The Study On Chemical Constituents And Antioxidant Activity In Clerodendrum Cyrtophyllum Turcz Leaves

Clerodendrum cyrtophyllum Turcz(Clerodendrum,, Verbenaceae) is extensively used as traditional folk medicine for the treatment of various aliments, such as prevention of tooth pain, dysentery, clearing away heat and toxic material, cooling blood and diuresis and centipede bites, clinically used for influenza, mumps, hepatitis and tumor, tonsillitis, mumps, acute laryngitis, periodontitis, bacterial dysentery, worms, snake bites, swollen drugs, cold fever, antiviral, antibacterial, with hypotensive effect.Several studies have already been conducted and it was reported that the pharmacological effects of leaves of C. cyrtophyllum leaves which are similar with flavonoids that have already reported in it. Based on the above statements, the optimal experimental parameters for antioxidant extraction from C. cyrtophyllum leaves were measured using single-factor experimentation combined with response surface methodology (RSM). Among the various factors contributed to the extraction efficiency, three factors were investigated, the ethanol concentration, extractionl time and extraction temperature. Our goal was to extract useful components from the leaves of C. cyrtophyllum leaves while retaining optimal total phenolic content (TPC), total flavonoid content (TFC), and scavenging activity on2,2’-diphenyl-1-picrylhydrazyl (DPPH) and2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid)(ABTS). Ultrasonic-assisted extraction(UAE) were used to exctract C. cyrtophyllum leaves,and using petroleum, chloroform, ethyl acetate and n-Butanol to extract the crude extraction, and spectrophotometric methods were used for the antioxidant evaluation. In the present study,6different antioxidant tests were employed in order to evaluate the antioxidant activities of extracts.They are scavenging activity on DPPH and ABTS, reducing capacity, chelating ability, superoxide radical-scavenging activity, and hydroxyl radical-scavenging activity. We also took a further study in chemical consituents of C. cyrtophyllum leaves with antioxidant activity. The results obtained are as follows:(1) Optimization of ultrasonic-assisted extraction of antioxidant consititutions from C. cyrtophyllum leaves:From single-factor experiments, the range for each independent variable was preliminarily determined as ethanol concentration(40%), extract time(80min), extract temperature(60℃), and being later used in subsequent experiments to test additional independent variables. Optimized extraction conditions for UAE from C. cyrtophyllum leaves were as follows:60.9%ethanol,85.4min, and63.3℃for maximal TPC extraction (16.8±0.2mg GAE/g DW);67.7%ethanol,82.9min, and63.0℃for maximal TFC extraction (49.36±0.4mg RT/g DW);48.8%ethanol,85.1min, and63.9℃ for maximal DPPH radical-scavenging capacity (86.8±0.2%); and50.6%ethanol,81.3min, and63.4℃for maximal ABTS radical-scavenging capacity (92.9±0.5%). Our studies have revealed that ethanol concentration was the most important factor in the extraction process, the TFC and their antioxidant activity was positively correlated with better consistency.(2) Antioxidant evaluation of crude extract of C. cyrtophyllum leaves and each extracted fractions:Fraction n-Butanol has the maximal TFC and TPC, which are57.52±0.13mgRT/gDW,5.1±0.019mgGAE/gDW. Scavenging ability was expressed by IC50and EC50. Ethyl acetate fraction had the highest scavenging DPPH radical, ABTS radical, superoxide radical ability, which IC50of0.36±0.006mg.ml-1and0.102±0.052mg.ml-1; fraction n-Butanol has the highest scavenging effect on hydroxyl radical, which IC50is0.653±0.08mg.ml-1; fraction chloroform has the highest chelating ability, which IC50of0.742±0.014mg.ml-1; fraction ethyl acetate has the highest reducing power, which EC50of0.203±0.008mg.ml-1.Our studies have shown that TFC greater than TPC in each fractions and crude extract of C. cyrtophyllum leaves, and TFC and TPC has a positive correlation(P=0.1097>0.05). Ethyl acetate fraction and n-Butanol fraction had the higher antioxidant activity.(3) Chemical investigation of C. cyrtophyllum leaves:6compounds were isolated from chloroform fraction of C. cyrtophyllum leaves using various chromatographic methods, such as silica gelcolumn chromatography, polyamide column chromatography and polydextran gel SephadexLH-20column chromatography. Four compounds were identified as Cirsilineol(1), Cirsimartin(2), Cirsilineol-4’-glucoside(3) and22-dehydroclerosterol3p-O-β-D-glucopyranoside(4). Their structures were elucidated on the basis of spectroscopic data (HPLC、 ESI-MS、 IDNMR、2DNMR). Amongst, cirsimartin and22-dehydroclerostero13β-O-β-D-glucopyranoside were isolated from C. cyrtophyllum leaves for the first time. This study offers theoretic basis for utilization of C. cyrtophyllum leaves as a potential resource of natural antioxidants and its antioxidant mechanism was primary esTablelished.