Studies On The Structure Of Active Polysaccharide From Glechoma Longituba(Nakai)Kupr And Metabolism Of Rosmarinic Acid

The dried aerial part of Glechoma longituba?Nakai?Kupr.The main effects are beneficial to dampness and drenching,clearing away heat and detoxification,dispersing blood stasis and detumescence,etc.Clinically,it is mainly used for urination,treatment of bladder stones,kidney stones,ureteral stones,nephritis and edema,damp-heat jaundice,cholecystitis,cholelithiasis,mumps,burns,etc.Most Chinese medicines with heat-clearing and detoxifying effects have anti-complement activity.When we screened the anti-complement activity of the antipyretic and detoxicating drug,we found that the 75%alcohol extract had poor anti-complement activity,while the crude polysaccharide had obvious anti-complement activity.Therefore,in order to elucidate the anti-complement active ingredients of Glechoma longituba?Nakai?Kupr.The crude polysaccharide of Glechoma longituba?Nakai?Kupr was further studied.Two homogeneous polysaccharides of GLP-1 and GLP-2 with anti-complement activity were separated.The structural characteristics of the two homogeneous polysaccharides were systematically characterized by various physical and chemical methods.GLP-1,brown and loose,is a heteropolysaccharide mainly composed of two monosaccharides.The molar ratio of arabinose to galactose in GLP-1 is 1:6.3.Molecular weight was5.37 KDa,protein content was 0.94%,and sugar content was 94.8%.Methylation and NMR results showed that GLP-1was one main long chain of 1,6-linked galactose?Gal?and 1,3,6-linked Gal,the branched links of GLP-1 was composed of 1-linked arabinose?Ara?,1-linked terminal Gal.The result of atomic force microscopy shows that spatial structure of GLP-1 is looser and its curl degree is higher.GLP-2 is similar to GLP-1,which is light yellow and loose.It is a heteropolysaccharide mainly composed of two monosaccharides.The molar ratio of arabinose to galactose in GLP-2 is 1:4.9.Molecular weight is 20.04 KDa.Protein content is 0.95%,and sugar content is 93.5%.Methylation and NMR results showed that GLP-2 was one main long chain of 1,6-linked Gal and 1,4,6-linked Gal,the branched links of GLP-2 was composed of 1-linked Ara,1,4-linked terminal Gal.The result of atomic force microscopy shows that spatial structure of GLP-2 is less curled,and it has dense network surface characteristics.The results of anti-complement activity test showed that GLP-1 and GLP-2 had obvious anti-complement activity.The CH₅₀ of GLP-1 and GLP-2 are 0.028±0.011mg/mL and 0.056±0.015 mg/mL,respectively,which were closed to heparin(CH₅₀was 0.041 mg/mL),and GLP-1 had better anti-complement activity than GLP-2,which might be related to its spatial structure.The complement system is closely related to the induction of inflammation.We will further study the anti-inflammatory activity of two homogeneous polysaccharides.Macrophages play an important role in inflammatory response.Inflammatory factors are closely related to the occurrence of inflammation.Therefore,lipopolysaccharide?LPS?stimulates macrophages to induce inflammatory response to produce inflammatory factors.NO release inhibition rate was used as evaluation indicators.IL-6,IL-1?and TNF-?were used to investigate the mechanism of the anti-inflammatory activity.The results showed that the homogeneous polysaccharide could inhibit the release of NO from inflammatory cells significantly.It indicated that the homogeneous polysaccharide GLP-1 and GLP-2 had obvious anti-inflammatory activity,and the effect of GLP-1 was better than that of GLP-2.Homogeneous polysaccharides GLP-1 and GLP-2 inhibit the release of inflammatory factors IL-6 and TNF-?significantly,but they inhibit the release of inflammatory factors IL-1?were weak.It is speculated that the anti-inflammatory mechanism of homogeneous polysaccharides GLP-1 and GLP-2 may be mainly through the inhibition of inflammatory factors IL-6 and TNF-?.Previous studies found that the main ingredient of Glechoma longituba?Nakai?Kupr is rosmarinic acid,rosmarinic acid is one of the most important components of cholelithiasis drugs,which is widely used in clinical treatment of jaundice,urinary calculi and other diseases.However,there are few reports on the metabolism of rosmarinic acid in rats.The metabolites of rosmarinic acid in the urine,plasma,feces and viscera of rats?heart,liver,spleen,lung and kidney?were identified by UHPLC-QTOF/MS.The metabolites of rosmarinic acid in rats were identified by the techniques of filtering out exogenously sourced ions,diagnostic ion loss and neutral ion loss techniques,and improved quality defect techniques.A total of 24 metabolites were identified in plasma,urine,bile and feces.It was found that rosmarinic acid mainly undergoes methylation,sulfonation and glucuronidation in rats.Metabolites are mainly excreted from the body through urine and bile,mainly distributed in the kidney.The metabolic study of rosmarinic acid in vivo further showed that the target of rosmarinic acid was in kidney,which provided a scientific basis for its diuretic and drenching effect.