金银花黄酮活性成分分离纯化与生物学效应研究
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摘要
金银花为常用的传统中草药,功能清热解毒,临床上主要治疗细菌感染、感冒等症。现代药理研究显示金银花中主要化学成分有黄酮类、有机酸类、苷类及挥发油等。其中苷类中三萜皂苷类成分主要与金银花保肝利胆作用有关,而以绿原酸为主的有机酸类化合物和挥发油被认为与其抗菌活性相关。但金银花黄酮是其组成中仍未经充分药理学研究的成分,其相关的生物学活性研究甚少。对其它植物药黄酮的研究提示,该类成分多具有抗菌和抗氧化作用,因此本研究在国家自然科学基金的支持下,对金银花中黄酮类物质进行分离纯化,并考察其生物学效应,为说明金银花发挥药理作用的机制奠定实验基础。
第一部分金银花总黄酮的分离纯化及抑菌活性考察
一、实验方法:
1.金银花总黄酮的提取金银花总黄酮以醇提法提取,并采用均匀设计对提取条件进行优化。
2.金银花总黄酮初步分离及抑菌活性考察金银花总黄酮浸膏以DM130型大孔吸附树脂柱采用不同浓度乙醇(0,20%,40%,60%)梯度洗脱进行初步分离,对获得的分离组分按A、B、C等依序命名。微量稀释法考察各分离组分对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌的MIC值。
3.金银花黄酮组分分离纯化及抑菌活性考察对方法2中分离所得且体外抑菌活性较好的黄酮组分采用制备型HPLC分离制备,对制备组分按A1、A2、…B1、B2等依序命名,并采用微量稀释法考察各制备组份对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌体外抑菌活性。
4.金银花黄酮抑菌活性成分结构鉴定对方法3中制备所得且体外抑菌活性较好组分采用HPLC分离提纯,并对纯化分离所得纯度较高单一成分进行LC-MS、1H NMR、13C NMR等鉴定。
二、实验结果:
1.金银花总黄酮醇提法最佳提取工艺为:在10g金银花粉末(60目)中加入10倍体积50%乙醇溶液于90℃条件下提取1h。
2.金银花总黄酮浸膏经大孔吸附树脂柱分离后共收集到5组含有黄酮的洗脱液(0%洗脱组分按洗脱液的澄清与否分为A、B两组分;C为20%洗脱组分,D为40%洗脱组分,E为60%洗脱组分),各分离的洗脱组分对金黄色葡萄球菌、肺炎克雷伯菌、大肠杆菌、铜绿假单胞菌等细菌均有抑制作用,组分B、C抑菌作用优于其他组分,对耐甲氧西林金黄色葡萄球菌(Methicillin Resistant Staphylococcus aureus, MRSA)的MIC值均≤4 mg·mL-1。
3. B组分经HPLC初步纯化制备共获得6个组分(B1～B6)、C组分共获得2个组分(C1～C2),其中C2组分抑菌效果较好且稳定,对MRSA的MIC值≤2 mg·mL-1。
4. C2组分经HPLC进一步分离纯化,得到一纯度较高单一化合物,经LC-MS、1H NMR、13C NMR等进行结构鉴定为金丝桃苷。
第二部分金丝桃苷体外抑菌活性的研究
一、实验方法:
1.金丝桃苷体外抑菌活性初步考察微量稀释法考察金丝桃苷对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌的体外抑菌活性。
2.金丝桃苷与抗菌药物联用后对金黄色葡萄球菌体外抑菌活性考察采用棋盘法考察金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物联用后对金黄色葡萄球菌(n=20)抑菌活性,计算FIC值。
3.金丝桃苷与绿原酸联用后体外抑菌活性考察棋盘法考察金丝桃苷与绿原酸联用后对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌的抑菌活性,计算FIC值。
4.金丝桃苷对金黄色葡萄球菌粘附能力的影响采用半定量粘附法从20株金黄色葡萄球菌中筛选具有较强粘附能力的菌株;再对所筛选出的细菌采用粘附抑制实验考察不同浓度(0.03125、0.0625、0.125、0.25、0.5mg.mL-1)金丝桃苷对其粘附能力的影响。
5.金丝桃苷对金黄色葡萄球菌NorA外排泵的影响
采用携带NorA基因的金黄色葡萄球菌SA26592(pUT-norA)菌株为工具菌,通过PCR扩增的方法确认该菌株携带NorA基因。采用微量稀释法考察金丝桃苷与常用喹诺酮类药物联用后对金黄色葡萄球菌SA26592(pUT-norA)菌株的抑菌活性。
二、实验结果:
1.金丝桃苷对金黄色葡萄球菌体外抑菌作用最强,MIC为0.5～1mg·mL-1,对大肠杆菌,铜绿假单胞菌,肺炎克雷伯菌作用相当,均为2 mg·mL-1。
2.金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物联用,对20株金黄色葡萄球菌的抑菌实验结果显示,金丝桃苷与苯唑西林钠、青霉素钠、加替沙星和左氧氟沙星联用后,表现为相加作用的菌株分别占55%、30%、25%和15%。表明金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物存在一定的联用效果。
3.金丝桃苷与绿原酸联用,仅对铜绿假单胞菌ATCC27853菌株表现为明显的相加作用(FIC为0.75),其余多表现为无关作用。
4.金丝桃苷浓度低于MIC时(<0.5mg.mL-1),不能抑制金黄色葡萄球菌的生长,对其粘附能力亦无明显影响。
5. SA26592(pUT-norA)菌株药敏结果显示,该菌株对新霉素、卡那霉素耐药,对其它抗生素敏感。SA26592(pUT-norA)菌株对环丙沙星、氧氟沙星等亲水性喹诺酮类药物的MIC,较之于司帕沙星等疏水性喹诺酮类药物增加了32~64倍。
6.金丝桃苷在1/2 MIC浓度时(0.25mg·mL-1),能显著降低环丙沙星、氧氟沙星等亲水性喹诺酮类药物对SA26592(pUT-norA)菌株的MIC值,而对疏水性喹诺酮类药物影响不大。
第三部分金丝桃苷对CCl4诱导肝损伤模型的保护作用实验研究
一、实验方法:
1.确定CCl4诱导L-02人肝细胞损伤的最佳浓度在L-02人肝细胞培养液中加入不同浓度(2.5,5,10,15,20,30mM)的CCl4,MTT法检测存活率,确定CCl4诱导肝细胞损伤的最佳浓度。
2.金丝桃苷对CCl4诱导L-02人肝细胞损伤的影响向肝细胞培养液中加入CCl4及不同浓度金丝桃苷(5～160μg·mL-1),用MTT比色法检测细胞存活率;用赖氏法检测培养上清中ALT、AST含量;裂解细胞后,用硫代巴比妥酸法检测MDA含量,用比色法检测GSH水平。
3.金丝桃苷对CCl4诱导大鼠急性肝损伤模型的保护作用成年雄性SD大鼠36只随机分为6组(每组6只),①正常对照组、②CCl4损伤组、③金丝桃苷组(30 mg·kg-1)、④～⑥金丝桃苷+CCl4组(金丝桃苷剂量分别为10 mg·kg-1, 20 mg·kg-1, 30 mg·kg-1)。①和②给予生理盐水、③给予金丝桃苷30 mg·kg-1、④～⑥给予设定剂量的金丝桃苷,连续灌胃三天,于末次给药后6h灌胃给予1.25 mL·kg-1 CCl4液致急性中毒。24h后断颈处死所有大鼠,收集大鼠血液及肝脏组织进行检测。
4.大鼠生化指标检测用赖氏法检测大鼠血清中ALT、AST含量;肝脏组织中MDA、GSH及8-OHdG水平检测分别采用硫代巴比妥酸法、比色法及ELISA法。
5.病理检查取大鼠肝脏组织常规HE染色,光学显微镜下观察。
二、实验结果:
1. CCl4浓度为15mM时为肝细胞损伤模型最理想的造模浓度。
2.金丝桃苷5、10、20、40、80、160μg·mL-16个剂量组中, 80μg·mL-1剂量组肝细胞保护作用显著,明显降低ALT、AST、MDA含量,抑制GSH水平降低,且光镜下细胞结构未见明显损伤,MTT结果显示肝细胞存活率增加。
3.金丝桃苷10 mg·kg-1, 20 mg·kg-1, 30 mg·kg-1剂量组均有一定肝保护效应,肝脏组织病理检查和各项生化指标与模型组比较均有明显改善(p<0.05),其中30 mg·kg-1剂量组各项生化指标与正常对照组最接近。
4.金丝桃苷组(30 mg·kg-1)连续灌胃三天对大鼠肝脏无明显毒性。
全文结论
1.金银花中黄酮类物质具有较强的抗菌活性,其中金丝桃苷具有较强的体外抑菌作用,特别是对MRSA作用明显,MIC达0.5 mg.mL-1。因此金银花黄酮应成为评价金银花抗菌活性的重要指标。
2.金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物合用,对临床常见致病菌部分菌株有一定增效作用。
3.金丝桃苷浓度低于MIC时(<0.5mg.mL-1),对金黄色葡萄球菌的粘附能力无明显影响。但能显著增强环丙沙星、氧氟沙星等亲水性喹诺酮类药物对多药外排耐药菌株SA26592(pUT-norA)的抗菌作用,而对疏水性喹诺酮类药物影响不大。
4.金丝桃苷能明显减轻CCl4诱导的L-02肝细胞株的损伤,显著降低ALT、AST、MDA水平,增加GSH水平,增加细胞存活率。
5.金丝桃苷对于CCl4诱导的SD大鼠急性肝损伤具有保护效应,组织学和各项生化指标均有明显改善(p<0.05),其中30 mg·kg-1剂量组各项生化指标已接近正常。
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 japonica
Methods:
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 hydrophobic
quinolones.
Section 3. Hepatoprotection effect of hyperoside against carbon tetrachloride- induced acute liver injury in vitro and in vivo
Methods:
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.
第一部分金银花总黄酮的分离纯化及抑菌活性考察
一、实验方法:
1.金银花总黄酮的提取金银花总黄酮以醇提法提取,并采用均匀设计对提取条件进行优化。
2.金银花总黄酮初步分离及抑菌活性考察金银花总黄酮浸膏以DM130型大孔吸附树脂柱采用不同浓度乙醇(0,20%,40%,60%)梯度洗脱进行初步分离,对获得的分离组分按A、B、C等依序命名。微量稀释法考察各分离组分对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌的MIC值。
3.金银花黄酮组分分离纯化及抑菌活性考察对方法2中分离所得且体外抑菌活性较好的黄酮组分采用制备型HPLC分离制备,对制备组分按A1、A2、…B1、B2等依序命名,并采用微量稀释法考察各制备组份对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌体外抑菌活性。
4.金银花黄酮抑菌活性成分结构鉴定对方法3中制备所得且体外抑菌活性较好组分采用HPLC分离提纯,并对纯化分离所得纯度较高单一成分进行LC-MS、1H NMR、13C NMR等鉴定。
二、实验结果:
1.金银花总黄酮醇提法最佳提取工艺为:在10g金银花粉末(60目)中加入10倍体积50%乙醇溶液于90℃条件下提取1h。
2.金银花总黄酮浸膏经大孔吸附树脂柱分离后共收集到5组含有黄酮的洗脱液(0%洗脱组分按洗脱液的澄清与否分为A、B两组分;C为20%洗脱组分,D为40%洗脱组分,E为60%洗脱组分),各分离的洗脱组分对金黄色葡萄球菌、肺炎克雷伯菌、大肠杆菌、铜绿假单胞菌等细菌均有抑制作用,组分B、C抑菌作用优于其他组分,对耐甲氧西林金黄色葡萄球菌(Methicillin Resistant Staphylococcus aureus, MRSA)的MIC值均≤4 mg·mL-1。
3. B组分经HPLC初步纯化制备共获得6个组分(B1～B6)、C组分共获得2个组分(C1～C2),其中C2组分抑菌效果较好且稳定,对MRSA的MIC值≤2 mg·mL-1。
4. C2组分经HPLC进一步分离纯化,得到一纯度较高单一化合物,经LC-MS、1H NMR、13C NMR等进行结构鉴定为金丝桃苷。
第二部分金丝桃苷体外抑菌活性的研究
一、实验方法:
1.金丝桃苷体外抑菌活性初步考察微量稀释法考察金丝桃苷对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌的体外抑菌活性。
2.金丝桃苷与抗菌药物联用后对金黄色葡萄球菌体外抑菌活性考察采用棋盘法考察金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物联用后对金黄色葡萄球菌(n=20)抑菌活性,计算FIC值。
3.金丝桃苷与绿原酸联用后体外抑菌活性考察棋盘法考察金丝桃苷与绿原酸联用后对金黄色葡萄球菌(n=7)、肺炎克雷伯菌(n=5)、大肠杆菌(n=6)、铜绿假单胞菌(n=4)等细菌的抑菌活性,计算FIC值。
4.金丝桃苷对金黄色葡萄球菌粘附能力的影响采用半定量粘附法从20株金黄色葡萄球菌中筛选具有较强粘附能力的菌株;再对所筛选出的细菌采用粘附抑制实验考察不同浓度(0.03125、0.0625、0.125、0.25、0.5mg.mL-1)金丝桃苷对其粘附能力的影响。
5.金丝桃苷对金黄色葡萄球菌NorA外排泵的影响
采用携带NorA基因的金黄色葡萄球菌SA26592(pUT-norA)菌株为工具菌,通过PCR扩增的方法确认该菌株携带NorA基因。采用微量稀释法考察金丝桃苷与常用喹诺酮类药物联用后对金黄色葡萄球菌SA26592(pUT-norA)菌株的抑菌活性。
二、实验结果:
1.金丝桃苷对金黄色葡萄球菌体外抑菌作用最强,MIC为0.5～1mg·mL-1,对大肠杆菌,铜绿假单胞菌,肺炎克雷伯菌作用相当,均为2 mg·mL-1。
2.金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物联用,对20株金黄色葡萄球菌的抑菌实验结果显示,金丝桃苷与苯唑西林钠、青霉素钠、加替沙星和左氧氟沙星联用后,表现为相加作用的菌株分别占55%、30%、25%和15%。表明金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物存在一定的联用效果。
3.金丝桃苷与绿原酸联用,仅对铜绿假单胞菌ATCC27853菌株表现为明显的相加作用(FIC为0.75),其余多表现为无关作用。
4.金丝桃苷浓度低于MIC时(<0.5mg.mL-1),不能抑制金黄色葡萄球菌的生长,对其粘附能力亦无明显影响。
5. SA26592(pUT-norA)菌株药敏结果显示,该菌株对新霉素、卡那霉素耐药,对其它抗生素敏感。SA26592(pUT-norA)菌株对环丙沙星、氧氟沙星等亲水性喹诺酮类药物的MIC,较之于司帕沙星等疏水性喹诺酮类药物增加了32~64倍。
6.金丝桃苷在1/2 MIC浓度时(0.25mg·mL-1),能显著降低环丙沙星、氧氟沙星等亲水性喹诺酮类药物对SA26592(pUT-norA)菌株的MIC值,而对疏水性喹诺酮类药物影响不大。
第三部分金丝桃苷对CCl4诱导肝损伤模型的保护作用实验研究
一、实验方法:
1.确定CCl4诱导L-02人肝细胞损伤的最佳浓度在L-02人肝细胞培养液中加入不同浓度(2.5,5,10,15,20,30mM)的CCl4,MTT法检测存活率,确定CCl4诱导肝细胞损伤的最佳浓度。
2.金丝桃苷对CCl4诱导L-02人肝细胞损伤的影响向肝细胞培养液中加入CCl4及不同浓度金丝桃苷(5～160μg·mL-1),用MTT比色法检测细胞存活率;用赖氏法检测培养上清中ALT、AST含量;裂解细胞后,用硫代巴比妥酸法检测MDA含量,用比色法检测GSH水平。
3.金丝桃苷对CCl4诱导大鼠急性肝损伤模型的保护作用成年雄性SD大鼠36只随机分为6组(每组6只),①正常对照组、②CCl4损伤组、③金丝桃苷组(30 mg·kg-1)、④～⑥金丝桃苷+CCl4组(金丝桃苷剂量分别为10 mg·kg-1, 20 mg·kg-1, 30 mg·kg-1)。①和②给予生理盐水、③给予金丝桃苷30 mg·kg-1、④～⑥给予设定剂量的金丝桃苷,连续灌胃三天,于末次给药后6h灌胃给予1.25 mL·kg-1 CCl4液致急性中毒。24h后断颈处死所有大鼠,收集大鼠血液及肝脏组织进行检测。
4.大鼠生化指标检测用赖氏法检测大鼠血清中ALT、AST含量;肝脏组织中MDA、GSH及8-OHdG水平检测分别采用硫代巴比妥酸法、比色法及ELISA法。
5.病理检查取大鼠肝脏组织常规HE染色,光学显微镜下观察。
二、实验结果:
1. CCl4浓度为15mM时为肝细胞损伤模型最理想的造模浓度。
2.金丝桃苷5、10、20、40、80、160μg·mL-16个剂量组中, 80μg·mL-1剂量组肝细胞保护作用显著,明显降低ALT、AST、MDA含量,抑制GSH水平降低,且光镜下细胞结构未见明显损伤,MTT结果显示肝细胞存活率增加。
3.金丝桃苷10 mg·kg-1, 20 mg·kg-1, 30 mg·kg-1剂量组均有一定肝保护效应,肝脏组织病理检查和各项生化指标与模型组比较均有明显改善(p<0.05),其中30 mg·kg-1剂量组各项生化指标与正常对照组最接近。
4.金丝桃苷组(30 mg·kg-1)连续灌胃三天对大鼠肝脏无明显毒性。
全文结论
1.金银花中黄酮类物质具有较强的抗菌活性,其中金丝桃苷具有较强的体外抑菌作用,特别是对MRSA作用明显,MIC达0.5 mg.mL-1。因此金银花黄酮应成为评价金银花抗菌活性的重要指标。
2.金丝桃苷与β-内酰胺类及喹诺酮类抗菌药物合用,对临床常见致病菌部分菌株有一定增效作用。
3.金丝桃苷浓度低于MIC时(<0.5mg.mL-1),对金黄色葡萄球菌的粘附能力无明显影响。但能显著增强环丙沙星、氧氟沙星等亲水性喹诺酮类药物对多药外排耐药菌株SA26592(pUT-norA)的抗菌作用,而对疏水性喹诺酮类药物影响不大。
4.金丝桃苷能明显减轻CCl4诱导的L-02肝细胞株的损伤,显著降低ALT、AST、MDA水平,增加GSH水平,增加细胞存活率。
5.金丝桃苷对于CCl4诱导的SD大鼠急性肝损伤具有保护效应,组织学和各项生化指标均有明显改善(p<0.05),其中30 mg·kg-1剂量组各项生化指标已接近正常。
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 japonica
Methods:
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 hydrophobic
quinolones.
Section 3. Hepatoprotection effect of hyperoside against carbon tetrachloride- induced acute liver injury in vitro and in vivo
Methods:
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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