Fractionation Coupled to Molecular Networking: Towards Identification of Anthelmintic Molecules in Terminalia leiocarpa (DC.) Baill
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cytotoxicity and Anthelminthic Activities of Fractions
2.2. Molecular Networking and Major Compounds in the Most Active Fractions
Code | RT (min) | Molecular Formula | Quasi- Molecular Ion | MS/MS Fragment | Molecular Mass | Error (ppm) | Identification | Isolated Previously from | Source | References | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Observed | Theoretical | F8# | F9# | |||||||||
1 | 3.02 | C6H14O6 | 181.0715 [M-H]- | 179.0560 144.0665 101.0245 163.0610 | 182.0794 | 182.0790 | 1.99 | Hexitol | √ | |||
2 | 3.17 | C6H12O6 | 179.0564 [M-H]- | 161.0460 113.0248 | 180.0643 | 180.0634 | 5.06 | Fructose * | √ | √ | ||
3 | 3.29 | C7H12O6 | 191.0562 [M-H]- | 181.0715 179.0569 173.0465 189.8369 | 192.0641 | 192.0634 | 3.71 | Quinic acid * | Terminalia ferdinandiana | √ | √ | [35] |
4 | 3.40 | C7H10O5 | 173.0456 [M-H]- | 119.0353 129.0198 137.0243 155.0348 | 174.0535 | 174.0528 | 3.89 | Shikimic acid | Anogeissus latifolia | √ | √ | [3] |
5 | 3.44 | C20H18O14 | 481.0605 [M-H]- | 331.0672 421.1343 173.0456 300.9998 375.1294 | 482.0684 | 482.0697 | −2.70 | 2,3- (S)-Hexahydroxydiphenoyl-D-glucose | T. calamansanai, T. myriocarpa | √ | [29,30] | |
6 | 4.97 | C9H18O7 | 283.1037 [M+HCOO]- | 243.0630 273.0739 179.0564 | 238.1061 | 238.1053 | 3.56 | n.i | √ | |||
7 | 5.51 | C17H26O12 | 421.1360 [M-H]- | 375.1310 287.0888 267.0739 357.1195 331.0686 | 422.1439 | 422.1424 | 3.49 | n.i | √ | |||
8 | 9.02 | C₇H₆O₅ | 169.0144 [M-H]- | 125.0247 168.0070 124.0173 126.0283 | 170.0223 | 170.0215 | 4.57 | Gallic acid * | A. leiocarpa, T. ferdinandiana | √ | √ | [2,25,35] |
9 | 20.43 | 447.1860 [M-H]- | 401.1822 300.9978 179.0560 | Ellagic acid derivative | √ | √ | ||||||
10 | 20.90 | 477.1626 [M-H]- | 431.1540 445.1712 300.9982 169.0147 | Ellagic acid derivative | √ | |||||||
11 | 21.60 | 447.1515 [M-H]- | 300.9987 289.0723 387.1662 169.0150 | Ellagic acid derivative | √ | |||||||
12 | 22.43 | 431.1910 [M-H]- | 387.1653 169.0145 300.9980 327.1093 | Ellagic acid derivative | √ | √ | ||||||
13 | 22.78 | 387.1660 [M-H]- | 169.0149 301.0005 | Ellagic acid derivative | √ | |||||||
14 | 23.56 | 453.1048 [M-H]- | 387.1666 289.0226 439.0686 169.0143 300.9990 125.0252 | Ellagic acid derivative | √ | √ | ||||||
15 | 24.73 | 431.1912 [M-H]- | 300.9982 169.0146 289.0718 125.0248 205.1234 | Ellagic acid derivative | √ | √ | ||||||
16 | 24.86 | 453.1979 [M-H]- | 433.2072 300.9979 407.1930 169.0145 | Ellagic acid derivative | √ | |||||||
17 | 25.24 | C27H30O16 | 609.1453 [M-H]- | 459.1496 301.0353 313.0574 567.2086 169.0144 | 610.1532 | 610.1534 | −0.30 | Rutin * | A. leiocarpa | √ | [21] | |
18 | 25.44 | C28H24O16 | 615.0955 [M-H]- | 301.0353 313.0553 565.2844 463.0887 169.0144 | 616.1034 | 616.1064 | −4.93 | Quercetin-3-O-(6-O-galloyl)-β-D-galactopyranoside | T. guianensis | √ | √ | [26] |
19 | 26.22 | C14H6O8 | 300.9978 [M-H]- | 163.0398 169.0144 | 302.0057 | 302.0063 | −1.88 | Ellagic acid * | A. leiocarpa Terminalia brownii | √ | √ | [4,7] |
20 | 26.59 | C21H18O13 | 477.0677 [M-H]- | 301.0354 302.0383 169.0145 439.0670 151.0035 289.0715 287.0564 | 478.0756 | 478.0747 | 1.80 | Quercetin-3-O-glucuronide | √ | |||
21 | 26.79 | C48H68O5 | 723.5013 [M-H]- | 439.0679 169.0140 463.0896 303.0508 289.0721 677.5002 125.0249 | 724.5092 | 724.5067 | 3.48 | n.i | √ | |||
22 | 27.02 | C28H24O15 | 599.1047 [M-H]- | 435.1282 285.0406 473.1672 313.0556 | 600.1126 | 600.1115 | 1.80 | Kaempferol linked to gallate and deoxy-hexose | √ | √ | ||
23 | 27.35 | C21H20O11 | 447.0932 [M-H]- | 285.0404 284.0323 439.0670 442.7359 289.0715 | 448.1011 | 448.1006 | 1.20 | Astragalin * | P. suberosa | √ | √ | [27] |
24 | 27.85 | C37H60O14 | 727.3909 [M-H]- | 565.3358 519.3334 439.0675 477.1035 343.2121 | 728.3988 | 728.3983 | 0.68 | n.i | √ | |||
25 | 28.53 | C37H60O13 | 711.3926 [M-H]- | 343.2126 371.1710 531.1526 439.0681 583.1072 289.0722 | 712.4005 | 712.4034 | −4.06 | n.i | √ | |||
26 | 29.36 | C22H24O10 | 447.1303 [M-H]- | 439.0685 303.0513 169.0151 287.0574 289.0725 125.0251 | 448.1382 | 448.1369 | 2.80 | n.i | √ | |||
27 | 30.90 | C30H62O18 | 709.3831 [M-H]- | 507.2063 547.3296 501.3242 597.1829 461.1088 | 710.3910 | 710.3936 | −3.68 | n.i | √ | √ | ||
28 | 31.96 | C37H60O13 | 711.3934 [M-H]- | 549.3431 697.3820 503.3406 695.4033 702.6718 | 712.4013 | 712.4034 | −2.94 | n.i | √ | |||
29 | 32.82 | C36H58O10 | 695.4020 [M + HCOO]- | 487.3446 173.9751 686.9651 533.3465 | 650.4043 | 650.4030 | 2.00 | Rosamultin or isomer | T. alata | √ | √ | [31] |
30 | 33.88 | C18H32O5 | 327.2178 [M-H]- | 324.4868 211.1343 289.0727 171.1030 229.1447 | 328.2257 | 328.2250 | 2.21 | Oxo-dihydroxy-octadecenoic acid | Globularia spp. Bituminaria bituminosa Sasa veitchii | √ | √ | [32,33,34] |
31 | 35.28 | C18H33O5 | 329.2329 [M-H]- | 211.1343 116.0257 229.1447 326.4767 169.0144 | 330.2408 | 330.2406 | 0.53 | Trihydroxy-octadecenoic acid | Globularia spp. B. bituminosa S. veitchii | √ | √ | [32,33,34] |
32 | 43.40 | C34H58O16 | 721.3667 [M-H]- | 675.3602 397.1340 712.4544 277.2173 | 722.3746 | 722.3725 | 2.93 | n.i | √ | √ | ||
33 | 46.81 | C34H60O16 | 723.3777 [M-H]- | 677.3750 116.0257 397.1347 167.0363 119.0260 | 724.3856 | 724.3881 | −3.50 | n.i | √ |
2.3. Prediction of Anthelmintic Activity of Features Detected in T. leiocarpa Fractions by Pearson Correlation
2.4. Anthelmintic Activity of Major Compounds and Their Quantification
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Plant Collection
3.3. Extraction Procedure
3.4. Open Column Chromatography (OCC) Fractionation of T. leiocarpa MeOH Extract
3.5. Fractions Cytotoxicity
3.6. Anthelmintic Activities of Fractions
3.6.1. Viability of C. elegans Adult Worms Treated with Fractions and Pure Compounds
3.6.2. Larval Migration Inhibition Assay (LAMIA)
3.7. HPLC-PDA-HRMS/MS Analysis
3.8. Data Processing on MZmine
3.9. Dereplication on GNPS
3.10. Quantification of Major Compounds
3.11. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample/Control | H. contortus (%) | C. elegans (%) | Cytotoxicity, IC50 (µg/mL) |
---|---|---|---|
MeOH | 63.4 ± 0.8 | 70.0 ± 7.1 | >100 |
1 | - | - | 64.5 ± 5.7 |
2 | 29.8 ± 17.5 | 2.5 ± 4.3 | 58.9 ± 2.9 |
3 | - | - | 64.1 ± 2.2 |
4 | 15.1 ± 6.2 | 12.5 ± 4.3 | 60.3 ± 10.0 |
5 | 21.0 ± 3.9 | 22.5 ± 4.3 | 59.6 ± 4.9 |
6 | 35.7 ± 11.7 | 20.0 ± 7.1 | 78.9 ± 2.0 |
7 | 39.9 ± 7.9 | 22.5 ± 4.3 | >100 |
8 | 69.4 ± 10.4 | 75.0 ± 5.0 | >100 |
9 | 76.4 ± 1.8 | 85.0 ± 5.0 | >100 |
DMSO (0.5%) | 3.1 ± 0.8 | 0 | - |
LEV (25µM) | 100.0 ± 0.0 | 100.0 ± 0.0 | - |
Compound (ID) | Equation | R2 | LOD (µg/mL) | LOQ (µg/mL) | F8 | F9 | ||
---|---|---|---|---|---|---|---|---|
µg/mL | µg/mg of Fraction | µg/mL | µg/mg of Fraction | |||||
Ellagic acid (19) | y = 228815x + 926041 | 0.999 | 5.5 | 16.5 | 86.4 ± 6.9 | 8.6 ± 0.7 | 71.4 ± 2.6 | 7.1 ± 0.3 |
Astragalin (23) | y = 45696x + 15964 | 0.997 | 3.3 | 9.9 | 7.7 ± 1.2 | 0.8 ± 0.1 | 9.6 ± 0.4 | 1.0 ± 0.0 |
Gallic acid (8) | y = 8197.3x + 26875 | 0.995 | 10.3 | 31.1 | 96.5 ± 7.8 | 9.7 ± 0.8 | 19.9 ± 0.5 | 2.0 ± 0.5 |
Compound (ID) | F8 | F9 | ||
---|---|---|---|---|
µg/mL | µg/mg of Fraction | µg/mL | µg/mg of Fraction | |
Ellagic derivative (9) | 1.0 ± 0.4 | 0.1 ± 0.0 | 13.6 ± 1.1 | 1.4 ± 0.1 |
Ellagic derivative (10) | 2.1 ± 0.3 | 0.2 ± 0.0 | - | - |
Ellagic derivative (11) | 4.2 ± 2.0 | 0.4 ± 0.2 | 35.9 ± 3.6 | 3.6 ± 0.4 |
Ellagic derivative (12) | - | - | - | - |
Ellagic derivative (13) | 2.4 ± 0.8 | 0.2 ± 0.1 | 0.5 ± 1.4 | 0.1 ± 0.2 |
Ellagic derivative (14) | - | - | 8.0 ± 0.1 | 0.8 ± 0.0 |
Ellagic derivative (15) | 0.3 ± 0.5 | 0.1 ± 0.1 | 0.4 ± 1.0 | <LOQ |
Ellagic derivative (16) | 11.1 ± 0.6 | 1.1 ± 0.1 | 1.8 ± 0.2 | 0.2 ± 0.0 |
Solvent System | Ratio | Volume (mL) |
---|---|---|
DCM-MeOH | 100–0 | 300 |
DCM-MeOH | 99.5–0.5 | 400 |
DCM-MeOH | 99–1 | 600 |
DCM-MeOH | 98–2 | 600 |
DCM-MeOH | 96–4 | 600 |
DCM-MeOH | 94–6 | 1000 |
DCM-MeOH | 92–8 | 600 |
DCM-MeOH | 90–10 | 1000 |
DCM-MeOH | 85–15 | 1200 |
DCM-MeOH | 80–20 | 1200 |
DCM-MeOH | 70–30 | 1200 |
DCM-MeOH | 60–40 | 1200 |
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Tchetan, E.; Ortiz, S.; Olounladé, P.A.; Hughes, K.; Laurent, P.; Azando, E.V.B.; Hounzangbe-Adote, S.M.; Gbaguidi, F.A.; Quetin-Leclercq, J. Fractionation Coupled to Molecular Networking: Towards Identification of Anthelmintic Molecules in Terminalia leiocarpa (DC.) Baill. Molecules 2023, 28, 76. https://doi.org/10.3390/molecules28010076
Tchetan E, Ortiz S, Olounladé PA, Hughes K, Laurent P, Azando EVB, Hounzangbe-Adote SM, Gbaguidi FA, Quetin-Leclercq J. Fractionation Coupled to Molecular Networking: Towards Identification of Anthelmintic Molecules in Terminalia leiocarpa (DC.) Baill. Molecules. 2023; 28(1):76. https://doi.org/10.3390/molecules28010076
Chicago/Turabian StyleTchetan, Esaïe, Sergio Ortiz, Pascal Abiodoun Olounladé, Kristelle Hughes, Patrick Laurent, Erick Virgile Bertrand Azando, Sylvie Mawule Hounzangbe-Adote, Fernand Ahokanou Gbaguidi, and Joëlle Quetin-Leclercq. 2023. "Fractionation Coupled to Molecular Networking: Towards Identification of Anthelmintic Molecules in Terminalia leiocarpa (DC.) Baill" Molecules 28, no. 1: 76. https://doi.org/10.3390/molecules28010076