4.3. Chromatographic purification of organic extract of T. conoides The solvent extract of T. conoides (30 g) was fractionated by sequential chromatographic procedures. The crude was mixed with the silica gel (60–120 mesh, 3.0 g) and loaded into a column of glass (0.1 m × 40 mm) with silica gel (60–120 mesh, 50 g). The elution was initiated with less polar solvent (n -hexane), followed by an increase of polarity (EtOAc to MeOH) to collect twenty-five fractions (10 mL/fraction), which were combined to five (TC 1 through TC 5) (Table S1) based upon TLC (8:2 v/v, n -hexane:EtOAc) and RP C 18 -HPLC (MeOH/acetonitrile MeCN, 3:2 v/v) analysis, and those fractions (TC 1 -TC 5) were assessed for their bioactivities. The fractions possessing potential bioactive properties were chosen for further purification. The fractions TC 3 (EtOAc: n - hexane 2:3 v/v; 4.65 g, 15.5% yield) and TC 4 (EtOAc: n -hexane 7:3 v/v; 4.26 g, 14.2% yield) displayed greater attenuation potential against COX-2 and 5-LOX enzymes. The radical scavenging potential of TC 3 and TC 4 against 1,1-diphenyl-2-picryl-hydrazil (DPPH) and 2, 2 ′ -azino-bis-3- ethylbenzothiozoline-6-sulfonic acid (ABTS +) were found to be greater compared to others (Table S1), and therefore, were selected for downstream purification. Likewise, the sub-fraction TC 3 was subjected to fractionation on a silica gel (230–400 mesh) loaded glass column (450 mm × 30 mm). Step-wise gradient elution with n -hexane-EtOAc (9:1 to 3:2, v/v) yielded twenty sub-fractions (20 mL/fraction), which were combined to four (TC 3-1 -TC 3-4) after TLC (n -hexane-EtOAc, 4:1 v/v) and RP-C 18 HPLC (MeOH–MeCN, 3:2 v/v) experiments. The fraction TC 3-4 (1.19 g, 3.96% yield) eluted at n -hexane-EtOAc (6:4, v/v) displayed potential bioactivities (Table S1). Therefore, TC 3-4 was further subfractionated on fine silica gel (SP 1 –B 1A, 230–400 mesh, 12 g) loaded into a column connected to the flash chromatography system (SP 1 –B 1A, Biotage, Sweden) by increasing gradient of n -hexane/EtOAc/MeOH to acquire twelve sub-fractions (15 mL/fraction). The latter fractions were combined to three (TC 3-4-1 -TC 3-4-3) after TLC (n -hexane-EtOAc, 8:2 v/v) and RP-C 18 HPLC (MeOH/MeCN, 3:2 v/v). TC 3-4-1 (0.768 g, 2.56% yield) was purified on silica gel GF 254 by n -hexane: EtOAc (25:1, v/v) solvent system using the preparatory TLC (PTLC) system to yield conoidecyclic A (85 mg, 0.28%, based on dry weight) and conoidecyclic B (73 mg, 0.24%, based on dry weight), and their homogeneity was confirmed by TLC (EtOAc/ n -hexane, 1:9 v/v) and RP-C 18 HPLC (MeOH/ MeCN, 3:2 v/v). The fraction TC 4 was subjected to flash chromatography on a silica gel column (230–400 mesh) with a gradient elution of n -hexane/EtOAc/MeOH to yield sixteen fractions (15 mL/fraction), which were combined into three (TC 4-1 -TC 4-3) after TLC (EtOAc- n -hexane, 1:4 v/v). TC 4-2 (1.15 g, 3.83% yield) displayed greater bioactive potential than other column sub-fractions (Table S1), and flash chromatographic separation using a gradient elution of n -hexane/EtOAc yielded eleven fractions (13 mL/fraction), which were combined into three (TC 4-2-1 -TC 4-2-3). The bioactive fraction TC 4-2-3 (0.442 g, 1.47% yield) was a mixture, and was further purified by PTLC (EtOAc: n -hexane, 1:26, v/v) to obtain conoidecyclic C (65 mg, 0.22%, based on dry weight). The homogeneity of the latter was confirmed by RP-C 18 HPLC (MeOH–MeCN 3:2, v/v) and TLC (EtOAc/ n -hexane 1:4, v/v).

Published as part of Chakraborty, Kajal & Dhara, Shubhajit, 2021, Conoidecyclics A-C from marine macroalga Turbinaria conoides: Newly described natural macrolides with prospective bioactive properties, pp. 1-14 in Phytochemistry (112909) (112909) 191 on page 10, DOI: 10.1016/j.phytochem.2021.112909, http://zenodo.org/record/8258118