Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD
Abstract
:1. Introduction
2. Results and Discussion
2.1. Large-Scale Preparation of Cycloastragenol
2.2. Structural Elucidation of the Main Isolated Impurities (≥0.05%) Derived from Fraction B
2.3. UPLC-LTQ-Orbitrap-MS Conditions Required for the Determination of Other Related Compounds (<0.05%)
2.4. Anti-Inflammatory Activity of Two New Impurities
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Large-Scale Preparation of Cycloastragenol
3.3. Sample Preparation and Enrichment for Impurities
3.4. Isolation of the Main Impurities (≥0.05%) Derived from Fraction B
3.5. Analysis of the Isolated Impurities (≥0.05%)
3.6. HRESIMS, NMR, UV, and IR Spectra of Isolated Impurities
3.7. UPLC-LTQ-Orbitrap-MS Conditions for Determination of Related Compounds (<0.05%)
3.8. Anti-Inflammatory Activity of Two New Impurities
3.8.1. LPS-Induced Lymphocyte Cells [29]
3.8.2. CES-Induced MLE-12 Cells [30]
4. Conclusions
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
- Shu, C.C.; Lee, J.H.; Tsai, M.K.; Su, T.C.; Wen, C.P. The ability of physical activity in reducing mortality risks and cardiovascular loading and in extending life expectancy in patients with COPD. Sci. Rep. 2021, 11, 21674. [Google Scholar] [CrossRef] [PubMed]
- Rehman, A.U.; Muhammad, S.A.; Tasleem, Z.; Alsaedi, A.; Dar, M.; Iqbal, M.O.; Rasoolet, M.F.; Shah, S.; Abbas, G.; Shakeel, S.; et al. Humanistic and socioeconomic burden of COPD patients and their caregivers in Malaysia. Sci. Rep. 2021, 11, 22598. [Google Scholar] [CrossRef] [PubMed]
- Zhou, Y.; Zhong, N.S.; Li, X.; Chen, S.; Zheng, J.; Zhao, D.; Yao, W.; Zhi, R.; Wei, L.; He, B.; et al. Tiotropium in early-stage chronic obstructive pulmonary disease. N. Engl. J. Med. 2017, 377, 923–935. [Google Scholar] [CrossRef] [PubMed]
- Fu, J.; Wang, Z.H.; Huang, L.F.; Zheng, S.H.; Wang, D.W.; Chen, S.L.; Zhang, H.T.; Yang, S.H. Review of the botanical characteristics, phytochemistry, and pharmacology of Astragalus membranaceus (Huangqi). Phytother. Res. 2014, 28, 1275–1283. [Google Scholar] [CrossRef] [PubMed]
- Chen, Z.J.; Liu, L.J.; Gao, C.F.; Chen, W.J.; Vong, C.T.; Yao, P.F.; Yang, Y.H.; Li, X.Z.; Tang, X.D.; Wang, S.P.; et al. Astragali radix (huangqi): A promising edible immunomodulatory herbal medicine. J. Ethnopharmacol. 2020, 258, 112895. [Google Scholar] [CrossRef] [PubMed]
- Li, S.S.; Sun, Y.; Huang, J.; Wang, B.; Gong, Y.N.; Fang, Y.X.; Liu, Y.Y.; Wang, S.J.; Guo, Y.; Wang, H.; et al. Anti-tumor effects and mechanisms of Astragalus membranaceus (AM) and its specific immunopotentiation: Status and prospect. J. Ethnopharmacol. 2020, 258, 112797. [Google Scholar] [CrossRef] [PubMed]
- Gong, A.G.W.; Duan, R.; Wang, H.Y.; Kong, X.P.; Dong, T.T.X.; Tsim, K.W.K.; Chan, K. Evaluation of the Pharmaceutical Properties and Value of Astragali Radix. Medicines 2018, 5, 46. [Google Scholar] [CrossRef] [PubMed]
- Shan, H.; Zheng, X.P.; Li, M. The effects of Astragalus membranaceus active extracts on autophagy-related diseases. Int. J. Mol. Sci. 2019, 20, 1904. [Google Scholar] [CrossRef]
- Li, L.; Hou, X.J.; Xu, R.F.; Tu, M.; Liu, C. Research review on the pharmacological effects of Astragaloside IV. Fundam. Clin. Pharmacol. 2017, 31, 17–36. [Google Scholar] [CrossRef]
- Zang, Y.B.; Wan, J.J.; Zhang, Z.; Huang, S.; Liu, X.; Zhang, W.D. An updated role of astragaloside IV in heart failure. Biomed. Pharmacother. 2020, 126, 110012. [Google Scholar] [CrossRef]
- Yu, Y.J.; Zhou, L.M.; Yang, Y.J.; Liu, Y.Y. Cycloastragenol: An exciting novel candidate for age-associated diseases. Exp. Ther. Med. 2018, 16, 2175–2182. [Google Scholar] [CrossRef] [PubMed]
- Zhu, X.; Cao, Y.; Su, M.; Chen, M.; Li, C.; Yi, L.; Qin, J.; Tulake, W.; Teng, F.; Zhong, Y.; et al. Cycloastragenol alleviates airway inflammation in asthmatic mice by inhibiting autophagy. Mol. Med. Rep. 2021, 24, 805. [Google Scholar] [CrossRef] [PubMed]
- Yao, J.C.; Li, H.H. Medical Application of Cycloastrogenol. CN113116907A, 18 December 2020. [Google Scholar]
- Feng, L.M.; Lin, X.H.; Huang, F.X.; Cao, J.; Qiao, X.; Guo, D.A.; Ye, M. Smith degradation, an efficient method for the preparation of cycloastragenol from astragaloside IV. Fitoterapia 2014, 95, 42–50. [Google Scholar] [CrossRef] [PubMed]
- ICH. Impurities in new drug substances Q3A(R2). In ICH Harmonised Tripartite Guideline; International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; ICH: Geneva, Switzerland, 2006. [Google Scholar]
- ICH. Impurities in new drug products Q3B(R2). In ICH Harmonised Tripartite Guideline; International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; ICH: Geneva, Switzerland, 2006. [Google Scholar]
- Isaev, I.M.; Agzamova, M.A.; Isaev, M.I. Triterpene glycosides from Astragalus and their genins. XCI. Chemical transformation of cycloartanes. X. Syntheses based on cycloalpioside D and cycloalpigenin D. Chem. Nat. Compd. 2012, 47, 947–954. [Google Scholar] [CrossRef]
- Yang, W.Z.; Ye, M.; Huang, F.X.; He, W.N.; Guo, D.A. Biocatalysis of cycloastragenol by filamentous fungi to produce unexpected triterpenes. Adv. Synth. Catal. 2012, 354, 527–539. [Google Scholar] [CrossRef]
- Isaev, I.M.; Iskenderov, D.A.; Isaev, M.I. Triterpene glycosides from Astragalus and their genins. LXXXVII. Chemical transformation of cycloartanes. IX. Partial synthesis of cycloasalgenin. Chem. Nat. Compd. 2010, 46, 407–411. [Google Scholar] [CrossRef]
- Feng, L.M.; Ji, S.; Qiao, X.; Li, Z.W.; Lin, X.H.; Ye, M. Biocatalysis of cycloastragenol by Syncephalastrum racemosum and Alternaria alternata to discover anti-aging derivatives. Adv. Synth. Catal. 2015, 357, 1928–1940. [Google Scholar] [CrossRef]
- Hirotani, M.; Zhou, Y.; Rui, H.; Furuya, T. Cycloartane triterpene glycosides from the hairy root cultures of Astragalus membranaceus. Phytochemistry 1994, 37, 1403–1407. [Google Scholar] [CrossRef]
- Isaev, I.M.; Iskenderov, D.A.; Isaev, M.I. Triterpene glycosides from Astragalus and their genins. LXXVIII. Chemical transformation of cycloartanes. VI. Partial synthesis of cycloadsurgenina. Chem. Nat. Compd. 2008, 44, 732–737. [Google Scholar] [CrossRef]
- Cheng, X.D.; Wei, M.G. Profiling the Metabolism of Astragaloside IV by Ultra Performance Liquid Chromatography Coupled with Quadrupole/Time-of-Flight Mass Spectrometry. Molecules 2014, 19, 18881–18896. [Google Scholar] [CrossRef]
- Qi, W.L.; Cao, J.; Li, P.; Yu, Q.T.; Wen, X.D.; Wang, Y.X.; Li, C.Y.; Bao, K.D.; Ge, X.X.; Cheng, X.L. Qualitative and quantitative analysis of Radix Astragali products by fast high-performance liquid chro-matography-diode array detection coupled with time-of-flight mass spectrometry through dynamic adjustment of fragmentor voltage. J. Chromatogr. A 2008, 1203, 27–35. [Google Scholar] [CrossRef] [PubMed]
- Chu, C.; Cai, H.X.; Ren, M.T.; Liu, E.H.; Li, B.; Qi, L.W.; Li, P. Characterization of novel astragaloside malonates from Radix Astragali by HPLC with ESI quadrupole TOF MS. J. Sep. Sci. 2010, 33, 570–581. [Google Scholar] [CrossRef] [PubMed]
- Song, H.T.; Li, C.Y.; Wan, Y.Y.; Ding, X.S.; Tan, X.Y.; Dai, G.L.; Liu, S.J.; Ju, W.Z. Screen astragalosides from Huangqi injections by LC-TOF-MS-based mass defect filtering approach. China J. Chin. Mater. Medica 2017, 42, 686–695. [Google Scholar] [CrossRef]
- He, M.T.; Wang, K.; Che, H.J.; Wang, H.F.; Yang, K.; Zhang, G.M.; Yao, J.C.; Wang, J.X. A comprehensive review of cycloastragenol: Biological activity, mechanism of action and structural modifications. Eur. J. Med. Chem. 2022, 5, 100060. [Google Scholar] [CrossRef]
- Takeuchi, D.M.; Kishino, S.; Ozeki, Y.; Fukami, H.; Ogawa, J. Analysis of astragaloside IV metabolism to cycloastragenol in human gut microorganism, bifidobacteria, and lactic acid bacteria. Biosci. Biotechnol. Biochem. 2022, 86, 1467–1475. [Google Scholar] [CrossRef] [PubMed]
- Du, B.Y.; Zhang, L.; Sun, Y.; Zhang, G.M.; Yao, J.C.; Jiang, M.M.; Pan, L.H.; Sun, C.H. Phillygenin exhibits anti-inflammatory activity through modulating multiple cellular behaviors of mouse lymphocytes. Immunopharmacol. Immunotoxicol. 2019, 41, 76–85. [Google Scholar] [CrossRef] [PubMed]
- Zhou, F.; Cao, C.; Chai, H.; Hong, J.; Zhu, M. Circ-HACE1 aggravates cigarette smoke extract-induced injury in human bronchial epithelial cells via regulating Toll-Like receptor 4 by sponging miR-485-3p. Int. J. Chronic Obstr. 2021, 16, 1535–1547. [Google Scholar] [CrossRef]
NO. | δH (J in Hz) a | δC (Type) b | 1H-1H COSY | HMBC |
---|---|---|---|---|
1 | 1.67 (1H, m) 2.42 (1H, m) | 35.5 (CH2) | H-2 | C-10 |
2 | 1.64 (1H, m) 1.92 (1H, m) | 26.5 (CH2) | H-1, 3 | - |
3 | 3.68 (1H, d, 4.9) | 82.7 (CH) | H-2 | C-2, 5, 10, 28 |
4 | - | 46.1 (C) | - | - |
5 | 1.46 (1H, m) | 61.4 (CH) | H-6 | C-6, C-10 |
6 | 4.40 (1H, m) | 65.9 (CH) | H-5, 7 | - |
7 | 1.61 (1H, m) 2.38 (1H, m) | 34.6 (CH2) | H-6, 8 | - |
8 | 2.42 (1H, m) | 46.6 (CH) | H-7 | - |
9 | - | 35.5 (C) | - | - |
10 | - | 93.2 (C) | - | - |
11 | 1.47 (1H, m) 1.97 (1H, m) | 32.8 (CH2) | H-12 | - |
12 | 1.23 (1H, m) 1.71 (1H, m) | 32.4 (CH2) | H-11 | - |
13 | - | 45.6 (C) | - | - |
14 | - | 45.7 (C) | - | - |
15 | 1.62 (1H, m) 2.04 (1H, m) | 46.0 (CH2) | H-16 | C-14, 17, 30 |
16 | 5.02 (1H, m) | 73.0 (CH) | H-15, 17 | C-14 |
17 | 2.43 (1H, m) | 57.4 (CH) | H-16 | C-13, 16, 18, 20, 21, 22 |
18 | 1.34 (3H, s) | 19.0 (CH3) | - | C-12, 13, 14, 17 |
19 | 1.40 (3H, s) | 35.8 (CH3) | - | C-8, 9, 10, 11 |
20 | - | 87.1 (C) | - | - |
21 | 1.33 (3H, s) | 28.6 (CH3) | - | C-17, 20, 22 |
22 | 1.63 (1H, m) 3.08 (1H, m) | 34.8 (CH2) | H-23 | C-20, 21, 23 |
23 | 2.09 (1H, m) 2.27 (1H, m) | 26.5 (CH2) | H-22, 24 | - |
24 | 3.89 (1H, dd, 8.7, 4.4) | 81.6 (CH) | H-23 | C-25 |
25 | - | 71.4 (C) | - | - |
26 | 1.30 (3H, s) | 27.0 (CH3) | - | C-24, 25, 26 |
27 | 1.57 (3H, s) | 28.1 (CH3) | - | C-24, 25, 27 |
28 | 1.41 (3H, s) | 23.1 (CH3) | - | C-3, 4, 5, 29 |
29 | 1.32 (3H, s) | 26.1 (CH3) | - | C-3, 4, 5, 28 |
30 | 0.97 (3H, s) | 19.8 (CH3) | - | C-8, 13, 14, 15 |
NO. | δH (J in Hz) a | δC (Type) b | 1H-1H COSY | HMBC |
---|---|---|---|---|
1 | 5.59 (1H, brs) | 115.2 (CH) | H-2 | C-3, 9, 10 |
2 | 2.42 (1H, m) 2.66 (1H, m) | 33.4 (CH2) | H-1, 3 | C-1, 3, 4, 10 |
3 | 3.80 (1H, dd, 9.6, 6.0) | 72.6 (CH) | H-2 | C-28, 29 |
4 | - | 40.1 (C) | - | - |
5 | - | 142.3 (C) | - | - |
6 | 5.59 (1H, brs) | 119.6 (CH) | H-7 | C-5 |
7 | 2.03 (1H, m) 2.40 (1H, m) | 25.2 (CH2) | H-6, 8 | C-5, 6, 8 |
8 | 2.06 (1H, m) | 42.2 (CH) | H-7 | C-6, 7, 9, 10, 19 |
9 | - | 37.2 (C) | - | - |
10 | - | 139.7 (C) | - | - |
11 | 1.69 (1H, m) 2.04 (1H, m) | 31.0 (CH2) | H-12 | - |
12 | 1.46 (1H, m) 1.82 (1H, m) | 31.3 (CH2) | H-11 | - |
13 | - | 46.9 (C) | - | - |
14 | - | 47.8 (C) | - | - |
15 | 1.59 (1H, m) 2.06 (1H, m) | 45.4 (CH2) | H-16 | C-14, 17, 30 |
16 | 5.07 (1H, dd, 13.7, 7.0) | 73.3 (CH) | H-15, 17 | C-13 |
17 | 2.48 (1H, m) | 56.8 (CH) | H-16 | C-13, 16, 18, 20, 21, 22 |
18 | 1.41 (3H, s) | 19.2 (CH3) | - | C-12, 13, 14, 17 |
19 | 1.05 (3H, s) | 32.2 (CH3) | - | C-8, 9, 10, 11 |
20 | - | 87.2 (C) | - | - |
21 | 1.34 (3H, s) | 28.6 (CH3) | - | C-17, 20, 22 |
22 | 1.65 (1H, m) 3.07 (1H, dd, 21.2, 10.5) | 34.8 (CH2) | H-23 | C-20, 21, 23 |
23 | 2.09 (1H, m) 2.29 (1H, m) | 26.5 (CH2) | H-22, 24 | - |
24 | 3.88 (1H, dd, 9.1, 4.5) | 81.6 (CH) | H-23 | C-25 |
25 | - | 71.4 (C) | - | - |
26 | 1.29 (3H, s) | 27.0 (CH3) | - | C-24, 25, 27 |
27 | 1.53 (3H, s) | 28.1 (CH3) | - | C-24, 25, 26 |
28 | 1.41 (3H, s) | 23.8 (CH3) | - | C-3, 4, 5, 29 |
29 | 1.13 (3H, s) | 20.0 (CH3) | - | C-3, 4, 5, 28 |
30 | 0.92 (3H, s) | 18.1 (CH3) | - | C-8, 13, 14, 15 |
NO. | tR (min) | Formula | CAS | m/z | Fragment Ion | |||
---|---|---|---|---|---|---|---|---|
[M+H]+ | [M+Na]+ | [M+HCOOH-H]− | [M-H]− | |||||
1 * | 9.83 | C35H58O9 | 86541-83-5 | 645.3965 (1.2) | 667.4053 (0.6) | 125.0960, 143.1065, 279.0928[Xyl+3Ac]+, 419.3302, 437.3408, 455.3512, 473.3618 | ||
2 | 11.99 | C36H60O10 | 86764-12-7 | 675.4068 (1.6) | 697.4161 (0.3) | 651.4112 (0.6) | 125.0961, 143.1065, 224.1277[Glu+Ac+H]+, 419.3302, 437.3408, 455.3513, 473.3618, 645.3959[M+2H2O-Ac]+ | |
3 | 21.34 | C35H58O9 | 1638800-96-0 | 645.3967 (0.9) | 667.4056 (0.1) | 621.4002 (0.1) | 143.1065, 297[Xyl+H2O+3Ac]+, 419.3302, 437.3408, 455.3512 | |
4 * | 22.36 | C30H50O5 | 86541-79-9 | 513.3356 (1.1) | 535.3638 (0.5) | 489.3586 (1.2) | 125.0962, 143.1067, 419.3307, 437.3414, 455.3519, 473.3627, 981.7380[2M+H]+ | |
5 | 25.92 | C30H46O5 | - | 509.3243 (1.1) | 531.3329 (1.3) | 485.3275 (1.6) | 125.0964, 143.1068, 437.3419, 469.3317 [M-H2O]+ | |
6 | 26.42 | C32H52O6 | 80604-30-4 | 555.3663 (1.2) | 125.0964, 143.1059, 419.3312, 437.3419, 455.3524, 473.3631, 495.3450[M-H-2H2O]+ | |||
7 | 26.96 | C30H48O5 | 2305765-07-3 | 511.3399 (0.9) | 533.3478 (0) | 107.0860, 125.0964, 143.1068, 435.3262, 453.3366, 471.3473[M+H-H2O]+ | ||
8 | 29.80 | C30H46O5 | 145497-67-2 | 509.3246 (1.7) | 531.3326 (0.7) | 485.3271 (0.8) | 107.0860, 125.0964, 419.1069, 437.3421, 455.3525, 469.3319[M+H-H2O]+ | |
9 * | 33.76 | C30H50O5 | - | 491.3734 (0.6) | 513.3557 (1.3) | 535.3636 (0.2) | 107.0860, 125.0963, 143.1068, 419.3311, 437.3418, 455.3523, 473.3629 | |
10 * | 34.11 | C32H52O6 | 199680-24-5 | 533.3840 (0.5) | 555.3659 (0.5) | 577.3743 (0.5) | 107.0859, 125.0963, 143.1068, 419.3310, 437.3416, 455.3521, 497.3625[M+H-2H2O]− | |
11 | 37.40 | C30H48O4 | 1807473-07-9 | 495.3448 (0.6) | 517.3528 (0.2) | 107.0860, 125.0963, 143.1068, 419.3311, 437.3417, 455.3522 | ||
12 | 38.47 | C30H48O4 | 819821-43-7 | 473.3630 (0) | 495.3450 (0) | 107.0860, 125.0963, 143.1068, 419.3311, 437.3417, 455.3522 | ||
13 | 41.40 | C30H50O5 | 97682-74-1 | 513.3557 (1.4) | 535.3644 (1.7) | 489.3590 (2.0) | 419.3312, 437.3420, 453.3368[M-H-2H2O+]+ | |
14 * | 43.58 | C30H48O4 | - | 473.3627 (0.4) | 495.3449 (0.8) | 517.3529 (0) | 107.0860, 125.0963, 143.1068, 419.3311, 437.3418, 455.3523 | |
15 * | 46.36 | C32H52O6 | 1252686-32-0 | 555.3660 (0.7) | 577.3742 (0.3) | 107.0860, 125.0963, 143.1068, 419.3311, 437.3418, 455.3523, 479.3523[M+H-3H2O]+, 515.3735[M+H-H2O]+ |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Zhu, F.; Zhang, X.; Du, B.-Y.; Zhu, X.-X.; Zhao, G.-F.; Sun, Y.; Yao, Q.-Q.; Liang, H.-B.; Yao, J.-C.; Liu, Z.; et al. Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD. Molecules 2023, 28, 6382. https://doi.org/10.3390/molecules28176382
Zhu F, Zhang X, Du B-Y, Zhu X-X, Zhao G-F, Sun Y, Yao Q-Q, Liang H-B, Yao J-C, Liu Z, et al. Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD. Molecules. 2023; 28(17):6382. https://doi.org/10.3390/molecules28176382
Chicago/Turabian StyleZhu, Feng, Xiao Zhang, Bing-Yuan Du, Xiang-Xia Zhu, Gui-Fang Zhao, Ying Sun, Qing-Qiang Yao, Hong-Bao Liang, Jing-Chun Yao, Zhong Liu, and et al. 2023. "Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD" Molecules 28, no. 17: 6382. https://doi.org/10.3390/molecules28176382
APA StyleZhu, F., Zhang, X., Du, B. -Y., Zhu, X. -X., Zhao, G. -F., Sun, Y., Yao, Q. -Q., Liang, H. -B., Yao, J. -C., Liu, Z., Zhang, G. -M., & Qin, G. -F. (2023). Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD. Molecules, 28(17), 6382. https://doi.org/10.3390/molecules28176382