The present study was aimed at investigation of the chemical composition, antioxidant and cytotoxic activities of topinambur Helianthus tuberosus L. extracts for its further application in medicine. Crude methanol extract from the tubers of H. tuberosus L. cultivated in Tajikistan, was analyzed by liquid chromatography–mass spectrometry (LC-MS/MS) method. Chlorogenic and 1,5-dicaffeoylquinic acids were identified as major components of the tuber methanol extract by NMR and peak matching in LC-MS/MS chromatogram with defined standards. Total phenolic and flavonoid contents of the methanol and aqueous extracts were determined using Folin – Ciocalteu and aluminum colorimetric methods, respectively. The methanol extract showed substantial phenolic and flavonoid contents in comparison to the aqueous extract. Total phenolic and flavonoid contents of 67.12 ± 1.2 mg caffeic acid equivalent and 3.53 ± 0.1 mg quercetin equivalent per gram of the methanol extract were determined. Their antioxidant capacity was evaluated using DPPH, ABTS, and FRAP assays. Half –inhibitory (IC50) values of the methanol extract were 13.61 ± 0.1 μg/mL for DPPH and 30.23 ± 0.4 μg/mL for ABTS test, respectively. The FRAP value of the methanol extract and positive control were 850.24 ± 14 and 2380 ± 46 mM FeSO4/mg extract, respectively. The aqueous extract exhibited low antioxidant activity. In addition, cytotoxicity of the methanol extracts was evaluated using MTT assay on CCRF-CEM and P-gp overexpressing CEM/ADR5000 cancer cell lines. The methanol extract showed a rather weak activity with IC50 values 265.0 μg/mL for CCRF-CEM and 345.9 μg/mL for CEM/ADR5000. As CEM/ADR5000 cells overexpress the ABC transporter P-gp, this result indicated that the extract apparently contained substances which affect P-gp, which need further investigations. In conclusion, methanol extract of H. tuberosus is rich with chlorogenic and 1,5-dicaffeoylquinic acid and exhibits strong antioxidant activity and weak cytotoxicity.
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Acknowledgements
The authors thank Dr. Martin Gartner (IPMB, Department of Pharmaceutical Chemistry, Heidelberg University) very much for performing the NMR measurements and his help in data assessment.
APPENDIX
SUPPLEMENTARYMATERIAL
Helianthus tuberosus L. is the best known inulin-producing plant that is widely used in many food products, including antidiabetic diets. The crude methanol extract from the tubers of H. tuberosus, cultivated in Tajikistan, was analyzed by LC-MS/MS. Chlorogenic and 1,5-dicaffeoylquinic acids were identified as major components of the tuber extract by NMR and peak matching in LC-MS/MS chromatogram with defined standards. Total phenolic and flavonoid contents of the methanol and aqueous extracts from the plant tubers were determined using Folin-Ciocalteu and aluminum calorimetric methods, respectively. Their antioxidant capacity was evaluated using DPPH-, ABTS-, and FRAP- assays. Moreover, the cytotoxicity of the extracts was evaluated using MTT-assay in CCRF-CEM and CEM/ADR5000 cancer cell lines.
Fig. S1. Structures of standards which were confirmed by NMR.
Determination of Standards by NMR
The standards of cynarine (1,3-dicaffeoylquinic acid) and 1,5-dicaffeoylquinic acid were determined by NMR because of ambiguities in the stereochemical nomenclature of both compounds. Cynarine, from our natural products collection, could be confirmed as 1,3-dicaffeoylquinic acid (Figure S1 A); the structure of 1,5-dicaffeoylquinic acid could be confirmed as well (Fig. S1 B). The NMR results agree with literature data (see section NMR results).
Characterization of Standards by LC-MS/MS
Chlorogenic acid and the authenticated standards cynarine and 1,5-dicaffeoylquinic acid and the methanol extract of Helianthus tuberosus were characterized by LC-MS/MS (Figs. S2 and S3).
NMR Results for Cynarine
1H NMR (500 MHz, methanol-[d4]): δ 7.49 (d, J = 15.9 Hz, 1H), 7.47 (d, J = 15.9 Hz, 1 H), 6.93 (d, J = 2.1 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.3 Hz, 2.1 Hz, 1H), 6.64 (d, J = 8.2 Hz, 1H), 6.60 (dd, J = 8.2 Hz, 2.1 Hz, 1H), 6.52 (d, J = 8.2 Hz, 1H), 6.19 (d, J = 15.9 Hz, 1H), 6.12 (d, J = 15.9 Hz, 1H), 5.37 (q, J = 3.4 Hz, 1H), 4.23 (ddd, J = 11.1 Hz, 9.5 Hz, 4.4 Hz, 1H), 3.63 (dd, J = 9.5 Hz, 3.6 Hz, 1H), 2.89 (dt, J = 16.0 Hz, 3.3 Hz, 1H), 2.53 (ddd, J = 13.7 Hz, 4.5 Hz, 3.0 Hz, 1H), 2.31 (dd, J = 16.0 Hz, 3.3 Hz, 1H), 1.84 (dd, J = 13.7 Hz, 11.2 Hz, 1H).
13C NMR (125 MHz, methanol-[d4]): δ 174.56 (s), 168.82 (s), 167.79 (s), 149.65 (s), 149.25 (s), 147.70 (d), 147.13 (d), 146.71 (s), 146.47 (s), 127.46 (s), 127.41 (s), 122.92 (d), 122.00 (d), 116.57 (d), 116.55 (d), 116.07 (d), 115.43 (d), 115.37 (d), 115.10 (d), 81.09 (s), 75.29 (d), 72.97 (d), 67.79 (d), 41.31 (t), 32.94 (t).
Fig. S2. Mass spectra and MS2 fragmentation patterns of the standards chlorogenic acid, cynarine (1,3-dicaffeoylquinic acid), and 1,5-dicaffeoylquinic acid.
1H NMR (500 MHz, D2O): δ 7.45 (dd, J = 16.0 Hz, 1.3 Hz, 1H), 7.34 (dd, J = 16.0 Hz, 1.2 Hz, 1H), 6.75 – 6.81 (m, 2H), 6.66 – 6.69 (m, 1H), 6.61 (dd, J = 8.8 Hz, 1.6 Hz, 1H), 6.27 (dd, J = 16.0 Hz, 1.4 Hz, 1H), 6.17 (dd, J = 16.0 Hz, 1.4 Hz, 1H), 5.27 (dt, J = 3.9 Hz, 2.0 Hz, 2.0 Hz, 1H), 4.38 (dddd, J = 11.4 Hz, 9.9 Hz, 4.5 Hz, 1.4 Hz, 1H), 3.79 (ddd, J = 9.9 Hz, 3.7 Hz, 1.4 Hz, 1H), 3.04 (dt, J = 16.4 Hz, 3.3 Hz, 3.3 Hz, 1H), 2.46 (dt, J = 13.6 Hz, 3.9 Hz, 3.9 Hz, 1H), 2.29 – 2.37 (m, 1H), 1.79 (ddd, J = 13.2 Hz, 11.5 Hz, 1.4 Hz, 1H).
Fig. S3. Mass spectra and MS2 fragmentation patterns of prominent peaks of the methanol extract of H. tuberosus.
NMR Results for 1,5-Di-O-caffeoylquinic Acid
1H NMR (500 MHz, D2O): δ 7.67 (d, J = 16.0 Hz, 1H), 7.65 (d, J = 16.0 Hz, 1H), 7.21 (d, J = 2.1 Hz, 1H),7.19 (d, J = 2.0 Hz, 1H), 7.14 (dd, J = 8.4 Hz, 1.9 Hz, 1H), 7.12 (dd, J = 8.5 Hz, 2.0 Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.46 (d, J = 16.0 Hz, 1H), 6.39 (d, J = 16.0 Hz, 1H), 5.38 (td, J = 9.8 Hz, 9.8 Hz, 4.2 Hz, 1H), 4.37 (ddd, J = 3.9 Hz, 3.8 Hz, 3.8 Hz, 1.4 Hz, 1H), 3.95 (dd, J = 9.2 Hz, 3.5 Hz, 1H), 2.64 (ddd, J = 13.9 Hz, 4.3 Hz, 2.8 Hz, 1H), 2.55 (ddd, J = 15.5 Hz, 3.8 Hz, 2.8 Hz, 1H), 2.8 Hz, 1H), 2.55 (ddd, J = 15.5 Hz, 3.8 Hz, 2.8 Hz, 1H), 2.36 (dd, J = 15.4 Hz, 3.6 Hz, 1H), 2.12 (dd, J = 13.9 Hz, 10.5 Hz, 1H).
Fig. S4. 1H NMR (500 MHz, methanol-[d4]). Scaling after Fulmer, et al. [6].
Comparison of 1 H NMR in methanol with literature data for1,3-di-O-caffeoylquinic acid (quinic acid part only, COOH at C-1 equatorial)
TABLE S1. Comparison of NMR Results for Cynarine Standard with Literature Data
Proton | Our cynarine standard | Pauli, et al. [1] | Wu, et al. [2] |
2-Haxial | 2.31 (dd, 16.0, 3.3 Hz) | 2.30 (dd, 16.0, 3.3 Hz) | 2.31 (dd, 12.9, 3.2 Hz) |
2-Hequatorial | 2.89 (dt, 16.0, 3.3, 3.3 Hz) | 2.89 (dt, 16.0, 3.2, 3.2 Hz) | 2.89 (ddd, 10.0, 3.2, 2.8 Hz) |
3-Hequatorial | 5.37 (q, 3.4, 3.4, 3.4 Hz) | 5.37 (ddd, 3.6, 3.2, 3.3 Hz) | 5.36 (m) |
4-Haxial | 3.63 (dd, 9.5, 3.6 Hz) | 3.63 (dd, 9.5, 3.6 Hz) | 3.62 (dd, 9.6, 3.7 Hz) |
5-Haxial | 4.23 (ddd, 11.1, 9.5, 4.4 Hz) | 4.23 (ddd, 11.2, 9.5, 4.5 Hz) | 4.23 (td, 9.7, 9.7, 4.6 Hz) |
6-Haxial | 1.84 (dd, 13.7, 11.2 Hz) | 1.84 (dd, 13.7, 11.2 Hz) | 1.85 (dd, 11.4, 11.0 Hz) |
6-Hequatorial | 2.53 (ddd,13.7, 4.5, 3.0 Hz) | 2.53 (ddd, 13.7, 4.5, 3.2 Hz) | 2.50 (ddd, 9.7, 4.1, 3.2 Hz) |
Fig. S5. 1H NMR (500 MHz, D2O). Scaling after Fulmer, et al. [6].
Comparison of 1 H NMR in D 2 O with Literature data for 1,5-di-O-caffeoylquinic acid (quinic acid part only)
TABLE S2. Comparison of NMR Results for Cynarine Standard with Literature Data for 1,5-Di-O-caffeoylquinic Acid (Quinic Acid Part Only)
Proton | Our cynarine standard | Tolonen, et al. [5] |
2-Haxial | 2.29 – 2.37 (m) | 2.22 (dd, 15.5, 3.6 Hz) |
2-Hequatorial | 2.46 (dt, 13.6, 3.9, 3.9 Hz) | 2.50 (ddd, 15.5, 3.8, 2.8 Hz) |
3-Hequatorial | 4.38 (dddd, 11.4, 9.9, 4.5, 1.4 Hz) | 4.27 (ddd, 3.8, 3.6, 3.5 Hz) |
4-Haxial | 3.79 (ddd, 9.9, 3.7, 1.4 Hz) | 3.86 (dd, 9.6, 3.5 Hz) |
5-Haxial | 5.27 (dt, 3.9, 2.0, 2.0 Hz) | 5.29 (ddd, 10.8, 9.6, 4.3 Hz) |
6-Haxial | 1.79 (ddd, 13.2, 11.5, 1.4 Hz) | 1.99 (dd, 13.9, 10.8 Hz) |
6-Hequatorial | 3.04 (dt, 16.4, 3.3, 3.3 Hz) | 2.56 (ddd, 13.9, 4.3, 2.8 Hz) |
Poor accordance (probably, different substance.
Comparison of 1 H NMR in D 2 O with literature data for 1,5-di-O-caffeoylquinic acid
TABLE S3. Comparison of NMR Results for 1,5-Di-O-caffeoylquinic Acid Standard with Literature Data (Quinic Acid Part Only)
Proton | Our 1,5-di-O-caffeoylquinic acid standard | Tolonen, et al. [5] |
2-Haxial | 2.36 (dd, 15.4, 3.6 Hz) | 2.22 (dd, 15.5, 3.6 Hz) |
2-Hequatorial | 2.55 (ddd, 15.5, 3.8, 2.8 Hz) | 2.50 (ddd, 15.5, 3.8, 2.8 Hz) |
3-Hequatorial | 4.37 (ddd, 3.9, 3.8, 3.8 Hz) | 4.27 (ddd, 3.8, 3.6, 3.5 Hz) |
4-Haxial | 3.95 (dd, 9.2, 3.5 Hz) | 3.86 (dd, 9.6, 3.5 Hz) |
5-Haxial | 5.38 (ddd, 9.8, 9.8, 4.2 Hz) | 5.29 (ddd, 10.8, 9.6, 4.3 Hz) |
6-Haxial | 2.12 (dd, 13.9, 10.5 Hz) | 1.99 (dd, 13.9, 10.8 Hz) |
6-Hequatorial | 2.64 (ddd, 13.9, 4.3, 2.8 Hz) | 2.56 (ddd, 13.9, 4.3, 2.8 Hz) |
Good agreement (δ-scale shifted by different calibrations).
TABLE S4. Comparison of NMR Results for 1,5-Di-O-caffeoylquinic Acid Standard with Literature Data (Caffeic Acid Part Only)
Proton | Our 1,5-di-O-caffeoylquinic acid standard | Tolonen, et al. [5] |
2′ a | 7.19 (d, 2.0 Hz) | 7.12 (d, 2.1 Hz) |
2′ b | 7.21 (d, 2.1 Hz) | 7.15 (d, 2.1 Hz) |
5′ a | 6.93 (d, 8.3 Hz) | 6.86 (d, 8.3 Hz) |
5′ b | 6.95 (d, 8.9 Hz) | 6.88 (d, 8.2 Hz) |
6′ a | 7.12 (dd, 8.5, 2.0 Hz) | 7.05 (dd, 8.3, 2.1 Hz) |
6′ b | 7.14 (dd, 8.4, 1.9 Hz) | 7.08 (dd, 8.2, 2.1 Hz) |
7′ a | 7.65 (d, 16.0 Hz) | 7.58 (d, 16.2 Hz) |
7′ b | 7.67 (d, 16.0 Hz) | 7.59 (d, 16.1 Hz) |
8′ a | 6.39 (d, 16.0 Hz) | 6.32 (d, 16.2 Hz) |
8′ b | 6.46 (d, 16.0 Hz) | 6.39 (d, 16.1 Hz) |
Good agreement (δ-scale shifted by different calibrations).
SUPPLEMENTARY LITERATURE
1. G. F. Pauli, F. Poetsch, and A. Nahrstedt, Phytochem. Anal., 9, 177 – 185 (1998).
2. C.Wu, F. Chen, X.Wang, et al., Phytochem. Anal., 18, 401 – 410 (2007).
3. X. Shu, M. Wang, D. Liu, et al., Quimica Nova, 36, 836 – 839 (2013).
4. I. Horman, R. Badoud, and W. Ammann, J. Agric. Food Chem., 32, 538 – 540 (1984).
5. A. Tolonen, T. Joutsamo, S. Mattlla, et al., Phytochem. Anal., 13, 316 – 328 (2002).
6. G. R. Fulmer, A. J. M. Miller, N. H. Sherden, et al., Organometallics, 29, 2176 – 2179 (2010).
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Sharopov, F., Wetterauer, B., Gulmurodov, I. et al. Chlorogenic and 1,5-Dicaffeoylquinic Acid-Rich Extract of Topinambur (Helianthus tuberosus L.) Exhibits Strong Antioxidant Activity and Weak Cytotoxicity. Pharm Chem J 54, 745–754 (2020). https://doi.org/10.1007/s11094-020-02265-0
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DOI: https://doi.org/10.1007/s11094-020-02265-0