白桦树皮提取物肝保护及降血脂作用研究
|
摘要
目的:1.建立高效液相色谱法测定白桦树皮中白桦酯醇和白桦酯酸含量的方法:2.探讨白桦树皮提取物对小鼠爆发性肝损伤的保护作用;3.通过建立高脂血症小鼠模型研究白桦树皮提取物(BHSP)的降血脂和抗氧化作用。方法:1.色谱柱:Shim-pack VP-ODS(150L×4.6),流动相为甲醇—水(86:14),检测波长210 nm,流速:1.0 ml/min,柱温30℃;2.采用D-氨基半乳糖(D-Galactosamine)和内毒素(Lipopolysaccharide),造小鼠爆发性肝损伤模型,观察白桦树皮提取物对爆发性肝损伤小鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST);同时观察白桦树皮提取物对急性肝损伤小鼠生存率的影响;肝组织中组织中丙二醛(MDA)、谷胱甘肽(GSH)等水平来进行评价,病理组织切片和免疫组化来观察白桦树皮提取物对肝损伤的保护作用;3.先用不同剂量的白桦树皮提取物(100 mg/kg,200 mg/kg和300 mg/kg)预防给药7天,末次给药2 h后采用腹腔注射75%蛋黄乳液制作小鼠高脂血症模型,造模12 h后测定不同剂量的白桦树皮提取物对75%蛋黄乳液诱导的小鼠血脂水平、血清生化指标和肝脏中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性及脂质过氧化产物丙二醛(MDA)含量的影响。结果:1.白桦酯醇在0.54～3.78μg范围内呈良好的线性关系(r=0.9997),白桦酯酸在0.045～0.315μg范围内呈良好的线性关系(r=0.9995),平均回收率为白桦酯醇104.5%,白桦酯酸99.9%,白桦酯醇RSD=0.94%,白桦酯酸RSD=1.03%;2.白桦树皮提取物明显降低因D-氨基半乳糖和内毒素合用导致中毒小鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)活力升高;同时可提高肝损伤小鼠的生存率;明显抑制了肝组织中MDA的升高和GSH的降低,HIF-1α和Caspase阳性表达率明显降低,减少了肝损伤程度;3.白桦树皮提取物能够显著降低小鼠血清中TC、TG、LDL-C浓度和ALT、AST活性,显著升高HDL-C浓度;显著降低肝脏中MDA含量,提高肝脏中SOD和GSH-Px活性。结论:1.结果表明此方法操作简单,准确,灵敏度高,重现性好,可以同时对白桦树皮提取物中的白桦酯醇和白桦酯酸的含量进行测定;2.白桦树皮提取物对D-氨基半乳糖和内毒素所致小鼠爆发性肝衰竭具有良好的保护作用;3.白桦树皮提取物具有明显的降血脂和抗氧化作用。
Objective 1.A simple procedure is described for the simultaneous extraction and determination of betulin and betulinic acid in white birch bark;2.To study the protective effect of white birch bark extract(BH) against experimental acute liver injury in mice;3.Establish the mice model of hyperlipidemia to research the blood lipid-lower and antioxidant effects of white birch bark extract.Methods 1.The analytical column Supelcosil RP-18(250 mm×4.6 mm×5μm) was used,with methanol-water 86:14(v/v),at a flow rate of 1.0 ml/min.The column temperature was set at 30℃.UV detection was set at 210 nm;2.Liver injury model induced by D-GalN/LPS,pretreated with extract of BH in mice.After 8 hour,the mice were decapitated and their blood and livers were collected.The enzyme activities of serum ALT,AST by automatic biochemistry analyzer,Simultaneously,the histopathological change and the expression of HIF-1αand Caspase-3 were investigated;3.i.g.different doses of the white bark extract(100 mg/kg,200 mg/kg and 300 mg/kg) and zhinbiticose(300 mg/kg) for 7days for prevention,i.p 75%egg yolk emulsion 2 hours after the last time of give the drug to establish the mice model of hyperlipidemia.The lipid levels and enzyme activities of serum,and the activities of MDA,SOD and GSH-Px in mice liver tissue were measured 12 h after the model established.Results 1.The linear range of betulin was 0.54μg～3.78μg,(r= 0.9995),and the linear range of betulin acid was 0.045μg～0.315μg(r=0.9997). Average recovery rate of betulin was 104.5%,and average recovery rate of betulinic acid was 99.9%.Relative standard deviation of betulin was 0.94%,and relative standard deviation of betulinic acid was 1.03%;2.Compared with control group,BH not only decreased the activities of ALT,AST in serum but also decreased the production of MDA level in liver tissue and kept the level of GSH activities in normal,The pathological section shows that BH has protective effect on mice,which suffered liver injury from liver injury.The expression of HIF-1αand Caspase-3 were also suppressed by BH;3.White birch bark extract can be a significant reduction in serum TC,TG,LDL-C levels,and significantly increased HDL-C concentration; Decrease activities of marker enzymes(AST) in serum and MDA in liver tissue,and increased activity of SOD and GSH-Px levels in liver tissue.Conclusion 1.The HPLC method mentioned here represented an excellent technique for simultaneous determination of betulin and betulinic acid in the extract of white birch bark,with good sensitivity,precision and reproducibility;2.White birch bark extract can protect mice from acute liver injury efficiently;3.White birch bark extract showed significantly hypolipidemic and antioxidant effects.
Objective 1.A simple procedure is described for the simultaneous extraction and determination of betulin and betulinic acid in white birch bark;2.To study the protective effect of white birch bark extract(BH) against experimental acute liver injury in mice;3.Establish the mice model of hyperlipidemia to research the blood lipid-lower and antioxidant effects of white birch bark extract.Methods 1.The analytical column Supelcosil RP-18(250 mm×4.6 mm×5μm) was used,with methanol-water 86:14(v/v),at a flow rate of 1.0 ml/min.The column temperature was set at 30℃.UV detection was set at 210 nm;2.Liver injury model induced by D-GalN/LPS,pretreated with extract of BH in mice.After 8 hour,the mice were decapitated and their blood and livers were collected.The enzyme activities of serum ALT,AST by automatic biochemistry analyzer,Simultaneously,the histopathological change and the expression of HIF-1αand Caspase-3 were investigated;3.i.g.different doses of the white bark extract(100 mg/kg,200 mg/kg and 300 mg/kg) and zhinbiticose(300 mg/kg) for 7days for prevention,i.p 75%egg yolk emulsion 2 hours after the last time of give the drug to establish the mice model of hyperlipidemia.The lipid levels and enzyme activities of serum,and the activities of MDA,SOD and GSH-Px in mice liver tissue were measured 12 h after the model established.Results 1.The linear range of betulin was 0.54μg～3.78μg,(r= 0.9995),and the linear range of betulin acid was 0.045μg～0.315μg(r=0.9997). Average recovery rate of betulin was 104.5%,and average recovery rate of betulinic acid was 99.9%.Relative standard deviation of betulin was 0.94%,and relative standard deviation of betulinic acid was 1.03%;2.Compared with control group,BH not only decreased the activities of ALT,AST in serum but also decreased the production of MDA level in liver tissue and kept the level of GSH activities in normal,The pathological section shows that BH has protective effect on mice,which suffered liver injury from liver injury.The expression of HIF-1αand Caspase-3 were also suppressed by BH;3.White birch bark extract can be a significant reduction in serum TC,TG,LDL-C levels,and significantly increased HDL-C concentration; Decrease activities of marker enzymes(AST) in serum and MDA in liver tissue,and increased activity of SOD and GSH-Px levels in liver tissue.Conclusion 1.The HPLC method mentioned here represented an excellent technique for simultaneous determination of betulin and betulinic acid in the extract of white birch bark,with good sensitivity,precision and reproducibility;2.White birch bark extract can protect mice from acute liver injury efficiently;3.White birch bark extract showed significantly hypolipidemic and antioxidant effects.
引文
[1] 崔艳霞，郑志方．白桦皮化学组成的研究．东北林业大学学报，1994，22(4)：268-270
[2] 韩英，李智纬，李岩等．桦树皮抗肿瘤作用及对免疫功能的影响．中药材，2000，23(6)：343-345．
[3] 李薇，李岩，金雄杰．白桦三萜类物质的抗肿瘤作用及其对免疫功能的增强效应．中国免疫学杂志[J]，2000，16(9)：485-487．
[4] Hwang, J.M., Tseng, T.H., Tsai, Y.Y., Lee, H.J., Chou, F.P., Wang, C.J., Chu, C.Y.. Protective effects of baicalein on tert-butyl hydroperoxide-induced hepatic toxicity in rat hepatocytes. Journal of Biomedical Science, 2005,12,389-397.
[5] Zou, Y.H., Yang, Y., Li, J., Li, W.P., Wu, Q.. Prevention of hepatic injury atraditional Chinese formulation, BJ-JN, in mice treated with Bacille-Calmette-Guerin and lipopolysaccharide. Journal of Ethnopharmacology, 2006,107, 442-448.
[6] Park, Y.W., Zhu,S., Palaniappan L., et.al.. The metabolic syndrome:prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey. Arch.Intern.Med.2003,163.1988-1994.
[7] Aronow,W.S..Managing hyperlipidaemia in the elderly: special considerations for a population at high risk. Drug Aging ,2006,23,181-189.
[8] Guoling Zhao, Weidong Yan, Dan Cao.Simultaneous determination of betulin and betulinic acid in white birch bark using RP-HPLC [J]. Pharmaceutical and Biomedical Analysis. 2007 (43): 959-962.
[9] 付荣，朱蓓薇．用二苯代苦昧酰基自由基分光光度法测定川芎、红景天和黄芪的抗氧化活性．大连轻工业学院报．200423：114-117．
[10]黄梅王学军杨凯，中药抗氧化成分及抗氧化活性的体外评价方法．重庆科技学院学报(自然科学版)．2006,8(3)：109-112
[11] Hu, H.L., Chen, R.D.. Changes in free radicals, trace elements, by dgalactosamine.Biological Trace Element Research , 1992,34, 19-25.
[13]金焕荣，张越，刘军，等．乙二醛对小鼠谷胱甘肽过氧化物酶活力及巯基；含量的影响[J]．工业卫生与职业病，2004，30(1)：25-28．
[12] Vaca, C.E., Wilhelm, J., Harms-Rihsdahl, M.. Interaction of lipid peroxidation product with DNA. A review. Mutation Research-Reviews in Genetic Toxicology, 1988,195,137-149.
[14] Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia[J]. J Appl Physiol, 2000, 88(4): 1474-1480.
[15] Kaplowitz N. Biochemical and cellular mechanisms of toxic liver injury [J].Semin Liver Dis, 2002, 22(2): 137-144.
[16]张弛，王继峰，钱家骏．中药治疗高脂血症实验研究的新进展(综述)．北京中医药大学学报，Res．2000，4(23)：23-25
[17] Park, Y.W., Zhu,S., Palaniappan, L., Heshka , S.,Carnethon, M.T., Heymsfield, S.D.,. The metabolic syndrome:prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey. Arch.Intern.Med. 2003163.1988-1994.
[18] Aronow,W.S.. Managing hyperlipidaemia in the elderly: special considerations for a population at high risk. Drug Aging .200623,181-189.
[19] Girotti A W. Lipid hydroperoxide generation, turnover, and effector action in biological systems[J]. J Lipid Res, 1998, 39: 1529-1542.
[20] Brown MS, Goldstein JI. Areceptor-mediated path way for cholesterol homeostasis. Science. J. 1986, 232, 34-37.
[21] Fhomas JP, Geiger PG, Girotti AM. Lethal damage to endothelial cell by oxidized low density lipoprotein: role of selenoperoxidases in cyctoprotection against lipid hydroperoxide and iron-mediated reactions, J. lipid Res. 1993,34 (37), : 479-490.
[1]崔艳霞,郑志方.白桦皮化学组成的研究.东北林业大学学报,1994,22(4):268-270
[2]王建华,黄文哲,张增辉.白桦皮镇咳去痰有效成分的研究.中国药学志,1994,29(5):268
[3]李薇,李岩,会雄杰.白桦三萜类物质的抗肿瘤作用及其对免疫功能的增强效应.中国免疫学杂志,2000,16(9):485-487
[4]李丹,周金培,吴晓明.白桦酸及其衍生物的研究进展.药物化学,2004,28(3):120-125
[5]王素娟,裴月湖.白桦叶化学成分研究.沈阳药科大学学报,2000,17(4):256-257
[6]王素娟,裴月湖.白桦叶中的二苯基庚烷类化合物.中草药,2001,32(2):99-101
[7]Pisha E,Chai H,Lee IS.Discover of betlinic acid as a selective inhibitor of human melanoma that function by induction of apoptosis.Nat Med1995,1(10):1046-1049.
[8]Fulda S,Scaflidi C,Susin SA.Activation of mitohondriaand release of mitochondrial.Boil Chem1998,273:33942-33946
[9]Fulda S,Sieverts H,Friescn C.The CD95(APO-1/Fas)sysmedies drug-indured apoptosis in neuroblastoma cells.Cancer Res,1997,57:3823-3825
[10]Fulda S,Jeremias ISteiner HH.Betulinc acid:a new cytotoxic-agent against malignant brain-tumor cellsInt J Cancer1999,82:435-439
[11]Valentina Z,Rasanna S,Sabina C.Seletive eyto-toxocity of bemlinic acid on tuinor cell linesbut not on normaleels.Cancer letters,2002,175:17-20
[12]Costantini P,Jacotot E.Mithecondrion as a noveltarget of anticancer chemotherapy.Natl Cancer Inst,2000,92:1042-1045
[13]Chowdhury,Mandal S,Mittra B.Betulinic acid,a potentinhibitor of eukaryotic topoisomerase Ⅰ:identification of the inhibitory step,the major functional group responsible and development of molepotent derivatives.Med Monit,2002,8(7):254-256
[14]ChouKJ,FangHC,ChungHM.Effect of betulinic acid tracellular-free levelsin Madin Darby canine kidney cells.Eur JPhannacol,2000,408:99-103
[15]叶银英,何道伟,叶文才.23-羟基桦木酸体外和体内抗黑色素瘤作用的研究.中国肿瘤临床与康复,2000,7(1):5-8
[16]WachsbergerPR,BuM R,Wahl ML.Betulinic acid send-tizationof low pH adapted human me lanoma cells to hyperthermial.Int J hyperthermia,2002,18(2):153-154
[17]韩淑英,吴子忠,熊建新.桦树皮提取物对荷瘤小鼠免疫抑瘤效应的研究.中成药,2002,24(3):200-203
[18]Melzig MF,Bonnann H.Betulinic acid inhibits aminopeptidase activity.Panta Med1998,64(7):655
[19]汪红,王强,余国奠.近十年抗肿瘤中药的研究进展.中国野生植物资源,2000,19(3):66-69.
[20]刘志芹,张庆云.白桦皮和白桦叶抗真菌作用研究.中国中医药杂志,2004,12(8):76-78
[2] 韩英，李智纬，李岩等．桦树皮抗肿瘤作用及对免疫功能的影响．中药材，2000，23(6)：343-345．
[3] 李薇，李岩，金雄杰．白桦三萜类物质的抗肿瘤作用及其对免疫功能的增强效应．中国免疫学杂志[J]，2000，16(9)：485-487．
[4] Hwang, J.M., Tseng, T.H., Tsai, Y.Y., Lee, H.J., Chou, F.P., Wang, C.J., Chu, C.Y.. Protective effects of baicalein on tert-butyl hydroperoxide-induced hepatic toxicity in rat hepatocytes. Journal of Biomedical Science, 2005,12,389-397.
[5] Zou, Y.H., Yang, Y., Li, J., Li, W.P., Wu, Q.. Prevention of hepatic injury atraditional Chinese formulation, BJ-JN, in mice treated with Bacille-Calmette-Guerin and lipopolysaccharide. Journal of Ethnopharmacology, 2006,107, 442-448.
[6] Park, Y.W., Zhu,S., Palaniappan L., et.al.. The metabolic syndrome:prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey. Arch.Intern.Med.2003,163.1988-1994.
[7] Aronow,W.S..Managing hyperlipidaemia in the elderly: special considerations for a population at high risk. Drug Aging ,2006,23,181-189.
[8] Guoling Zhao, Weidong Yan, Dan Cao.Simultaneous determination of betulin and betulinic acid in white birch bark using RP-HPLC [J]. Pharmaceutical and Biomedical Analysis. 2007 (43): 959-962.
[9] 付荣，朱蓓薇．用二苯代苦昧酰基自由基分光光度法测定川芎、红景天和黄芪的抗氧化活性．大连轻工业学院报．200423：114-117．
[10]黄梅王学军杨凯，中药抗氧化成分及抗氧化活性的体外评价方法．重庆科技学院学报(自然科学版)．2006,8(3)：109-112
[11] Hu, H.L., Chen, R.D.. Changes in free radicals, trace elements, by dgalactosamine.Biological Trace Element Research , 1992,34, 19-25.
[13]金焕荣，张越，刘军，等．乙二醛对小鼠谷胱甘肽过氧化物酶活力及巯基；含量的影响[J]．工业卫生与职业病，2004，30(1)：25-28．
[12] Vaca, C.E., Wilhelm, J., Harms-Rihsdahl, M.. Interaction of lipid peroxidation product with DNA. A review. Mutation Research-Reviews in Genetic Toxicology, 1988,195,137-149.
[14] Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia[J]. J Appl Physiol, 2000, 88(4): 1474-1480.
[15] Kaplowitz N. Biochemical and cellular mechanisms of toxic liver injury [J].Semin Liver Dis, 2002, 22(2): 137-144.
[16]张弛，王继峰，钱家骏．中药治疗高脂血症实验研究的新进展(综述)．北京中医药大学学报，Res．2000，4(23)：23-25
[17] Park, Y.W., Zhu,S., Palaniappan, L., Heshka , S.,Carnethon, M.T., Heymsfield, S.D.,. The metabolic syndrome:prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey. Arch.Intern.Med. 2003163.1988-1994.
[18] Aronow,W.S.. Managing hyperlipidaemia in the elderly: special considerations for a population at high risk. Drug Aging .200623,181-189.
[19] Girotti A W. Lipid hydroperoxide generation, turnover, and effector action in biological systems[J]. J Lipid Res, 1998, 39: 1529-1542.
[20] Brown MS, Goldstein JI. Areceptor-mediated path way for cholesterol homeostasis. Science. J. 1986, 232, 34-37.
[21] Fhomas JP, Geiger PG, Girotti AM. Lethal damage to endothelial cell by oxidized low density lipoprotein: role of selenoperoxidases in cyctoprotection against lipid hydroperoxide and iron-mediated reactions, J. lipid Res. 1993,34 (37), : 479-490.
[1]崔艳霞,郑志方.白桦皮化学组成的研究.东北林业大学学报,1994,22(4):268-270
[2]王建华,黄文哲,张增辉.白桦皮镇咳去痰有效成分的研究.中国药学志,1994,29(5):268
[3]李薇,李岩,会雄杰.白桦三萜类物质的抗肿瘤作用及其对免疫功能的增强效应.中国免疫学杂志,2000,16(9):485-487
[4]李丹,周金培,吴晓明.白桦酸及其衍生物的研究进展.药物化学,2004,28(3):120-125
[5]王素娟,裴月湖.白桦叶化学成分研究.沈阳药科大学学报,2000,17(4):256-257
[6]王素娟,裴月湖.白桦叶中的二苯基庚烷类化合物.中草药,2001,32(2):99-101
[7]Pisha E,Chai H,Lee IS.Discover of betlinic acid as a selective inhibitor of human melanoma that function by induction of apoptosis.Nat Med1995,1(10):1046-1049.
[8]Fulda S,Scaflidi C,Susin SA.Activation of mitohondriaand release of mitochondrial.Boil Chem1998,273:33942-33946
[9]Fulda S,Sieverts H,Friescn C.The CD95(APO-1/Fas)sysmedies drug-indured apoptosis in neuroblastoma cells.Cancer Res,1997,57:3823-3825
[10]Fulda S,Jeremias ISteiner HH.Betulinc acid:a new cytotoxic-agent against malignant brain-tumor cellsInt J Cancer1999,82:435-439
[11]Valentina Z,Rasanna S,Sabina C.Seletive eyto-toxocity of bemlinic acid on tuinor cell linesbut not on normaleels.Cancer letters,2002,175:17-20
[12]Costantini P,Jacotot E.Mithecondrion as a noveltarget of anticancer chemotherapy.Natl Cancer Inst,2000,92:1042-1045
[13]Chowdhury,Mandal S,Mittra B.Betulinic acid,a potentinhibitor of eukaryotic topoisomerase Ⅰ:identification of the inhibitory step,the major functional group responsible and development of molepotent derivatives.Med Monit,2002,8(7):254-256
[14]ChouKJ,FangHC,ChungHM.Effect of betulinic acid tracellular-free levelsin Madin Darby canine kidney cells.Eur JPhannacol,2000,408:99-103
[15]叶银英,何道伟,叶文才.23-羟基桦木酸体外和体内抗黑色素瘤作用的研究.中国肿瘤临床与康复,2000,7(1):5-8
[16]WachsbergerPR,BuM R,Wahl ML.Betulinic acid send-tizationof low pH adapted human me lanoma cells to hyperthermial.Int J hyperthermia,2002,18(2):153-154
[17]韩淑英,吴子忠,熊建新.桦树皮提取物对荷瘤小鼠免疫抑瘤效应的研究.中成药,2002,24(3):200-203
[18]Melzig MF,Bonnann H.Betulinic acid inhibits aminopeptidase activity.Panta Med1998,64(7):655
[19]汪红,王强,余国奠.近十年抗肿瘤中药的研究进展.中国野生植物资源,2000,19(3):66-69.
[20]刘志芹,张庆云.白桦皮和白桦叶抗真菌作用研究.中国中医药杂志,2004,12(8):76-78