| Objective: This study aims to investigate the efficacy factors of Gardenia jasminoides Ellis in treating Diabetes Mellitus,to clarify the hypoglycemic components and to study the mechanism,and to establish a rapid method for the preparation of effective components from Gardenia jasminoides Ellis and construct a compound sample library based on pharmacophore model matching.Methods:1.Using solvent extraction method to obtain the ethanol extract of Gardenia jasminoides Ellis;using macroporous resin column chromatography to obtain different chemical fractions(A～F)of gardenia extract;using physical and chemical identification method,HPLC method combined with literature reports to analyze the composition of each chemical fraction;using IR-Hep G2 model induced with high concentration insulin and CCK-8 method to measure cell survival rate,glucose consumption and hypoglycemic activity of each chemical fractions.2.A homogeneous acidic polysaccharides named as GJPP1 was isolated and purified using mixed ion-exchange resins and Sephadex G-50 chromatography from fraction A(polysaccharides)of the crude polysaccharide.The contents of neutral sugar,acidic sugar and protein of GJPP1 were investigated by the methods of phenol-sulfuric,m-hydroxybiphenyl and Bradford.The monosaccharide composition was determined through PMP pre-column derivatization.The molecular weight of GJPP1 was determined by high performance size exclusion chromatography(HPSEC).The degree of esterification and triple helix structure were determined by titration and Congo Red.Methylation,UV,FT-IR and NMR methods were used to analyze the structure of GJPP1.3.The method of high efficient and rapid separation and preparation of small molecular compounds was established to prepare the main effective components in large scale from the effective fractions.NMR and MS methods were used to analyze the structures of the compounds.4.The effect verification and mechanism research of the isolated effect factors were carried out by evaluation of the inhibition on ATP-citrate lyase(ACL),nuclear transcription factor-κB(NF-κB)signaling pathway,death associated related apoptotic protein kinase 2(Drak 2),and the effects on gluconeogenesis and Glucose-stimulated insulin secretion(GSIS)function.5.Molecular docking technology and Western blotting(WB)were used to detect the effects of GJPP1,geniposide,gardenoside,genipin-1-β-gentiobioside,crocin I and crocin Ⅱon the expression of PI3 K and AKT protein in pancreatic βTC3 cells.6.The hypoglycemic ligand-based pharmacophore model was set up with genipin,geniposide,gardenoside,genipin-1-β-gentiobioside and the molecular library of the compounds with hypoglycemic activity were established by the virtual screen.Results:1.Four effective fractions(Fr.A/B/C/E)with hypoglycemic activity were isolated from Gardenia jasminoides.Four effective fractions had certain effects on glucose consumption of IR-Hep G2 cells.Compared with the blank control group,the glucose consumption of IR model group was significantly decreased(P<0.01).Compared with the IR model group,the glucose consumption of the positive control group was significantly increased(P<0.01).Compared with IR model group,the glucose consumption of the crude extract,Fr.A and Fr.E fractions of Gardenia jasminoides decreased significantly(P<0.01)in a dose-dependent manner.The low concentration(10 μg/m L)of Fr.B and Fr.C had a certain promoting effect on glucose consumption(P<0.05),and the medium and high concentrations(50 μg/m L,100 μg/m L)had a significant promoting effect(P<0.01).2.A homogeneous acidic polysaccharide named as GJPP1 was isolated from Fr.A.The content of Gal A was 83.33%,and the total content of uronic acids was as high as 90.06%.GJPP1 was composed of mannose(Man),rhamnose(Rha),glucuronic acid(Glc A),galactose(Gal A),glucose(Glc),galactose(Gal)and arabinose(Ara)with the molar ratio of2.05 : 0.26 : 1.39 : 92.56 : 0.23 : 1.97 : 1.55.The methylation results showed that GJPP1 was composed of seven residues,and the main linkage modes were 1-Ara(p),1-Man(p),1-Gal(p),1,4-Gal(p)A,1,4-Glc(p)A,1,3,4-Gal(p)A and 1,4,6-Gal(p)A with the molar ratios of 1.80 : 5.98 : 2.16 : 77.58 : 6.73 : 1.34 : 4.41.The relative molecular weight of GJPP1 was 41.9 k Da.The protein content was 2.47 %.The degree of esterification was75.5% and GJPP1 had not the triple helical conformation.3.With only two chromatographic steps,macroporous resin column chromatography combined with high-speed counter-current chromatography(HSCCC)was used to purify crocins I and II from Fr.E.In an effort to shorten the isolation time and reduce solvent usage,forward and reverse rotations were successively utilized in the HSCCC isolation procedure.Crocins I and II were simultaneously obtained from a herbal resource with high recoveries of 0.5% and 0.1%,respectively,and high purities of 98.7% and 99.1%,respectively,by HPLC analysis.Simultaneously,geniposide,gardenoside,genipin-1-β-gentiobioside were isolated from Fr.B and C,and the purity was above 98%.4.Five models of different mechanisms were used for evaluating the hypoglycemic activity of GJPP1,geniposide,gardenoside,genipin-1-β-gentiobioside,Crocin I,and Crocin II.When the concentration of the compound is 2 m M(geniposide,gardenoside,genipin-1-β-gentiobioside,crocin I and crocin II)and 1 mg/m L(GJPP1),the above compounds have a certain degree of inhibitory effect on ACL,respectively.The results of NF-κB signaling pathway inhibition experiment showed that geniposide,genipin-1-β-D-gentiobiside,crocin I,crocin II and GJPP1 have no cytotoxicity;Gardenoside has cytotoxicity.The transcription factor activities of the tested samples were all above 50%.The results of the Drak2 inhibition experiment showed that geniposide,gardenoside,genipin-1-β-gentiobioside possessed very poor Drak2 inhibitory activity compared with the blank group,while crocins I,crocins II and GJPP1 showed moderate inhibitory activity.Experimental results of mouse primary hepatic cytoplasmic neoplasia model showed that,compared with the blank group,except for GJPP1,other compounds had no gluconeogenesis inhibitory activity.The high and low concentration groups of GJPP1 compared with the blank group,had statistical difference(p<0.05).The effect of glucose in promoting insulin secretion in INS-1E cells showed that geniposide and genipin-1-β-D-gentiobiside didn’t promote glucose-stimulated insulin release;gardenoside,crocin I and crocin II can promote glucose-stimulated insulin release;and the six substances did not damage the integrity of the cell membrane at the experimental concentration.5.The results of molecular docking showed that the binding energy of geniposide,gardenoside,and genipin-1-β-gentiobioside with PI3 K protein is-2.9～-3.2kcal/mol;the binding energy with AKT protein is-1.6～-6.7 kcal/mol,and the binding activity is weak.The binding energy of crocin I and II with PI3 K protein is-8.7 and-8.9 kcal/mol,and their binding energy with AKT protein is-13.2 and-13.5 kcal/mol,indicating that crocin I and II have a positive effect on PI3 K.The protein has good binding activity and strong binding activity with AKT protein.The results of WB experiment showed that compared with the blank control group,the expression of AKT and PI3 K in the cells of model group was significantly reduced(P<0.01).The two protein expressions of AKT and PI3 K in the metformin hydrochloride group,the high-dose group of crocin II(10 m M),and GJPP1(10mg/m L and 50 mg/m L)were significantly higher than those in the model group(P<0.01),and present a dose-dependent.Compared with the model group,the expression of AKT in each dose group of crocin I(2 m M and 10 m M)and the low dose group of crocin II(2 m M)was significantly increased(P<0.01).Regarding the expression of PI3 K protein,compared with the model group,the low-dose group(2 m M)of crocin I and II had a regulatory effect(P<0.05),while the high-dose group of crocin I(10 m M)had a significant regulatory effect(P<0.01).6.Based on the compound information of genipin,geniposide,gardenoside,and genipin-1-β-gentiobioside,a pharmacophore model based on hypoglycemic ligands was established.And the main pharmacophore was selected which consists of one hydrophobic group,one hydrogen bond acceptor,and two hydrogen bond donors;The hypoglycemic active ingredients were virtually screened in the MCE Bioactive Compound Library and Discovery Diversity Set 50 databases with the preferred pharmacophore,andthe top 200 compounds with good pharmacophore matching were selected from each library to establish a compound library.Conclusion:1.The polysaccharide GJPP1 from Gardenia jasminoides was a new kind of polysaccharide and had hypoglycemic effects.2.The results showed that Gardenia polysaccharide(GJPP1)could reduce blood glucose by regulating carbohydrate and lipid metabolism,protecting islet cells,inhibiting gluconeogenesis and insulin resistance.Crocin I and crocin II may achieve positive regulation of blood glucose by regulating carbohydrate and lipid metabolism,protecting islet cells and inhibiting insulin resistance.Geniposide B had a moderate protective effect on islet cells,and geniposide,geniposide B and genipin-1-β-D-gentianoside had a certain degree of control on glucose and lipid metabolism.GJPP1,crocin I and crocin II may participate in blood glucose regulation by regulating the expression of PI3 K and AKT proteins. |
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