Subcritical Water Extraction,Composition And Activity Evaluation Of Polyphenols From Lotus Seedpod

In this paper,we extracted the lotus seedpod polyphenols?LSPP?by subcritical water?subcritical water extraction,SWE?,and compared with traditional hot water extraction?HWE?technology.The composition of LSPP was analyzed by HPLC-ESI-MSⁿ.Antioxidant activity,antiproliferative ability and storage stability of LSPP were evaluated.The antioxidant activity of LSPP with different degrees of polymerization was investigated.The composition of the low polymerization fraction of LSPP was also analyzed.The dynamic behavior of SWE polyphenols was studied,and the kinetic equation was established,which may provide a theoretical foundation for LSPP SWE process control,and provides theoretical guidance for the resource development and utilization of LSPP.The detailed research contents and results of this paper are as follow:1.Optimization of subcritical water extraction process,activity evaluation and composition analysis of lotus seedpod polyphenols.?1?Based on results of the single factor experiment,the response surface method was used to optimize the SWE process of LSPP.The optimal extraction conditions for LSPP were:extraction temperature of 140°C,extraction time of 20 min,liquid-solid ratio of 70 mL/g,and sodium bisulfite addition of 4‰?W/V?.Under optimal conditions,the yield of LSPP was 178.32mg GAE/g DW,which was significantly higher than HWE?146.15 mg GAE/g DW?.?2?Separation/enrichment of polyphenols by AB-8 macroporous adsorption resin column chromatography,and lyophilization to obtain LSPP powder.The total polyphenols?815.4 mg GAE/g DW?and total flavonoids?1012.05 mg RE/g DW?content of LSPP obtained by SWE were higher than HWE.The in vitro free radicals scavenging and antioxidant activities of LSPP were evaluated by scavenge of DPPH·,ABTS^·+and NO₂^-and reducing power.The results showed that compared with HWE,LSPP?SWE?has better reducing power and free radicals scavenging activity[the scavenging rates of DPPH·,ABTS^·+and NO^2-were 49.9%?7.5?g/mL?,62.4%?50?g/mL?and 52.8%?100?g/mL?,respectively].The results indicate that LSPP has good antioxidant activity and lotus seedpod is a potential resource for natural antioxidants.?3?The composition of LSPP was analyzed by HPLC-ESI-MSⁿ,and the difference in composition of LSPP obtained by two extraction methods was compared.Through detailed HPLC-ESI-MSⁿ information analysis,compared with the literature,and using standard to verify,identified eight polyphenolic compounds?proanthocyanidin dimer 1,proanthocyanidin dimer 2,catechin,cyanidin-3-O-glucoside,quercetin-3-O-glucuronoside,isoquercetin,kaempferol-3-O-glucuronide and isorhamnetin?.Four main compounds were quantified.Compared with HWE,the content of proanthocyanidin dimer 1,proanthocyanidin dimer 2 and catechin in LSPP obtained by SWE is higher,but the content of quercetin-3-O-glucuronoside is lower than that of HWE.This may be the reason of the difference in antioxidant activity of LSPP obtained by two extraction methods.In addition,the effect of LSPP?SWE?on the proliferation of human hepatoma HepG2 cells was also investigated.The results showed that the antiproliferative rate reached 57.21%?P<0.05?when the concentration of LSPP was 200?g/mL,indicating that LSPP has good antiproliferative ability to HepG2 cells.2.Analysis of composition,stability and antioxidant activity of different polymerization components in LSPP.?1?LSPP was fractionated by ethyl acetate to obtain ethyl acetate fraction?EAF?of lower degree of polymerization and water fraction?WF?of higher degree of polymerization,respectively.The antioxidant activity(reducing power,Scavenge of DPPH·and ABTS^·+)of LSPP of two degrees of polymerization was evaluated.The low polymerization component has better antioxidant activity than the high polymerization component.However,the free radical scavenging ability of both polymerization component is lower than the whole component?SWE?,the free radical scavenging ability is from high to low:SWE>EAF>WF,indicating that there is synergistic effect between polyphenols with different polymerization degrees.?2?HPLC-ESI-MSⁿ was used to analyze the composition of low-polymerization of LSPP,and 24 polyphenolic compounds[catechin,epicatechin,?epi?gallocatechin,proanthocyanidin dimer?1,2,3 and 4?,proanthocyanidin trimer?1,2,3 and 4?,?epi?GC-?epi?C?1,2?,?epi?C-?epi?GC?1,2?,?epi?C-?epi?C-?epi?GC?1,2 and 3?,cyanidin-3-O-glucoside,quercetin-3-O-glucuronoside,isoquercetin,kaempferol-3-O-glucuronide,kaempferol-3-O-glucoside and isorhamnetin-3-O-glucoside]were identified,and 22 compounds were quantified.?3?The storage stability of LSPP was initially evaluated.LSPP was stable under low temperature,dark and acidic conditions.The effect of salt on the stability of LSPP was significant.Sucrose and glucose had certain protective effects on LSPP.3.Kinetic study on the extraction of polyphenols by subcritical waterThe kinetics behavior of LSPP extracted by subcritical water were modeled using a"two-site kinetic model".The model is based on Fick's second law of diffusion and is an extension of the“one-site kinetic model”.The model assumes that the matrix particles are uniformly spherical.This model assumes that the solute is initially uniformly distributed in the solid matrix,and that the extraction is not limited by solubility?i.e.the solute never saturates the solvent?.C_t?mg/g?is the mass of analyte extracted per mass of substrate,after time t.C₀?mg/g?is the initial mass of analyte per mass of substrate.At the same extraction time,as the extraction temperature increased,the C_t/C₀ of polyphenols,flavonoids and proanthocyanidins also increased.At the same extraction time,as the extraction temperature increased,the C_t/C₀ of polyphenols,flavonoids and proanthocyanidins also increased.The relative extraction rate of polyphenols,flavonoids and proanthocyanidins reached to a range between 48%⁷3%in the first 5 minutes of the extraction process.During the 5¹9 minutes of the extraction process,the relative extraction rate increased to a range between 85%⁹8%.This indicates that the process of extracting polyphenols,flavonoids and proanthocyanidins by subcritical water is divided into two parts:faster and slower.The two-site kinetic model is a good description of the process of SWE of polyphenols,flavonoids and proanthocyanidins?R²?0.992?.The faster desorption rate constant?k₁?,the slower desorption rate constant?k₂?,faster extracted fraction?F?and the R² values obtained using the two-site kinetic model.According to Fick's second law and Arrhenius equation,the effective diffusion coefficient?D_e?and the reference activation energy?E_a?of the three components were obtained.