Chemical profiling of root bark extract from Oplopanax elatus and its in vitro biotransformation by human intestinal microbiota
- Published
- Accepted
- Received
- Academic Editor
- Rogerio Sotelo-Mundo
- Subject Areas
- Biochemistry, Microbiology
- Keywords
- Oplopanax elatus, Intestinal microbiota, UPLC-Q-TOF/MS, Metabolic profiles, Biotransformation
- Copyright
- © 2021 Wan et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
- Cite this article
- 2021. Chemical profiling of root bark extract from Oplopanax elatus and its in vitro biotransformation by human intestinal microbiota. PeerJ 9:e12513 https://doi.org/10.7717/peerj.12513
Abstract
Oplopanax elatus (Nakai) Nakai, in the Araliaceae family, has been used in traditional Chinese medicine (TCM) to treat diseases as an adaptogen for thousands of years. This study established an ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) method to identify chemical components and biotransformation metabolites of root bark extract from O. elatus. A total of 18 compounds were characterized in O. elatus extract, and 62 metabolites by human intestinal microbiota were detected. Two polyynes, falcarindiol and oplopandiol were recognized as the main components of O. elatus, whose metabolites are further illustrated. Several metabolic pathways were proposed to generate the detected metabolites, including methylation, hydrogenation, demethylation, dehydroxylation, and hydroxylation. These findings indicated that intestinal microbiota might play an essential role in mediating the bioactivity of O. elatus.
Introduction
Oplopanax elatus (Nakai) Nakai is the plant of genus Oplopanax, which belongs to the Araliaceae family. It is mainly distributed in northeast China, Korea and far east of Russia (Dou et al., 2009; Yang et al., 2010). As a traditional medicinal plant, O. elatus is being utilized as a ginseng-like herbal medicine and has been long used as an adaptogen to treat arthritis, diabetes mellitus, rheumatism, neurasthenia, and cardiovascular diseases (Dai et al., 2016; Eom et al., 2017; Knispel et al., 2013; Moon et al., 2013; Panossian et al., 2021). Previous studies have identified several components derived from O. elatus, such as the lignans, saponins, phenolic glycosides, and polyynes (Huang et al., 2010; Shao et al., 2016). To date, polyynes have been chiefly reported with high contents in the root of O. elatus (Huang et al., 2014a). Increasing attention has been paid to two main polyynes facarindiol (FAD) and oplopandiol (OPD), because of their significant anti-tumor activities (Purup, Larsen & Christensen, 2009; Qiao et al., 2017; Sun et al., 2016). However, most studies remain focused on the pharmacological and chemical constituents of O. elatus, while its metabolic profiles are rather obscured.
It is widely known that human beings live in symbiotics with coevolutionary microbiota (Thursby & Juge, 2017; Yang & Lao, 2019). The human gastrointestinal tract is the primary habitat for trillions of microbes. The gut microbiota serves metabolic functions crucial for the human host (Bäckhed et al., 2004; Chen et al., 2016; Rajilić-Stojanović & de Vos, 2014) and influences the biofunctions (Barko et al., 2018; Defois et al., 2018; Pagliari et al., 2017). Like most herbal medicines, O. elatus products are orally administered. The multiple constituents of O. elatus are typically brought into contact with intestinal bacteria and subsequently transformed in the digestive tract (Gao et al., 2018; Huang et al., 2014b; Koppel, Maini Rekdal & Balskus, 2017; Shikov et al., 2014; Teschke et al., 2015). However, existing reports did not address intestinal microflora’s biotransformed metabolites of O. elatus. Therefore, elucidating how gut microbes treat these complex components may contribute to a complete understanding of the metabolic profiles and biological activities of O. elatus.
Recently, various analytical platforms are typically applied to identify metabolic profiles in the complex extracts of TCMs. Most notably, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is one of the powerful analytical tools (Jin et al., 2018; Wu et al., 2019; Yang et al., 2016). With the newly developed chromatographic technique, the UPLC system allows significant improvements in the resolution, analysis speed, and reduction of solvent waste (Chekmeneva et al., 2018; Du et al., 2017; Wang et al., 2008). Meanwhile, high-resolution Q-TOF/MS can give more specific and accurate mass information on characteristic molecular ions and fragment ions, providing a reliable basis for the qualitative analysis of complex samples (Li et al., 2019; Lou et al., 2015; Wewer et al., 2011). Based on these characteristics, UPLC-Q-TOF/MS was ultimately selected for fast identification of constituents in O. elatus.
In the present study, we focused on the metabolic behavior of human intestinal microflora on O. elatus. A highly selective and sensitive UPLC-Q-TOF/MS method was established to characterize the chemical and metabolic profiles of O. elatus. Furthermore, the proposed metabolic pathways were also summarized. This work will provide a better understanding for exploring the bioactivities of O. elatus in vivo.
Materials & methods
Materials and reagents
The general anaerobic medium for bacteria culture was obtained from Shanghai Kayon Biological Technology Co. Ltd. (Shanghai, China). Formic acid and HPLC-grade acetonitrile were purchased from Merck (Darmstadt, Germany). Deionized water (18 MΩ·cm) was supplied with a Millipore Milli-Q water system (Milford, MA, USA). All other reagents were from standard commercial sources and of analytical purity.
Preparation of Oplopanax elatus extract
Root bark of O. elatus was obtained from Benxi city (Liaoning, China). The voucher samples were deposited at the Tang Center for Herbal Medicine Research at the University of Chicago (Chicago, IL, USA). The air-dried root bark of O. elatus was pulverized into powder and sieved through an 80-mesh screen. Eight g of the powder were extracted twice by heat-reflux with 70% ethanol for 2 h. The combined extract was evaporated under vacuum and lyophilized with a yield of 28%. The samples were stored at 4 °C until use.
Preparation of human intestinal microflora
The Institutional Review Board approved the present study protocol at the University of Chicago (IRB protocol number: 12536). Fresh fecal samples were collected from six healthy adult volunteers (male, aged 20–55, non-smokers without antibiotic consumption for more than 6 months, and written consent was obtained). All the fecal samples were mixed for analysis. A total of five g of samples were homogenized in 30 ml cold physiological saline, and centrifuged at 13,000 rpm for 10 min to obtain the resulting fecal supernatant.
Incubation of sample in intestinal bacteria
Two microliters of the fecal supernatant were added with eight ml anaerobic dilution medium containing five mg of O. elatus extract, which were then anaerobically incubated at 37 °C for 24 h in an anaerobic workstation (Electrotek, UK). The reaction mixtures were extracted three times with water-saturated n-butanol. All the n-butanol layers were mixed and dried under a nitrogen stream and then dissolved in one ml methanol. The solutions were centrifuged at 13,000 rpm for 10 min for analysis.
UPLC-Q-TOF/MS analysis
Data were collected as previously described (Wang et al., 2020). The Agilent 1290 Series UPLC system (Agilent Technologies, Santa Clara, CA, USA) was applied to perform the chromatographic analysis, and a binary pump, an online degasser, an auto plate-sampler, and a thermostatically controlled column compartment were also equipped for this system. The separation was carried out on UPLC ACQUITY HSS C8 column (2.1 mm × 100 mm × 1.7 μm, Waters) with a constant flow rate of 0.4 mL/min, and the column temperature was kept at 40 °C. A gradient mobile phase system of 0.1% formic acid in water (phase A) and acetonitrile (phase B) was applied as follows: 5% B at 0–1 min, 5–20% B at 1–18 min, 20–30% B at 18–27 min, 30–35% B at 27–32 min, 35–60% B at 32–40 min, 60–95% B at 40–50 min, 95% B at 50–53 min, 95–5% B at 53–55 min. The injection volume of samples was set at two μL for MS mode and five μL for MS/MS mode.
The Agilent 6545 Q-TOF-MS system with a Dual electrospray ionization source was used to conduct the detection. Nitrogen (purity > 99.999%) served as a sheath gas and drying gas, and the flow velocities were set at 11 and 8 L/min. The temperatures of sheath gas and drying gas were set at 350 and 320 °C respectively. Positive and negative ion modes were both employed in this study. The other parameters were set as follows: nebulizer pressure, 35 psig; voltage, 3,500 V; fragmentor voltage, 175 V; mass range, m/z 100–1,700; data acquisition rate, 1.5 scans/s; MS/MS spectra collision energy, 50 eV (Wang et al., 2020).
Data analysis
Mass data were analyzed by the Agilent MassHunter Workstation software (Version B.06.01), based on the accurate measurements of m/z values with online databases (MassBank, etc.), to screen probable compounds. The empirical molecular formula was deduced by comparing the theoretical mass of molecular ions at the mass accuracy of less than five ppm.
Results
Optimization of UPLC-Q-TOF/MS conditions
To obtain the chromatograms with better resolution and higher baseline stability of O. elatus extract and its primary metabolites, multiple mobile phases such as acetonitrile-water and methanol-water were detected. Acetonitrile-water was applied as the solvent, for its stronger separation ability, shorter retention time, and lower column pressure. Additionally, 0.1% formic acid added in the water as mobile phase adducts may help to achieve higher response and better peak sensitivity (Tao et al., 2016). Therefore, the optimal solvent system consisting of acetonitrile-water (0.1% formic acid), which remarkably enhanced the efficiency of ionization and satisfactory sensitivity, was ultimately selected as mobile phase with a gradient elution.
In addition, the factors related to MS performance, including ionization mode and collision energy, were further improved. The positive ion mode was ultimately employed to gain comprehensive data for structural characterization and metabolite assignment with much lower background noise. The collision energy was optimized to obtain the higher ionization efficiency and relative abundance of precursor and product ions.
Chemical profiling of O. elatus extract
In total, 18 ingredients of O. elatus were detected in this study, and their chemical structures are shown in Fig. 1. There are six types of compounds, including nine polyynes, three lignans, one phenylpropanoid, two sesquiterpenes, one triterpenoid, and two fatty acids. The total ion chromatogram (TIC) of O. elatus extract is shown in Fig. 2A in the positive ion mode by UPLC-Q-TOF-MS. Table 1 shows the detailed information, including retention time, signal intensity, molecular formula, calculated and experimental mass m/z, ppm error, and fragment ions of these 18 components (Schymanski et al., 2014; Wang et al., 2020).
Figure 1: The chemical structures of bioactive compounds detected in Oplopanax elatus extract.
(A) Polyynes; (B) Lignans; (C) Phenylpropanoid; (D) Sesquiterpenes; (E) Triterpenoid; (F) Fatty acids.Figure 2: UPLC-TOF/MS profiles of O. elatus extract in the positive ion mode.
(A) Total ion chromatogram (TIC) of O. elatus extract. (B) TIC of blank sample including dilution medium and human fecal microflora. (C) TIC of biotransformed O. elatus sample by intestinal bacteria.No. | Compound | Formula | tR (min) | Signal intensity (×105) | [M+H]+ or [M+Na]+ | Fragment ions in the positive mode with the energy 50 V CID | ||
---|---|---|---|---|---|---|---|---|
m/z | Calc m/z | Diff (ppm) | ||||||
1 | 4-(3-hydroxyprop-1-en-1-yl)-2,6-dimethoxyphenyl β-D-glucopyranoside | C17H24O9 | 5.24 | 2.86 ± 0.82 | 373.1495 | 373.1493 | −0.51 | 211.1526[M-glc+H]+, 373.1495[M+H]+ |
2 | 4’,7-epoxy-4,9,9’-trihydroxy-3,3’-dimethoxy-5’,8-lignan-4,9-bis[O-β-D-glucopyranoside] | C32H44O16 | 10.92 | 1.74 ± 0.34 | 707.2521 | 707.2522 | 0.08 | 545.1981[M-glc+Na]+, 707.2521[M+Na]+ |
3 | isolariciresinol 3-O-β-D-glucopyranoside | C26H34O11 | 14.14 | 0.55 ± 0.17 | 545.1997 | 545.1993 | −0.70 | 383.1428[M-glc+Na]+, 545.1997[M+Na]+ |
4 | 5-methoxylariciresinol 4-O-β-D-glucopyranoside | C27H36O12 | 15.04 | 0.89 ± 0.30 | 575.2112 | 575.2099 | −2.36 | 412.1434[M-C10H11O2+Na]+, 250.0381[M-C10H11O2-glc+Na]+, 575.2112[M+Na]+ |
5 | 2-decenoic acid | C10H18O2 | 23.41 | 5.62 ± 1.74 | 171.1378 | 171.1380 | 0.92 | 55.9342[M-HCOOH-C5H10+H]+, 171.1378[M+H]+ |
6 | oploxyne B | C18H30O4 | 36.91 | 2.73 ± 0.36 | 311.2212 | 311.2217 | 1.57 | 107.0527[M-H2O-C11H22O2+H]+, 79.0547[M-H2O-C11H22O2-C2H4+H]+, 311.2212[M+H]+ |
7 | oploxyne A | C17H26O3 | 37.62 | 2.62 ± 0.44 | 279.1957 | 279.1955 | −0.82 | 107.0485 [M-H2O-C10H18O+H]+, 79.0545[M-H2O-C10H18O-C2H4+H]+, 279.1957[M+H]+ |
8 | oplopantriol B | C18H28O3 | 37.89 | 1.28 ± 1.07 | 293.2110 | 293.2111 | 0.41 | 107.0491[M-H2O-C11H20O+H]+, 79.0538[M-H2O-C11H20O-C2H4+H]+, 293.2110[M+H]+ |
9 | 9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate | C20H28O4 | 38.63 | 1.57 ± 1.21 | 333.2048 | 333.2060 | 3.72 | 105.0700[M-H2O-C13H22O2+H]+, 79.0546[M-H2O-C13H22O2-C2H2+H]+, 333.2048 [M+H]+ |
10 | oplopandiol acetate | C20H30O4 | 39.18 | 3.93 ± 1.41 | 335.2215 | 335.2217 | 0.56 | 107.0503[M-H2O-C13H22O2+H]+, 79.0545[M-H2O-C13H22O2-C2H4+H]+, 335.2215[M+H]+ |
11 | 6,9-octadedicenoic acid | C18H32O2 | 39.46 | 5.13 ± 1.95 | 281.2470 | 281.2475 | 1.81 | 65.0396[M-CH3COOH-C11H20+H]+, 281.2470[M+H]+ |
12 | falcarindiol | C17H24O2 | 40.76 | 101.47 ± 12.16 | 261.1848 | 261.1849 | 0.41 | 105.0713[M-H2O-C10H18+H]+, 79.0548[M-H2O-C10H18-C2H2+H]+, 261.1848 [M+H]+ |
13 | oplopandiol | C17H26O2 | 41.23 | 119.24 ± 9.28 | 263.2010 | 263.2006 | −1.69 | 107.0459[M-H2O-C10H18+H]+, 79.0563[M-H2O-C10H18-C2H4+H]+, 263.2010[M+H]+ |
14 | falcarinol | C17H24O | 42.85 | 62.85 ± 7.26 | 245.1899 | 245.1900 | 0.38 | 105.0699[M-C10H18+H]+, 79.0556[M-C10H18-C2H2+H]+, 245.1899 [M+H]+ |
15 | oplopantriol A | C18H26O3 | 43.31 | 67.83 ± 4.40 | 291.1956 | 291.1955 | 0.44 | 105.0659[M-H2O-C11H20O+H]+, 79.0567[M-H2O-C11H20O-C2H2+H]+, 291.1956 [M+H]+ |
16 | curcumene | C15H22 | 44.92 | 4.37 ± 2.22 | 203.1797 | 203.1794 | −1.35 | 134.1063[M-C5H9+H]+, 65.0382[M-2×C5H9+H]+, 203.1797[M+H]+ |
17 | muurolene | C15H24 | 45.27 | 8.27 ± 3.82 | 205.1950 | 205.1951 | 0.38 | 65.0550[M-2×C5H9+H]+, 205.1950[M+H]+ |
18 | oleanolic acid | C30H48O3 | 46.88 | 10.83 ± 3.28 | 479.3508 | 479.3496 | −2.70 | 231.1715[M-C16H24O2+Na]+, 479.3508[M+Na]+ |
Polyynes have been found as the main constituents in the root of O. elatus (Yang et al., 2014). Among them, falcarindiol and oplopandiol were determined to have very high contents in the air-dried root bark. As shown in Table 1, polyynes exhibit the same elemental composition and similar MS/MS behaviors, with the characteristic fragment ions at m/z 79.05 in the positive ion mode.
For example, the typical protonated molecular ion [M+H]+ of FAD was observed at m/z 261.1848 in the mass spectrum. The fragment ion at m/z 105.0713 was formed by the losses of H2O and C10H18, with m/z 79.0548 by further loss of C2H2. OPD was identified by the protonated molecular ion [M+H]+ at m/z 263.2010 compared with calculated m/z 263.2006. The fragment ion at m/z 107.0459 was produced by the losses of H2O and C10H18, and m/z 79.0563 was formed by further loss of C2H4.
In addition, phenylpropanoid compound 4-(3-hydroxyprop-1-en-1-yl)-2,6-dimethoxyphenyl β-D-glucopyranoside was determined to be C17H24O9 at m/z 373.1495 ([M+H]+, C17H24O9+; calculated as 373.1493). The neutral loss of 1 × Glc moiety formed fragment ion at m/z 211.1526. Similarly, three lignans were determined by the neutral loss of 1 × Glc moiety in the [M+Na]+ mode.
Detection and identification of metabolites of O. elatus extract
The control sample was prepared in parallel, which used in the dilution medium and human fecal microflora, as shown in Fig. 2B. The biotransformed O. elatus sample by intestinal bacteria is shown in Fig. 2C. Samples were incubated, pretreated, and analyzed under the same conditions as mentioned in “Incubation of sample in intestinal bacteria”. The potential metabolites were detected from the TIC of the transformed O. elatus sample compared to the control group. All the metabolites were further confirmed by the extracted ion chromatograms (EICs) and their MS/MS corresponding fragments. A total of 62 metabolites were identified by UPLC-Q-TOF-MS in the positive mode. Table 2 shows the retention time, signal intensity, experimental and calculated mass m/z, difference between m/z and calculated m/z in ppm, and fragment ions in the MS/MS stage of these 62 metabolites (M1-M62). All these metabolites could not be observed or only in trace amounts in control samples (Schymanski et al., 2014).
No. | Description | Formula | tR (min) |
Signal intensity (×105) | [M+H]+ or [M+Na]+ | Fragment ions in the positive mode with the energy 50 V CID | ||
---|---|---|---|---|---|---|---|---|
m/z | Calc m/z | Diff (ppm) | ||||||
M1 | deglycosylation product of 4-(3-hydroxyprop-1-en-1-yl)-2,6-dimethoxyphenyl β-D-glucopyranoside | C11H14O4 | 9.90 | 2.83 ± 0.32 | 211.0962 | 211.0965 | 1.36 | 92.0582[M-C3H5O-OCH3×2+H]+, 211.0962[M+H]+ |
M2 | acetylization product of 9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate | C22H30O5 | 16.55 | 16.27 ± 2.46 | 375.2170 | 375.2166 | −1.07 | 79.0544[M-C13H24O3-C2H2+H]+, 375.2170[M+H]+ |
M3 | deglycosylation product of 4’,7-epoxy-4,9,9’-trihydroxy-3,3’-dimethoxy-5’,8-lignan-4,9-bis[O-β-D-glucopyranoside] | C20H24O6 | 20.06 | 11.37 ± 2.01 | 383.1470 | 383.1465 | −1.36 | 188.1603 [M-C10H11O4+Na]+, 383.1470 [M+Na]+ |
M4 | deglycosylation product of isolariciresinol 3-O-β-D-glucopyranoside | C20H24O6 | 20.08 | 11.32 ± 0.98 | 383.1464 | 383.1465 | 0.30 | 167.4648 [M-C10H12O3-2×H2O+Na]+, 383.1464[M+Na]+ |
M5 | deglycosylation product of 5-methoxylariciresinol 4-O-β-D-glucopyranoside | C21H26O7 | 22.65 | 14.28 ± 1.42 | 413.1578 | 413.1571 | −1.86 | 231.8773 [M-C9H10O4+Na]+, 413.1578 [M+Na]+ |
M6 | hydroxylation product of oplopandiol acetate | C20H30O5 | 30.58 | 0.72 ± 0.43 | 351.2153 | 351.2166 | 3.71 | 107.0840 [M-H2O-C13H22O3+H]+, 79.0547[M-H2O-C13H22O3-C2H4+H]+, 351.2153[M+H]+ |
M7 | hydroxylation product of oploxyne B | C18H30O5 | 30.64 | 3.02 ± 0.55 | 327.2163 | 327.2166 | 0.92 | 107.0851 [M-H2O-C11H22O3+H]+, 79.0543 [M-H2O-C11H22O3 -C2H4+H]+, 327.2163[M+H]+ |
M8 | acetylization product of oploxyne B | C20H32O5 | 31.64 | 1.82 ± 0.63 | 353.2321 | 353.2323 | 0.43 | 107.0847 [M-H2O-C13H24O3+H]+, 79.0545[M-H2O-C13H24O3-C2H4+H]+, 353.2321[M+H]+ |
M9 | hydroxylation product of curcumene | C15H22O | 32.61 | 42.47 ± 4.29 | 219.1745 | 219.1743 | 0.73 | 63.0237[M-2×C5H9-H2O+H]+, 219.1745 [M+H]+ |
M10 | hydroxylation product of 9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate | C20H28O5 | 34.08 | 0.98 ± 0.26 | 349.1994 | 349.2010 | 4.45 | 105.0700[M-H2O-C13H22O3+H]+, 79.0573[M-H2O-C13H22O3-C2H2+H]+, 349.1994 [M+H]+ |
M11 | demethylation product of oploxyne B | C17H28O4 | 34.24 | 15.83 ± 3.11 | 297.2061 | 297.2060 | −0.22 | 107.0508 [M-H2O-C10H20O2+H]+, 79.0554[M-H2O-C10H20O2-C2H4+H]+, 297.2061[M+H]+ |
M12 | dehydroxylation product of 2-decenoic acid | C10H18O | 34.27 | 1.36±0.42 | 155.1431 | 155.1430 | −0.38 | 56.9427[M-CHO-C5H10+H]+, 155.1431[M+H]+ |
M13 | hydroxylation product of oplopantriol A | C18H26O4 | 34.90 | 1.54 ± 0.72 | 307.1902 | 307.1904 | 0.61 | 105.0682[M-H2O-C11H20O2+H]+, 79.0539 [M-H2O-C11H20O2-C2H2+H]+, 307.1902[M+H]+ |
M14 | dehydroxylation product of oplopantriol A | C18 H26O2 | 34.92 | 52.73 ± 3.40 | 275.2003 | 275.2006 | 0.94 | 105.0698[M-H2O-C11H20+H]+, 79.0541[M-H2O-C11H20-C2H2+H]+, 275.2003[M+H]+ |
M15 | methylation product of falcarindiol | C18H26O2 | 34.98 | 49.23 ± 2.49 | 275.2008 | 275.2006 | −0.89 | 105.0704[M-H2O-C11H20+H]+,79.0548[M-H2O-C11H20-C2H2+H]+, 275.2008[M+H]+ |
M16 | dehydroxylation product of oplopandiol acetate | C20H30O3 | 35.25 | 37.62 ± 4.21 | 319.2256 | 319.2268 | 3.68 | 107.0525 [M-H2O-C13H22O+H]+, 79.0538[M-H2O-C13H22O-C2H4+H]+, 319.2256[M+H]+ |
M17 | hydrogenation product of falcarindiol | C17H26O2 | 35.89 | 1.36 ± 0.77 | 263.2008 | 263.2006 | −0.93 | 105.0695[M-H2O-C10H20+H]+,79.0535[M-H2O-C10H20-C2H2+H]+, 263.2008[M+H]+ |
M18 | acetylization product of oplopantriol B | C20H30O4 | 37.06 | 12.48 ± 2.41 | 335.2202 | 335.2217 | 4.45 | 107.0855 [M-H2O-C13H22O2+H]+,79.0543[M-H2O-C13H22O2-C2H4+H]+,335.2202[M+H]+ |
M19 | hydrogenation product of oploxyne A | C18H32O4 | 37.63 | 7.37 ± 2.49 | 313.2375 | 313.2373 | −0.53 | 107.0575[M-H2O-C11H24O2+H]+, 79.0541[M-H2O-C11H24O2-C2H4+H]+, 313.2375[M+H]+ |
M20 | hydroxylation product of muurolene | C15H24O | 37.76 | 13.47 ± 3.57 | 221.1901 | 221.1900 | −0.49 | 65.0374[M-2×C5H9-H2O+H]+, 221.1901[M+H]+ |
M21 | hydrogenation product of 9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate | C20H30O4 | 37.77 | 17.38 ± 4.76 | 335.2204 | 335.2217 | 3.85 | 105.0694[M-H2O-C13H24O2+H]+, 79.0543[M-H2O-C13H24O2-C2H2+H]+, 335.2204 [M+H]+ |
M22 | acetylization product of oplopantriol A | C20H28O4 | 37.90 | 2.55 ± 0.77 | 333.2047 | 333.2060 | 4.02 | 105.0705[M-H2O-C13H22O2+H]+, 79.0524[M-H2O-C13H22O2-C2H2+H]+, 333.2047[M+H]+ |
M23 | hydrogenation product of oplopandiol acetate | C20H32O4 | 37.91 | 12.42 ± 3.28 | 337.2358 | 337.2373 | 4.57 | 79.0538[M-C13H26O3-C2H4+H]+, 337.2358[M+H]+ |
M24 | hydroxylation product of 6,9-octadedicenoic acid | C18H32O3 | 37.92 | 17.52 ± 2.71 | 297.2423 | 297.2424 | 0.41 | 77.0379[M-CH3COOH-C10H20O+H]+, 297.2423[M+H]+ |
M25 | hydrogenation product of oplopantriol B | C18 H30O3 | 38.22 | 24.52 ± 3.98 | 295.2263 | 295.2268 | 1.60 | 79.0557[M-C11H24O2-C2H4+H]+, 295.2263[M+H]+ |
M26 | demethylation product of falcarindiol | C16H22O2 | 38.59 | 2.26 ± 0.74 | 247.1689 | 247.1693 | 1.45 | 105.0686[M-H2O-C9H16+H]+, 79.0528[M-H2O-C9H16-C2H2+H]+, 247.1689[M+H]+ |
M27 | hydroxylation product of oplopandiol | C17H26O3 | 38.64 | 50.12 ± 4.62 | 279.1958 | 279.1955 | −1.18 | 107.0496[M-H2O-C10H18O+H]+, 79.0541[M-H2O-C10H18O-C2H4+H]+, 279.1958[M+H]+ |
M28 | acetylization product of oplopandiol acetate | C22H32O5 | 39.27 | 4.92 ± 0.44 | 377.2326 | 377.2323 | −0.93 | 79.0543[M-C15H26O4-C2H4+H]+, 377.2326[M+H]+ |
M29 | hydroxylation product of falcarinol | C17H24O2 | 39.54 | 4.39 ± 0.58 | 261.1851 | 261.1849 | −0.74 | 79.0538[M-C10H20O-C2H2+H]+, 261.1851 [M+H]+ |
M30 | hydroxylation product of oploxyne A | C17H26O4 | 39.60 | 2.74 ± 0.94 | 295.1902 | 295.1904 | 0.63 | 79.0557[M-C10H20O3-C2H4+H]+, 295.1902[M+H]+ |
M31 | demethylation product of falcarinol | C16H22O | 39.61 | 1.02 ± 0.57 | 231.1744 | 231.1743 | −0.25 | 79.0544[M-C9H18-C2H2+H]+, 231.1744[M+H]+ |
M32 | demethylation product of oplopantriol B | C17H26O3 | 39.69 | 33.79 ± 4.95 | 279.1965 | 279.1955 | −3.70 | 79.0542[M-C10H20O2-C2H4+H]+, 279.1965[M+H]+ |
M33 | hydrogenation product of oploxyne A | C17H28O3 | 40.06 | 10.32 ± 1.45 | 281.2113 | 281.2111 | −0.64 | 79.0541[M-C10H22O2-C2H4+H]+, 281.2113[M+H]+ |
M34 | hydroxylation product of oplopantriol B | C18H28O4 | 40.07 | 5.28 ± 1.82 | 309.2062 | 309.2060 | −0.53 | 79.0560[M-C11H22O3-C2H4+H]+, 309.2062[M+H]+ |
M35 | demethoxy product of oploxyne B | C17H28O3 | 40.07 | 9.24 ± 1.23 | 281.2112 | 281.2111 | −0.28 | 79.0541[M -C10H22O2-C2H4+H]+, 281.2112[M+H]+ |
M36 | hydrogenation product of oplopantriol A | C18 H28O3 | 40.09 | 2.83 ± 0.75 | 293.2109 | 293.2111 | 0.76 | 79.0538 [M-C11H24O2-C2H2+H]+, 293.2109[M+H]+ |
M37 | demethylation product of oleanolic acid | C29H46O3 | 40.18 | 0.54 ± 0.26 | 443.3524 | 443.3520 | −0.97 | 165.0912[M-C16H24O2-2×CH3+H]+, 443.3524[M+H]+ |
M38 | demethylation product of curcumene | C14H20 | 40.18 | 2.42 ± 0.29 | 189.1636 | 189.1638 | 0.94 | 51.0229[M-2×C5H9+H]+, 189.1636[M+H]+ |
M39 | dehydroxylation product of oploxyne A | C17H26O2 | 40.29 | 0.51 ± 0.23 | 263.2005 | 263.2006 | 0.22 | 79.0552[M-C10H20O-C2H4+H]+, 263.2005[M+H]+ |
M40 | demethylation product of oplopandiol | C16H24O2 | 40.48 | 7.12 ± 0.44 | 249.1851 | 249.1849 | −0.78 | 107.0863[M-H2O-C9H14+H]+, 79.0543[M-H2O-C9H14-C2H4+H]+, 249.1851[M+H] |
M41 | hydrogenation product of falcarinol | C17H26O | 40.50 | 22.15 ± 2.35 | 247.2053 | 247.2056 | 1.39 | 79.0528[M-C10H22-C2H2+H]+, 247.2053 [M+H]+ |
M42 | methylation product of oplopandiol acetate | C21H32O4 | 40.51 | 1.10 ± 0.77 | 349.2373 | 349.2373 | 0.10 | 79.0541[M-C14H26O3-C2H4+H]+, 349.2373[M+H]+ |
M43 | dehydroxylation product of oplopandiol | C17H26O | 40.53 | 20.63 ± 3.86 | 247.2060 | 247.2056 | −1.45 | 107.0513[M-C10H20+H]+, 79.0528[M-C10H20-C2H4+H]+, 247.2060[M+H]+ |
M44 | dehydroxylation product of falcarinol | C17H24 | 40.53 | 4.76 ± 1.42 | 229.1953 | 229.1951 | −0.98 | 77.0398[M-C9H18-C2H2+H]+, 229.1953 [M+H]+ |
M45 | demethylation product of muurolene | C14H22 | 40.55 | 4.97 ± 0.99 | 191.1796 | 197.1794 | −0.91 | 53.0384[M-2×C5H9+H]+, 191.1796 [M+H]+ |
M46 | dehydroxylation product of falcarindiol | C17H24O | 41.18 | 67.22 ± 3.74 | 245.1903 | 245.1900 | −1.26 | 105.0692[M-C10H20+H]+, 79.0521[M-C10H20-C2H2+H]+, 245.1903[M+H]+ |
M47 | methylation product of oplopantriol A | C19H28O3 | 41.41 | 0.52 ± 0.28 | 305.2111 | 305.2113 | −0.59 | 79.0547[M-C12H24O2-C2H2+H]+, 305.2111 [M+H]+ |
M48 | demethylation product of oplopantriol A | C17H24O3 | 41.42 | 53.27 ± 3.42 | 277.1800 | 277.1798 | −0.65 | 79.0537[M-C10H20O2-C2H2+H]+, 277.1800[M+H]+ |
M49 | methylation product of oplopandiol | C18H28O2 | 41.43 | 51.35 ± 2.53 | 277.2160 | 277.2162 | 0.75 | 107.0871[M-H2O-C11H20+H]+, 79.0547[M-H2O-C11H20-C2H4+H]+, 277.2160[M+H]+ |
M50 | dehydroxylation product of oplopantriol B | C18H28O2 | 41.44 | 53.87 ± 4.21 | 277.2164 | 277.2162 | −0.70 | 79.0541[M-C11H22O-C2H4+H]+, 277.2164[M+H]+ |
M51 | methylation product of falcarinol | C18H26O | 41.59 | 3.75 ± 0.89 | 259.2057 | 259.2056 | −0.23 | 79.0524[M-C11H22-C2H2+H]+, 259.2057 [M+H]+ |
M52 | methylation product of oplopantriol B | C19 H30O3 | 41.69 | 1.26 ± 0.82 | 307.2262 | 307.2268 | 1.87 | 79.0543[M-C12H24O2-C2H4+H]+, 307.2262[M+H]+ |
M53 | methylation product of oploxyne B | C19H32O4 | 41.76 | 0.37 ± 0.20 | 325.2371 | 325.2373 | 0.73 | 79.0540[M-C12H26O3 -C2H4+H]+, 325.2371[M+H]+ |
M54 | dehydroxylation product of 9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate | C20H28O3 | 42.15 | 28.46 ± 1.55 | 317.2096 | 317.2111 | 4.81 | 79.0537[M-C13H24O2-C2H2+H]+, 317.2096 [M+H]+ |
M55 | dehydroxylation product of oploxyne B | C18H30O3 | 42.16 | 56.82 ± 2.47 | 295.2273 | 295.2268 | −1.80 | 79.0550 [M-C11H24O2 -C2H4+H]+, 295.2273[M+H]+ |
M56 | hydroxylation product of falcarindiol | C17H24O3 | 42.20 | 79.26 ± 1.34 | 277.1797 | 277.1798 | 0.44 | 105.0693[M-H2O-C10H18O+H]+,79.0541[M-H2O-C10H18O-C2H2+H]+, 277.1797[M+H]+ |
M57 | methylation product of 9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate | C21H30O4 | 42.52 | 0.42 ± 0.28 | 347.2213 | 347.2217 | 1.11 | 79.0541[M-C14H26O3-C2H2+H]+, 347.2213 [M+H]+ |
M58 | hydrogenation product of curcumene | C15H24 | 42.65 | 70.36 ± 4.29 | 205.1953 | 205.1951 | −1.09 | 67.0540[M-2×C5H9+H]+, 205.1953[M+H]+ |
M59 | demethylation product of 6,9-octadedicenoic acid | C17H30O2 | 42.75 | 0.48 ± 0.32 | 267.2327 | 267.2319 | −3.17 | 65.0390[M-CH3COOH-C10H18+H]+, 267.2327[M+H]+ |
M60 | dehydroxylation product of 6,9-octadedicenoic acid | C18H32O | 43.40 | 65.77 ± 5.21 | 265.2536 | 265.2526 | −3.81 | 69.0688[M-CH3CHO-C11H20+H]+, 265.2536[M+H]+ |
M61 | hydrogenation product of 6,9-octadedicenoic acid | C18H34O2 | 43.50 | 128.46 ± 8.42 | 283.2636 | 283.2632 | −1.57 | 65.0389[M-CH3COOH-C11H22+H]+, 283.2636[M+H]+ |
M62 | acetylization product of oleanolic acid | C32H50O4 | 48.23 | 2.48 ± 1.63 | 521.3606 | 521.3601 | −0.94 | 220.0842[M-C16H24O2-C2H3O+Na]+, 521.3606[M+Na]+ |
Polyynes
A total of 46 metabolites of nine polyynes generated by the transformation of human intestinal microflora were detected and identified. For each polyyne, at least four types of metabolites were identified. Due to the high biological activities, FAD and OPD selected as the representative compounds of polyynes were stated in detail.
The EICs and MS/MS spectrums of metabolites of FAD are shown in Fig. 3. Five metabolites including M15, M17, M26, M46, M56 were detected. M15 was assigned to be the methylation product of FAD with the molecular formula C18H26O2 at m/z 275.2008 ([M+H]+, C18H26O2+; calculated as 275.2006). The fragment ion at m/z 105.0704 was generated by the neutral losses of H2O and C11H20, and m/z 79.0548 was formed by further loss of C2H2. M17 was assigned to be the hydrogenation product of FAD with the characteristic fragment ions at m/z 105.0695 and 79.0535. In addition, metabolites M26, M46, and M56 were assigned as demethylation, dehydroxylation, and hydroxylation products of FAD, respectively.
Figure 3: Metabolites of falcarindiol using UPLC-TOF/MS in the positive ion mode.
(A) Extracted ion chromatograms (EICs); (B) MS/MS spectra and structural elucidation.Figure 4 presents the EICs and MS/MS spectrums of OPD metabolites (M27, M40, M43, and M49). M27 was assigned as the hydroxylation product of OPD, owing to the presence of [M+H]+ at m/z 279.1958. The characteristic fragment ion at m/z 107.0496 was formed by the neutral losses of H2O and C10H18O, and m/z 79.0541 was formed by further loss of C2H4. Similarly, three other metabolites like M40, M43, and M49 were supposed to be the demethylation, dehydroxylation, and methylation products of OPD.
Figure 4: Metabolites of oplopandiol using UPLC-TOF/MS in the positive ion mode.
(A) EICs; (B) MS/MS spectra and structural elucidation.Lignans
M3–M5 were the deglycosylation products of three lignans via the loss of glycose moieties. For example, the parent compound of M3 was determined to be C32H44O16 while M3 was C20H24O6, indicating M3 was the deglycosylation product via the loss of two glucose moieties. M4 and M5 were assigned as the products by losing a glucose moiety from their corresponding parent lignan compounds.
Phenylpropanoids
M1 was identified as the deglycosylation metabolite of phenylpropanoid compound 4-(3-hydroxyprop-1-en-1-yl)-2,6-dimethoxyphenyl β-D-glucopyranoside. The protonated molecular ion [M+H]+ of M1 at m/z 211.0962 was observed in the positive ion mode, providing the molecular formula of C11H14O4.
Others
For two sesquiterpenes, M9, M38, and M58 were identified as the hydroxylation, demethylation, and hydrogenation products of curcumene, while M20 and M45 were the hydroxylation and demethylation product of muurolene, respectively. M37 and M62 were identified as the demethylation and acetylization products of oleanolic acid. In addition, for 2 fatty acids, M12 was the dehydroxylation product of 2-decenoic acid, while M24 and M59-61 were the products of 6,9-octadedicenoic acid.
Proposed metabolic pathways of O. elatus extract
The proposed metabolic pathways of O. elatus extract by human intestinal microflora are presented in Fig. 5. Multiple major metabolite pathways can be observed in this study. The common pathways involved in the biotransformation of O. elatus extract include methylation, demethylation, hydroxylation, dehydroxylation, acetylation, hydrogenation, demethoxylation, and deglycosylation. Among them, polyynes were undoubtedly the most important compounds, as 46 out of 62 metabolites originated from polyynes. By comparing the signal intensity of metabolites, we could find that methylation, dehydroxylation and hydroxylation are major metabolic pathways of polyynes. Moreover, four metabolites of lignans and phenylpropanoid were produced by the loss of glucose. The other metabolites were generated from one triterpenoid and two fatty acids. This indicated that polyynes of O. elatus generated comprehensive biotransformation and were more readily metabolized than other compounds under the same conditions.
Figure 5: The proposed metabolic pathways of O. elatus extract by human intestinal microflora, including (A) Polyynes; (B) Lignans; (C) Phenylpropanoid; (D) Sesquiterpenes; (E) Triterpenoid; (F) Fatty acids. Methylation (1), demethylation (2), hydrogenation (3), hydroxylation (4), dehydroxylation (5), acetylization (6) and demethoxylation (7) were observed in this biotransformation.
In summary, the main metabolic pathways of O. elatus refer to hydrolytic and reductive reactions by gut microorganisms. Because of the complexity of active ingredients or constituent concentrations, in vivo exposure, and individual differences, the metabolic profiles of O. elatus might be affected by several factors.
Discussion
In this study, a UPLC-Q-TOF-MS/MS method was developed to screen and identify the chemical composition and metabolites from a traditional Chinese herb, the air-dried root bark of O. elatus. A total of 18 ingredients and 62 metabolites biotransformed by human intestinal microflora were characterized from O. elatus in UPLC-Q-TOF/MS positive ion mode. Two polyynes, falcarindiol and oplopandiol, as the main components of O. elatus and their metabolites by human intestinal microflora are mainly illustrated. It could be noted that the major metabolic pathways of O. elatus refer to methylation, dehydroxylation, and hydroxylation. Studies on the chemical and metabolic profiling of O. elatus by human intestinal microflora will be helpful for the understanding of mechanism research on the active components and further in vivo investigation.
Supplemental Information
Raw data.
The mass spectrum of Oplopanax elatus (the herbal medication we studied) and its metabolites by human intestinal microbiota, which were detected by an Agilent 6545 Q-TOF-MS system. Mass data including MS and MS/MS information could be analyzed by Agilent MassHunter Workstation software. The software was applied in the screening and identification of the probable compounds, based on the accurate measurements of m/z values with databases.