YILDIRIM A et al. JOTCSA. 2020; 7(2): 545-560. RESEARCH ARTICLE An improved isolation of trimyristin from Myristica fragrans as a renewable feedstock with the assistance of novel cationic gemini surfactant Ayhan YILDIRIM , Serkan ÖZTÜRK , Haluk TÜRKDEMIR , Atakan KOLALI , Beyza Gökçem ATALAY , Hatice KOCATAŞ Bursa Uludağ University, Faculty of Arts and Sciences, Department of Chemistry, 16059, Bursa, Turkey. Abstract: In the present work, a surfactant-assisted convenient extraction method was developed for the isolation of trimyristin from nutmeg. Commercially available monomeric surfactants and novel readily synthesized cationic dimeric surfactants were used as auxiliary chemicals. The improved isolation method herein revealed that the combination of dimeric surfactant with n-hexane allows selective extraction (without colored polar components of nutmeg) and maximum yield of triglyceride. Besides, the developed method is more practical than existing protocols and provides higher yields of trimyristin in a short period. Keywords: Nutmeg, triglycerides, surfactant-assisted extraction, renewable materials. Submitted: August 16, 2019. Accepted: May 18, 2020. Cite this: YILDIRIM A, ÖZTÜRK S, TÜRKDEMİR H, KOLALI A, ATALAY BG, KOCATAŞ H. An improved isolation of trimyristin from Myristica fragrans as a renewable feedstock with the assistance of novel cationic gemini surfactant. JOTCSA. 2020;7(2):545–60. DOI: https://doi.org/ 10.18596/jotcsa. 605805 . *Corresponding author. E-mail: [email protected]. INTRODUCTION known as triacylglycerol (3). Triglycerides are receiving increased interest owing to their potential The necessary chemical raw materials required by as a starting material for the production of biofuel the chemical, pharmaceutical, textile, and food and as a source for valuable renewable raw industries have been provided for a long time from chemicals (4-6). As a result of pyrolysis or thermal crude oil. However, the depletion of oil reserves, and catalytic cracking processes performed with the cost of production and derivatization processes triglycerides, bio-based versatile chemicals can be of different petrochemicals and excessive energy prepared suitable for different industrial fields (7- requirements have led both the academic and the 9). industrial community to the search for new sources of raw materials. It is becoming increasingly Lipids are often found in tissues with popular to find more environmentally friendly, carbohydrates and proteins, which makes their biodegradable, and renewable raw material isolation difficult. Triglycerides and many other resources. Among these feedstock sources, animal natural compounds of the lipid class are generally and vegetable-originated fats and oils are isolated from plant sources like canola and particularly remarkable (1, 2). Vegetable oils are Jatropha curcas by extraction techniques, and biomass that has the potential to replace many supercritical carbon dioxide is the widely used chemicals in oil refineries, and the main solvent along with other supercritical fluids (10- component of this biomass is triglycerides, also 12). The triglyceride ratios and compositions of 545 YILDIRIM A et al. JOTCSA. 2020; 7(2): 545-560. RESEARCH ARTICLE lipid extracts obtained from several vegetable sources show variability. For instance, Myristica fragrans seeds (nutmeg) are rich in myristicin as EXPERIMENTAL SECTION one of the necessary components of its essential oil and trimyristin as a major triglyceride Reagents and chemicals component (13-15). As known, trimyristin is a All reagents and solvents were purchased from neutral lipid component, and non-polar solvents either Merck or Sigma‐Aldrich (St. Louis, MO) and such as ether, n-hexane, and chloroform are used without further purification. Thin‐layer suitable for its extraction. Trimyristin is a chromatography (TLC) was performed using silica yellowish-white solid at room temperature, formed gel plates (60 F254, Merck, Darmstadt, Germany). by esterification of glycerol with myristic acid as a saturated fatty acid. It is a raw material for the Analytical techniques soap and oleochemical industries and widely used Melting points were recorded by BÜCHI melting in the cosmetic industry (16). In the cosmetic point B-540 apparatus (BÜCHI Labortechnik AG in industry, trimyristin can be classified as the Flawil, Switzerland). The Fourier Transformed following: emollient, refatting, skin conditioning, Infrared (FTIR) spectra were measured using a solvent, and viscosity controlling agent. It is used PerkinElmer Spectrum 100 spectrometer. The in makeup products, deodorants, creams and Nuclear Magnetic Resonance (NMR) spectra were lotions, suntan and sunscreen products, hair measured using Bruker Avance NEO 600 MHz NMR conditioners, skincare, and skin cleansing spectrometer (Santa Clara, CA, USA). Chemical products. Therefore, it is crucial to develop shifts (δ) are reported in ppm, and J values in efficient extraction procedures to obtain this Hertz. A Hitachi 3100H dual-beam particular triglyceride with high yields. spectrophotometer (Tokyo, Japan) was used for Unfortunately, it can be grown on an industrial the spectrophotometric analyses. Conductivity scale only in regions with a tropical climate. measurements were performed using a Thermo Scientific ORION 3 STAR digital conductometer Beal reported a convenient ether-based isolation (Madison, WI, USA). The elemental analyses were method with Soxhlet extractor, which requires a performed using a LECO CHNS-932 elemental long time (24 to 72 hrs.), and the yield of the analyzer (Saint Joseph, MI, USA). purified trimyristin was approximately 53% based on the mass of crude nutmeg extract (17). Preparation of the surfactant According to a procedure recommended by Ikan, extraction was carried out with chloroform as a N1,N1,N6,N6-tetramethyl-N1,N6-bis(4-((10-(5- solvent, and after the purification stage, trimyristin (octylthio)-1,3,4-oxadiazol-2- was obtained with a yield of 20% based on the yl)decyl)oxy)benzyl)hexane-1,6-diaminium mass of crushed nutmeg materials (18). Lugemwa chloride used water-organic solvent mixtures for the N,N,N′,N′-Tetramethyl-1,6-hexanediamine (0.15 g, isolation of trimyristin from nutmeg and obtained 0.87 mmol) and two equivalents of 2-(10-(4- the desired triglyceride with a yield of only 8% at (chloromethyl)phenoxy)decyl)-5-(octylthio)-1,3,4- the end of 1 hour of extraction (19). On the other oxadiazole (31) (0.85, 1.72 mmol) are heated in hand, some isolation methods designed as general MeCN at 80 ºC for 24 h. After completion of the chemistry experiments have also been developed reaction, the mixture was cooled, and the excess but with a lower yield of trimyristin based on the of solvent was removed under reduced pressure. mass of crude nutmeg extract (20, 21). The residue is washed successfully with n-hexane. A white waxy product is obtained quantitatively in As is known, surfactants are both hydrophilic and a sufficient purity. lipophilic compounds which make them unique Waxy solid (mp: 35–36 °C); FTIR (ATR): νmax tools in isolation of various natural compounds via 3351, 2919, 2852, 1667, 1612, 1586, 1514, 1484, extraction processes (22-30). At the extraction 1474, 1433, 1367, 1306, 1252, 1183, 1146, 1043, stage, the combination of solvent with surfactant 1018, 983, 958, 928, 865, 825, 795, 742, 718, molecules facilitates the penetration of solvent 682, 617, 555, 522 cm-1; 1H NMR (600 MHz, molecules throughout seed cell walls. To the best CDCl3) δ 7.50-7.47 (m, 4H, Ar), 6.87-6.85 (m, 4H, + of our knowledge, there has been no work Ar), 4.74 (s, 4H, 2 x ArCH2N ), 3.90 (t, J = 6.4 Hz, investigating the surfactant-assisted isolation of 4H, 2 x ArOCH2-), 3.60 (t, J = 7.4 Hz, 4H, 2 x + trimyristin from nutmeg. In this work, we report (CH3)3N CH2CH2-), 3.18 (t, J = 7.4 Hz, 4H, 2 x + the synthesis of a novel cationic gemini surfactant HetSCH2CH2-), 3.15 (s, 12H, 2 x –N (CH3)2), 2.77 and its evaluation in an efficient extraction process (t, J = 7.4 Hz, 4H, 2 x HetCH2CH2-), 2.04-2.01 (m, of trimyristin from nutmeg. 4H, 2 x HetSCH2CH2CH2 -), 1.77-1.70 (m, 12H, 6 x CH2), 1.43-1.32 (m, 16H, 8 x CH2), 1.28-1.23 (m, 32H, 16 x 1.36 CH2), 0.84 (t, J = 7.1 Hz, 6H, 2 x - 13 CH2CH3); C NMR (150 MHz, CDCl3) δ 167.91, 546 YILDIRIM A et al. JOTCSA. 2020; 7(2): 545-560. RESEARCH ARTICLE 164.24, 160.77, 134.57, 119.12, 114.90, 68.13, The Myristica fragrans seeds were ground using a 67.27, 64.17, 49.24, 42.96, 32.47, 31.69, 29.42, porcelain mortar and pestle at ambient 29.31, 29.29, 29.20, 29.11, 29.04, 29.03, 28.92, temperature just before extraction to isolate 28.91, 28.53, 26.36, 25.96, 25.35, 24.96, 22.56, triglycerides more efficiently. From this undried 21.70, 14.03; Anal calc for C64H110Cl2N6O4S2 crushed sample, exactly 1 gram was weighed, (1162.64): C 66.12, H 9.54, N 7.23, S 5.52. placed in a 50 mL single-necked flask, and was Found: C 66.15, H 9.49, N 7.27, S 5.50. extracted at 50 ºC for 20 min with 20 mL of the solvent indicated in the present work. In the case Conductivity measurements of surfactant-assisted extractions, 5 mg of the Conductivity measurements were performed using related surfactant was also added into the flask. a Thermo Scientific ORION 3 STAR digital After that, the obtained mixture was filtered, and conductometer apparatus (Madison, WI, USA). To the filtrate was evaporated in a vacuum. The measure the specific conductivity at each residue was weighed and recrystallized from surfactant, we followed the step-by-step dilution– acetone to afford pure trimyristin as a white solid extraction method. The critical micellar with mp: 55–56 ºC. However, the triglyceride is concentration (cmc) value was estimated from the sufficiently pure and can be used for many breakpoint on the curve of conductivity versus purposes without further purification.