Studies On Extraction, Purification And Bioactivities Of Flaxseed Lignan

Flaxseed (Linum usitatissimum L) is a rich source for several kinds of lignans. Secoisolariciresinol (SECO,C20H26O6) is the most abundant among these lignans and exists in flaxseed as a diglucoside (SDG). Researches have been carried out and flaxseed lignan (mainly SDG) was found to possess multiple bioactivities including anticancer, preventing cardiovascular disease, releasing postmenopausal symptoms, preventing osteoporosis and antioxidation. Exploitation of flaxseed lignan could make great contribution to prevent these dietary-dependent diseases and improve people's health. Extraction and purification of flaxseed lignan would add value to flaxseed and make economic benefit. Besides, comprehensive utilization of flaxseed needs deep and systematic research.This paper aims at accurate and fast determination of flaxseed lignan and development of a safe, economic and easy-to-scale-up process to extract and isolate SDG (with purity higher than 95%). Besides, an integrate process for utilization of flaxseed has also been taken into consideration. As to the inhibition effect on breast cancer and its mechanism, in vitro cell culture experiment and chemical calculation are combined to cross validation and to explore its dose-effect and structure-activity relationship.For the determination of SDG, we found combination of basic hydrolysis and solvent extraction would get high SDG yield and the result is more accurate. Acid hydrolysis of purified SDG would cause three main hydrolysates, namely secoisolariciresinol glucoside (SG), SECO and anhydrosecoisolariciresinol (ANHSEC). With lower HCl concentration, SECO and SG were produced mainly and ANHSEC became the main product when acid concentration and heating duration was improved. HPLC-PAD-MS was adopted to determine SDG, SG, SECO and ANHSEC, and NMR was used to identify configuration of SDG. The quantitative method using SDG as standard avoided artifacts caused by acid hydrolysis was considered as simple, reliable and sensible. A colorimetric method was also developed to determine SDG quickly and easily.To integrate recover bioactive components in flaxseed, a wet process was proposed to get rid of the mucilage. Yield and static viscosity of the extract were determined and produced as a comprehensive index, demucilage ratio, to evaluate different levels of each factor. Demucilage conditions were optimized as following: temperature of 70°C, liquid to solid ratio of 7:1, pH 6.0, soaking duration 1 h and soaking times four. Major components of crude flaxseed gum, i.e., polysaccharide and protein were determined as 65.4% and 8.50% respectively. Monosaccharide composition was determined as rhamnose: fucose: arabinose: xylose: glucose: galactose = 5.10: 1.00: 4.18: 8.98: 2.13: 2.75 in mole ratio. High content of xylose means that crude gum contains neutral polysaccharide of high content. Hull and kernel were separated with a wet process. Flaxseed were breaking with a grinding wheel under the conditions that wheel distance 0.40 mm and wheel speed 2800 rpm. Broken flaxseed was fractionated by hydrocyclone for two times. Kernel-rich fraction, hull-rich fraction and mixture fraction were recovered and the yields of them were 52.6%, 22.3% and 12.7% respectively. Kernel content of hull-rich fraction and hull content of kernel-rich fraction were 20.9% and 11.1% respectively. Effect of Demucilage and dehull on SDG was investigated, and it was found little amount of SDG (9.6%) leaked with fine hull, most of the SDG was enriched in hull-rich fraction, mixture fraction has higher content of SDG than kernel-rich fraction.Conditions for stirring extraction of SDG from defatted flaxseed hull flour (DFHF) were optimized using orthogonal experimental design. Under the optimized conditions, i.e., 50% (v/v) aqueous ethanol as solvent, temperature of 60°C, liquid to solid ratio 20 mL/g and stirring duration of 3 h, 1.80 g SDG was obtained from 100 g DFHF. When adding NaOH to solvent with final concentration of 0.25 mol/L, SDG yield was improved to 1.90 g /100 g DFHF. Ultrasound-assisted extraction (UAE) of SDG was investigated and under the conditions that ultrasonic power as 160 W, time duration as 30 min, temperature as 40°C, SDG yield of 2.01 g/100 g DFHF. Central composite design and response surface analysis were carried out to optimize a microwave-assisted extraction (MAE) process for SDG. With ethanol concentration of 40.9 % (v/v), liquid to solid ratio of 21.9: 1 (mL/g), presoaking with ultrasound treatment for 5 min, microwave power of 130 W and irradiation time of 90.5 s, SDG yield of 2.19 g/100 g DFHF was obtained. MAE was superior to stirring extraction and UAE in SDG yield and time-saving. Circular dichroism of SDG with MAE was determined and it was found MAE didn't change its configuration.Partition of crude SDG extract was carried out with 4 kind of solvent, i.e., n-hexane, chloroform, acetic ether and water, SDG was found mostly in water fraction. By Judging UV spectra, SDG was purified on a silica gel column for 2 times. Purity of the eluate was determined by HPLC-PAD-MS as 91.85%, and 92.4% SDG was recovered. Static adsorption and desorption capacity pf several kinds of macroporous adsorption resin (MAR) were determined and X-5 was found to possess a high adsorption and desorption rate as 86.4% and 95.1% respectively. X-5 was used to isolate SDG and dynamic elution characteristics were investigated. Gradient concentration of aqueous ethanol was adopted to separate SDG from the crude extract. SDG was eluted with 20% (v/v) aqueous ethanol and purity was determined with HPLC-PAD-MS as 65.7%. 80.8% SDG was recovered after MAR separation. Sephadex LH-20 column chromatography was used to purify SDG. Elution conditions were investigated and water was found to give the best resolution. After separation on Sephadex LH-20 column for two times, 97.2% SDG was recovered and eluate purity was determined as 96.6%. Effect of different concentration of SECO and its metabolites, enterodiol (END) and enterolactone (ENL) on proliferation of an estrogen receptor (ER) positive cancer cell, MCF-7 was studied. Genistein (GEN) was used as the counter. We found that SECO and GEN could inhibit proliferation of MCF-7 at high concentration (100 and 40μmol/L respectively). However, proliferation of MCF-7 was promoted when 10~40μmol/L SECO or 10~20μmol/L GEN was added to culture media. Two kinds of mammalian lignans, END and ENL showed dose-dependent inhibitory effect on proliferation of MCF-7 in concentration range of 10~100μmol/L. Flow cytometry method was adopted to analyze the changes of MCF-7 cell cycle after treatment with 100μmol/L SECO, 40μmol/L END, 40μmol/L ENL and 40μmol/L GEN. Results showed cell ratio of both G0/G1 and G2/M stages increased while that of S stage decreased. Therefore, SECO, END, ENL and GEN may exert its inhibitory effect by control proliferation of DNA proliferation.The method by competitive binding of irradiative ligand was used to determine relative binding affinity (RBA) of SECO to ERα. It was found that at concentration of 10μmol/L, SECO could not bind to ERα. Docking study of different ligands including SECO, END, ENL with two types of ER, i.e., ERαand ERβwas carried out. Energy change (ΔG) of the system during docking was compared and we found that there was no obvious linear relationship betweenΔG and log (RBA). However, ligand binding domain (LBD) was determined and possible interactions between ligands and ER were analyzed. RBA of ligands to ERαand ERβwere predicted by comparative molecular field analysis (CoMFA). It was found that hydrogen bond plays an important role in the interaction between ligands and both ER. Predictive values of optimized CoMFA model was close to observed values. RBA of END, ENL and SECO to ERαand ERβwere predicted by the CoMFA models, we found that RBA of ENL was much higher than that of SECO for both estrogen receptors. And all three ligands showed higher RBA to ERβthan that to ERα. Therefore, we suppose that in the tissues where ERβwas distributed, SECO may exert anticancer bioactivity by its metabolite ENL.