| Lonicera japonica, a traditional Chinese herb medicine known as its effects of heat-cleaning and detoxification, has been used to mainly treat bacterial infections and common cold in clinic practice. There were several active components including flavonoids, organic acids, glycosides and volatile has been found in this herb medicine by modern pharmacological studies. Among them, the triterpenoid saponin in glycosides, caffeotannic acid in organic acids and aetherolea are believed to be associated with the hepatoprotective and antibacterial effects, respectively. However, little investigation on the bioactivities of flavonoids of Lonicera japonica was carried out to date. Since the results that there had been a antibacterial and antioxidation effects found with flavonoids extracted from other herb medicine, in order to understand better the pharmacological basis of lonicera japonica, the active flavonoid components were extracted and then the bioactivities were investigated in the present study. This work was supported by grants from National Natural Science Foundation, China (30572366).Section 1. Isolation and antimicrobial effects test of crude flavonoids from lonicera japonicaMethods:1. Extraction of crude flavonoids from lonicera japonica The crude flavonoids in lonicera japonica was extracted by the method"alcohol extract technology"and optimized by"Even design".2. Preliminary separation of different components in crude flavonoids and antibacterial effect test The crude flavonoids was separated by macroporous adsorption resin and eluted with gradient concentrations (0, 20, 40, and 60%) of alcohol, then the different elution that contained components were designated as A, B…and so on, respectively. The minimal inhibitory concentration (MIC) of the components for Staphylococcus aureus(n=7), Klebsiella pneumonia(n=5), Escherichia coli(n=6) and Pseudomonas Aeruginosa(n=4) were determined by micro-dilution method, respectively.3. Separation of active flavonoid components and antibacterial effect test The components which exhibited significant antimicrobial effects obtained from method 2 was further separated by HPLC, then the prepared components were designated as A1, A2…B1,B2 and so on, respectively. The minimal inhibitory concentration (MIC) of each prepared components for Staphylococcus aureus(n=7), Klebsiella pneumonia(n=5), Escherichia coli(n=6) and Pseudomonas Aeruginosa(n=4) were determined by micro-dilution method, respectively.4. Structure identification of active flavonoid monomer The components which exhibited significant antimicrobial effects obtained from method 3 was further purified by HPLC, then the structure of monomer component with highest purification was identified by LC-MS, 13CNMR,1HNMR.Results:1. The optimum extraction method of crude flavonoids in lonicera japonica was established as follows: The crude flavonoids was extracted from lonicera japonica powder (10.0g, 60 mesh) by added 10 times solvent of ethanol concentration 50% (weight : volume = 1 : 10), at 90℃for 1 h.2. There had been total 5 flavonoids elutions contained flavonoids obtained with macroporous adsorption resin and eluted with gradient concentrations of alcohol. Elution A and B was obtained by 0% alcohol with a difference of clarity between them. Elution C by 20%, D by 40% and E by 60%l, respectively. All 5 elutions contained flavonoids were found with a significant bactericidal effects, especially B and C, the both had a similar low MIC value of≤4 mg·mL-1 for methicillin resistant staphylococcus aureus (MRSA) strains.3. There were 6 components (B1~B6) from the elution B and 2 (C1~C2) from elution C were prearaed by HPLC, respectively. Among them, component C2 had a MIC of≤2 mg·mL-1 on MRSA strains.4. A monomer component with highest purification was isolated from C2 by HPLC and identified as hyperoside by LC-MS, 1H NMR and 13C NMR. Section 2. Study of antibacterial effects of hyperoside in vitro.Methods: 1. Preliminary study of antibacterial effects of hyperoside in vitro The MICs of hyperoside for the strains of Staphylococcus aureus(n=7), Klebsiella pneumonia(n=5), Escherichia coli(n=6) and Pseudomonas Aeruginosa(n=4) were determined by micro-dilution method.2. Study of antibacterial effects of hyperoside combined with antibiotics in vitro The antimicrobial effects of hyperoside combined withβ-lactams or quinolones for the strains of Staphylococcus aureus(n=20) were measured by checkerboard method, respectively. The FIC values were calculated to indicate the combined antibacterial effect.3. Study of antibacterial effects of hyperoside combined with chlorogenic acid in vitro The antibacterial effects of hyperoside combined with chlorogenic acid for the strains of Staphylococcus aureus (SA)(n=7), Klebsiella pneumonia(n=5), Escherichia coli(n=6) and Pseudomonas Aeruginosa(n=4) were evaluated by checkerboard method. The FIC values were calculated to indicate the combined antibacterial effect.4. The effect of hyperoside on adhesion ability of SA 11strains of SA with a obvious adhesion ability were elected from 20 clinical isolations by demi-quantitation of bacterial adhesion. Then the adhesion of elected SA strains were observed when treated with hyperoside at concentrations of 0.03125,0.0625,0.125,0.25,0.5mg.mL-1.5. Study of antibacterial effects of hyperoside combined with quinolones for SA26592 strain(pUT-norA) in vitro The expression of NorA gene in strain SA26592 was confirmed by PCR. The antimicrobial resistant characteristics of SA26592 was examined by K-B slip. The antibacterial effects of hyperoside combined with quinolones for SA26592 were measured by micro-dilution method.Results:1. Hyperoside showed a excellent antibacterial effect on SA strains with a low MIC of 0.5~1mg/ml, and the MIC of 2mg/ml for strains of Escherichia Coli, Pseudomonas Aeruginosa, Klebsiella pneumonia were observed.2. The FIC index indicated that the addition effects were found in 55%,30%,25% and 15% of MRSA strains ( n=20) when hyperoside combined with oxacillin, benzylpenicillin, gatifloxacin and levofloxacin, respectively. The results suggested that hyperoside could enhance the anti-MRSA efficency of theseβ-lactams or quinolones. 3. Hyperoside combined with chlorogenic acid showed an obvious cumulate bactericidal action on Pseudomonas aeruginosa ATCC27853 with a FIC index of 0.75, but not for other strains.4. Hyperoside had no significant effects on bacteria growth and adhesion at sub-MIC concentration under 0.5mg.mL-1.5. SA26592(pUT-norA)was only resistant to neomycin and Kanamycin. The MICs of hydrophilic quinolones (ciprofloxacin, ofloxacin) were 32~64 times higher than that of hydrophobic quinolones (sparfloxacin) on SA26592.6. Hyperoside at the half-MIC (0.25mg.mL-1) could make MICs of ciprofloxacin or ofloxacin for SA26592 decrease obviously, but little changes found for the hydrophobicquinolones.Section 3. Hepatoprotection effect of hyperoside against carbon tetrachloride- induced acute liver injury in vitro and in vivoMethods:1. Determination of the appropriate CCl4 concentration to induce the L-02 hepatocytes injury The appropriate CCl4 concentration to induce the hepatocytes injury was determined according to the survival rates of L-02 hepatocytes incubated with series concentrations (2.5,5,10,15,20,30mmol·L-1)of CCl4. The cell survival rate was measured by MTT method.2. The effects of hyperoside on the L-02 hepatocytes injuried by CCl4 CCl4 solutions were added to hepatocytes in present of various concentrations of hyperoside (5～160μg·mL-1) and the MTT method was used to test the survival rate of cells. The concentrations of ALT and AST in supernatants were determined by King's method. After cells lysis, the MDA and GSH in hepatocytes were measured by TBARS and colorimetric method, respectively.3. Hepatoprotection effect of hyperoside against CCl4 -induced acute liver injury in rats 36 male SD rats were divided into six groups (six in each) including①normal control,②the injury model,③hyperoside alone(30 mg·kg-1), and④～⑥injury rats treated with hyperoside (10 mg·kg-1, 20 mg·kg-1, 30 mg·kg-1).The same volume of saline for①and②, hyperoside of 30 mg·kg-1 for③and the designed concentrations of hyperoside for④～⑥were administered by gavage, respectively, once a day for 3 days. Then rats in②and④～⑥were received CCl4 1.25 ml·kg-1 by gavage at 6 h after the administration of saline or hyperoside, and 24 h after which, all rats were sacrificed under ether anesthesia, and their blood and liver samples were collected and analyzed.4. Test of biochemical index of rats The concentrations of ALT and AST in serum were determined by King's method. The levels of MDA, GSH and 8-OHdG in hepatocytes were measured by TBARS, colorimetric method and ELISA, respectively.5. Pathological examination of liver tissue The histopathology examination of liver tissue samples was observed under microscope by HE stain.Results:1. The appropriate concentration of CCl4 that could induce hepatocytes injury was determined as 15mM.2. In the 6 concentrations of hyperoside, 80μg·mL-1 exhibited the best protective effects for hepatocytes injuried by CCl4, characterized as the levels of ALT, AST and MDA decreasing, elevation of GSH level and survival hepatocytes increasing with little damage in cell structure.3. The significant hepatoprotective effects for CCl4-attatcked rats were found in three dosages of hyperoside (10 mg·kg-1, 20 mg·kg-1, and 30 mg·kg-1) with the obvious improve biochemical indexes and liver histopathology examination. The results of animals received hyperoside of 30 mg/kg were almost similar to that of normal controls.4.There were not any liver toxic reactions observed in rats gavaged hyperoside of 30 mg·kg-1 per day for 3 days.Conclusions:1. Flavonoids from lonicera japonica had a strong antibacterial action, especially for MRSA (MIC≤5 mg·mL-1). This result suggested that flavonoids should be regarded as an important index in the bacterical effects evaluation of lonicera japonica.2. Hyperoside combined withβ-lactams or quinolones could make the antibacterial effects better on some strains of common pathogenic bacteria.3. At the sub-MIC concentration (<0.5mg.mL-1), hyperoside had no significant inhibitory effects on the adhesion ability of MRSA, but could enhance the antibacterial effects of hydrophilic quinolones on bacteria SA26592(pUT-norA).4. Hyperoside could relieve cell injury induced by CCl4 in hepatocyte L-02 with the level of ALT, AST and MDA decrease, GSH and cell survival rate increase.5. Hyperoside showed a significant hepatoprotective effect in CCl4-attatcked rats at three dosages used in this study. The biochemical indexes and liver histopathology examination of rats treated with hyperoside of 30 mg·kg-1 were almost similar to that of untreated animals.
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