| Purpose: Polygonatum odoratum (Mill.) Druce is a plant in Liliaceae family which is used in treating diabetic. Chloroform fraction from Polygonatum odoratum (Mill.) Druce have protective effects in streptozotocin-induced experimental diabetic rats, which might be related to inhibiting AGEs formation by previous study. This research study effective component which have inhibiting AGEs formation, establish determination method and extraction technology, study the effects on diabetic nephropathy by the effection extraction .Methods: Compounds are isolated from Polygonatum odoratum by ODS column chromatography and HPLC separation techniqueand. Their structures are identified by the data of MS and NMR. Reference substance of effective component are isolated by macroporous adsorption resin and HPLC. We determined content of effective component from Polygonatum odoratum by HPLC-DAD method. Extraction technology of effective component are optimized by orthogonal test. Effective component are researched on the kidney protective effect by STZ induction of diabetic rats model.Results: 3 compounds have been isolated from the effective fraction on inhibiting AGEs formation. The structures of compounds have been identify as 3-(4′- hydroxybenzyl)-5,7-dihydroxy-6-methyl-8-methoxychroman-4-one (I), 3-(4′-hydroxybenzyl)- 5,7-dihydroxy-6,8-dimethylchroman-4-one(II), 3-(4′-methoxybenzyl)-5,7-dihydroxy-6-methyl -8-methoxychroman-4-one(III), 3 compounds have inhibitory activities on AGEs formation. It was found that IC50 values were 56.30μM,46.05μM and 107.10μM. In this research, we isolated reference substance of three homoisoflavones by macroporous adsorption resin and HPLC, the purity are more than 98 %. We determined contents of three homoisoflavones isolated from Polygonatum odoratum by HPLC-DAD method. The optimization HPLC conditions: column, Diamonsil ODS 250 mm×4.6 mm I.D.; column temperature: 30℃; mobile phase: CH3OH-H2O containing 0.1 % acetic acid; gradient program: 0-5 min: 60 % CH3OH→60 % CH3OH, 5-25 min: 60 % CH3OH→80 % CH3OH, 25-60 min:80 % CH3OH→80 % CH3OH; flow rate: 1.0 mL·min-1; detection wavelength: 297 nm. The optimization extraction method: 80 % methanol, reflux extraction 120 minutes; the methodological study showed good linear correlations existed in the range of 0.1068~2.1360μg of injection volume of compound I, 0.0564~1.1280μg of injection volume of compound II, and 0.0108~0.2160μg of injection volume of compound III; The mean recovery of low, middle, and high concentration of compound I were 99.37 %, 98.67 %, and 100.03 % respectively with RSD being 2.11 %, 1.85 %, and 1.81 % respectively. The mean recovery of low, middle, and high concentration of compound II were 99.46 %, 99.60 %, and 100.10 % respectively with RSD being 1.73 %, 1.93 %, and 2.07 % respectively. The mean recovery of low, middle, and high concentration of compound III were 99.63 %, 100.52 %, and 99.29 % respectively with RSD being 1.97 %, 1.78 %, and 2.28 % respectively. The extraction technology is established, the optimization extraction technology of three homoisoflavones is 80 % ethanol, reflux extraction 2 times, 1 h each times. The extraction of homoisoflavones have protective in streptozotocin-induced experimental diabetic rats, reduce serum creatinine, urea nitrogen and urinary protein excretion rate; reduce AGEs content of renal cortical; inhibit TGF-β1 expression; pathological changes of kidney have different degrees of improvement.Conclusions: Material base and action mechanism of P. odoratum are interpreted by modern science and technology. It is founded that homoisoflavones have treatment for diabetic nephropathy on inhibiting AGEs formation, establish determination method and extraction technology, all the results of this paper provided development and utilization for Polygonatum odoratum. |
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