Metabolomic Analysis of Phytochemical Compounds from Ethanolic Extract of Lime (Citrus aurantifolia) Peel and Its Anti-Cancer Effects against Human Hepatocellular Carcinoma Cells
Abstract
:1. Introduction
2. Results
2.1. LC-qTOF/MS Screening and Identification
2.2. GC-HRMS Screening and Identification
2.3. The Concentration of Hesperidin and Limonin in the Extract
2.4. Cytotoxicity on PLC/PRF/5 Hepatoma Cells
2.5. Anti-Invasion Effect on PLC/PRF/5 Hepatoma Cells
2.6. Induction of Apoptosis in PLC/PRF/5 Hepatoma Cells
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Preparation of Extract
4.3. Metabolomics Analysis
4.3.1. Using LC-MS/MS
4.3.2. Using GC-HRMS Analysis
4.4. Quantitative Measurement of Hesperidin and Limonin
4.5. Cell Culture
4.6. Cell Viability Assay
4.7. Cell Invasion Assay
4.8. Cell Apoptosis Assay
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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No. | RT (min) | Compounds | Mode | Molecular Mass | Precursor Mass | Molecular Formula | Library Score | Relative Area (%) |
---|---|---|---|---|---|---|---|---|
1. | 0.93 | D-Aspartic acid | M+ | 133.0382 | 134.0449 | C4H7NO4 | 90.3 | 0.10 |
2. | 0.97 | N-Oxalylglycine | [M + CH3OH + H]+ | 115.0272 | 148.0602 | C4H5NO5 | 100.0 | 0.06 |
3. | 1.05 | Benzothiazole, 2-methylmercapto- | M+ | 181.0507 | 182.0575 | C8H7NS2 | 99.2 | 0.04 |
4. | 1.05 | Melibiose | [M + NH4]+ | 342.1164 | 360.1497 | C12H22O11 | 86.3 | 0.10 |
5. | 1.20 | 3-Pyridinecarboxaldehyde | M+ | 107.0378 | 108.0445 | C6H5NO | 85.7 | 0.10 |
6. | 1.28 | Synephrine | M+ | 167.0588 | 168.0655 | C9H13NO2 | 83.7 | 0.14 |
7. | 1.57 | 2,5-Dihydroxy benzaldehyde | M+ | 137.9961 | 139.0028 | C7H6O3 | 82.3 | 0.08 |
8. | 1.57 | trans-Aconitic acid | M+ | 174.0170 | 175.0237 | C6H6O6 | 72.1 | 0.85 |
9. | 1.57 | Citric acid | [M + NH4]+ | 192.0279 | 210.0611 | HOC (CH2CO2H)2 | 76.3 | 0.64 |
10. | 3.34 | Adenosine | M+ | 267.0976 | 268.1044 | C10H13N5O4 | 100.0 | 0.40 |
11. | 4.14 | 2,3-Desisopropylidenetopiramate | M+ | 321.0845 | 322.0913 | C9H17NO8S | 89.3 | 0.30 |
12. | 4.28 | Phenprobamate | M+ | 165.0794 | 166.0861 | C10H13NO2 | 99.7 | 0.22 |
13. | 5.30 | Poly-L-asparagine | M+ | 114.1026 | 115.0387 | C4H6 H2O2 | 98.9 | 0.03 |
14. | 6.49 | Stachydrine | M+ | 143.1732 | 144.0799 | C7H13NO2 | 83.6 | 0.02 |
15. | 7.32 | Isoorientin | M+ | 448.1002 | 449.1069 | C21H20O11 | 99.1 | 0.04 |
16. | 7.40 | Lactobionic acid | M+ | 380.1087 | 381.1155 | C12H22O12 | 97.9 | 0.07 |
17. | 7.83 | Engeletin | M+ | 435.1177 | 435.1244 | C21H22O10 | 80.0 | 0.06 |
18. | 7.91 | trans-3-Coumaric acid | [M + H]+ | 146.0374 | 147.0441 | C9H7O3 | 96.9 | 0.25 |
19. | 7.91 | cis-3-Coumaric acid | M+ | 164.0476 | 165.0543 | C9H7O3 | 80.1 | 0.05 |
20. | 7.91 | Quercetin | M+ | 302.0433 | 303.0501 | C15H10O7 | 97.9 | 0.03 |
21. | 7.99 | Isovitexin | [M + H]+ | 432.1077 | 433.1144 | C21H20O10 | 98.0 | 0.80 |
22. | 8.11 | Rutin | M+ | 610.4776 | 611.4843 | C27H30O16 | 99.6 | 0.02 |
23. | 8.54 | Quercetin 3′-methyl ether | M+ | 316.0587 | 317.0654 | C16H12O7 | 98.9 | 0.09 |
24. | 8.54 | Isorhoifolin | M+ | 578.1632 | 579.1699 | C27H30O14 | 100.0 | 0.19 |
25. | 8.54 | Narirutin | M+ | 580.1779 | 581.1846 | C27H32O14 | 92.0 | 0.09 |
26. | 8.54 | Narcissoside | M+ | 624.1702 | 625.1769 | C28H32O16 | 97.9 | 0.66 |
27. | 8.58 | Bisdemethoxy curcumin | M+ | 308.0900 | 309.0967 | C19H16O4 | 71.3 | 0.14 |
28. | 8.83 | Diosmin | M+ | 608.1755 | 609.1823 | C28H32O15 | 99.9 | 0.62 |
29. | 8.92 | Hesperetin | M+ | 302.0799 | 303.0867 | C16H14O6 | 99.5 | 0.69 |
30. | 8.92 | Hesperidin * | M+ | 610.1898 | 611.1966 | C28H34O15 | 88.8 | 4.98 |
31. | 8.92 | Neohesperidin | M+ | 610.2029 | 611.2097 | C28H34O15 | 82.4 | 4.29 |
32. | 8.92 | Hyperin | [M + CH3OH + H]+ | 432.1057 | 465.1387 | C21H19O12 | 91.7 | 0.30 |
33. | 9.32 | Isorhamnetin 3-O-glucoside | M+ | 478.1113 | 479.1181 | C22H22O12 | 98.9 | 0.11 |
34. | 9.32 | Syringetin 3-glucoside | M+ | 508.1228 | 509.1296 | C23H24O13 | 93.8 | 0.57 |
35. | 9.76 | Mono-2-ethylhexyl phthalate | M+ | 278.1169 | 279.1237 | C16H21O4 | 82.8 | 0.04 |
36. | 9.84 | Scoparone | M+ | 206.0261 | 207.0328 | C11H10O4 | 92.5 | 0.06 |
37. | 10.02 | Heraclenin | M+ | 286.0855 | 287.0922 | C16H14O5 | 95.0 | 0.68 |
38. | 10.48 | 2′,6′-Dihydroxy-4- methoxychalcone-4′- O-neohesperidoside | M+ | 594.1943 | 595.2010 | C28H34O14 | 79.3 | 0.04 |
39. | 10.60 | Oxypeucedanin hydrate | M+ | 304.1034 | 305.1102 | C16H16O6 | 98.1 | 4.24 |
40. | 10.83 | Byakangelicol | M+ | 316.0955 | 317.1022 | C17H16O6 | 93.0 | 0.45 |
41. | 11.32 | 7-Methoxycoumarin | M+ | 176.0489 | 177.0556 | C10H8O3 | 85.1 | 1.22 |
42. | 12.07 | Angelicin | M+ | 186.0316 | 187.0383 | C11H6O3 | 98.3 | 0.03 |
43. | 13.06 | 5,7-Dimethoxycoumarin | M+ | 206.0661 | 207.0729 | C11H10O4 | 79.2 | 14.62 |
44. | 13.38 | Isopimpinellin | [M + NH4]+ | 246.0529 | 264.0862 | C13H10O5 | 100.0 | 0.07 |
45. | 13.98 | Limonin * | M+ | 470.2057 | 471.2125 | C26H30O8 | 95.3 | 5.01 |
46. | 14.83 | Nomilin | [M + H]+ | 514.2206 | 515.2273 | C28H34O9 | 91.8 | 0.24 |
47. | 15.88 | Obacunone | M+ | 454.1990 | 455.2058 | C26H30O7 | 86.1 | 0.03 |
48. | 16.62 | Monolinolenin (9c,12c,15c) | M+ | 352.2618 | 353.2685 | C21H36O4 | 75.7 | 0.04 |
49. | 18.98 | 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine | M+ | 453.2863 | 454.2931 | C21H44NO7P | 88.0 | 0.66 |
50. | 19.47 | 1-Palmitoyl-sn-glycero-3-phosphocholine | M+ | 495.3338 | 496.3405 | C26H52NO8P | 94.4 | 1.40 |
51. | 19.74 | Esculetin | M+ | 178.0269 | 179.0336 | C9H6O4 | 70.1 | 0.04 |
52. | 20.08 | 1-Oleoyl-sn-glycero-3-phosphocholine | M+ | 521.3492 | 522.3560 | C26H52NO7P | 97.2 | 1.20 |
53. | 20.63 | Cinnamic acid | M+ | 148.0167 | 149.0234 | C9H8O2 | 79.6 | 1.45 |
54. | 21.13 | 8-Allyloxypsoralen | M+ | 242.0583 | 243.0650 | C14H10O4 | 90.2 | 0.06 |
55. | 21.17 | 3-Propyl- 2-methylpyrazine | M+ | 136.1255 | 137.1322 | C8H12N2 | 81.3 | 0.49 |
56. | 21.17 | Xanthotoxol | M+ | 202.0275 | 203.0342 | C11H6O4 | 95.3 | 1.20 |
57. | 22.51 | Bergaptol | M+ | 202.0350 | 203.0417 | C11H6O4 | 85.8 | 14.30 |
58. | 22.55 | Bergamotin | M+ | 338.1583 | 339.1651 | C21H22O4 | 82.2 | 8.35 |
59. | 22.66 | 5-Geranoxy- 7-methoxy-coumarin | M+ | 328.1823 | 329.1890 | C20H24O4 | 91.7 | 24.36 |
60. | 23.61 | 8-O-Acetylharpagide | M+ | 406.0821 | 407.0888 | C17H26O11 | 95.0 | 0.17 |
61. | 23.75 | Oleamide | M+ | 281.2732 | 282.2799 | C18H35NO | 76.9 | 2.39 |
62. | 25.67 | Hexadecyltrimethylammonium cation | M+ | 284.2878 | 284.2945 | C16H33N(CH3)3 | 98.5 | 0.03 |
No. | RT (min) | Compounds | Molecular Mass | Molecular Formula | SI | RSI | Relative Area (%) |
---|---|---|---|---|---|---|---|
1. | 3.0932 | 1,1-Diethoxyethane | 118.1742 | C6H14O2 | 622 | 884 | 1.06 |
2. | 4.8921 | Furfural | 96.0841 | C5H4O2 | 752 | 888 | 1.36 |
3. | 7.1966 | α-pinene | 136.2340 | C10H16 | 776 | 866 | 1.43 |
4. | 7.2959 | Furan | 68.0740 | C4H4O | 849 | 911 | 9.35 |
5. | 7.7484 | 5-Methyl furfural | 110.1106 | C6H6O2 | 803 | 905 | 1.71 |
6. | 7.900 | 1-Methylcyclopentanol | 100.1589 | C6H12O2 | 689 | 792 | 2.04 |
7. | 9.1655 | D-Limonene | 136.2340 | C10H16 | 867 | 871 | 20.92 |
8. | 9.7108 | 3-Carene | 136.2340 | C10H16 | 792 | 842 | 1.67 |
9. | 11.8210 | Terpinen-4-ol | 154.2493 | C10H18O | 757 | 832 | 1.05 |
10. | 12.0373 | α-Terpineol | 154.2493 | C10H18O | 817 | 897 | 1.99 |
11. | 12.3772 | 5-Hydroxymethy furfural | 126.1100 | C6H6O3 | 736 | 758 | 4.59 |
12. | 15.3505 | Caryophyllene | 204.3511 | C15H24 | 888 | 896 | 7.60 |
13. | 15.4631 | Cis-α-Bergamotene | 204.3511 | C15H24 | 876 | 899 | 6.34 |
14. | 15.8405 | Aristolochene | 204.3511 | C15H24 | 762 | 835 | 1.71 |
15. | 16.3901 | α-Farnesene | 204.3511 | C15H24 | 733 | 747 | 6.31 |
16. | 16.4895 | Aromandendrene | 204.3511 | C15H24 | 867 | 881 | 8.61 |
17. | 17.0126 | 6,9-Guaiadene | 204.3511 | C15H24 | 815 | 857 | 1.47 |
18. | 17.0899 | Selina-3,7(11)-diene | 204.3511 | C15H24 | 823 | 879 | 1.38 |
19. | 24.1909 | Citraptene | 206.1947 | C11H10O4 | 878 | 896 | 7.11 |
20. | 25.5329 | Bergapten | 216.1895 | C12H8O4 | 840 | 937 | 3.12 |
21. | 28.3627 | Isopimpinellin | 246.2155 | C13H10O5 | 860 | 873 | 8.43 |
22. | 31.6207 | Palmitic acid β-monoglyceride | 330.5026 | C19H38O4 | 667 | 789 | 0.95 |
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Phucharoenrak, P.; Muangnoi, C.; Trachootham, D. Metabolomic Analysis of Phytochemical Compounds from Ethanolic Extract of Lime (Citrus aurantifolia) Peel and Its Anti-Cancer Effects against Human Hepatocellular Carcinoma Cells. Molecules 2023, 28, 2965. https://doi.org/10.3390/molecules28072965
Phucharoenrak P, Muangnoi C, Trachootham D. Metabolomic Analysis of Phytochemical Compounds from Ethanolic Extract of Lime (Citrus aurantifolia) Peel and Its Anti-Cancer Effects against Human Hepatocellular Carcinoma Cells. Molecules. 2023; 28(7):2965. https://doi.org/10.3390/molecules28072965
Chicago/Turabian StylePhucharoenrak, Pakkapong, Chawanphat Muangnoi, and Dunyaporn Trachootham. 2023. "Metabolomic Analysis of Phytochemical Compounds from Ethanolic Extract of Lime (Citrus aurantifolia) Peel and Its Anti-Cancer Effects against Human Hepatocellular Carcinoma Cells" Molecules 28, no. 7: 2965. https://doi.org/10.3390/molecules28072965
APA StylePhucharoenrak, P., Muangnoi, C., & Trachootham, D. (2023). Metabolomic Analysis of Phytochemical Compounds from Ethanolic Extract of Lime (Citrus aurantifolia) Peel and Its Anti-Cancer Effects against Human Hepatocellular Carcinoma Cells. Molecules, 28(7), 2965. https://doi.org/10.3390/molecules28072965