Structural Elucidation Of The Polysaccharides Of Chuan Mingshen And Its Effect On The Activity Of Digestive Enzymes

Plant polysaccharides is a kind of macromolecular compound,it not only participate in various physiological and biochemical reactions of cells,but also have a lot of benefits,including immune regulation,anti-tumor,hypoglycemic and other antioxidant activities.As a kind of traditional Chinese medicine,Chuanminshen violaceum（C.violaceum）has been widely used in the production of medicated food,which has a far-reaching edible history.It belongs to umbelliferae family,and mainly distributed in Sichuan and Hubei.And it is rich in polysaccharides,coumarins,amino acids and fatty acids.Polysaccharides,as the main component of active substances in C.violaceum,has good biological activity,including immune,antiviral and antioxidant activities.In this paper,ultrasonic-assisted water extraction and alcohol precipitation method was used to extract crude polysaccharide（CVPs）from C.violaceum.Herein,CVPs were purified to yield two polysaccharide fractions（CVPs-1-G and CVPs-2-G）using DEAE Cellulose-52and Sephadex G-100.Ion chromatography（IC）,gel permeation chromatography（HPGPC）,ultraviolet（UV）spectroscopy,infrared spectroscopy（IR）,gas chromatography-mass spectrometry（GC-MS）,nuclear magnetic resonance spectroscopy（NMR）and mass spectrometry（ESI-MS）were used to identify the main components（CVPs-1-G）of polysaccharides from C.violaceum.And the effects of CVPs,CVPs-1-G and CVPs-2-G on the activities ofα-amylase,lipase and pepsin were investigated.In addition,the structural changes of CVPs-1-G and its effect on digestive enzyme activity during simulated digestion in vitro were investigated.The results are as follows:（1）Structural characterization of polysaccharide from C.violaceum.CVPs were purified to yield two polysaccharide fractions（CVPs-1-G and CVPs-2-G）,the yield was 63.72±1.76%and 32.14±2.05%respectively,using DEAE Cellulose-52 and Sephadex G-100.Ion chromatography showed that CVPS,CVPs-1-G and CVPs-2-G were mainly composed of glucose,and the content of glucose was 93.09%,98.75%and 96.96%respectively.HPGPC results showed that the molecular weights of CVPs-1-G and CVPs-2-G were 8.1 k Da and 187.5 k Da,respectively,and they were homogeneous polysaccharides.In addition,the results of methylation,NMR and MS showed that The main chain of CVPs-1-G was mainly composed of（1→4）-linked-α-D-Glcp and contained a certain amount of（1→6）-linked-α-D-Glcp;the branch chain was connected with the main chain through the O-6 or O-3 of→3,6)-α-D-Glcp-(1→.（2）Effects of polysaccharides from C.violaceum on the activities of digestive enzymes and their mechanisms.In vitro experiments showed that CVPs CVPs-1-G and CVPs-2-G inhibited the activities ofα-amylase,lipase and pepsin,and CVPs-1-G had the highest inhibitory activities onα-amylase,lipase and pepsin,with IC₅₀ values of 3.100±0.276 mg/m L,2.480±0.359mg/m L and 6.723±0.180 mg/m L,respectively.In addition,kinetic studies show that CVPs,CVPs-1-G,and CVPs-2-G all exhibited competitive inhibition ofα-amylase and noncompetitive inhibition of lipase and pepsin.（3）Structural changes of polysaccharides from C.violaceum during simulated digestion in vitro and their effects on digestive enzyme activities.The results of simulated digestion in vitro showed that the molecular weight and reducing sugar content of CVPs-1-G decreased and increased to a certain extent during saliva digestion and small intestine digestion.In addition,the digestive products of CVPs-1-G had no significant effect on pepsin inhibitory activity in gastric digestion stage,but compared with CVPs,the gastric digestive products of CVPs-1-G had significantly higher than that of pepsin inhibitory activity.The inhibitory activities ofα-amylase and lipase of digestive products of CVPs-1-G in small intestine increased to a certain extent,and reached a stable level after a period of time.The results showed that the inhibitory activity of digestive products of CVPs-1-G onα-amylase,pancreatic lipase and pepsin was affected by its structural changes.