Extraction, Purification And Biological Activities Of Phenylpropanoid Amides Of 5-Hydroxytryptamine From Safflower Seeds

Phenylpropanoid amides of 5-hydroxytryptamine (PAHA) from safflower (carthamus tinctorius L.) seeds have a number of biological effects including free radical scavenging, anti-oxidant, anti-tumor, and UV absorption activities, and thus have attracted gaining attention globally in academia. However, previous researches have been concentrated primarily on the separation, characterization and pharmacological aspects, yet have paid scarce attention to larger scale extraction and purification of PAHA. This work is mainly concerned with N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS), two principal components of PAHA from safflower (carthamus tinctorius L.) seeds. Methods for quantitative determination of PAHA were established, followed consecutively by extraction of crude PAHA with ethanol aqueous solution, bioconversion, purification of PAHA, macroporous resin chromatography and silicon gel chromatography, and the process conditions was established for recovering PAHA at high yields and purity.A reversed phase high performance liquid chromatographic (RP-HPLC) method was established for rapid analysis of PAHA in safflower seed, which shows an average added standard recovery of 99.0% and a constant of variation of <3.2% in a linear range of 50-2000 ng. And a novel spectrophotometric method using p-dimethylaminobenzaldehyde (Ehrlich's reagent), was also developed for the determination of total PAHA in safflower (carthamus tinctorius L.) seeds, which shows an average added standard recovery of 99.5% and a constant of variation of <2.0% in a linear range of 0.05-0.25 mmol/L. Both methods exhibit good accuracy and precision.The process for crude PAHA extraction by 60% aqueous ethanol under reflux was established through orthogonal experiments, which gave a PAHA yield, purity, and extraction ratio of 9.96%, 7.87%, and 0.784%, respectively. The extraction process fitted well to the shrinking unreacted core model, with the pertaining kinetics governed by intra-particle diffusion. When the particle sizes of safflower cake meal is ranged between 35-80 meshes, this model can define the extraction kinetics of PAHA with an error below 20%.Deglycosidation of PAHA extracts from safflower cake meal withβ-D-glucosidase was optimized using response surface design to convert N-(p-coumaroyl)serotonin glucoside (CSG) and N-feruloylserotonin glucoside (FSG) to CS and FS, which gave conversion rates of >90% for both CSG and FSG, and remarkable reduction of impurities such as 2-hydroxyarctrin.A process of XDA-1 macroporous resin column chromatography for purifying PAHA was established. Conditions for both isocratic and stepwise elution were determined, which provided 7.5- and 12.8-fold concentration of PAHA with a recovery of 84.4% and 72.9%, respectively. A silica gel chromatographic process was then developed, using petroleum ether/acetone system as the eluent, to further purify PAHA to a purity and recovery of 90.4% and 87.2%, respectively.The two isoforms of PAHA as obtained were characterized with melting point determination, UV and IR spectroscopy, HPLC/MS, and NMR spectroscopy and, with reference to relevant literature, identified as N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS), both in trans configuration. It was suggested that the chemical structure of CS and FS were little changed in the extraction, purification, and biotransformation procedures.The free radical scavenging activities of FS, CS, CSG, and FSG were evaluated using DPPH·system, Fe²⁺/phen system, and pyrogallol auto-oxidation system, and by Rancimat assay. The free radical scavenging activities exhibited are dose-dependent and follow the order of CS≈FS>FSG>CSG.The cytotoxic effects of four isoforms of PAHA, i.e., FS, CS, CSG, and FSG, on various human malignant tumor cell lines were tested in vitro by MTT assay. FS and CS showed dose-dependent anti-proliferative activities to the lung tumor cell line H446, liver tumor cell line HepG-2, and the melanoma cell line B16, while had little such activities towards the breast tumor cell lines MCF-7 and MB-231.