Bioactivity-guided isolation of alkamides from a cytotoxic fraction of the ethyl acetate extract of Anacyclus pyrethrum (L.) DC. roots.

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Introduction. The alcohol extract of Pellitory (Anacyclus pyrethrum) roots has been previously shown to exert anticancer activities on the Human Colorectal Cancer Cell Line (HCT) by targeting apoptosis, metastasis and cell cycle arrest. However, the nature of the cytotoxic molecules associated with this activity remains unexplored.

Aims. This study aims to reinvestigate Pellitory root extract as regard to its cytotoxic activity and to proceed to a bioguided fractionation to explore its active fraction and to give new insight in their phytochemical constituents.

Methods. Powdered roots were subjected to repeated extraction with Petroleum ether (Pe), Chloroform (Ch), Ethyl acetate (Ea) and Methanol (Me). Pellitory extracts were then screened for cytotoxic activity using the Brine Shrimp Lethality (BSL) bioassay.

Results. Ea extract exhibited a marked cytotoxic activity, with LC50 of 249.26 μg/mL in the BSL bioassay. The remaining extracts (Pe,Ch,Me) treated groups exhibited no or low mortality in the range of tested concentrations (1-1000 µg/mL). BSL assay-guided chromatographic fractionation of Ea active Extract revealed a highly cytotoxic fraction (F11) with LC50 of 42.5 µg/mL. Multistep purifications of the active F11 fraction afforded four alkamides, namely N-isobutyldeca-2,4-dienamide or Pellitorine (I), N-propyldodeca- -2,8-dienamide (II), N-isobutyltetradeca-2,4-dienamide (III) and N-propylnona-2,5- -dienamide (IV).

Conclusions. This study suggests that cytotoxic activity is localized mainly in the ethyl acetate extract (Ea) of pellitory roots. BSL assay fractionation of this active extract leads to the isolation of four alkamides, including pellitorine (I). While this isobutyl alkamide has previously shown strong cytotoxic activities against human cancer cell lines, the other compounds (II to IV) were not previously reported as cytotoxic. Subsequently, the isolated alkamides will be considered in future study as candidates for in depth in-vitro evaluation of their cytotoxicity against cancer and normal cell lines. Finally, through this study, BSL assay demonstrate again its usefulness as bench-top assay in exploring plant extracts for cytotoxic compounds.

1. Kishor KV, Lalitha GK. Pharmacognostical studies on the root of Anacyclus pyrethrum DC. Indian Journal of Natural Products and Resources. 2012;3:518-26.

2. Puri HS. Rasayana – Ayurvedic herbs for longevity & rejuvenation. Vol. 2. London: Taylor & Francis; 2003:20-1.

3. Sharma V, Thakur M, Chauhan NS, Dixit VK. Effects of petroleum ether extract of Anacyclus pyrethrum DC. on sexual behavior in male rats. Journal of Chinese Integrative Medicine. 2010;8(8):767-72.

4. Pahuja M, Mehla J, Reeta KH, Joshi S, Gupta YK. Root extract of Anacyclus pyrethrum ameliorates seizures, seizure-induced oxidative stress and cognitive impairment in experimental animals. Epilepsy Research. 2012;98(2-3):157-65.

5. Pahuja M, Mehla J, Reeta KH, Tripathi M, Gupta YK. Effect of Anacyclus pyrethrum on pentylenetetrazole-induced kindling, spatial memory oxidative stress and rho- kinase II expression in mice. Neurochem Res. 2013;38(3):547-56.

6. Veryser L, Taevernier L, Roche N, Peremans K, Burvenich C, De Spiegeleer B. Quantitative transdermal behavior of pellitorine from Anacyclus pyrethrum extract. Phytomedecine. 2014;21(14): 1801-07.

7. Usmani A, Khushtar M, Arif M, Siddiqui MA, Sing SP, Mujahid M. Pharmacognostic and phytopharmacology study of Anacyclus pyrethrum: an insight. Journal of Applied Pharmaceutical Science. 2016;6(3):144-50.

8. Singh A. Compendia Of World’s Medicinal Flora. Boca Raton: CRC Press; 2006: 58-9.

9. Bellakhdar J. La pharmacopée marocaine traditionnelle: Médecine arabe ancienne et savoirs populaires. Paris: Ibis Press; 1997.

10. Boulos L. Medicinal plants of North Africa. Michigan: Reference Publications, Inc; 1983.

11. IUCN. A guide to medicinal plants in North Africa. Malaga: Centre for Mediterranean Cooperation; 2005.

12. Sujiet K, Darwin CR, Suba V. Antioxidant activity of ethanolic root extract of Anacyclus pyrethrum. International Research Journal of Pharmacy. 2011;2(12):222-6.

13. Singh DK, Nirwan S, Babbar SB. Micropropagation of Anacyclus pyrethrum and chemical profiling of the regenerated plants for pellitorine, the active principle. Plant Cell Tiss Organ Cult. 2015; 122(1):249-55.

14. Boonen J, Sharma V, Dixit VK, Burvenich C, De Spiegeleer B. LC-MS N-alkylamide profiling of an etanolic Anacyclus pyrethrum root extract. Planta Med. 2012;78:1784-95.

15. Hinz B, Woelkart K, Bauer R. Alkamides from Echinacea inhibit cyclooxygenase-2 activity in human neuroglioma cells. Biochemical and Biophysical Research Communications. 2007;360(2): 441-6.

16. Shahraki MR, Shahraki S, Arab MR, Shahrakipour M. The Effects of Aqueous Extract of Anacyclus pyrethrum on sperm count and reproductive organs in adult male rats. Zahedan Journal of Research in Medical Sciences. 2015;17(2):42-6.

17. Mohammadi A, Mansoori B, Chaghakaboodi K, Baradaran B. Cytotoxic effects of Anacyclus pyrethrum plant extract in oral cancer cell (KB cell line). J Urmia Univ Med Sci. 2016;27(4): 257-65.

18. Mohammadi A, Mansoori B, Baradaran PC, Baradaran SC, Baradaran B. Anacyclus Pyrethrum extract exerts anticancer activities on the human colorectal cancer cell line (HCT) by targeting apoptosis, metastasis and cell cycle arrest. J Gastrointest Cancer. 2017;48(4):333-40.

19. McLaughlin JL. Assays for bioactivity. In: Hostettmann K (Ed), Methods in Plant Biochemistry, Vol.6, London: Academic Press; 1991:1-33.

20. Wagner H, Bladt S. Plant drug analysis: a thin layer chromatography atlas. 2 edition. Berlin: Springer-Verlag; 2011.

21. Harborne JB. Phytochemical methods. A guide to modern techniques of plant analysis. London: Chapman and Hall ltd; 1984.

22. Bidau CJ, Amat AG, Yajia ME, Marti DA, Gimenez MD, Riglos AG and al. Evaluation of cytotoxic and mitodepressive activity of aqueous extracts from thirteen Argentine medicinal plants. Acta Farm. Bonaerense. 2006;25(4):555-9.

23. Lagnika L, Anago E, Sanni A. Screening for antibacterial, antioxidant activity and toxicity of some medicinal plants used in Benin folkloric medicine. Journal of Medicinal Plants Research. 2011;5(5):773-7.

24. Syahmi AR, Vijayarathna S, Sasidharan S, Latha LY, Kwan YP, Lau YL and al. Acute oral toxicity and brine shrimp lethality of Elaeis guineensis Jacq., (oil palm leaf) methanol extract. Molecules. 2010; 15(11):8111-21.

25. Salawu KM, Ajaiyeoba EO, Ogbole OO, Adeniji JA, Faleye TC, Agunu A. Antioxidant, brine shrimp lethality and antiproliferative properties of gel and leaf extracts of Aloe schweinfurthii and Aloe vera. Journal of Herbs, Spices & Medicinal Plants. 2017;23(4):1-10.

26. Pisutthanana S, Plianbangchang P, Pisutthanana N, Ruanruaya S, Muanrita O. Brine shrimp lethality activity of Thai medicinal plants in the family Meliaceae. Naresuan University Journal. 2004; 12(2):13-8.

27. Sahgal G, Ramanathan S, Sasidharan S, Mordi MN, Ismail S, Mansor SM. Brine shrimp lethality and acute oral toxicity studies on Swietenia mahagoni (Linn.) Jacq. seed methanolic extract. Pharmacognosy Research. 2010;2(4):215-20.

28. Hartl M, Humpf HU. Toxicity assessment of fumonisins using the brine shrimp (Artemia salina) bioassay. Food and Chemical Toxicology. 2000;38(12):1097-102.

29. Hamidi MR, Jovanova B, Panovska TK. Toxicological evaluation of the plant products using brine shrimp (Artemia salina L.) model. Macedonian Pharmaceutical Bulletin. 2014;60(1):9-18.

30. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica. 1982;45(5):31-4.

31. Oladimeji HO, Nia R, Essien EE. In vitro antimicrobial and brine shrimp lethality potential of the leaves and stem of Calotropis procera (Ait). African Journal of Biomedical Research. 2006;9:205-11.

32. Rosario SL, Da Silva AJ, Parente JP. Alkamides from Cissampelos glaberrima. Planta Med. 1996;62(4):376-7.

33. Rios-Chavez P, Ramirez-Chavez E, Armenta-Salinas C, Torres-Molina J. Acmella radicans var. radicans: In vitro culture establishment and alkamide content. In vitro Cellular & Developmental Biology-Plant. 2003;39:37-41.

34. Wu S, Sun C, Pei S, Lu Y, Pan Y. Preparative isolation and purification of amides from the fruits of Piper Longum L. by upright counter – current chromatography and reversed – phase liquid chromatography. Journal of Chromatography. 2004;1040(2):193-204.

35. Olah Z, Redei D, Pecze L, Vizler C, Josvay K, Forgo P and al. Pellitorine, an extract of Tetradium daniellii, is an antagonist of the ion channel TRPV1. Phytomedicine. 2017;34: 44-9.

36. Althaus JB, Kaiser M, Brun R, Schmidt TJ. Antiprotozoal activity of Achillea ptarmica (Asteraceae) and its main alkamide constituents. Molecules. 2014;19(5): 6428-38.

37. Cheng YB, Liu RH, Ho MC, Wu TY, Chen CY, Lo IW and al. Alkamides of Acmella oleracea. Molecules. 2015;20:6970-7.

38. Silveira N, Saar J, Santos AD, Barison A, Sandjo LP, Kaiser M and al. A new alkamide with an Endoperoxide structure from Acmella ciliate (Asteraceae) and its in vitro antiplasmodial activity. Molecules. 2016;21(6):765-70.

39. Elazzouzi H, Khennouchi S, Bentayeb A, Elhilali F, Zair T. Effets biocides des alcaloides extraits des raciness d’Anacyclus pyrethrum L. (Astéracées) sur Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae). International Journal of Innovation and Applied Studies. 2015;13(4):756-74.

40. Chen Y, Fu T, Tao T, Yang J, Chang Y, Wang M and al. Macrophage activating effects of new alkamides from the roots of Echinacea species. J. Nat. Prod. 2005;68(5):773-6.

41. Boonen J, Bronselaer A, Nielandt J, Veryser L, De Tré G, De Spiegeleer B. Alkamid database: Chemistry, occurrence and functionality of plant N-alkylamides. J Ethnopharmacol. 2012;142 (3):563-90.

42. Rios MY. Natural Alkamides: Pharmacology, Chemistry and Distribution. In: Drug Discovery Research in Pharmacognosy, Omboon Vallisuta and Suleiman M. Olimat (edts). China: InTech; 2012:107-44.

43. Ee GC, Lim CM, Rahmani M, Shaari K, Bong CF. Pellitorine, a potential anti-cancer lead compound against HL60 and MCT-7 cell lines and microbial transformation of piperine from Piper Nigrum. Molecules. 2010;15(4):2398-404.

44. Bezerra DP, Pessoa C, de Moraes MO, Saker-Neto N, Silveira ER, Costa-Lotufo LV. Overview of the therapeutic potential of piplartine (piperlongumine). European Journal of Pharmaceutical Sciences. 2013;48(3):453-63.

45. Meegan MJ, Nathwani S, Twamley B, Zisterer DM, O’Boyle NM. Piperlongumine (Piplartine) and Analogues: Antiproliferative Microtubule-Destabilising Agents. European Journal of Medicinal Chemistry. 2017;125:453-63.

46. Luo XJ, Peng J, Li YJ. Recent advances in the study on capsaicinoids and capsinoids. European Journal of Pharmacology 2011;650:1-7.

47. Kim KH, Choi SU, Son MW, Lee KR. Two new phenolic amides from the seeds of pharbitis nil. Chemical and Pharmaceutical Bulletin. 2010;58:1532-5.

48. Carballo JL, Hernández-Inda ZL, Pérez P, García-Grávalos MD. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BMC Biotechnol. 2002;2:17-21.

49. Harwig J, Scott PM. Brine shrimp (Artemia salina L.) larvae as a screening system for fungal toxins. Appl Microbiol. 1971;21(6): 1011-6.

50. Mclaughlin JL, Rogers LL. The use of biological assays to evaluate botanicals. Drug Information Journal. 1988;32:513-24.

51. Anderson JE, Goetz CM., Mclaughlin JL, Suffness M. A blind comparison of simple benchtop bioassay and human tumour cell cytotoxicities as antitumor prescreens. Phytochem Analysis. 1991;2:107-11.

52. Solís PN, Wright CW, Anderson MM, Gupta MP, Phillipson JD. A microwell cytotoxicity assay using Artemia salina. Plant Med. 1993;59:250-2.

53. Mackeen MM, Ali AM, Lajis NH, Kawazu K, Hassan Z, Amran M and al. Antimicrobial, antioxidant, antitumour-promoting and cytotoxic activities of different plant part extracts of Garcinia atroviridis Griff. Ex T. Anders. J Ethnopharmacol. 2000;72:395-402.

54. Zhao G, Hui Y, Rupprecht JK, McLaughlin JL, Wood KV. Additional bioactive compounds and trilobacin, a novel highly cytotoxic acetogenin, from the bark of Asimina triloba. Journal of Natural Products. 1992;55:347-56.

55. Rieser MJ, Gu ZM, Fang XP, Zeng L, Wood KV, McLaughlin JL. Five novel mono-tetrahydrofuran ring acetogenins from the seeds of Annona muricata. Journal of Natural Products. 1996;59: 100-8.

56. Mongelli E, Pomilio AB, Sanchez JB, Guerra FM, Massanet GM. ent-Kaur-16-en-19-oic acid, a KB cells cytotoxic diterpenoid from Elaeoselinum foetidum. Phytotherapy Research. 2002;16:387-8

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