| We demonstrated that glucaricate existed in Liuwei dihuang pills (LWPs) inprevious work. Multiple studies have established that glucarate has a wide range ofpharmacological effects including anti-cancer and anti-inflammation. Therefore, wedeveloped a novel method for measuring glucaric acid and its1,4-lactone usinghigh-performance liquid chromatography (HPLC) couple with ultraviolet (UV)detector, evaporative light-scattering detector (ELSD) and mass spectrometry (MS)detector in this study. Interconversion between glucaric acid and its lactones was alsoinvestigated in vitro and in vivo. On this basis, nuclear magnetic resonance (1H NMR)based metabonomics was employed to study on their anti-inflammatory effects. Themain findings are as follows:1. Development of a novel method for determination of glucaric acid and its1,4-lactone in liuwei dihuang pills.1) Development of HPLC-HILIC-UV method for determination of glucaric acidand its1,4-lactone in Liuwei dihuang pills. LWPs was extracted by20%methanolultrasonically as the sample for HPLC analysis. The analytes were separated by ahilic column. Acetonitrile-5mM ammonium dihydrogen phosphate and0.1%phosphoric acid was the mobile phase with a gradient elution process, flow speed was0.8ml/min; temperature of column was set at35℃; Injection volume was10μl;detected wavelength was at205nm. The results show that the linear range ofD-glucaric acid is range from25to400μg/ml with linear regression equation A=0.0848C+0.6958and r=0.9993(n=5); that of D-glucaro-1,4-lactone is range from25to400μg/ml with linear regression equation A=0.1430C+0.5917and r=0.9996（n=5）.The results indicate that the method is specific, accurate, sensitive andreproducible. The contents of GA and1,4-GL in Water-honeyed pills are7.10±3.2mg/g and3.96±1.6mg/g respectively, those in Concentrated pill are15.60±4.8mg/g and8.34±4.3mg/g respectively.2) Development of HPLC-HILIC-ELSD method for determination of glucaric acid in Liuwei dihuang pills. LWPs was extracted by20%methanol ultrasonically asthe sample for HPLC analysis. The analytes were separated by a hilic column,,Acetonitrile-20mM ammonium acetate was the mobile phase with a gradient elutionprocess,0-5min (80:20),5-25min (80:20-75:25),25-30min (75:25-50:50),30-35min (50:50-80:20),35-45min (80:20); flow speed was0.8ml/min; temperatureof column was set at35℃;injection volume was10μl; Drift tube temperature60℃,gas flow speed was1.2L/min. The results show that the linear range of D-glucaricacid is range from50to400μg/ml, with linear regression equation A=0.5882C-7.0275r=0.9991（n=6）. The results suggest that this method is specific, accurate,sensitive and reproducible, stable results. The contents of GA in Water-honeyed pillsare8.18±2.4mg/g, The contents of GA in Concentrated pills are19.52±3.8mg/g.3) Development of UPLC-MS/MS method for determination of glucaric1,4-lactone in Liuwei dihuang pills. LWPs was extracted by20%methanol ultrasonicallyas the sample for HPLC analysis. The analytes were separated by Waters BEH C18column, Acetonitrile-10mM formic acid（10：90）was the mobile phase;flow speedwas0.4ml/min; temperature of column was set at40℃;. The results show that thelinear range of glucaric1,4-lactone is range from0.83to830μg/ml with linearregression equation A=12.771C+15.47and r=0.9990（n=5）;.The results show that thismethod is specific, accurate, sensitive and reproducible, stable results. The contentsof1,4-GL in Water-honeyed pills are4.03±3.1mg/g, The contents of1,4-GL inConcentrated pills are8.69±2.9mg/g.Our results strongly suggest that the established method for GA and1,4-GLmeasurement in Liuwei dihuang pills is accurate and reliable, it can be used todetermination of GA and1,4-GL quantitatively.2. Investigation of the inter-conversion of glucaric acid between its lactonesin vitro and in vivo. Investigating convert1,4-GL into GA at pH1.0,3.0,7.0,10.0,and pH12.0; investigating convert GA into1,4-GL at pH1.0,3.0,7.0. Then thetemperature is37℃and100℃respectively. The results show that1,4-GL can convertcompletely into GA immediately under alkaline conditions (pH=12.0) at roomtemperature. In contrast, the conversion of GA into1,4-GL is slow at roomtemperature, more than2weeks are required to gain for equilibrium. The process is faster (less than10min) with higher conversion rate (33.3%) only at acidic conditions(pH=1.0) and100℃; Meanwhile, we found the same process of converting GA into1,4-GL in the stomach only a small amount of GA convert into1,4-GL (less than5%)after the rats administration of GA. The results indicates that the conversion of GAinto1,4-GL in vitro is alternative to ensure the pharmacological effects of glucoseacid derivatives.3. Metabonomic study on glucaric acid and its1,4-lactoneanti-inflammatory effects.1H NMR based profilings of rat urine, blood, extract ofkidney tissue and liver tissue from carrageenan-induced inflammation group, GA andits1,4-lactone treatment group and the control group were established and analysedby principal component analysis（PCA）. Differences of metabolic phenotype of theinflammation group from the control group were observed. The metabolites related tothe differences were detected by orthogonal partial least squares discriminant analysis(OPLS-DA). Comparing to the control group, plasma pyruvic acid and3-hydroxybutyric acid is seen to elevate. A rise in urinary concentration of lactic acid,alanine, acetic acid, citric acid, succinic acid, glycine, uridine, formate and a decreasein plasma hippuric acid, allantoin, dimethylaminewas observed. The level of acetateis higher, and allantoin is lower in kidney tissue. No significant differences ofmetabolite in liver were observed. Meanwhile, we found that rat paw swelling wasinhibited after the rats were administered with the mixture of GA and1,4-GL,suggesting GA and1,4-GL have the anti-inflammation effect. The metabolomicresults show that the levels of urinary lactate and alanine are normalized after theinflammation rats administered with the mixture of GA and1,4-GL. The glucosemetabolism, nucleic metabolism and intestinal flora are regulated by GA and1,4-GL. |
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