Studies On Hypoglycemic Activity Of Banaba (Lagerstroemia Specious) Leaves And Its Mechanism

Banaba(Lagerstroemia specios) belongs to family Lythraceae. In the Phillippines,Banaba leaves have been used for the treatment and prevention of diabetes mellitus. Greatattention has been paid on hypoglycemic activity of banaba leaves in recently years and manystudies have been done. There were plentiful of banaba resources in Guangdong and Guangxiprovinces in china. However, fewer researches about its hypoglycemic activity have beendone. The studies on the hypoglycemic activity of Chinese banaba leaves were very importantfor the elucidation of mechanism of hypoglycemic activity of Chinese banaba leaves and thedevelopment of functional food in an academic and practical view.Firstly, 3T3-L1 adipocytes were used to the analysis of the hypoglycemic activity ofBanaba extracts. The aqueous and ethanolic extracts of Banaba leaves were separated intoseveral different fractions by solvent extraction and resin adsorption. The activity of differentfractions was tested against 3T3-L1 adipocytes. The results showed that the extracts ofethanol and petroleum ether, which contained more triterpenoid components, have morepotent hypoglycemic activity.The TLC-densitometry method used to determine the amount of total triterpenes and theTLC-HPLC method used to determine the amount of corosolic acid in the extract of Banabaleaves were developed and established. The TLC-densitometry method and the TLC-HPLCmethod were used as a control method in extraction and purification. The methanol extract ofBanaba leaves was separated by thin layer chromatography on silica gel to wipe offdisturbance. The amount of total triterpenes and corosolic acid was quantified byspectrophotometric and HPLC methods, respectively. The developing solvent system for TLCwas a mixture of CHCl3: acetone (4:1, v/v). Conditions of spectrophotometric method were asfollows: 0.3 ml of 5% vanillin/glacial acetic acid, 1 ml perchloric acid solution, heating 15minutes at 60 oC and the absorbance at 542 nm. The HPLC method and condition were asfollows: Luna C18 column (5 μm, 250 x 4.60 mm, UV detection: λ=204 nm, mobile phase:acetonitrile-0.2% phosphoric acid(45:55), flow rate: 1.0 ml/min, and injection volumn10 μl.Both methods were established for the first time and found to be accurate, sensitive andreliable in the analysis of various Banaba extracts.The technology of the ultrasonic wave extraction of triterpenes components in banabaleaves was studied. The results were as follows: pre-marinate 120 min, extraction 25 min at200W, 95% of alcohol content and the ratio of material to alcohol with1:10(w:w).The yieldsof total triterpenes and corosolic acid were 2.12% and 0.17%, respectively.The triterpenoid constituents in the extract of Banaba leaves were separated by resinadsorption. HPD100 resin was found to be the best for the separation of triterpenecomponents in Banaba leaves among five different types of resins. The adsorption isothermalcurves, kinetic curves and breakthrough curve were obtained through the studies of adsorptionproperties of HPD100 resin. The multiple purity of total triterpenes and corosolic acid reached2.08 and 2.15, respectively. The recovery of total triterpenes and corosolic acid were up to83.2% and 86.3%, respectively.Two isomers with high hypoglycemic activity were identified by TLC separation guidedby 3T3-L1 adipocytes. The structures of the two monomers were identified by DSC , UV ,MS , 1HNMR , 13CNMR and DEPT-NMR. The results showed that the two monomers werecorosolic acid and ursolic acid. Ursolic acid was identified in the Banaba leaves extract for thefirst time.The hypoglycemic activity of corosolic acid and total triterpenes were compared. It wasfound that the hypoglycemic activity of total triterpenes is stronger than the hypoglycemicactivity of corosolic acid at the same concentration. The results suggested that the totaltriterpenes are responsible for the hypoglycemic effect of Banaba leaves.After the determination of hypoglycemic activity of Banaba leaves by 3T3-L1adipocytes in vitro, the total triterpenes of Banaba leaves demonstrated the hypoglycemiceffect in ALX diabetic mice in vivo. When ALX diabetic mice were fed a diet containing totaltriterpenes of Banaba leaves for 4 weeks, their related diabetes index (blood glucose,urineglucose,blood lipids,weighe and glycosylatde hemoglobin) were significantly improved.The hypoglycemic mechanism of triterpenes of Banaba leaves was studied at the cellularand molecular levels. By analysis of the effect of triterpenes of Banaba leaves on glucoseconsumption, the effect of triterpenoids components of Banaba leaves against 3T3-L1adipocytes differentation and the effect of triterpenoids of Banaba leaves on PPARγ mRNAand C/EBPα mRNA expression. The results showed that an induction time anddose-dependent response of the triterpenoids of Banaba leaves stimulated glucoseconsumption in 3T3-L1 adipocytes, which is similar to that of insulin. Furthermore, there wasa synergistic effect between triterpene components and insulin on glucose consumption. Theinhibitory activity was further demonstrated by drastic reductions of PPARγmRNA andC/EBPαmRNA in 3T3-L1 cells in the presence of triterpene components.To make good use of Banaba resource, the TLC fingerprints and HPLC/UV/MSfingerprints of triterpenoids in Banaba leaves and fruits were compared. The result showedthat the leaves and the seeds of Banaba contained both corosolic acid and maslinic acid. Butthe leaves of Banaba contained ursolic acid only and the seeds of Banaba contained theoleanolic acid only.