Extraction,separation And Purification,structural Characterization And Bioactivity Of Effective Components From Gardenia Jasminoides Ellis

Gardenia（Gardenia jasminoides Ellis）is a natural product that can be both edible and medicinal.It contains many chemical components with research and application value.Its effective components,geniposide and gardenia yellow pigment（GYP）are the main research objects for the deep development of gardenia.Polysaccharides are commonly found in plants and have a wide range of bioactivity.However,it is currently researched lacking on gardenia polysaccharide.To learn more about gardenia and make it better developed and utilized,this paper took gardenia as the raw material to investigate the preparation process of geniposide,GYP,and polysaccharides.Then,the physical and chemical analysis and structural identification were performed for the obtained components.Moreover,the bioactivities of gardenia polysaccharides were investigated.The specific research methods and results are as follows:1.Extraction and purification of geniposide and GYP.（1）The geniposide and GYP were extracted by ultrasonic extraction from the gardenia.The extraction process were optimized by a single factor experiment and an orthogonal experiment design.The optimal extraction conditions as shown below:the ratio of raw materials to 70%（v/v）ethanol-aqueous solution is 1∶10（g∶ml）,and it was extracted at 40℃for 40 min.（2）The extracting solution contained geniposide and GYP were separated and purified using macroporous adsorption resins.The GYP was refined by a HPD722 resin with the process parameters as follows:loading the extracting solution with a concentration of 15 mg/ml onto the resin at a flow rate of 2 ml/min.Then distilled water,20%ethanol,and 80%ethanol were successively used for elution at a flow rate of 2.5ml/min.The geniposide was purified by a H103 resin with the process parameters as follows:loading the geniposide rich solution（0.078 mg/ml）onto the resin at a flow rate of 2 ml/min,then distilled water and 20%ethanol were successively used for elution at a flow rate of 2.5 ml/min.2.The separated and purified geniposide and GYP were identified by ultraviolet-visible spectrophotometry（UV-VIS）,thin layer chromatography（TLC）,high performance liquid chromatography（HPLC）,and fourier transform infrared（FTIR）spectroscopy.The results showed that the color value of the GYP was 489.44 and the OD value was 0.16（<0.4）;the geniposide and GYP were both single compounds with purities of 85.4%and 74.0%,respectively.3.Extraction and purification of gardenia polysaccharides.（1）The polysaccharides were extracted by ultrasonic extraction from the dried gardenia dregs.An extraction yield of the crude polysaccharides was used as the assessment index.The extraction process were optimized by a single factor experiment and an orthogonal experiment design.The optimal extraction conditions as shown below:the ratio of raw materials to water is 1∶20（g∶ml）,and it was extracted once for 50 min at50℃.The yield of crude polysaccharides under this extraction condition is 11.5%.（2）The crude polysaccharides prepared by the optimal extraction process were subjected to protein removal,decolorization,and dialysis.The resulting polysaccharides were labeled as GP.Thereafter,the GP was separated by DEAE-sepharose FF ion-exchange chromatography and were further purified and enriched using Chromdex200 PG gel-filtration chromatography.The purified polysaccharides were collected and labeled as GP2a.The total carbohydrate content of GP2a was 27.1%,and the uronic acid content of it was 97.0%,indicating that GP2a is an acidic polysaccharide.4.Structural characterization of GP2a（1）The weight-average molecular weight（Mw）and the number-average molecular weight（Mn）of GP2a were 44.8 and 20.8 k Da,respectively,as determined by size exclusion chromatography（SEC）connected with multi-angle laser light scattering（MALLS）detector and refractive index（RI）detector.The PDI of GP2a is 2.2（Mw/Mn）,indicating that GP2a is a polymer with moderate dispersion.Additionaly,the molecular conformation plot of root mean square（RMS）radius versus molar mass（Mw）yields a line whose slope is 0.18,displaying that GP2a is a compact and curly spherical molecule in Na NO₃aqueous solution.（2）The FTIR spectrum showed that GP2a had acidic groups,indicating that GP2a was an acidic polysaccharide.Also,it could be inferred the presence of anα-glycosidic linkage in GP2a,and the type of sugar ring is mainly pyranose,which may contain part of furanose.（3）The monosaccharide composition of GP2a was determined by ion chromatography（IC）using trifluoroacetic acid（TFA）method.The results showed that GP2a was mainly composed of galacturonic acid（Gal A,63.1%）.Other small amounts of arabinose（Ara,11.1%）,galactose（Gal,10.9%）,glucose（Glc,7.4%）,rhamnose（Rha,2.7%）,mannose（Man,2.2%）,glucuronic acid（Glc A,1.2%）,xylose（Xyl,0.8%）,and fucose（Fuc,0.2%）were found.（4）The bond structure of GP2a was analyzed by gas chromatography–mass spectrometry（GC–MS）with methylation method.The results showed that the main sugar residues of GP2a were 4-Galp A（74.8%）,and it also contains a small percentage of t-Araf,t-Galp,t-Galp A,4-Glcp,5-Araf,3-Galp,3,6-Galp,2,4-Glcp A,and 4,6-Galp A,as well as tiny 3,4-Galp and 3,4-Galp A.（5）Scanning electron microscope（SEM）was used to perform microscopic imaging of GP2a.It was observed that the GP2a ultrastructure was piled together in a lamellar or clastic form,with circular pores of different sizes on the smooth surface,and annular protrusions around the pores,indicating that the intermolecular force of GP2a might be weak.（6）Nuclear magnetic resonance（NMR）spectroscopy was performed to obtain more detailed structural information of GP2a.It indicated that GP2a is possibly a pectic polysaccharide whose backbone contains the homogalacturonan（HG）region（consists of→4）-α-D-Galp A-（1→）and rhamnogalacturonide（RG）-I region（consists of→4）-α-D-Galp A-（1→and→2）-α-L-Rhap-（1→alternating linkage）,with galactan and arabinans linked to the C-4 position of→2)-α-L-Rhap-(1→residue as branched chains.In addition,the HG structure of GP2a were determined with a degree of methyl esterification（DM）of around 60%,and it also contains a small amount of acetylation substitution at the O-2 and O-3 positions.5.Antioxidant activity and immunoregulatory activity of GP2a（1）The scavenging ability of GP2a against DPPH free radicals（DPPH·）and ABTS free radicals（ABTS+·）was determined in vitro to evaluate its antioxidant activity.The results indicated that GP2a has good antioxidant activity in vitro,which shows a scavenging ability comparable to the ascorbic acid（VC）for DPPH·and ABTS+·at the concentration of 5.0 mg/ml.And the median effective concentration（EC50）for DPPH·and ABTS+·were 0.5 mg/ml and 1.4 mg/ml,respectively.（2）The effect of GP2a on the viability of macrophages RAW 264.7 cultured in vitro was detected by cell counting kit 8（CCK8）.The effect of GP2a on the cytophagocytosis was analyzed by neutral red phagocytosis experiment.The effect of GP2a on the production and release of nitric oxide（NO）and the effect of GP2a on the secretion level of cytokines were determined by enzyme-linked immunosorbent assay（ELISA）.GP2a showed no obvious toxicity to macrophages RAW 264.7 while enhancing cell viability in a dose-and time-dependent manner.Furthermore,GP2a concentration of 120～300μg/ml could significantly improve the phagocytic activity of macrophages RAW 264.7,promote the production and release of NO,and increase the secretion level of cytokines.