobiotic compounds reported in previous studies has been compared in PMRPW and PMRPE [1,40,41]. The representative chromatograms of potentially xenobiotic compounds are shown in Figure 11, and the specific parameters are listed in Table 2.
Figure 11.
The chromatograms of potentially xenobiotic compounds in PMRPW and PMRPE.
Table 2.
The peak area of potentially xenobiotic compounds in PMRPW and PMRPE.
| Peak No. | RT | Compound Name | Molecular Formula | Area | Percentage of the Area | Mechanisms of Hepatotoxicity | ||
|---|---|---|---|---|---|---|---|---|
| PMRPW | PMRPE | PMRPW | PMRPE | |||||
| 9 | 1.99 | Gallic acid | C7H6O5 | 4,237,639,440 * | 3,642,690,762 | 99.93% | 99.91% | CYP1A2 ↓; Caspase-3 ↑ |
| 28 | 4.79 | Catechin | C15H14O6 | 22,672,980 * | 16,781,588 | 95.54% | 96.05% | UGT1A6 ↓; UGT2B1 ↓ |
| 31 | 5.93 | Epicatechin | C15H14O6 | 10,315,546 * | 6,669,699 | 92.05% | 90.44% | Caspase-3 ↑; UGT1A6 ↑; UGT2B1 ↓ |
| 47 | 7.76 | Resveratrol | C14H12O3 | 5,525,111 | 6,868,739 * | 76.05% | 74.84% | Cyp7a1 ↑ |
| 52 | 7.94 | THSG | C20H22O9 | 32,436,422 | 49,753,744 * | 96.08% | 95.11% | Cyp7a1 ↑; Hmgcr ↑ CytP-450 ↓; UDP ↓ |
| 85 | 12.12 | EG | C21H20O10 | 5,266,457 | 10,448,815 * | 97.11% | 96.42% | UDP ↓; CYP2A ↓; UGT1A1 ↓; CYP3A4 ↓ |
| 90 | 13.65 | Physcion | C16H12O5 | 187,344,509 | 394,975,616 * | 94.04% | 95.77% | Cyp8b1 ↓; Cyp7a1 ↑ |
| 103 | 20.37 | Emodin | C15H10O5 | 4,512,341,510 | 773,950,089 * | 99.71% | 99.28% | CytP-50 ↓; Cyp8b1 ↓ Cyp7a1 ↑; CYP3A4 ↓ |
*: means the peak area of this group is higher than the other group; “↑”: means promoting expression; “↓”: means inhibiting expression.
3. Materials and Methods
3.1. Materials and Chemicals
HPLC grade acetonitrile (ACN) and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA), and Dikma Technology Co., Ltd. (Beijing, China). Ethanol (industrial grade) was obtained from Shandong Yuwang Industry Co., Ltd. (Shandong, China). Purified water was obtained from the Hangzhou Wahaha Corporation (Hangzhou, China). PMRP was bought from GuoDa Pharmacy (Shenyang, Liaoning, China) and was identified as the processed root of Polygonum multiflorum Thunb. by professor Zhiguo Yu of the Shenyang Pharmaceutical University. The reference standards of emodin, physcion, and gallic acid were purchased from Chengdu MUST Bio-Technology Co., Ltd. (Sichuan, China). Chrysophanol, rhein and polydatin were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). Emodin-8-O-β-d-glucoside and 2,3,5,4-tetrahydroxystilbene-2-O-β-d-glucoside (THSG) were obtained from Sichuan Victory Bio-Technology Co., Ltd. (Sichuan, China). The purity of all the reference substances was higher than 98%.
3.2. Standard Solutions and Sample Preparation
Standard solution: each reference standard was accurately weighed and dissolved in methanol as the stock solutions. Afterward, appropriate amounts of eight stock solutions were mixed and diluted into a suitable working solution with methanol before it was used.
Sample solution: 100 g of PMRP was soaked for 1 h with 1000 mL 70% aqueous ethanol solution as soaking solvent and then refluxed for 2 h. After being filtered with gauze, the residue was refluxed twice with another 800 mL of 70% aqueous ethanol solution for 1h. Finally, the filtrates were mixed and evaporated to 0.5 g/mL as PMRPE in a rotary evaporator. The preparation method of PMRPW was the same as that of PMRPE, except that 70% aqueous ethanol solution was replaced with water. Appropriate PMRPE and PMRPW extract were diluted with 50% aqueous methanol solution to obtain 15 mg/mL solution, respectively (calculated as raw herbs). The solution was centrifuged at 13000 rpm for 10 min and filtered through a 0.22 µm membrane. An aliquot of 5 µL was injected for analysis.
3.3. LC System and Mass Spectrometry
LC analysis was performed on a Vanquish Flex UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters Corporation, Milford, UK). The sample chamber and column temperatures were maintained at 10 °C and 35 °C, respectively. The gradient elution with mobile phase acetonitrile (A)—0.1% formic acid in water (B) was set as follows: 5–15% (A) from 0 to 4 min; 15–50% (A) from 4 to 10 min; 50–60% (A) from 10 to 15 min; 60–95% (A) from 15 to 25 min; 95% (A) from 25 to 28 min; 95–5% (A) from 28 to 28.1 min; 5% (A) from 28.1 to 31 min. The flow rate was 0.3 mL/min, and the injection volume was 5 μL.
A Q-Exactive Orbitrap mass spectrometry instrument (Thermo Fisher Scientific, USA) was used to identify the constituents of PMRPE and PMRPW in negative modes. The mass spectrometer was set with the following parameters: spray voltage, 3.0 kV; capillary temperature, 350 °C; auxiliary gas heater temperature, 350 °C; sheath gas flow rate, 35 Arb; auxiliary gas flow rate, 10 Arb; S-lens RF level, 55 V. The full scan and fragment spectra were collected at a resolution of 70,000 and 17,500, respectively. Full scan spectra were measured in a range from m/z 80 to 1200. The automatic gain control (AGC) target and maximum injection time (IT) were 3 × 106 ions capacity and 50 ms, respectively. For the dd MS2 mode, the automatic gain control (AGC) target and maximum injection time (IT) were 1 × 105 ions capacity and 50 ms. In each cycle, the top 10 precursor ions were chosen for fragmentation at collision energy (CE) of 20, 40 and 60 V. Data were analyzed by using Xcalibur™ version 2.2.1 and TraceFinder 4.1 version (Thermo Fisher Scientific, Waltham, MA, USA).
4. Conclusions
In this study, a rapid, sensitive, and specific analytical method was established using UHPLC-Q-Exactive Orbitrap-MS to identify the chemical constituents of PMRP. A total of 103 compounds, including 24 anthraquinones, 21 stilbenes, 15 phenolic acids, 14 flavones, and 29 other types were identified or tentatively characterized. There were 101 components in PMRPE and 91 components in PMRPW. Moreover, we have compared the peak areas of several significant components in PMRPW and PMRPE. The results showed that PMRPE has a higher content of anthraquinones and stilbenes than that of PMRPW. Previous studies have suggested that the hepatotoxicity of ethanol extract was stronger than that of water extract, indicating that anthraquinones and stilbenes might be the crucial xenobiotic components of liver injury induced by PMRP. Meanwhile, the specific toxicity compounds and mechanisms of hepatotoxicity also need further exploration. Considering the complex absorption and metabolism after oral administration, the characterization of PMRP’s composition in vitro research was not enough. Therefore, the identification of chemical constituents in vivo and the verification of hepatotoxicity mechanisms of PMRP are still under investigation. In conclusion, the profiles of the constituents provide more information to understand PMRP from a chemical viewpoint and establish a substantial basis for further studies. The results also demonstrated that the novel method would be meaningful to the characterization of components in other botanical extracts.
Supplementary Materials
The following are available online, Figure S1: Chemical structures of compounds identified in PMRP.
Author Contributions
S.W. and Z.Y.; formal analysis, X.S.; investigation, S.W., S.A. and F.S.; software, S.W. and X.S.; writing—original draft, S.W. writing—review and editing, S.W.; supervision, Y.Z. and Z.Y.; All authors have read and agreed to the published version of the manuscript.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Conflicts of Interest
The authors declare no conflict of interest.
Sample Availability
Samples of the compounds are available from the authors.
Footnotes
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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