Study On The Chemical Constituents From Averrhoa Carambola L. Root And Its Hypoglycemic Activities

Objective:The study was conducted to understand the chemical constituents from the Averrhoa carambola L.(Oxalidaceae) root, and to screen the hypoglycemic activity and the relative machnisms of the main compounds, and to establish the determination method of the marker compounds with HPLC. Methods:1. The Averrhoa carambola L. root (ACLR) was extracted in turn by petroleum ether, ethanol and water. Then the chemical components were concluded via the phenomena on the corresponding reactions which were carried out for the extracts mentioned above.2. The60%ethanolic solution of ACLR was concentrated, then suspended in H2O and extracted with cyclohexane, EtOAc and n-BuOH, respectively. And the compounds were isolated by the repeated silica gel, Sephadex-LH20, ODS column chromatography or PHPLC, and elucidated on the basis of various spectral analysis (’H-NMR,13C-NMR, HMBC or HSQC, IR, etc).3. The hypoglycemic effect of three compounds, including PNS and (±)LGP, were investigated by STZ-induced mice. In the animals test, the mice were randomly divided into12groups (n=10) mentioned as follows:Group1,2were normal and T2DM mice (control), which were both given saline (vehicle). Group3were diabetic mice of intragastric treating with Pioglitazone10mg.kg-1.d-1. However, the diabetic mice of groups4-12were intragastric treated with20,40,80mg·kg-1.d-1of PNS,(±)LGP for14days, respectively. The body weight, death, FBG, FINS and ISI, etc. of the mice were recorded during these days. Then, the mice were sacrificed, and their kidneys were harvested, fixed in10%formaldehyde solution immediately, embedded in paraffin and sectioned at a thickness of5μm for histopathological, apoptosis (TUNEL) and immunohistochemical (NF-κB, caspase-3,-8,-9, etc) analysis.4.2-methoxy-6-nonylcyclohexa-2,5-diene-1,4-dione (MNDD) and2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD), were determined by HPLC under the following chromatographic conditions:Thermo ODS-2HYPERSIL column (4.6×250mm,5μm), Methanol-H2O solution (90:10) as the mobile phase, the flow rate of1.0ml/min and the detection wavelength at270nm. Results:1. There were phenols, organic acids, tannins, cumarins, terpenoids, steroids or terpene lactones, polysaccharides and glycosides, etc. in the ACLR.2. Twenty four compounds were separated from the60%ethanol extract of ACLR, including2benzoquinone compounds,1steriod compound,1alkanes compound and20glycocide compounds. All of the compounds, namely2-methoxy-6-nonylcyclohexa-2,5-diene-1,4-dione (1),2-dodecyl-6-methoxycyclo-hexa-2,5-diene-1,4-dione (2), daucosterol (3),4-hydroxy-3-methoxyphenol-1-β-D-[6-O-(4-hydroxyl-3,5-dimethoxylbenzoate)]-glucopyranoside (4), methyl-cyclohexane (5), prunasin (6), benzyl-1-O-β-D-glucopyranoside (7),3,4,5-tri-methoxyphenol-1-O-β-D-glucopyranoside (8),(+)-5’-methoxyisolariciresinol3a-O-β-D-glucopyranoside (9),(+)-isolariciresinol3a-O-β-D-glucopyranoside (10), koaburaside (11), compound (12),3,4,5-trimethoxyphenyl1-O-β-apio-furanosyl (1"-+6’)-β-glucopyranoside (13), cuneataside C (14),(+)-lyoni-resinol3a-O-β-D-glucopyranoside (15),(-)-lyoniresinol3a-O-β-D-glucopyran-oside (16),(-)-5’-methoxyisolariciresinol3a-O-β-D-glucopyranoside (17),(-)-isolariciresinol3a-O-β-D-glucopyranoside (18), methoxyhydroquinone-4-β-D- glucopyranoside (19), compound (20),2-O-β-D-glucopyranosyl-2-hydroxyl-phenyl-acetic acid (21),3-hydroxy-4-methoxyphenol1-O-β-D-apiofuranosyl-(1â†'6)-O-β-D-glucopyranoside (22),4-hydroxy-3-methoxyphenol1-O-β-D-apiofuranosyl-(1-6)-O-β-D-glucopyranoside (23),3,5-dimethoxy-4-hydroxy-phenyl-1-O-β-apiofuranosyl (1"â†'6’)-O-β-D-gluopyranoside (24), were isolated from this plant for the first time. Among the isolated compounds, compound12and20were two novel phenolic glycosides. And DMDD and MNDD also have never been reported in the natural products.3. The FBG level in the groups of PNS (20,40,80mg.kg-1.d-1) and (+)LGP (20,40,80mg.kg-1.d-1) deceased23.3%,26.9%,27.5%and23.0%,24.5%,32.2%, respectively. The results of cell apoptosis and immunohistochemistry test showed that PNS,(+)LGP could inhibit the cell apoptosis in the kidney and the activity of NF-κB pathway, which should be related with the machnism of their hypoglycemic effects. But,(-)LGP had no hypoglycemic effect in the dosage of no more than80mg.kg-1.d-1for the STZ-induced diabetic mice.4. HPLC can be used for the determination of MNDD and DMDD in the ACLR for its convenience, rapidity, stability, accuracy, reliability and good reproducibility. The highest level of MNDD and DMDD from different batches herb were8.49mg/g and3.34mg/g, respectively. However, the two compounds couldn’t be detected in some bacthes of ACLR.