Study On The Fingerprints Of Polysaccharides From Lotus Root And Their Profile-activity Relationships

As one of the aquatic vegetables,lotus root is the most widely cultivated and has the highest yield among them.The germplasm resources of lotus root are very abundant,and its genetic polymorphism has attracted great attentions.However,the diversity of chemical component in lotus roots has been rarely reported.Lotus root is also a kind of functional food recorded in Chinese Pharmacopeia,and its biological functions have been confirmed with several modern pharmacological methods.Polysaccharides has been identified as one of the bioactive components contributing to certain functions,such as antioxidant,hyperglycemic,hypolipidemic,antiobesity and immunomodulatory activities.So far,the systemic evaluation on the bioactive polysaccharides from lotus root resources has not been carried out.In this work,39 polysaccharides from different parts of lotus root varieties were isolated and analyzed.To reveal the physicochemical diversity of lotus root polysaccharides(LRPs),their physicochemical characteristics were analyzed,followed by the establishment of multi-dimensional fingerprints.Meanwhile,their antioxidant,antitumor and immunomodulatory activities in vitro were evaluated,and the potential relationship between fingerprint and activity were explored.The main contents and results of this work were presented as the followings(1)Optimization of hot-water extraction technology of polysaccharide from lotus root.Based on single factor experiment,three independent variables,including extraction temperature,extraction time and shearing speed,were chosen to establish the quadric regression equation for predicting the extraction yield of polysaccharides from lotus root.The optimum extraction conditions were obtained as follows: extraction temperature 89 ?,extraction time 2 h and shear speed rate 11800 r/min,which led to estimated and observed values of maximal yield of polysaccharides of 5.64% and 5.88%,respectively.In addition,the effects of experimental factors on the extraction yield of LRPs could be ordered as extraction temperature > extraction time > shearing speed.(2)Physicochemical characteristic analyses of LRPs.13 lotus root varieties were collected to isolate polysaccharides from different parts.The yield of polysaccharides from different parts of lotus root could be ordered as flesh > peel > node,but on the contrary,and the protein content in the polysaccharides was flesh < peel < node.39 LRPs showed typical bands of polysaccharide and protein in Fourier Transform Infrared(FTIR)spectra.The measurement of monosaccharide composition by a high performance liquid chromatography(HPLC)method with pre-column PMP derivatization indicated that LRPs were mainly composed of Man,Rib,Rha,GlcUA,GalUA,Glc,Gal and Ara.The measurement of molecular weight distribution by a high performance size exclusion chromatography(HPSEC)method with photodiode array(PDA)detector and refractive index(RI)detector indicated that LRPs mainly contained 5 fractions.Their molecular weights were 18.45,11.57,9.18,5.94 and 5.30 kDa,respectively.Specially,the 18.45 and 9.18 kDa fractions were polysaccharide-protein complexes.(3)Fingerprint establishments of LRPs.The HPLC,FTIR,HPSEC-RI and HPSEC-PDA fingerprints of LRPs were established by chemometric resolution software,followed by the analysis of similarity,two-dimensional clustering,principal component(PCA),partial least squares-discrimination(PLS-DA).Both the HPLC fingerprints and FTIR fingerprints were highly similar,which could be used as models for identifying polysaccharides from lotus root.The HPSEC fingerprints of polysaccharides from different varieties or different parts showed significant differences,and had the potentials to be developed for tracing the source of LRPs.(4)In vitro activity evaluations of LRPs.39 LRPs from three parts of 13 lotus root varieties showed well antioxidant,antitumor and immunomodulatory activities,according to the in vitro activity evaluations.And the differences in antioxidant and antitumor activities among different lotus root varieties and parts were obvious.The DPPH free radical scavenging abilities and ferric reducing antioxidant powers(FRAP)of LRPs from different parts could be ordered as node > peel > flesh,the hydroxyl radical scavenging capacity was flesh > node ? peel,and the antitumor activity was peel ? node > flesh.LRPs could effectively stimulate the NO production and TNF-? secretion of macrophages in the dose range of 100~400 ?g/mL,with relatively small difference among them.(5)Correlation analysis of in vitro activities of LRPs.The DPPH free radical scavenging ability,FRAP total antioxidant capacity and macrophage NO production stimulating activity of LRPs displayed significant correlations(P<0.05).LRPs of node could be effectively distinguished from those of flesh and peel in the scatter plot formed by the PCA of activities.Node had advantage to develop active polysaccha-rides,as its LRPs had relatively higher activities of scavenging DPPH free radical,reducing ferric,inhibiting tumor cell growth and stimulating macrophage NO production.In the 13 lotus root varieties,the overall advantage of ‘E Lian' No.7 on developing active LRP was obvious.(6)Analysis on the profile-activity relationship of LRPs.The relationship between fingerprinting characteristics and activity of LRPs was investigated by multiple linear regression analysis.The content of GalUA in LRPs was in direct proportion to their antioxidant activities,but the content of GlcUA performs the opposite.The high content of Man contributed to strong inhibition on HepG2 cells.Molecular weight was not the main factor related to the activities of LRPs.The content of protein or binding protein was closely related to the antioxidant activity of LRPs,but showed not significant relationship with the immunostimulating activity.This work established the multidimensional fingerprints of LRPs for the discrimination of lotus root variety and the identification of LRPs,based on thestructural complexity of polysaccharide.The bioactive diversity of polysaccharides from lotus root resources were overall evaluated,followed by the analysis on the profile-activity relationship to exploring the physicochemical characteristics related to bioactivities.The results will be useful for the breeding of lotus root and its utilization for high value.