Study On The Extraction, Isolation, Purification, Structure Characterization And Immunomodulatory Activity Of Inulin From Dahlia Pinnata Cav.

Dahlia pinnata Cav.is a plant of the Dahlia genus in the Asteraceae family,also known as Dahlia in English,and originated in Mexico.In the early 20th century,Dahlias were introduced to China as ornamental plants,and now there are more than700 varieties.In addition to being used as ornamental plants,the petals and tubers of Dahlias can also be used as food.The petals can be eaten raw and used as an ingredient in salads,while the tubers contain a sweet substance（dacopa）that can be used to make beverages and added to butter or cheese as a seasoning.The tubers also contain a large amount of inulin,a functional food ingredient that has been widely used in the development of new foods.The tuberous roots of Dahlia also have certain medicinal value,with research focusing on its polysaccharide,inulin.Inulin is a natural,edible functional polysaccharide and a soluble dietary fiber,and therefore has various physiological functions such as promoting the proliferation of probiotics in the intestinal tract,stabilizing intestinal flora,preventing intestinal infections,promoting the absorption of nutrients such as proteins,vitamins,and minerals,regulating blood sugar and blood lipid levels,as well as exhibiting anti-cancer,antioxidant,anti-obesity,and laxative activities.In addition,it can greatly help improve human health and enhance immunity and is commonly used in functional foods,drugs,and health products.Therefore,further in-depth research on the chemical structure and pharmacological activity of Dahlia inulin will help develop new functional foods and drugs,and provide better protection for human health.This article extracts,separates,purifies,and structurally characterizes inulin from the tubers of Dahlia,and studies its immunoregulatory activity.The main research contents and results are as follows:1.Polysaccharides were extracted from Dahlia by hot water reflux extraction method.The Sevage method was used to investigate the extraction times of Sevage in the enzymatic hydrolysis+Sevage combined method by employing the Kumas test.The results showed that the enzymatic hydrolysis combined with Sevage for three times could effectively remove the protein in the crude polysaccharides,with a protein content of 4.10%.Finally,88.6 g of crude polysaccharides were obtained from 496.13 g of dried Dahlia tubers by water extraction and alcohol precipitation method,with an extraction yield of 17.72%.After the removal of protein by the enzymatic hydrolysis+Sevage method,71.2 g of crude polysaccharides were obtained,with a protein removal rate of80.36%.DEAE cellulose column was used to perform gradient elution on the samples with distilled water,0.2 mol/L,0.5 mol/L,and 1 mol/L Na Cl solutions.The Dahlia polysaccharide components eluted with distilled water and different concentrations of Na Cl solutions were sequentially named F1,F2,and F3,with masses of 15 g,1.3 g,and0.5 g,respectively.F2 and F3 polysaccharides were obtained in small amounts and were not studied for the time being.F1 was further purified by Sephadex G-200 gel column,and finally,the Dahlia polysaccharide DLH-A with a purity of 98.12%was obtained,with a yield of 88.0%.2.A series of methods,including molecular weight determination,monosaccharide composition analysis,methylation analysis,Fourier transform infrared analysis,and nuclear magnetic resonance analysis,were used to characterize the structure of DLH-A.The results showed that the molecular weight of DLH-A was Mp（peak molecular weight）3633 Da,Mw（weight average molecular weight）3956 Da,and Mn（number average molecular weight）3108 Da.Its monosaccharides were composed of glucose（Glc）,galactose（Gal）,arabinose（Ara）,fructose（Fru）,and mannosyl glucuronic acid（Man A）,with mole percentages of 55.7%,8.2%,4.4%,28.6%,and 3.2%,respectively.Fourier transform infrared analysis showed that the absorption peaks at 985 cm^-1,935cm^-1,873 cm^-1,and 817 cm^-1 were characteristic absorption peaks of inulin.The structure of DLH-A was inferred from nuclear magnetic resonance spectra asα-D-Glcp-1→（2-β-D-Fruf-1）₁₉→2-β-D-Fruf.The structural characterization of DLH-A was consistent with the characteristics of inulin,and therefore DLH-A is a type of inulin.3.Chicory inulin has immunomodulatory activity.This thesis further studied the immunomodulatory activity of DLH-A through cell experiments.（1）Cell viability assay was used to detect its effect on cell proliferation.By examining the effect of different concentrations of DLH-A on the proliferation activity of RAW264.7 macrophages,it was found that DLH-A chicory inulin at 25μg/m L could significantly promote the proliferation of RAW264.7 macrophages,while DLH-A at 50μg/m L and 125μg/m L had no significant effect,indicating that DLH-A had no toxic side effects on macrophages.（2）The cell phagocytic ability experiment reflects its effect on cell activity and immune function.The results showed that DLH-A chicory inulin at low,medium,and high concentrations（25,50,125μg/m L）could enhance（P<0.05,marked*）the phagocytic ability of RAW264.7 cells,and showed a concentration-dependent characteristic.（3）NO production experiment reflects its effect on the secretion of NO by cells.The results showed that DLH-A chicory inulin at concentrations of 25～125μg/m L could significantly stimulate the secretion of NO by RAW264.7 cells（P<0.05）,and showed a clear dose-effect relationship.（4）ROS release experiment was used to detect its effect on the level of intracellular reactive oxygen species.DLH-A at a concentration of 25μg/m L could effectively promote the production of ROS in RAW264.7 cells（P<0.05）,showing good immunomodulatory activity.DLH-A at 50μg/m L and 125μg/m L had no significant difference in the experimental results（P>0.05）,and may have an inhibitory effect on the level of ROS in RAW264.7 cells.In summary,DLH-A of chicory inulin has certain immunomodulatory activity according to all the cell experiment results.