Isolation of Alpha-linolenic acid (ALA) from tasar, Antherea mylitta pupal oil and its anticancer activity
Abstract
Previously, we reported that tasar silkworm pupal oil (TPO) is a rich source of alpha-linolenic acid (ALA) (37%). In this study, we have used three chromatographic techniques, namely preparative HPLC, gas chromatography-mass spectroscopy (GC-MS), and gas chromatography flame ionization detection (GC-FID), to purify, determine, and quantify alpha-linolenic acid (ALA), respectively, from TPO. Additionally, to determine the potential of the purified ALA as an anticancer drug, the sulforhodamine B (SRB) assay was utilised for in vitro investigation. Results revealed that ALA was successfully purified in a fraction of TPO by preparatory HPLC. It was discovered that only ALA was present in the purified fraction of ALA when it underwent GC-MS analysis, further confirming its purity. In addition, the quantification analysis using GC-FID showed that the concentration of ALA was 11760 ppm. Furthermore, anticancer analysis revealed purified ALA significantly inhibited the growth of a colon cancer cell line, (COLO-205) with a GI 50 ≤20. This is the initial investigation into isolating and purifying ALA from Tasar pupal oil.
Keywords: Alpha-linolenic acid; GC-FID; HPLC; Omega-3Preparative, Tasar pupal oil.
Keywords:
Alpha-linolenic acid, GC-FID, HPLC, Omega-3Preparative, Tasar pupal oilDOI
https://doi.org/10.22270/jddt.v13i5.5823References
Gebauer SK, Psota TL, Harris WS, Kris-Etherton PM. n-3 fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits. Am J Clin Nutr., 2006; 83 https://doi.org/10.1093/ajcn/83.6.1526S
Huang W, Guo X, Wang C, Alzhan A, Liu Z, Ma X et al., Xiaofeng et al. α-linolenic acid induces apoptosis, inhibits the invasion and metastasis, and arrests cell cycle in human breast cancer cells by inhibiting fatty acid synthase. J Funct Foods, 2022; 92. https://doi.org/10.1016/j.jff.2022.105041
Srivastava D, Singh V, Kumar U, R. VK. Alpha-linolenic acid: A Pharmacologically Active Ingredient from Nature. Indian J Nutr Diet., 2020; 58: 534-553. https://doi.org/10.21048/IJND.2021.58.4.28086
Pan WJ, Liao AM, Zhang JG, Dong Z, Wei ZJ. Supercritical carbon dioxide extraction of the oak silkworm (Antheraea pernyi) pupal oil: process optimization and composition determination. Int J Mol Sci., 2012; 13:2354-2367. https://doi.org/10.3390/ijms13022354
Thirupathiaiah YV, Sivaprasad E, Bhuvaneswari MM, Reddy V, Kumar M, Chandrashakharaiah. Extraction and characterization of mulberry silkworm, Bombyx mori L., pupae oil. Indian J Seric., 2016;55: 31-37.
Longvah T, Manghtya K, Qadri SSYH. Eri silkworm: a source of edible oil with a high content of α-linolenic acid and of significant nutritional value. J Sci Food Agric., 2012; 92:1988-1993. https://doi.org/10.1002/jsfa.5572
Ramappa VK, Singh V, Srivastava D, Kumar D, Verma A, Verma D et al. Fabrication of mulberry leaf extract (MLE)- and tasar pupal oil (TPO)-loaded silk fibroin (SF) hydrogels and their antimicrobial properties. 3 Biotech, 2023; 13: 37. https://doi.org/10.1007/s13205-022-03443-5
Wang X, Zhang Z-J, Li H-Z, Hou T-Y, Zhao Y-N, Li, He. Urea complexation combined with rapid preparative reversed-phase liquid chromatography to separate α-linolenic acid from perilla seed oil: purity, yield, and oxidation stability. Ind Crops Prod., 2022; 187. https://doi.org/10.1016/j.indcrop.2022.115473
Coskun O. Separation techniques: chromatography. North Clin Istanb., 2016; 11; 3:156-160. https://doi.org/10.14744/nci.2016.32757
Oh CE, Kim GJ, Park SJ, Choi S, Park M, Lee O et al. Purification of high purity docosahexaenoic acid from Schizochytrium sp. SH103 using preparative-scale HPLC. Appl Biol Chem., 2020; 63:56. https://doi.org/10.1186/s13765-020-00542-w
Kim GJ, Son HJ, Whang WS, Koo YM, Kim JI, Yang JH. High-purity purification method for omega-3 highly unsaturated fatty acids. 2011;U.S Patent No. 20110091947A1.
Vidhathri BS, Parama VRR, Subbarayappa CT, Narayanaswamy TK, Muthuraju R, Mahesh DS et al. Isolation and detection of alpha linolenic acid from silkworm pupal residue oil (Bombyx mori L.) using HPLC. Int J Curr Microbiol App Sci., 2017; 6:2202-2206. https://doi.org/10.20546/ijcmas.2017.607.258
Latif Z, Sarker SD. Isolation of natural products by preparative high performance liquid chromatography (prep-HPLC). Methods Mol Biol., 2012; 864: 255-274. https://doi.org/10.1007/978-1-61779-624-1_10
Chahardoli A, Jalilian F, Memariani Z, Farzaei MH, Shokoohinia Y. Chapter 26. Analysis of organic acids. In: Sanches Silva A, Nabavi SF, Saeedi M, Nabavi SM, editors. Recent advances in natural products analysis. Elsevier; 2020; 767-823. https://doi.org/10.1016/B978-0-12-816455-6.00026-3
Bindu MRLH, Angala Parameswari S, Gopinath C. A review on GC-MS and method development and validation. Int J Pharm Qual Assur., 2013; 4:42-51.
Zhang H, Wang Z, Liu O. Development and validation of a GC-FID method for quantitative analysis of oleic acid and related fatty acids. J Pharm Anal., 2015; 5:223-230. https://doi.org/10.1016/j.jpha.2015.01.005
Tuama AA, Mohammed AA. Phytochemical screening and in vitro antibacterial and anticancer activities of the aqueous extract of Cucumis sativus. Saudi J Biol Sci., 2019; 26(3):600-604. https://doi.org/10.1016/j.sjbs.2018.07.012
Deshpande R, Mansara P, Suryavanshi S, Kaul-Ghanekar R. Alpha-linolenic acid regulates the growth of breast and cervical cancer cell lines through regulation of NO release and induction of lipid peroxidation. J Mol Biochem., 2013; 2:6-17.
Yuan Q, Xie F, Huang W, Hu M, Yan Q, Chen Z et al. The review of alpha-linolenic acid: sources, metabolism, and pharmacology. Phytother Res., 2022; 36:164-188. https://doi.org/10.1002/ptr.7295
Roy S, Rawat AK, Sammi SR, Devi U, Singh M, Gautam S et al. Alpha-linolenic acid stabilizes HIF-1 and downregulates FASN to promote mitochondrial apoptosis for mammary gland chemoprevention. Oncotarget. 2017; 8:70049-70071. https://doi.org/10.18632/oncotarget.19551
Bligh EG, Dyer WJA. A Rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959; 37:911-917. https://doi.org/10.1139/y59-099
Vichai V, and Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc., 2006; 1:1112-1116. https://doi.org/10.1038/nprot.2006.179
Kode J, Kovvuri J, Nagaraju B, Jadhav S, Barkume M, Sen S et al. Synthesis, biological evaluation, and molecular docking analysis of phenstatin based indole linked chalcones as anticancer agents and tubulin polymerization inhibitors. Bioorg Chem., 2020; 105:104447. https://doi.org/10.1016/j.bioorg.2020.104447
Fisk HL, West AL, Childs CE, Burdge GC, Calder PC. The use of gas chromatography to analyze compositional changes of fatty acids in rat liver tissue during pregnancy. J Vis Exp., 2014; 13(85):51445. https://doi.org/10.3791/51445
Alinafiah MS, Azlan A, Ismail A, Mahmud Ab Rashid NK. Method Development and Validation for Omega-3 Fatty Acids (DHA and EPA) in Fish Using Gas Chromatography with Flame Ionization Detection (GC-FID). Molecules, 2021; 30:26(21):6592. https://doi.org/10.3390/molecules26216592
Zhang H, Wang Z, Liu O. Development and validation of a GC-FID method for quantitative analysis of oleic acid and related fatty acids. J Pharm Anal., 2015; 5:223-230. https://doi.org/10.1016/j.jpha.2015.01.005
Jarukas L, Kuraite G, Baranauskaite J, Marksa M, Bezruk I, Ivanauskas L. Optimization and validation of the GC/FID method for the quantification of fatty acids in bee products. Appl Sci., 2021; 11:83. https://doi.org/10.3390/app11010083
Kumar GS, Das UN. Cytotoxic action of alpha-linolenic and eicosapentaenoic acids on myeloma cells in vitro. Prostag, Leukot Essen fatty acids, 1997; 56:285-293. https://doi.org/10.1016/S0952-3278(97)90572-X
Dai J, Shen J, Pan W, Shen S, Das UN. Effects of polyunsaturated fatty acids on the growth of gastric cancer cells in vitro. Lipids Health Dis., 2013; 12:71. https://doi.org/10.1186/1476-511X-12-71
Chamberland JP, Moon HS. Down-regulation of malignant potential by alpha linolenic acid in human and mouse colon cancer cells. Fam Cancer., 2015; 14:25-30. https://doi.org/10.1007/s10689-014-9762-z
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