Collection of the plant materials The roots of the plant were collected from Gazipur, Bangladesh and taxonomic identification was made by the Department of Botany, University of Dhaka. The collected roots were cleaned to remove mud and dust particles. The roots were dried at room temperature followed by in an oven below 40°C. A grinder (Cyclotec 200 meshes) was used to grind the dried roots to powder. The root powder was stowed in an airtight bottle and used during the investigations.
Phytochemical screening To identify the phytoconstituents such as tannin, phlabotannins, alkaloid, saponin, flavonoid, steroid, terpenoid, and cardiac glycoside etc. different phytochemical tests were done using standard protocols.
Extraction The powdered roots of A. spinosus (360 g) were extracted with n-hexane followed by ethyl acetate (EtOAc). Using filter paper through a funnel these extracts were filtered separately and the filtrates were evaporated to dryness with a rotary evaporator (Stuart, UK) under reduced pressure temperature of 40oC. Ethyl acetate extract (~3.10 g) was used for phytochemical investigation and n-hexane extract was kept for fatty acid analysis.
Isolation of phytochemicals from EtOAc extract The crude EtOAc extract was exposed to TLC screening and it demonstrated several spots in iodine chamber and vanillin sulfuric acid spray on TLC plate. The dry mass of EtOAc extract (3.10 g) was subjected to column chromatography over column grade silica gel (Kiesel gel 60G). At first the column was eluted with 100% hexane and then eluted with mixtures of hexane with increasing amount of dichloromethane, and finally with increasing methanol. The effluents were collected in 250 mL conical flask where 12 fractions marked as P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, and P12 were obtained according to TLC pattern. Among the fractions P8 was observed as a single spot. So, the fraction P8 was allowed to stand for hours and white crystalline compound was obtained which was marked as 1. The fraction P9 appeared to contain three spots. The fraction P9 was further subjected to sub column for re-fractionation by column chromatography. A total of seven fractions were collected based on their TLC pattern and marked as F1, F2, F3, F4, F5, F6 and F7. Among them, F5 produced another white crystalline compound which was marked as 2. The fraction F6 was observed two spots with distinct Rf value. From F6 a compound was separated and purified by preparative thin layer chromatography (PTLC) and was marked as 3.
Analysis of fatty acids n-hexane extract of A. spinosus was subjected to fatty acid (FA) analysis. Both free fatty acids (FFAs) and bound fatty acids (BFAs) were extracted from the plant and converted into their corresponding methyl ester to make the volatile to be capable of being analyzed by gas liquid chromatography (GC).The prepared methyl ester of FFA and BFA along with standard fatty acids ester samples were analyzed by GLC (Shimadzu 9A, Column-BP-50, Detector-FID, 105oC+5oC/min-150oC+2oC/min-280) and their retention time was recorded. The relative percentages of the FFAs and BFAs were calculated from peak area.
Membrane stabilizing activity 200 g dried powder of A. spinosus root was soaked in 1000 mL of methanol for 7 days with occasional shaking and filtered through a cotton plug followed by Whatman filter paper number 1. The filtrate was dried using a rotary evaporator under reduced pressure evaporator at low temperature. 5 g of the dried extract of A. spinosus was subjected to solvent-solvent partitioning following the modified Kupchan method 15 to yield n-hexane, dichloromethane, chloroform and aqueous soluble fractions. Then the crude methanol extract and its concentrated Kupchan fractions were evaluated for membrane stabilizing activity.