Abstract
Objective
To further explore the unknown chemical composition and aimed to develop a reliable strategy for characterizing compounds in Rhodobryum roseum (R. roseum).
Methods
This study employed an efficient, high-sensitivity, and high-resolution analysis method. The method utilized UHPLC-Q-Exactive Orbitrap MS, along with trace data acquisition mode (parallel reaction monitoring scanning, PRM), and multiple data processing methods.
Results
A total of 77 chemical substances were identified in R. roseum, including 16 amino acids, 33 organic acids including 6 aromatic acids, 8 phenolics compounds, 2 flavonoid glycosides, 4 flavonoids and 14 other compounds including 4 fatty alcohols by comparing with reference substances, secondary characteristic ion fragments, public databases, and literature. Among these, 74 compounds were found in this plant for the first time.
Conclusion
This result significantly expands the knowledge of the chemical constituents of R. roseum and contributes to the understanding of its active constituents for quality control.
Introduction
Rhodobryum roseum (Hedw.) Limpr. (R. roseum) belongs to the Bryaceae family, also known as Herba huixincao, sun flower or stone chrysanthemum. This is a traditional folk medicine, which is widely distributed in YunNan and other regions. 1 It is often used alone or in combination with jujube, pepper, pig heart, etc to make a decoction with water to treat coronary heart disease (heartache, palpitation, shortness of breath, etc). 2 Currently, it is known that R. roseum contains bioactive compounds such as piperine and kaempferol,3,4 etc, have hepatoprotective, anti-tumor and cardioprotective effects. In addition, it also has anti-inflammatory, 5 antioxidant6,7 and antibacterial 8 effects. In recent years, the research on the chemical composition of R. roseum, which is relatively scarce, has mainly focused on the pharmacological activity study of the total extract 5 and Phenolic, 9 and there is no detailed report on the chemical composition of the plant. Because of its good pharmacological material basis and practical application, it is necessary to systematically study the chemical composition of R. roseum, which will help to understand the active compounds of R. roseum, explore its further applications, and provide references for quality control and drug use.
In recent years, more and more studies have been conducted using liquid chromatography-mass spectrometry (LC-MS) to identify and quantify the chemical constituents of different botanicals, compound preparations and metabolites metabolised in vivo. 10 It combines the high efficiency separation capability of liquid chromatography and the high sensitivity detection capability of mass spectrometry to become a powerful analytical technique. Because of its high selectivity, high sensitivity, fast analytical speed, and ability to accurately provide secondary fragment ions, it has been widely used in the modernisation studies of herbal medicines. 11 In this study, UHPLC-Q-Exactive Orbitrap MS combined with PRM was used to characterise the chemical constituents in R. roseum. More detailed information about its chemical composition will be obtained in this study. More detailed information about its chemical composition will be obtained through this study.
Materials and Methods
Chemicals and Reference Standards
Chromatographic grade formic acid was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Methanol and acetonitrile were chromatography grade supplied by Merck (Branchburg, NJ, USA). Water as the mobile phase solvent was obtained from Watson Water Company, Guangzhou, China, and the ethanol used in the study was of analytical grade. Phloretin (Lot: 200-488-7), supplied by Spring Autumn. Cis-aconitic acid (batch number: 20190512), Suberic acid (batch number: 20190620) and Azelaic acid (batch number: CM1980000) provided by Shandong West Asia Chemical Co., Ltd L-malic acid (lot number: M105695) was supplied by aladdin. Salicylic acid (batch number: H31A10B96399) was provided by Shanghai Yuanye Biotechnology Co., Ltd 4-Coumaric acid and Adenine was supplied by Acmec. R. roseum(20220320) was obtained from Xiang yun County Dali and authenticated by Professor Wei Cai, and dried under vacuum at 45 °C. The specimens were deposited in the School of Pharmacy, Hunan University of Medicine, Huaihua, China.
Preparation of Standards and Samples
The dry powder of R. roseum (1 g) was ultrasonically extracted in 10 mL of 70% methanol aqueous solution for 1 h, and then the extracted solution was centrifuged at 12 000 rpm for 30 min. The obtained supernatant was dried under nitrogen flow at room temperature to obtain a residue, which was reconstituted with 0.1 mL of methanol. Finally, the reconstituted solution was placed in a UHPLC-Q-Exactive orbitrap MS and injected into 2 µL for analysis. All reference standards (0.1 mg/mL) were accurately weighed and dissolved in methanol, then stored in a refrigerator at 4 °C until further analysis.
Instruments and Conditions
An Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific, San Jose, CA, USA) and the Q-Exactive Focus Orbitrap MS, equipped with an electrospray ionization (ESI) source, were used for acquiring the MS and MS2 data of R. roseum.An Thermo Scientific Hypersil GOLD™ aQ (100 mm × 2.1 mm, 1.9 μm) was applied for chromatographic separation with a column temperature of 40 °C with a flow rate of 0.3 mL/min. The mobile phase was water containing 0.1% formic acid (C) and acetonitrile (D). The gradient program is: 0 min, 5% D; 2 min, 10% D; 5 min, 16% D; 10 min, 22% D; 12 min, 55% D; 20 min, 80% D; 25 min, 95% D; 26 min, 5% D; The sample injection volume was 2 μL. MS analysis was running in both positive and negative ionization modes using an electrospray ionization (ESI). The key parameters were as follows: spray voltage, 3.5 kV (+); spray voltage, 3.2 kV (−); the sheath gas flow rate, 35 arb; aux gas flow rate, 10 arb; capillary temperature, 320 °C; heater temperature, 350 °C; S-lens RF level, 60. The scan modes were full MS with a resolution of 35 000 and MS2 spectra were obtained by PRM triggered by inclusion ions list 12 ; the scan range was m/z 100–1200. The stepped, normalized collision energies were 30%. Data acquisition and processing were carried out with the Xcalibur version 4.2 and Compound Discovery version 3.0 (Thermo Fisher Scientific, California, USA).
Results and Discussion
Analysis Strategy
To systematically screen and identify components in R. roseum, a UHPLC-Q-Exactive Orbitrap MS-PRM analysis strategy was established. First of all, R. roseum was extracted ultrasonically with 70% methanol/water (10 mL), and then dried and enriched under normal temperature nitrogen flow. Secondly, the sample was injected into the UHPLC-Q-Exactive Orbitrap MS to obtain high-resolution mass spectrometry data of trace components in R. roseum; MS2 data was obtained through parallel reaction monitoring mode (PRM). Thirdly, the improved metabolic process of Compound Discover version 3.0 was performed to process R. roseum data and predict expected compounds. Finally, the components were identified based on reference substances, secondary characteristic ion fragments, public databases, and literature.
The Chemical Constituents of Rhodobryum roseum Were Analyzed by LC-MS/MS
A total of 77 different compounds were discovered. 74 of these compounds were reported for the first time. The 77 compounds including 16 amino acids, 33 organic acids including 6 aromatic acids, 8 phenolics compounds, 2 flavonoid glycosides, 4 flavonoids and 14 other compounds including four fatty alcohols were identified through UHPLC-Q-Exactive Orbitrap MS and PRM analysis strategy, shown in Table 1. Figures 1 and 2 show extracted ion chromatograms in positive and negative ion mode and representative compounds fragmentation flow diagrams respectively.
The high-resolution extracted ion flow diagram of R. roseum (A): 137.02441,273.07684,283.06119,132.03023,151.06119,243.06225,131.03498,218.10339,153.01933,175.06119,197.08193,159.06628,163.04006,173.08193,435.12967,201.11323,229.14453,319.11871,187.13396,501.32216,257.17583,285.20713,243.19656,303.23295;(B): 181.07176, 135.02989, 173.00916, 188.05644, 145.05063, 187.09758, 299.05611, 271.22786, 191.01972, 133.01424; (C); 298.27405, 148.06043, 175.11895, 180.08664, 127.03897, 343.12348, 165.05462, 169.04953, 170.08116, 146.06004, 180.1019, 223.09648, 579.17083, 321.13326, 211.13287, 315.08631, 284.12811, 277.17982, 365.30502, 217.15869, 462.3115, 291.23185; (D); 147.07641, 130.04986, 136.06177, 182.08116, 132.1019, 166.08625, 205.09715;(A, B) EIC in negative mode. (C, D) EIC in positive mode.
Proposed selected fragmentation pattern of components identified in R. roseum: Phloretin (A), L-Malic acid (B), Jasmonic acid (C), Uridine (D), Cis-Aconitic acid (E).
Retention Times and Mass Spectral Data of R. roseum.
With standard references
Identification of Amino Acid Components
Compounds 5, 6, 15, 17, 27, 29, 32 and 38 were eluted at 0.82, 0.85, 0.90, 1.00, 1.01, 1.15, 1.23, 1.76 and 3.14 min, respectively, with the deprotonated molecular ion [M + H]− at m/z 148.0604 (C5H9NO4, −0.98 ppm), 147.0764 (C5H10N2O3, −0.74 ppm), 175.1189 (C6H14N4O2, −0.58 ppm), 147.0764 (C5H10N2O3, −0.94 ppm), 148.0604 (C5H9NO4, −1.11 ppm), 182.0811 (C9H11NO3, 0.28 ppm), 132.1019 (C6H13NO2, 0.49 ppm), 166.0862 (C9H11NO2, 0.45 ppm) and 205.0971 (C11H12N2O2, 0.37 ppm), respectively; For example, compound 12 has strong fragment ion peaks at m/z 116.0708 and 70.0658, after neutral loss calculations lost (CH5N3,59 Da) and (C2H7O2N3,105 Da), respectively, combined with the molecular ion peaks can be initially determined that the compound is Argininic acid; these were characterized as L-glutamic acid, D-glutamine,13,14 L-glutamine, D-glutamic acid, L-tyrosine, Isoleucine, L-phenylalanine and L-tryptophan respectively, by comparing the retention times and MS and MS2 information with those of the database and literature. Compounds 1 and 13 were eluted at 0.79 and 0.93 min respectively, with the deprotonated molecular ion [M-H]− at m/z 132.0302 (C4H7NO4 −7.73 ppm) and 132.0302 (C4H7NO4 −8.49 ppm), Compound 1 undergoes neutral loss (H2O, 18 Da) at m/z 115.0025 and loss (CO2, 44 Da) at m/z 88.0391, probably due to the presence of a dicarboxylic acid structure in the molecule, which undergoes a ring condensation according to Blanc G and loses one molecule of water, Also, they were identified as L-aspartic acid and D-aspartic acid 15 respectively.
Compounds 7, 19 and 24 were found at 0.85, 1.02 and 1.15 min with the quasimolecular ion [M + H]+ at m/z 130.0498. They have the same quasi-molecular ions and characteristic fragment ions as pyroglutamic acid and were initially identified as pyroglutamic acid isomers. 16
Compounds 11 and 26 have the same characteristic fragment ion [M-H]− at m/z 146.0450 as glutamic acid, and compounds 11 and 26 have a fragment ion [M-H]− at m/z 128.0343 The loss of (C2H4O2, 60 Da), then, can be initially identified as N-acetyl-L-glutamic acid and N-acetyl-D-glutamic acid. 17
Identification of Organic Acids Components
Compounds 9, 16, 48, 49, 51, and 53 were eluted at 0.86, 1.00, 6.44, 6.48, 7.62, and 9.22 min, respectively, and their deprotonated ions [M-H]− were also at m/z 133.0142 (C4H6O5, −7.79 ppm), 173.0091 (C6H6O6, −4.46 ppm), 163.0400 (C9H8O3, −4.28 ppm), 173.0819 (C8H14O4, −3.60 ppm), 137.0244 (C7H6O3, −6.48 ppm) and 187.0976 (C9H16O4, −3.22 ppm) . Compared with the retention time and MS/MS2 signal of the standard, they were identified as L-malic acid, cis-aconitic acid, 4-coumaric acid, Suberic acid, Salicylic acid and Azelaic acid respectively.
Compounds 10 and 14 have the same quasi-molecular ions and characteristic fragment ions as compounds 16 and 9, respectively. Therefore, they were tentatively identified as Trans-aconitic acid and D-malic acid.
Compounds 8 and 20 elute at 0.85 and 1.02 min, respectively, with the same quasi-molecular ion and fragment ion. Compound 8 formed a fragment ion at [M-H]- at m/z 173.0084, suggesting the loss of a water molecule (18 Da), which is likely due to steric effects that cause the hydrogen on the hydroxyl and carboxyl groups to condense and leave the carboxyl group. They were initially identified as isocitric acid and citric acid 18 by comparison with databases and literature. Compounds 31 and 33 have the same quasi-molecular ions and fragment ions. at m/s 87.0439, showing the loss of a carboxyl group (COOH, 44 Da), which can be confirmed as Ethylmalonic acid isome 19 according to the literature review and database. Similarly, compounds 43 and 52 can be determined as Camphanic acid isomer.
compound 28 has strong fragment ion peaks at m/z 123.0442 and 147.0440, and after neutral loss calculations (C2H2O, 42 Da) and (H2O,18 Da) were lost respectively, which may be due to double-kinetic fracture and phenolic hydroxyl group dropping, moreover, it can be judged to be the summed proton of phenol at m/z 95.0495, so it can be initially judged to be 2- hydroxycinnamic acid; Compound 35 has a strong fragment ion peak at m/z 83.0489, which is calculated to have lost (CH2O3, 62 Da) by neutral loss, and can be judged to be formed by further dehydration after the decarboxylation, and based on its molecular weight, it can be initially judged to be 2-methylglutaric acid. Similarly, Compounds 30, 40, 42, 47, 56, 65, 74, and 76 were eluted at 1.40, 3.27, 3.67, 5.11, 12.44, 14.22, 17.42, and 19.51 min, respectively, and their quasi-molecular ions were also at m/z 169.0495 (C8H8O4, −0.15 ppm), 175.0612 (C7H12O5, −3.64), 137.0244 (C7H6O3, −6.62 ppm), 159.0663 (C7H12O4, −4.67 ppm), 201.1132 (C10H18O4, −2.25 ppm), 187.1340 (C10H20O3, −2.61 ppm), 243.1966 (C14H28O3, 0.05 ppm) and 271.2279 (C16H32O3, 1.04 ppm). They were initially recognized as 4-methoxysalicylic acid,20,21 2-isopropylmalic acid, 22 4-hydroxybenzoic acid, 3,3-Dimethylglutaric acid, 3-tert-butyladipic acid, 3-hydroxydecanoic acid, 2-hydroxymyristic acid, and 16-hydroxyhexadecanoic acid by comparing them to database MS/MS2 information and literature.
Compound 3 has a strong fragment ion peak at m/z 75.0074 and has lost an o-diol structure by neutral loss calculation, and can be initially identified as Threonic acid based on its molecular weight. Compound 34 has a strong fragment ion peak at m/z 88.0391, combined with the fragment ion at m/z 71.0126, it can be judged that there may be an amide bond in the molecule of the compound, based on the rest of the ion fragments at m/z 146.0813 and the relevant literature, it can be preliminarily judged that the compound is Pantothenic acid. 18 Likewise, Compounds 36, 45, 59, 69, 71, and 77 were found at 1.92, 2.49, 4.21, 13.67, 14.48, 15.67, and 19.91 min, respectively, and their quasi-molecular ions [M-H]− were also at m/z 153.0193 (C7H6O4, −5.70 ppm), 159.0663 (C7H12O4, −4.67 ppm), 229.1445 (C12H22O4, −0.67 ppm), 257.1758 (C14H26O4, 0.77 ppm), 285.2071 (C16H30O4, 1.15 ppm) and 303.2330 (C20H32O2, 0.98 ppm). They were initially recognized as Protocatechuic acid, 23 Pimelic acid, Dodecanedioic acid, Tetradecanedioic acid, Hexadecanedioic acid, and Arachidonic acid 24 by comparing them to the database and literature. Compound 61 was identified in 13.80 min, and the positive ion mode quasi-molecular ion at [M + H]−m/z 211.1329 (C12H18O3, 0.52 ppm). By comparison with databases and literature, it was identified as Jasmonic acid. 25
Identification of Phenolics Components
Compound 46 had a characteristic fragment ion peak at m/z 121.0649, which was identified as acetyl (C2H5ON,59 Da), and combined with the molecular ion peaks, it could be initially determined that the compound was N-Acetyltyramine, Compound 60 exhibited both positive and negative ions, by comparison with databases and literature, it was identified as Mycophenolic acid. 26 Compounds 22, 37 and 64 were found at 1.03, 3.06 and 14.14 min, respectively, and their quasi-molecular ions [M + H]− were also at m/z 127.0390 (C6H6O3, 0.15 ppm), 170.0812 (C8H11NO3, 0.30 ppm) and 277.1798 (C17H24O3, 0.39 ppm). They were initially identified as Pyrogallol, Norepinephrine and Shogaol by comparing them to database MS/MS2 information and literature.
Compounds 39 and 44 observed at 3.14 and 3.91 min have the same precursor ion at m/z 119.0493 and a strong molecular ion peak at m/z 147.0441, which can be preliminarily judged that there is a benzene ring; The fragment ion generated by the loss of C2HON residue (55 Da) at m/z 91.0547 can be further considered to be the loss of the phenolic hydroxyl group and its attached carbon atom and nitrogen atom on another ring. These compounds were tentatively designated as Hydroxyquinoline isomers. 27 And the same, Compounds 41 and 76 responded in negative ion mode, and compared with the database and literature, they can be identified as Protocatechualdehyde. 28
Identification of Flavonoids Components
Compounds 54, 62, 57, 58, and 68 were eluted at 10.19, 13.91, 13.16, 13.44, and 14.39 min in positive and negative ion modes, respectively, and their quasi-molecular ions were also at m/z 579.1708 (C27H30O14, 0.33 ppm), 315.0863 (C17H14O6, 0.43 ppm), 273.0768 (C15H14O5, 2.21 ppm), 299.0561 (C16H12O6, 1.37 ppm), 283.0612 (C16H12O5, 1.46 ppm). By comparing with standard MS/MS2 information and literature, they were preliminary identification For Kaempferitrin, Scrophulein,29,30 Phloretin, Hispidulin 31 and Genkwanin.32,33 Compound 55 has a strong signal at m/z 121.0284. According to the secondary fragment ion molecules, it may be o-formylphenol (C7H5O2, 121 Da), according to the literature review and database, tentatively for Phloridzin isomer. 34
Identification of Other Components
Compounds 18 and 23 were eluted at 1.02 and 1.04 min, respectively, and compound 23 dropped a C6H15O8 group (216 Da) at [M + H]− at m/z 127.0390 in positive ion mode, according to MS/MS2 information, it can be speculated that a dehydroxylated glycoside is left. According to the standard and literature database, these compounds were tentatively designated as Adenine, α-Lactose. Compound 25 observed at 1.11 min has a fragment ion at m/z 110.0236 caused by the loss of C5H8O4 resi-due (133 Da), which can be judged to have lost a molecule of ribofuranose and can be tentatively identified as uridine by reviewing the literature, and its cleavage pattern may be the one shown in Figure 2, D. 35 Similarly, compounds 2, 4 and 66 were eluted at 0.80, 0.81 and 14.22 min, respectively, detected in negative ion mode and compared with literature and database MS/MS2, they can be initially identified as Dulcitol, 36 Arabitol and Retigeric acid B. 37
Compound 72 obtained a molecular ion peak at m/z 217.1587 in positive ion mode and a strong fragment ion peak at m/z 161.0961, according to the neutral loss calculation (C4H8, 56 Da) it can be judged that the loss of the tert-butyl substituent, and according to the database and the molecular weight it can be preliminarily judged that this compound is Turmerone. Similarly, Compounds 21, 50, 63, 67, 70, 73, and 75 were eluted at 1.03, 6.71, 13.91, 14.31, 15.24, 17.03, and 17.44 min, respectively, and their protons in positive ion mode Chloride ions [M + H]− were also at m/z 180.0866 (C6H13NO5, −0.38 ppm), 223.0965 (C12H14O4, −0.34 ppm), 284.1281 (C17H17NO3, −0.14 ppm), 365.3050 (C23H40O3, 0.35 ppm), 298.2741 (C18H35NO2, −0.02 ppm), 462.3115 (C29H39N3O2, −0.74 ppm) and 291.2319 (C19H30O2, −0.57 ppm). After comparing the database and literature MS/MS2, they were initially identified as D-glucosamine, 38 Resorcinol diglycidyl ether, methyl N-benzoylphenylalaninate, 39 2-Arachidonyl glycerol ether, 2-Aminooctadec-4-yne-1, 3-diol-, Echinulin 40 and 5α-Dihydrotestosterone. 41
Discussion
In this study, uridine and protocatechuic acid were reported in previous studies. Liuliu Zi et al 42 verified by in vitro and in vivo experiments that uridine inhibits the proliferation, invasion and migration of Hepatocellular Carcinoma Cells by the activation of the iron pendant pathway. The uridine acts as a precursor substance for uridine-5′-diphosphate, which activates mitoKATP and thus gives cardioprotection. 43 Liyan Bai et al 44 verified that protocatechuic acid inhibits the growth of cardiomyocytes by acting on kynurenine-3-monooxygenase through the down-regulation of its expression in a constructed animal model of heart failure, and reduced the number of cardiomyocytes by constructing an animal model of heart failure with protocatechuic acid as the delivery group. The use of protocatechuic acid as an inhibitor of cardiomyocytesis reactive oxygen species production to prevent and treat heart failure.
Among the compounds reported for the first time in this study, hispidulin and protocatechualdehyde also showed significant and relevant biological activities. Gao Hui et al 45 demonstrated that apoptosis of HepG2 cells was due to a decrease in Bcl-2/Bax ratio, disruption of mitochondrial membrane potential, and an increase in the release of cytochrome C and activated capase-3, possibly due to the pro-apoptotic effect of hispidulin through the dysfunction of mitochondria and inhibition of P13k/Akt signalling pathway. The mechanism of action may be hispidulin-mediated through mitochondrial dysfunction and inhibition of P13k/Akt signalling pathway. hispidulin also has effects on other tumour cells, and has a synergistic effect in combination with radiotherapy and chemotherapy drugs.46,47 Ning Wang et al 48 identified a possible pathway Wnt/β-catenin by PCR enrichment, Wnt/β-catenin pathway is positively correlated with hepatocellular carcinoma, and protocatechuic acid can indirectly regulate the Wnt/β-catenin pathway through the modulation of the WT1 protein to inhibit the growth of hepatocellular carcinoma tumour. In addition, protocatechuic aldehyde can regulate oxidative stress to fight against related diseases. 49
Therefore, the chemical discovery of R. roseum was very benefit for understanding their material basis, further development of new antitumour drugs and cardioprotective drugd and its usage in clinical.
Conclusions
In this study, a strategy based on UHPLC-Q-Exactive Orbitrap MS combined with PRM analysis technology was develop to identify the chemicals of R. roseum. A total of 77 chemical components were identified by comparison of retention time, MS and MS2 and reference data, including 16 amino acids, 33 organic acids, 8 phenolics compounds, 6 flavonoids and 14 other compounds. Among them, 74 compounds were discovered for the first time from R. roseum. The results of this study greatly expanded the understanding of the chemical constituents of R. roseum, contributed to the utilization of active components of R. roseum, and provided a basis for the quality control of R. roseum. However, the study did not explore further on the biological activities of the identified compounds and the mechanism of interaction between the chemical constituents.
Footnotes
Acknowledgments
This work was supported by the Scientific Research of Hunan Provincial Education Department and the Scientific research cultivation project of Hunan University of Medicine.
Author Contributions
S.-L.Y., writing—original draft preparation and data curation; K.-L.L., conceptualization, formal analysis, and validation; Q.-R.Y., data curation; C.H. and K.-Q.Y., investigation and formal analysis; L.-W.W. and L.L., resources; Q.L., software; H.L. and Y-N.L., writing—review and editing, conceptualization, and supervision. All authors have read and agreed to the published version of the manuscript.
Data Availability
Data will be made available on request.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
