Results
Mass fragmentation rule of phenylethanoid glycosides and iridoids
Phenylethanoid glycosides are the main chemical constituents of CD. The standard solutions of isoacteoside, cistanoside F, tableside A, echinacoside, acteoside, and 2’-actylacteoside were taken, followed by providing a different level of collision energies (Table 1), and then corresponding MS2 maps were obtained (Fig. 1).
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The mass spectrometric analysis revealed that phenylethanoid glycosides have similar mass spectrum fragmentation patterns, the cleavage pathways in the negative-ion mode mainly include (1) Ester bond cleavage: loss of neutral caffeoyl group (C9H3O6, 162.03) and neutral acetyl group (C2H2O, 42.00); (2) Glycosidic cleavage: loss of neutral rhamnose residues (C6H10O4, 146.05) and neutral glucose residue (C6H10O5, 162.05). From high-resolution mass spectrometry, caffeoyl (162.03) and glucose residue (162.05) could be distinguished.
Iridoids ajugol, catalpol, teniposide acid, geniposide, and 8-epideoxyloganic acid standard solutions were taken, followed by providing different collision energies, and corresponding MS2 maps were obtained (Fig. 2).
Iridoid glycosides have similar mass spectrum fragmentation patterns, the cleavage pathways in the negative-ion mode mainly include (1) Glycosidic cleavage: Loss of neutral glucose residue (C6H10O5, 162.05); (2) Loss of neutral CO2 (43.99) and H2O (18.01).
Identification of the compounds in CD, CD‑NP, and CD‑HP extracts
UPLC‑QTOF‑MSE analysis
The optimization of chromatographic conditions was carried out. Next, the compounds of Cistanche Herba were evaluated in both negative and positive ion modes with high as well as low CEs. The obtained results revealed that the compatibility of the negative mode was higher relative to the positive mode for these compounds. Figure 3 showed MS basic peak ion (BPI) chromatogram traced with numbered peaks. The intensity of each detected ion in UPLC-Q-TOF-MSE analysis was normalized concerning the whole ion count for the generation of a data matrix which comprised of m/z value, the normalized peak area, and retention time.
The evaluation of components from CD and its processed products on the UNIFI platform
A total of 97 compounds were identified with -SEM (n=6) mode from CD and its processed product (Table 2), including phenylethanoid glycosides (PhGs), iridoids, lignans, and oligosaccharides. The 95, 91, and 94 components were detected in CD, CD-NP, and CD-HP, accordingly. Among them, 64 were phenylethanoids, 13 were iridoids, and 20 other kinds of compounds were determined. There was a similarity in the chemical composition of CD and its processed product, however, the quantity of the components was found to be different between CD and its processed product.
Variations in chemical components of processed products
Te Simca-P 13.0 software was employed for analyzing the multivariate data matrix. Before PCA, all variables were mean-centered and Pareto-scaled, followed by the identification of potential discriminant variables. In a PCA score plot, every point showed an individual sample. Samples that showed similarity in their chemical components were scattered adjacent to each other, while those which showed variations in their components were divided. As seen in PCA (Fig. 4), the group of CD-HP was separated from the groups of CD and CD-NP.
To distinguish CD from CD-HP and CD-NP, OPLS-DA, permutation test, S-plot, and VIP value were developed. (Figs. 5, 6, 7) The obtained results revealed that many components were key characteristic components of each product. The screening condition was the VIP>1 and P<0.05. From the date of the S-plot, the characteristic components evaluated, which were commonly existing in the three groups were.
From Fig. 8, we found the intensity of acteoside (54), cistanoside C (74), campneoside II (43), osmanthuside (75), and 2’-actylacteoside (80) having the 4’-O-cafeoyl group in the 8-O-β-d-glucopyranosyl part (see Fig. 9) decreased after being processed by rice-wine, while the intensity of isoacetoside (60), isocistanoside (71), isocampneoside I (69), isomartynoside (86) having the 6’-O-cafeoyl group (see Fig. 9) increased, especially for the CD-NP group. Tough tubuloside B (72) having a 6’-O-cafeoyl group, the same as isoacteoside, the intensity decreased because of its 2’-actyl group. The intensity of echinacoside (38) and cistanoside B having 6’-O-β-d-glucopyranosyl moiety groups increased, but the intensity of tubuloside A (55) decreased also because of its 2’-actyl group.
Our research team also studied the thermal stability of acteoside and isoacteoside and found that acteoside was unstable in water, methanol, and yellow rice wine solution, and could be converted into isoacteoside partly under heating conditions. But the thermostability of isoacteoside was better, especially in yellow rice wine solution. Figure 10 showed the possible changes of PhGs in CD during processing:
Identification of the metabolites in rats
From high-resolution mass spectrometry data, the accurate molecular weight and elemental composition for metabolites and proto-molecule compounds were analyzed and compared. As the same kinds of compounds in TCM showed similarity in metabolic modifications, the correlations of phytochemical constituents in vitro can extend to their metabolites in vivo. Meanwhile, based on conventional biotransformation pathways, a reasonable change in molecular weight was inferred. Finally, the metabolites were identified by analyzing the MSE mass spectra of the metabolites and proto-compounds fragmentation pathway in the mass spectrum [21, 22]. Compared with the blank sample, its components were identified in vivo based on the information provided by the chromatogram-mass spectrum, the possibility of a metabolic reaction, the characteristics of the compound structure, and the fragmentation rule of its mass spectrum. See Table 3.
Identification of phenylethanoid glycosides-related metabolites
UNIFI platform was used for processing. Figure 11 showed the TIC chromatograph of urine, feces, and plasma for CD and its processed products. Compared with blank samples, a total of 54 metabolites were identified in rats, including 10 prototype components and 44 metabolites, of which 24, 49, and 6 were in feces, urine, and plasma, accordingly.
Based on accurate mass, fragmentation cascade, and predictable neutral losses by biotransformation, a total of 35 phenylethanoid glycosides-associated metabolites were tentatively evaluated. Te related metabolites of phenylethanoid glycosides have similar mass spectrum fragmentation patterns, like the typical caffeoyl fragment m/z 461.1605, then further hydrolyzed by glycosidic and ester bonds in vivo, and metabolized into hydroxytyrosol (HT) (m/z 153.0504, C8H10O3, 4.73 min) and cafes acid (CA)(m/z 179.0389, C9H7O4, 0.77 min), see Fig. 12A.
M11 indicated [M–H]− at m/z 153.0504 with formula i.e., C8H10O3, and identified as HT. M16 presented [M–H]− at m/z 329.0851, which was 176 Da elevated than that of HT, revealing that it might be a glucuronidated metabolite of HT. The [M–H]− of M26 was at m/z 343.1037, 14 Da higher than that of HT-glucuronide. Therefore, M26 was identified as HT-methylated glucuronide. M17 was identified as HT-sulfate based on its [M–H]− at m/z 233.0112, 80 Da over the HT, which could be further methylated, then produced M22, which showed the m/z 247.0278, indicating that it was HT-methylated sulfated metabolite. M7 (m/z 167.0335) and M5 (m/z 167.0762) were considered oxidation products and methylated HT, respectively (Fig. 12B).
M1 indicated [M–H]− at m/z 179.0389, the elucidated molecular formula was C9H7O4 and identified as caffeic acid (CA). M25 revealed [M–H]− at m/z 355.0704, which was 176 Da elevated than that of CA, showing that it might be a glucuronidated metabolite of CA. M27 had m/z 258.994, which was 80 Da higher than that of CA, so we elucidated it as CA sulfate, and it could produce M35 (m/z 273.0064). As M4 gives the [M–H]− at m/z 193.0524, 14 Da higher than CA, it was identified as CA methylated metabolite. M39 was a CA dehydroxylation metabolite, with m/z 163.04, and it could be sulfated into M32 (m/z 242.9951).
M33 (m/z 181.0491, C9H10O4, 9.06 min) was the reduction product of CA, that is 3,4-dihydroxy benzene propionic acid, which could be methylated into M19 (m/z 195.0623, C10H12O4, 0.93 min). M33 could be dehydrated into M43, that is 3-HPP (m/z 165.0558, C9H10O3, 11.29 min), and M31 (m/z 341.0942, C15H17O9, 8.90 min) and M29 (m/z 245.0125, C9H10O6S, 8.52 min) were the glucuronidated and sulfated products (Fig. 12C).
For the phenylethanoid glycosides-associated metabolites, the key metabolic cascades were phase II metabolic reactions, i.e., glucuronidation, methylation, and sulfation. The proposed metabolic cascades of phenylethanoids are depicted in Fig. 13.
Identification of iridoids related metabolites
By analyzing the elemental composition of the metabolites, MSE fragmentation, and associated literature, a total of 19 iridoid-associated metabolites were tentatively
evaluated. Iridoid glycosides were hydrolyzed by glycosidic bonds to form their corresponding aglycones. Te m/z 185.117 was for M8, 162 Da less than ajugol, which was yielded by the loss of glucose residue. M40 (m/z 199.0641, Rt 10.91 min) was the deglycosylated product of catalpol. M45 m/z 169.0487, Rt 12.15 min) was less than 30 Da that of catalpol deglycosylated metabolite, and was identified as the removal of a molecule of CH2O metabolite. M34 (m/z 151.0352, Rt 9.08 min), was further loss of H2O metabolite.
M44 (m/z 211.0665, Rt 11.31 min) was a deglycosylated metabolite of geniposide, and M37 (m/z 197.0833, Rt 15.03 min) was deglycosylation of 8-epideoxyloganic acid. Metabolic reactions for iridoids could be revealed as phase I metabolism of deglycosylation (Fig. 12D).
Comparison of metabolic profiling in plasma, urine, and feces between CD and its processed products
2 prototypes in plasma, 7 in urine, and 3 in feces were compared. There were 7 prototypes absorbed in CD, 7 prototypes absorbed in CD-NP, and 8 prototypes in CD-HP. M21 was only detected in the feces group of CD-NP, and M38 and M51 were detected just in the urine groups of CD-HP. Compared with metabolites, identical metabolites in plasma, urine, and feces were 4, 42, and 21, respectively. There were 34 metabolites absorbed in the CD group, 39 in CD-NP, and 40 in the CD-HP group. M5, M7, M40, and M52 were only detected in CD-NP groups, while M24, M41, and M48 were just detected in CD-HP groups.
Variations were observed in the absorption as well as the metabolism of active compounds in diverse processed products of CD. From Fig. 14, we found that the intensity of HT-sulfate conjugation (M17) was the highest in the urine, followed by 3-HPP sulfate conjugation (M29), methylated HT sulfate conjugation (M22), dehydroxylated CA sulfate conjugation (M32), and 3,4-dihydroxy benzene propionic acid sulfate conjugation (M19). The content of metabolic products in the processed group was higher than in the CD group, especially for M22, M29, M27, M16, M19, M1, and M2. Their precursor compounds, such as hydroxytyrosol have anti-tumor, anti-inflammatory, antibacterial, an tiviral, and antifungal properties [23]. Cafeic acid possesses anti-inflammatory, anti-cancer, and antiviral activities [24]. It was consistent with the clinical use of CD and its processed products.
Discussion
The CD is a TCM, and its major bioactive components, including PhGs, iridoids, and polysaccharides have been documented by various research studies. In TCM clinical practice, the processed products of CD have been widely used relative to raw ones. The chemical composition will be changed during the processing, which may lead to changes in the medicinal effects (Fig. 14).
PhGs are a type of phenolic compound characterized by a β-glucopyranoside structure bearing a hydroxyphenylethyl moiety as the aglycone. These compounds often comprise caffein acid and rhamnose attached to the glucose residue through ester or glycosidic linkages respectively. In the current study, the qualitative analyses of CD, CD-NP, and CD-HP were carried out, and a total of 97 compounds, including phenylethanoid glycosides (PhGs), iridoids, etc. were identified. The obtained results showed variations in chemical composition before and after processing. The intensity of PhGs having the 4’-O-caffeoyl group in the 8-O-β-d-glucopyranosyl part, like acteoside, cistanoside C, campneoside II, osmanthuside decreased after being processed, while PhGs with the 6’-O-cafeoyl group in the 8-O-β-d-glucopyranosyl part, such as isoacetoside, isocistanoside, isocampneoside I, isomartynoside increased, especially in the CD-NP group. Te intensity of echinacoside and cistanoside B whose structure possesses 6’-O-β-d-glucopyranosyl moiety also increased. PhGs having a 2’-actyl group often decreased because of hydrosis reactions during the process, like tubuloside B, and 2-acetylacteoside.
Investigation of metabolites absorbed in vivo was carried out after oral administration of CD and its processed products. The metabolic processes of phase II were the key cascades and most of the metabolites were sulfate, glucuronide, and methylated conjugates. Phenylethanol glycosides have low oral absorption and utilization. Tey is difficult to be absorbed into the blood, and act as progenitors to play their roles after metabolic activation in vivo. Phenylethanoids produced into phenylethanolaglycone, like hydroxytyrosine (HT) and caffein acid (CA) and its derivative 3-hydroxyphenylpropionic acid (3-HPP), these metabolites may be more easily absorbed into the plasma and have a better medicinal effect.
Most of the metabolites were found in their lower concentrations or not detected in rat plasma, however, higher concentration was observed in the urine, indicating that metabolites would get easily eliminated via the urine. As depicted in Table 3, the same compounds were determined in various groups, while considerable variations were found in the concentrations of the metabolites which might be associated with the unequal efficacy of CD and its processed products. HT-sulfate conjugation (M17) has the highest intensity in the urine, followed by 3-HPP sulfate conjugation (M29), methylated HT sulfate conjugation (M22), dehydroxylated CA sulfate conjugation (M32), and 3,4-dihydroxy benzene propionic acid sulfate conjugation (M19). The content of metabolic products in the processed group was higher than in the CD group, especially for M22, M29, M27, M16, M19, M1, and M2.
Generally, the components having high exposure in target organs could be effective. A sufficient amount of phenylethanoids and their derivatives have been evaluated and determined in vitro. Acteoside is the characteristic compound, whose content decreased after being processed by rice wine, and the content of isoacteoside, isocistanoside C, and isocampneoside I increased correspondingly. The degradation products of PhGs, like CA and HT derivatives, could be evaluated in the bio-samples, and rice-wine processing can enhance the absorption of metabolites in vivo.
Conclusion
In this study, 97 compounds were detected in the extracts of CD and its processed product. The degradation of a few glycosides occurred under an elevated temperature and as a result, some new isomers and complexes were synthesized. In in vivo study, prototype components (10) and metabolites (44) were determined or tentatively evaluated in rat plasma, feces, and urine. Phase II metabolic processes were the key cascades, most of the metabolites were associated with echinacoside or acteoside, like HT, CA, and their derivatives 3-hydroxyphenylpropionic acid 3-HPP. These metabolites may be more easily absorbed into the plasma and have a better medicinal effect. The obtained results showed that the chemical composition of CD was different and affected the disposition of the compound in vitro and in vivo.
Abbreviations
PhGs: Phenylethanoid glycosides; CD: Cistanche deserticola; CMM: Chinese Materia Medica; TCM: Traditional Chinese Medicine; CD-NP: Cistanche deserticola Processed by steaming with rice wine under normal pressure; CD-HP: Cistanche deserticola Processed by steaming with rice-wine under high pressure; UPLC-Q-TOF-MSE: Ultra-high performance liquid chromatography coupled with TOF-MSE; PCA: Principal component analysis; VIP: Variable importance for the projection; CA: Cafeic acid; HA: Hydroxytyrosol.
Acknowledgments
Not applicable.
Authors’ contributions
LZ, LBN, and SJ participated in drafting, and writing the manuscript. RJ, LPP assisted with the animal experiments and drafted and finalized all figures and tables. ZC, HY, and JTZ assisted with the design and performance of this study and reviewed the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the National Natural Science Foundation of China (Grant No: 81874345) and the Natural Science Foundation of Liaoning Province (Grant No: 2020-MS-223).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
Ethical approval for using experimental animals for this study had been obtained from the Medical Ethics Committee of Liaoning University of Traditional Chinese Medicine (Approval number: 2018YS(DW)-044-01). All experimental procedures in this study were under the ethical standards of the medical Ethics Committee of Liaoning University of Traditional Chinese Medicine.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no conflicts of interest to disclose.
Author Details
1Pharmaceutic Department, Liaoning University of Traditional Chinese Medicine, Dalian, Liaoning, China. 2Drug Research Institute of Monos Group, Ulaanbaatar 14250, Mongolia.
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