Study On Hypoglycemic Effect Of Rhizoma Anemarrhenae-Cortex Phellodendri Herb-pair And Its Chemical Components Based On LC-MS Method

Objective1. Study on hypoglycemic effect of Rhizoma Anemarrhenae(RA)-Cortex Phellodendri Chinesis (CP) herb-pair decoction.2. To study the mass fragmentation pathways of nine compounds in drug couple RA-CP decoction including mangiferin, neomangiferin, timosaponin A Ⅲ, timosaponin BⅡ, palmatine, jatrorrhizine, phellodendrine, magnoflorine and berberine.3. An UPLC-LTQ-Orbitrap method was developed to study the chemicals in RA-CP decoction.4. To establish a liquid chromatography-mass spectrometry/mass spect-rometry (LC-MS/MS) method for the determination of nine ingredients in RA-CP decoction and study the effects of differrnt proportions between RA with CP on contents of the nine ingredients in RA-CP decoctionMethods1.To establish type 2 diabetic rats model with streptozotocin and diet including high proportion of carbonhydrate and fat. Four weeks after treatment with RA-CP decoction, the fasting blood glucose(FBG), triglycerides, cholesterol and other physiological index are detected and the oral glucose tolerance test (OGTT) was done.2. UPLC-LTQ-Orbitrap method was used to study the mass fragmentation pathways of mangiferin, neomangiferin, timosaponin AⅢ, timosaponin BⅡ, palmatine, jatrorrhizine, phellodendrine, magnoflorine and berberine.3. An UPLC-LTQ-Orbitrap method was developed to analysis and identify the chemicals in RA-CP decoction with the help of related literature.4. A LC-MS/MS method was developed for the determination of mangiferin, neomangiferin, timosaponin AⅢ, timosaponin BⅡ, palmatine, jatrorrhizine, phellodendrine, magnoflorine and berberine in RA and CP decoction.Results1.After four weeks’treatment, the blood glucose of model group rats continue to rise. Blood glucose of RA-CP decoction group rats has been controlled. And RA-CP decoction can also control cholesterol and triglycerides levels, reflecting the advantage of Chinese medicine on treating diabetes by multi-target treatment.2. With the developed UPLC-LTQ-Orbitrap method and help of related literature, the mass fragmentation pathways of the nine compounds from RA-CP decoction was studied. In positive ion mode, losing a H20 molecular, a CH3 or a CH2 is an important pathway. In negative ion mode, timosaponin is easy to lose an glucose molecule. Isomers may create different fragment ions in each ion mode.3. Based on UPLC-LTQ-Orbitrap, we have identified 61 ingredients in RA-CP decoction, including 9 timosaponin ingredients,25 alkaloids and 27 other ingredients. Timosaponin is easy to combine formic acid producing fragment ion as M+46.4. The contents of mangiferin, neomangiferin, timosaponin AIII, timosaponin BII, palmatine, jatrorrhizine, phellodendrine, magnoflorine and berberine were 4.96,9.33,3.62,43.6andO.75,0.48,2.62,1.29,33.18 mg·g-1, when RA:CP was 1:1.The method was rapid, specific and sensitive.The contents of mangiferin and timosaponin AⅢ in group RA:CP(1:O) were the highest. Neomangiferin content was higher than other groups and Jatrorrhizine and Palmatine content were the lowest in group RA:CP(1:1). Timosaponin BⅡand jatrorrhizine content is similar in each group. The contents of Berberine and phellodendrine in RA:CP(2:1 and 3:l)were higher than other groups, magnoflorine content was highest in RA:CP(2:1).Conclusion:This paper proved the hypoglycemic effect of Rhizoma Anemarrhenae-Cortex Phellodendri Chinesis through animal experiment, explored the mass fragmentation pathways of the nine compounds from RA-CP decoction and Identified 61 ingredients with the developed UPLC-LTQ-Orbitrap method. Mangiferin, neomangiferin, timosaponin AⅢ, timosaponin BⅡ, palmatine, jatrorrhizine, phellodendrine, magnof lorine and berberine contents from RA-CP decoction were determined with a sensitive, accurate, simple, rapid LC-MS/MS method, respectively. RA used with different proportions of CP could affect the contents of each compound included. None of new compounds was found, change of main compound content may be the key how RA-CP herb-pair decoction work.