| ObjectiveMelicope ptelefolia (Champ.ex Benth.) Hartley (Rutaeeae) was early recorded in "Lin Nan Cai Yao Lu". Now, this medical plant is recorded in "Guangdong Chinese Medicine Criterion", used as traditional Chinese medicine in the southern region of the People's Republic of China, also named "San-cha-ku". It's a frequently used ingredient of Guang Dong herbal tea; also it serves as a medical herb for the treatment of injury, wounds, fester and eczema. Now, this plant use as the main ingredient in a lot of patent medicine in China, example Sanjiuweitai and Sanjiuganmaoling etc. The patent medicine including "Sanchaku" have good perspective marketing. The original plant of "Sanchaku" is distributed widespread in Guangdong province, China. However, Pharmacopoeia of the People's Republic of China hadnot been recorded this plant. The main reason is this plant was lacked of the chemical constituents or biological active constituents were unclear. So could not control the quality of this medicinal material. In order to better control the quality of the medicinal material because this medicinal material in clinical pharmacological effect, clearly labeled with the chemical composition and to find its main active ingredient or the nucleus of its main active ingredient structure-based drug lead compounds to determine the quality standard of medicine, the intensive research and development of this plant is value and meaningful.Currently, the study of Melicope ptelefolia mainly concentrated in the low polar fractions, but on the high polar parts of the basic blank. The water extracts of Melicope ptelefolia was used in traditional. So the bioactive part should be at high polarity part of the extracts from Melicope ptelefolia. Therefore, to find the inhibited nitric oxide active constituents of leaves from Melicope ptelefolia, and established the fingerprint of leaves from Melicope ptelefolia have important significance.MethodsAspects of chemical constituents:The powder of dried leaves of Melicope ptelefolia been roomtemperature immersion24hours with80%ethanol, and then diacolation, vacuum solvent, and then get extract. Mixing160-200mesh silica gel, the diatomite and the extract, followed by continuous reflux extraction with petroleum ether, ethyl acetate, acetone and methanol. Acetone part were separated and purified by silica gel, Sephadex LH-20, reverse the ODS silica gel separation technology to obtain monomeric compound. The chemical structure of the monomers were identification by ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-NMR and13C-NMR, DEPT,1H-1H-COSY, HSQC, HMBC) and other modern spectroscopy and spectroscopic techniques.Aspects of biological activity in vitro study, the RAW264.7were cultured3generation with DMEM complete medium (containing10%fetal bovine serum,100U· ml-1 penicillin and100μg ml-1 streptomycin) at37℃,5%CO2incubator, whichever is the logarithmic growth phase cells used in the experiment. In cells in the logarithmic growth phase, with0.25%trypsin digestion and cells, regulates cell concentration of2.5X104ml-1, inoculated in48well plates,250μl/hole. When the cells to80%confluence after grouping. Divided into:blank control group, LPS group, a variety of sample group (cells pretreated for1h), and then1μg-ml-1 of LPS stimulation the cells for24h. After24h, supernatant was collected, according to the kit instructions to determine NO content. Then NO inhibition rate calculated by the formula: NO inhibition rate (%)=100×Aspects of fingerprints:The fingerprints were establish of19batches of leaves of Melicope ptelefolia by HPLC, and the use of fingerprint parameters common peaks and similarity analysis, and the map were analysis with the cluster analysis and principal component analysis.Results19compounds were isolation and identification from acetone part of leave of Melicope ptelefolia. Among them,1acetophenones were identified as:1-[2,7-Bis-(3,4-dihydroxy-tetrahydro-furan-2-yl)-2,4,7-trihydroxy-2,3,7,8-tetrahydro-benzo[1,2-b;3,4-b']difuran-5-yl]-ethanone (compound1);12flavonoids were identified as:Kaempferol-3-O-alpha-L-rha-mnosyl(1→2)-beta-D-galactopyranoside (compound2), kaempferol-3-O-beta-D-pyran-glu-curonic acid methyl ester (compound3), kaempferol-3-O-alpha-D)-glucosyl(1→2)-beta-D-glucopyranoside (compound4), isorhamnetin (compound5), kaempferol (compound6), kaempferol-3-O-beta-D-glucopyranoside (compound7), Kaempferol-3-O-beta-D-pyran-gl-ucuronic acid (compound8), quercetin (compound9), kaempferol-3-O-α-L-arabinopyran-oside (compound10),3,5,7,3'-tetrahydroxy-8,4'-dimethoxy flavone (compound11),3,5,3'-trihydroxy-4'-methoxy-7-prenyloxycoumarin flavonoids (compounds12),3,5,4'-trihydro-xy-8,3'-dimethoxy-7-prenyloxycoumarin flavonoids (compounds13);2steroidal were identified as:P-sitosterol (compound15) and daucossterol (compounds16);4other compounds were identified as:inositol (compound14), glucose (compound17), rhamnose (compounds18), palmitic acid (compound the19).The compounds from Melicope ptelefolia were screened on inhibition nitric oxide activity. The results showed that fifteen compounds had significant inhibit murine macrophages to release nitric oxide at1μg·ml-1 and10μg·ml-1(P＜0.05). These compounds including:isorhamnetin, kaempferol,3,5,7,3'-tetrahydroxy-8,4'-dimethoxy flavones. inositol, quercetin, β-sitosterol, Tricosanoic acid tetradecyl ester, vanillic acid. Isorhamnetin-3-O-α-L-arabinopyranoside, kaempferol-3-O-rutinoside,3,7-dimethoxyl kae-mpferol,3,5,4'-trihydroxy-8,3'-dimethoxy-7-(3-methylbut-2-enyloxy)flavones,3,5,3'-trihy-droxy-4'-methoxy-7-(3-methylbut-2-enyloxy)flavones,3,5,3'-trihydroxy-8,4'-dimethoxy-7-(3-methylbut-2-enyloxy)flavones, erythro-3-(1',2',3'-trihydroxy)isopenthl-7-hydroxycoum-arin. Seven compounds had significant inhibit murine macrophages to release nitric oxide at1(μg-ml"1(P＜0.05). These compounds including palmitic acid, daucossterol, kaempferol-3-O-(3-D-glucopyranoside, kaempferol-3-O-beta-D-pyran-glucuronic acid methyl ester, kaempferol-3-O-a-L-arabinopyranoside, kaempferol-3-O-alpha-L)-glucosyl-(1→2)-beta-D-glucopyranoside, Kaempferol-3-O-beta-D-pyran-glucuronic acid, pteleifolosin D, S26, S27.HPLC fingerprint of leaves of Melicope ptelefolia were established and obtained12common peaks and similarity0.69-0.98.19batches of medicinal materials were clustered3classes.Conclusion19compounds were isolation and identification from acetone part of leave of Melicope ptelefolia by physicochemical properties and spectrum analysis. Among them,1acetophenones,12flavonoids,2steroids,4other compounds. Compound1is a new compound. Compound2,3,4,6,8,9,11,14,17,18were isolated from Melicope genus for the first time. Compound19were isolated from this species for the first time.The compounds from Melicope ptelefolia were screened on inhibition nitric oxide activity. The results showed that fifteen compounds had significant inhibit murine macrophages to release nitric oxide at1μg-ml"1and10μg-ml"1. Ten compounds had significant inhibit murine macrophages to release nitric oxide at1μg-ml-1.HPLC fingerprint and chemical pattern recognition of leaves of Melicope ptelefolia were established, and provide more competed reference for control quality of the medicinal materials. |
PDF Full Text Request