核桃外果皮抑制人宫颈癌HeLa细胞活性成分筛选
|
摘要
本文对新疆核桃外果皮抑制人宫颈癌HeLa细胞增殖的有效部位及活性成分进行筛选。采用四甲基偶氮唑盐(MTT)法从核桃外果皮石油醚萃取部位、乙酸乙酯萃取部位、氯仿萃取部位、正丁醇萃取部位和水萃取部位筛选出对人宫颈癌HeLa细胞生长有抑制作用的有效部位,采用柱色谱法对有效部位中的活性成分进行分离纯化,利用薄层色谱板检识,将分离纯化得到的活性成分通过液-质联用仪(LC-MS)及核磁共振仪(NMR)进行结构鉴定。采用MTT法对活性成分进行人宫颈癌HeLa细胞增殖抑制试验,并通过Hoechst 33342染色法及流式细胞术方法研究其对HeLa细胞凋亡的影响。通过小鼠的最大耐受量试验对活性成分的安全性能进行评价。同时研究了反相高效液相色谱法(RP-HPLC)检测活性成分的条件。
结果表明：石油醚萃取部位、乙酸乙酯萃取部位、氯仿萃取部位、正丁醇萃取部位和水萃取部位对人宫颈癌HeLa细胞抑制的IC50值分别为158.81 mg/L,40.43 mg/L,130.49 mg/L,137.67 mg/L,5.10E+07 mg/L。说明核桃外果皮的石汕醚提取物、乙酸乙酯提取物、氯仿提取物、正丁醇提取物对人宫颈癌HeLa细胞均有一定抑制作用,且随着浓度的提高抑制作用增强,其中氯仿提取物的抑制作用最强。采用硅胶柱色谱,洗脱液为氯仿∶乙酸乙酯(3∶7,V/V),从氯仿提取物中分出的成分,经LC-MS及NMR结构分析,确认为齐墩果酸。采用MTT法检测齐墩果酸对人宫颈癌HeLa细胞的抑制率,齐墩果酸浓度为10-4mol/L时,抑制率达40.22±3.24%；人宫颈癌HeLa细胞经40 mg/L齐墩果酸处理24小时经Hoechst33342染色,在荧光显微镜下可以观察到细胞出现凋亡小体；人宫颈癌HeLa细胞经40 mg/L齐墩果酸处理24小时后经Annexin V-FITC/PI染色后,在流式细胞仪上检测细胞的早期凋亡率为3.93%。对齐墩果酸进行小鼠的最大耐受量试验(MTD),给药方式采用灌胃。结果表明小鼠在给药7天后心、肝、脾、肺等主要的脏器系数均未发现异常,其对齐墩果酸的最大耐受量大于22.2 mg/kg,相当于体重为60 kg的成人每日用量200 mg的200倍。通过研究建立了核桃外果皮中齐墩果酸含量测定的RP-HPLC法,采用色谱柱为Intersil ODS(150 mm×4.6 mm,5μm)流动相为甲醇∶水∶冰乙酸(265∶35∶0.1,V/V)；流速0.8mL/min进样量20μL；检测波长为210 nm；柱温30℃。结果表明齐墩果酸进样量在0.728～7.28μg(r=0.9997)范围内呈现良好的线性关系,齐墩果酸平均回收率(n-9)为101.1%,相对标准偏差(RSD)为2.44%。测得核桃外果皮中齐墩果酸含量为2.1 mg/100g。该方法简便快速,结果可靠准确,重现性和稳定性好。
In this paper, both the effective parts and active components from the different solvent fractions which were petroleum ether part, ethyl acetate part, chloroform part, butanol part and water part in walnut pericarp were selected by MTT assay against human cervical cancer HeLa cells. The active ingredients were separated and purified by column chromatography, and identified by thin layer chromatography. The structure of the active ingredient was identified through LC-MS and NMR. The morphology of HeLa cells apoptosis was observed by Hoechst 33342 staining and data about HeLa cells apoptosis analyzed from flow cytometry. The safety performance was evaluated by MTD test in mice. In the same time, the method for examination of active components by RP-HPLC was studied.
The results showed that the IC50 of petroleum ether part, ethyl acetate part, chloroform part, butanol 1 part and water part against HeLa cells was 158.81 mg/L,40.43 mg/L,130.49 mg/L,137.67 mg/L,5.10E+07 mg/L, respectively. These findings provided evidence that petroleum ether, ethyl acetate, chloroform, and butanol had considerable inhibition and showed the dose-effect manner to human cervical cancer HeLa cells. The chloroform extraction showed the strongest activity. The chloroform extract was isolated and purified by silica gel column chromatography and TLC. The eluent was chloroform:ethyl acetate (3:7, V/V). The structure of the active component was identified by LC-MS and NMR. The result indicated it was oleanolic acid. The inhibition rate of oleanolic acid to HeLa cells was 40.22±3.24% by MMT assay when the concentration of oleanolic acid was 10-4 mol/L; The apoptotic bodies of HeLa cells were observed under a fluorescence microscope after treatment of 24h by 40 mg/L oleanolic acid and further Hoechst 33342 staining. The early apoptosis rate of HeLa cells was 3.93% by flow cytometry after treatment of 24h by 40 mg/L oleanolic acid and further Annexin V-FITC/PI staining. The maximum tolerated dose in mice test to oleanolic acid was carried out and the mode of drug delivery with lavage. The result pointed out that mice's major organ coefficient also presented normally after 7 days of drug delivery; the maximum tolerated dose of mice to oleanolic acid was greater than 22.2 mg/kg which was equivalent to 200 times of 200 mg daily dosage of adult in 60 kg body weight. The method of RP-HPLC for determination of oleanolic acid in walnut pericarp was established. The optimal conditions were:the chromatographic column was Intersil ODS (150 mm×4.6 mm,5μm); the mobile phase was methanol:water:acetic acid (265:35:0.1, V/V), velocity of flow was 0.8 mL/min, the injection volume was 20μL, detection wavelength was 210 nm, and temperature of column was 30℃. The result showed that good linear relationship was presented when the injection volume of oleanolic acid was among 0.728～7.28μg (r=0.9997), the average recovery of oleanolic acid (n=9) was 101.1%, RSD was 2.44%. The content of oleanolic acid in walnut pericarp was 2.1 mg/100g. The method is simple, rapid, reliable and accurate, reproducible and stable.
结果表明：石油醚萃取部位、乙酸乙酯萃取部位、氯仿萃取部位、正丁醇萃取部位和水萃取部位对人宫颈癌HeLa细胞抑制的IC50值分别为158.81 mg/L,40.43 mg/L,130.49 mg/L,137.67 mg/L,5.10E+07 mg/L。说明核桃外果皮的石汕醚提取物、乙酸乙酯提取物、氯仿提取物、正丁醇提取物对人宫颈癌HeLa细胞均有一定抑制作用,且随着浓度的提高抑制作用增强,其中氯仿提取物的抑制作用最强。采用硅胶柱色谱,洗脱液为氯仿∶乙酸乙酯(3∶7,V/V),从氯仿提取物中分出的成分,经LC-MS及NMR结构分析,确认为齐墩果酸。采用MTT法检测齐墩果酸对人宫颈癌HeLa细胞的抑制率,齐墩果酸浓度为10-4mol/L时,抑制率达40.22±3.24%；人宫颈癌HeLa细胞经40 mg/L齐墩果酸处理24小时经Hoechst33342染色,在荧光显微镜下可以观察到细胞出现凋亡小体；人宫颈癌HeLa细胞经40 mg/L齐墩果酸处理24小时后经Annexin V-FITC/PI染色后,在流式细胞仪上检测细胞的早期凋亡率为3.93%。对齐墩果酸进行小鼠的最大耐受量试验(MTD),给药方式采用灌胃。结果表明小鼠在给药7天后心、肝、脾、肺等主要的脏器系数均未发现异常,其对齐墩果酸的最大耐受量大于22.2 mg/kg,相当于体重为60 kg的成人每日用量200 mg的200倍。通过研究建立了核桃外果皮中齐墩果酸含量测定的RP-HPLC法,采用色谱柱为Intersil ODS(150 mm×4.6 mm,5μm)流动相为甲醇∶水∶冰乙酸(265∶35∶0.1,V/V)；流速0.8mL/min进样量20μL；检测波长为210 nm；柱温30℃。结果表明齐墩果酸进样量在0.728～7.28μg(r=0.9997)范围内呈现良好的线性关系,齐墩果酸平均回收率(n-9)为101.1%,相对标准偏差(RSD)为2.44%。测得核桃外果皮中齐墩果酸含量为2.1 mg/100g。该方法简便快速,结果可靠准确,重现性和稳定性好。
In this paper, both the effective parts and active components from the different solvent fractions which were petroleum ether part, ethyl acetate part, chloroform part, butanol part and water part in walnut pericarp were selected by MTT assay against human cervical cancer HeLa cells. The active ingredients were separated and purified by column chromatography, and identified by thin layer chromatography. The structure of the active ingredient was identified through LC-MS and NMR. The morphology of HeLa cells apoptosis was observed by Hoechst 33342 staining and data about HeLa cells apoptosis analyzed from flow cytometry. The safety performance was evaluated by MTD test in mice. In the same time, the method for examination of active components by RP-HPLC was studied.
The results showed that the IC50 of petroleum ether part, ethyl acetate part, chloroform part, butanol 1 part and water part against HeLa cells was 158.81 mg/L,40.43 mg/L,130.49 mg/L,137.67 mg/L,5.10E+07 mg/L, respectively. These findings provided evidence that petroleum ether, ethyl acetate, chloroform, and butanol had considerable inhibition and showed the dose-effect manner to human cervical cancer HeLa cells. The chloroform extraction showed the strongest activity. The chloroform extract was isolated and purified by silica gel column chromatography and TLC. The eluent was chloroform:ethyl acetate (3:7, V/V). The structure of the active component was identified by LC-MS and NMR. The result indicated it was oleanolic acid. The inhibition rate of oleanolic acid to HeLa cells was 40.22±3.24% by MMT assay when the concentration of oleanolic acid was 10-4 mol/L; The apoptotic bodies of HeLa cells were observed under a fluorescence microscope after treatment of 24h by 40 mg/L oleanolic acid and further Hoechst 33342 staining. The early apoptosis rate of HeLa cells was 3.93% by flow cytometry after treatment of 24h by 40 mg/L oleanolic acid and further Annexin V-FITC/PI staining. The maximum tolerated dose in mice test to oleanolic acid was carried out and the mode of drug delivery with lavage. The result pointed out that mice's major organ coefficient also presented normally after 7 days of drug delivery; the maximum tolerated dose of mice to oleanolic acid was greater than 22.2 mg/kg which was equivalent to 200 times of 200 mg daily dosage of adult in 60 kg body weight. The method of RP-HPLC for determination of oleanolic acid in walnut pericarp was established. The optimal conditions were:the chromatographic column was Intersil ODS (150 mm×4.6 mm,5μm); the mobile phase was methanol:water:acetic acid (265:35:0.1, V/V), velocity of flow was 0.8 mL/min, the injection volume was 20μL, detection wavelength was 210 nm, and temperature of column was 30℃. The result showed that good linear relationship was presented when the injection volume of oleanolic acid was among 0.728～7.28μg (r=0.9997), the average recovery of oleanolic acid (n=9) was 101.1%, RSD was 2.44%. The content of oleanolic acid in walnut pericarp was 2.1 mg/100g. The method is simple, rapid, reliable and accurate, reproducible and stable.
引文
[1]祁承经,林亲众.湖南树木志[M].湖南科学技术出版社,2000：545.
[2]江苏新医学院编.中药大辞典(下册)[M].上海科学技术出版社,1998：154.
[3]郗荣庭,张毅萍.中国果树志(核桃卷)[M].北京：中国林业科学出版社,1996.
[4]李志美.核桃的开发利用[J].林业调查规划,2004,29(增刊)：199-200.
[5]Kim S H, Lee K S, Son J k, et al. Cytotoxic compound from the roots of Juglans mandshurica[J]. J Nat Prod,1998,61 (5):6432-6451.
[6]Lee S W, Lee K S, Song J K. New Naphthalenyl Glycosides from the Roots of Juglans mandshurica[J]. Planta Med,2000,66 (2):184-186.
[7]兰英,房金波,张维晶,等.核桃楸青果皮(GPJM)水提物诱生小鼠肿瘤坏死因子的实验研究[J].东北师大学报自然科学报,2003,35(1)：78-81.
[8]李正生,王云彩,蒋东风,等BSLB法筛选青龙衣与核桃枝抗肿瘤作用有效部位[J].西北药学杂志,2000,15(3)：114.
[9]王春玲,曹小红.核桃青皮对S180实体瘤的作用研究[J].食品科学,2004,25(11)：285-287.
[10]曲中原,邹翔,崔兰,等.青龙衣不同萃取部位抗肿瘤活性研究[J].上海中医药杂志,2009,43(1)： 87-90.
[11]姜秀梅,郭虹.青龙衣乙醇提取液对乳腺癌细胞抑制作用的研究[J].特产研究,2009,1：34-36.
[12]季宇彬,曲中原,邹翔,等.青龙衣中胡桃醌对S180肉瘤小鼠的抑瘤作用研究[J].中国药学杂志,2009,44(3)：195-199.
[13]徐巍,高景泰,杨志红,等.青龙衣粉针剂抑瘤与免疫作用的实验研究[J].中国中医基础医学杂志,2002,10(8)：39-41.
[14]DU X, LIYW, NI Y, et al. Research and perspective of annlgesic effect of Chinese medicin green walnut[J]. Journal of Chinese Materia Medica,2000,25 (1):7-10.
[15]WAN L, CHEN X F, DU J. Studies on the anti inflammatory and analgesic Effect of Hu Tao Qing Pi[J]. pharmacological and Clinical of Chinese Medicines,1999,15 (2):29-30.
[16]宋君.植物间的他感作用[J].生态学杂志,1990,9(6)：43-47.
[17]翟明普,贾黎明.森林植物间的他感作用[J].北京林业大学学报,1993,15(3)：138-147.
[18]别智鑫,翟梅枝,周博,等.核桃青皮鲜样水提液对盆栽苗生长的影响[J].陕西农业科学,2007,2：59-60.
[19]翟梅枝,李鹏影,黄海玲.核桃青皮活性物质P4-2对烟草体内生理生化指标的影响[J].西北林学院学报,2009,24(4)：143-146.
[20]别智鑫,张国贞,蔡永社,等.核桃青皮化感物质初提溶剂的选择[J].杨凌职业技术学院学报, 2007,6(2)：5-8.
[21]朴仁哲,赵洪颜,朴京一,等.核桃楸青果皮提取液对植物生长的影响[J].天然产物研究与开发,2006,18：11-14.
[22]何衍彪,詹儒林,赵艳龙.植物源农药的研究和应用[J].热带农业科学,2004,24(3)：58-62.
[23]张兴.植物性农药在生防中的地位和作用[J].世界农业,2003,2(1)：31-32.
[24]翟梅枝,张凤云,魏花,等.核桃青皮中次生代谢物质的生物活性研究-杀虫活性[J].西北林学院学报,2006,21(1)：122-125.
[25]孙墨珑,袁海舰,宋湛谦,等.核桃楸树皮乙醇提取物的杀虫活性[J].东北林业大学学报,2007,35(1)：60-62.
[26]梁永峰.核桃青皮提取物对小麦蚜虫的毒杀和拒食活性研究[J].安徽农业科学,2010,38(27)：15020-15021.
[27]曹雁平.我国天然食用色素研究现状[J].食品与发酵工业,2007,33(1)：80-84.
[28]吕俊芳,张美丽,刘启瑞,等.核桃外果皮中棕褐色色素的提取及性质测试[J].化学研究与应用,2001,13(4)：387-390.
[29]李庆勇,王丽敏,祖元刚,等.抗肿瘤植物原料药的现状与发展趋势[J].中国药房,2009,20(1)：228-230.
[30]韩旭东,张皓楠,等.流式细胞仪工作原理及在中医药研究中的应用[J].实验室科学,2010,13(3)： 158-160.
[31]周金黄,刘干中.免疫药理学进展—基础与临床[M].北京：中国科学技术出版社,1993：367.
[32]Waggoner S E. Cervical cancer[J]. Lancet,2003,361 (9376):2217-2220.
[33]Dunne A L, Price M E, Mothersill C, et al. Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells[J]. Br J Cancer, 2003,89 (12):2277-2280.
[34]胡野,凌志强,单小云,等.细胞凋亡的分子医学[M].北京：军事医学科学出版社,2002：2
[35]彭黎明,姚震.妇科恶性肿瘤与细胞凋亡[J].医学综述,2002,8(1)：5-7.
[36]Tishler R B, Lampu D M, Park S, et al. Microtubule-active drug taxol, vinblastine, and nocodazole increase the levels of transcrptionally active P53[J]. Cancer Res,1995,55 (3):602-605.
[37]Samel D, Deana D A, De Witte P. The effect of purified extract of Fagopyrum esculent um(buckwheat)on protein kinases involved in signal transduction pathways[J]. Planta Med,1996,62 (2):106-110.
[38]程春旭,高颜茹.抗肿瘤药物作用机制的研究进展[J].吉林医学,2009,30(22)：3080-3083.
[39]张莉萍,蒋纪恺,谭荣安.苦参碱对K562细胞株端粒酶活性和细胞周期的影响[J].中华肿瘤杂志,1998,20(5)：328-330.
[40]徐秀,月杨戚.活化型高分子量激肽原潜在的抗肿瘤作用及其分子机制[J].中国生物化学与分子生物学报,2006,22(9)：694-697.
[41]王广秀.白黎芦醇抗肿瘤作用的研究进展[J].国外医学·肿瘤学分册,2006,32(12)：894.
[42]Takara K, Sakaeda T, Okumura K. An update on overcoming (MDR)mediated multidrug resistance in cancer chemotherapy [J]. Curr Pharm Des,2006,12(3):273-286.
[43]石宇雄,黄永明,许少键,等.参麦注射液对人成骨肉瘤多药耐药细胞株R-OS-732逆转作用[J].广东医学,2006,27(8)：1200-1201.
[44]李雪梅,吴运光,潘达鑫.新型抗肿瘤药槐定碱[J].中国新药杂志,2006,15(8)：655-658.
[45]Lin J, Dong H F, Oppenhein J J, et al. Effects of astragli radix on the growth of different cancer cell lines[J]. World J Gastroenterol,2003,9(3):670-673.
[46]黄自丽,黄修燕,郑起.中药抗肿瘤作用及其作用机制研究进展[J].医学综述,2010,16(3)：386-389.
[47]Mans D R, Rocha A B, Schwartsmann G, et al. Anti-cancer drug discovery and development in Brazil:targeted plant collection as a rational strategy to acquire candidate anti-cancer compounds[J]. Oncologist,2000,5:185-198.
[48]Duffin J. Poisoning the spindle:serendipity and discovery of the anti-tumor properties of the vinca alkaloids[J]. Canc Bull Med Hist,2000,17:155-192.
[49]赵锐,赵玮玮.抗癌植物药紫杉醇研究与动态[J].中草药,2009,40(7)：1172-附2.
[50]徐艳红.宫颈癌的致病因素及预防的研究综述[J].中国实用医药,2009,25(4)：252-253.
[51]李华,高国兰.HPV 与宫劲癌的研究进展[J].实用癌症杂志,2007,22(4)：420-422.
[52]De Villiers EM, Gunst K, Stein H, et al. Esophageal squamous cell cancer in patients with head and neck cancer prevalence of human papillomavirus DNA sequences[J]. Int J Cancer,2004,109 (2): 253.
[53]Rion G, Faver M, Jeannel D, et al. Association between poor prognosis in early stage invasion cervical carcinomas and non-detection of HPV DNA[J]. Lancet,1990,335:1171.
[54]乔玉环,张梦真,史记蓉.子宫颈癌及其癌前病变组织端粒酶活性的研究[J].中华妇产科杂志,2001,36(8)：483.
[55]Fu J, Jin Y, Arend LJ. Smac3, a novel smac/DIABLO splicing variant, attenuates the stability and apoptosis inhibiting activity of X-linked inhibitor of apoptosis protein[J]. J Biol Chem,2003,278 (52):52660.
[56]贾艳菊,代玲,张灿.龙葵生物碱诱导HeLa细胞凋亡的研究[J].动物医学进展,2010,31(8)：51-54.
[57]崔晓燕,罗琼,杨明亮,等.枸杞多糖对人宫颈爱细胞生长及细胞凋亡影响[J].中国公共卫生,2006,22(12)：1411-1412.
[58]赵令武,万福生.白英水提物诱导人宫颈癌HeLa细胞凋亡的实验研究[J].中国临床药理学与治疗学,2007,12(8)：883-886.
[59]李志玖,郑芳,蒋飞,等.自藜芦醇对宫颈癌HeLa细胞凋亡的诱导作用[J].郧阳医学院学报,2009,28(1)：16-17,
[60]陈艳,田莉,盛习峰,等.紫花牡荆素诱导人宫颈癌HeLa细胞凋亡[J].湖南师范大学学报(医学版),2010,7(2)：11-13.
[61]JI Y B, MA H T, YANG B, et al. Antitumor effects of different extract fractions from Qinglongyi[J]. China Tradit Herb Drugs,2004,35 (10):1145-1147.
[62]JI Y B, JI C F, MA H T. Study on the effects of cool and hot ethanol extracts from Qinglongyi on the biochemical function of tumor membrane in H22 mice[J]. China J Chin Mater Med,2005,30 (7): 531-534.
[63]LIU W, JI Y B. Study on acute toxicity experiment of mice and anti-tumor function in vivo of Chinese traditional medicine Qinglongyi[J]. J Harbin Univ Com(Nat Sci Ed),2004,20(1):4-10.
[64]LIU W, LIN W H, JI Y B. Study on the acute toxicity experiment of mice and anti-tumor function in vitro of the Qinglongyi[J]. China J Chin Mater Med,2004,29(9):887-890.
[65]张野平,杨志博,景永奎,等.胡桃醌对肿瘤细胞的增殖抑制作用和抗菌作用[J].沈阳药学院学报,1993,10(4)：271-274.
[66]孔陵生,邹翔,季宇彬.青龙衣中胡桃醌对A-549细胞周期及凋亡影响[J].哈尔滨商业大学学报(自然科学版),2008,24(6)：641-644.
[67]季宇彬,曲中原,邹翔,等.青龙衣中胡桃醌对S180肉瘤小鼠的抑瘤作用研究[J].中国药学杂志,2009,44(2)：195-199.
[68]周媛媛,王栋.青龙衣中二芳基庚烷类化学成分的研究[J].时珍国医国药,2009,20(8)：1936-1937.
[69]刘红兵.中草药东京枫杨Pterocarya tonkinesis (Franch.)Dode抗肿瘤活性成分的研究[D].青岛：中国海洋大学,2004,42：88.
[70]Liu J X, Di D L, Huang X Y, Li C. Two new diarylheptanoids from the pericarps of Juglans regia L.[J]. China Chem Lett,2007,18 (8):943-946.
[71]Liu J X, Meng M, Di D L, et al. Simultaneous determination of three diaryhptanoids and an a-tetralone derivative in the green walnut husks(Juglans regia L.)by high-performance liquid chromatography with photodiode array detector[J]. J Chrom A,2008,1190 (1):80-85.
[72]周媛媛,王栋,牛峰.抗肿瘤中药青龙衣化学成分的研究[J].中草药,2010,41(1)：11-14.
[73]卫小红,邵杰,王军辉,等.齐墩果酸诱导人肺腺癌细胞A549凋亡及其与细胞内钙离子的关系[J].同济大学学报(医学版),2009,30(5)：19-23.
[74]灵林蔚,蒲蔷,陈晓珍,等.齐墩果酸对卵巢癌细胞IGROV1和乳腺癌细胞MDA-MB-231生长的抑制作用[J].应用与环境生物学报,2010,16(2)：202-204.
[75]徐新伟,郭玲玲,顾振纶,等.熊果酸对乳腺癌SK-BR-3细胞增殖和凋亡的影响[J].苏州大学学报(医学版),2009,29(1)：68-70.
[76]卢丹,冯常炜,马彦娟.熊果酸对食管癌KYSE105细胞增殖、凋亡的影响[J].胃肠病学和肝病学杂志,2010,19(2)：130-133.
[77]王辉,方全华,李海军.熊果酸诱导人肝癌BEL-7404细胞凋亡的作用及其机制[J].肿瘤基础与临床,2010,23(2)：93-95.
[78]夏阳,苟欣.熊果酸诱导前列腺癌细胞株PC-3凋亡的实验研究[J].重庆医科大学学报,2009,34(12)：1661-1665.
[79]中国科学院上海药物研究所.中草药有效成分提取与分离[M].上海：上海科学技术出版社,1983.
[80]袁珂,刘延泽,冀春茹.中草药化学成分研究中几种不同提取方法的比较研究[J].天然产物研究与开发,1997,9(4)：57-61.
[81]向智敏,毕丽君,张骊,等.超临界二氧化碳萃取墨红花香气成分的研究[J].天然产物研究与开发,1996,8(4)：42-46.
[82]吴文君,刘惠霞,朱靖博,等.天然产物杀虫剂—原理·方法·实践[M].西安：陕西科学技术出版社,1998.
[83]储茂泉,古宏晨,刘国杰.中草药浸提过程的动力学模型[J].中草药,2000,31(7)：504-506.
[84]李才国,刘睿.木瓜中齐墩果酸的提取、分离及鉴定[J].现代商贸工业,2007,19(3)：159-162.
[85]韦国锋,程金生,黄祖良,等.大孔吸附树脂纯化女贞子中齐墩果酸的研究[J].中国现代应用药学杂志,2008,25(2)：90-92.
[86]李海鹏,徐怀德等.光皮木瓜齐墩果酸超声波辅助提取及纯化工艺[J].农业工程学报,2008,24(10)：222-226.
[87]黄晓君,殷志琦,叶文才,等.女贞子的化学成分研究[J].中国中药杂志,2010,35(7)：861-864.
[88]朱春林,杨洪元.亚微肝泰片中齐墩果酸的含量测定[J].中国中医药信息杂志,2010,17(11)：47-48.
[89]沈晓丹,袁杰,徐乃玉,等HPLC测定蓝萼香茶菜中齐墩果酸和熊果酸的含量[J].中成药,2010,32(12)： 2182-2183.
[90]陈亮,甘辉,刘洪.反相高效液相色谱法测定木瓜中齐墩果酸和乌索酸的含量[J].安徽农业科学,2007,35(32)：10183-10184.
[91]邓世星,孙志勇,周卿,等.紫外光谱法测定齐墩果酸含量[J].遵义医学院学报,2003,26(6)：556-557.
[92]张彦功,杨玉琴,李建,等.不同产地牛膝药材中齐墩果酸的含量测定[J]贵阳中医学院学报, 2010,32(1)：20-21.
[93]胡野,凌志强,单小云.细胞凋亡的分子医学[M].北京：军事医学科学出版社,2002：2210.
[94]中草药诱导人宫颈癌HeLa细胞凋亡及其分子机制[J].江西医学院学报,2006,46(5)：176-177.
[95]陈奇.中药药理研究方法学[M].北京：人民卫生出版社,1994：112.
[96]Zhu Y M. Synthesis and anti-HIV activity of oleanolic acid derivatives. Bioorganic and Medicinal Chemistry Letters,2001,11:3115-3118.
[97]张建平,包海花,刘海峰,等.薄层色谱法鉴别蛇莓中齐墩果酸[J].牡丹江医学院学报,2010,31 (1)： 57-58.
[98]闫花丽,赵陆华,朱丹妮,等.衍生化气相色谱法测定夏枯草中齐墩果酸和熊果酸的含量[J].中国中药杂志,1999,24(12)：744-745.
[99]袁晓环,王伟光,李春彦,等HPLC法检测蛇莓中齐墩果酸的含量[J].中国生化药物杂志,2010,31 (3)： 195-197.
[100]张兴旺,梅丽娟,邵赟,等HPLC法测定甘青铁线莲中的齐墩果酸[J].分析试验室,2009,28.Suppl：238-240.
[101]谭朝阳,袁宏佳HPLC法测定龙牙楤木药材中齐墩果酸的含量[J].中国中医药信息杂志2010,17(1)：46-47.
[102]陈婷婷,热娜·卡斯木HPLC法测定新疆鼠尾草三萜有效部位中熊果酸的含量[J].新疆医科大学学报,2008,31(4)：394-395.
[2]江苏新医学院编.中药大辞典(下册)[M].上海科学技术出版社,1998：154.
[3]郗荣庭,张毅萍.中国果树志(核桃卷)[M].北京：中国林业科学出版社,1996.
[4]李志美.核桃的开发利用[J].林业调查规划,2004,29(增刊)：199-200.
[5]Kim S H, Lee K S, Son J k, et al. Cytotoxic compound from the roots of Juglans mandshurica[J]. J Nat Prod,1998,61 (5):6432-6451.
[6]Lee S W, Lee K S, Song J K. New Naphthalenyl Glycosides from the Roots of Juglans mandshurica[J]. Planta Med,2000,66 (2):184-186.
[7]兰英,房金波,张维晶,等.核桃楸青果皮(GPJM)水提物诱生小鼠肿瘤坏死因子的实验研究[J].东北师大学报自然科学报,2003,35(1)：78-81.
[8]李正生,王云彩,蒋东风,等BSLB法筛选青龙衣与核桃枝抗肿瘤作用有效部位[J].西北药学杂志,2000,15(3)：114.
[9]王春玲,曹小红.核桃青皮对S180实体瘤的作用研究[J].食品科学,2004,25(11)：285-287.
[10]曲中原,邹翔,崔兰,等.青龙衣不同萃取部位抗肿瘤活性研究[J].上海中医药杂志,2009,43(1)： 87-90.
[11]姜秀梅,郭虹.青龙衣乙醇提取液对乳腺癌细胞抑制作用的研究[J].特产研究,2009,1：34-36.
[12]季宇彬,曲中原,邹翔,等.青龙衣中胡桃醌对S180肉瘤小鼠的抑瘤作用研究[J].中国药学杂志,2009,44(3)：195-199.
[13]徐巍,高景泰,杨志红,等.青龙衣粉针剂抑瘤与免疫作用的实验研究[J].中国中医基础医学杂志,2002,10(8)：39-41.
[14]DU X, LIYW, NI Y, et al. Research and perspective of annlgesic effect of Chinese medicin green walnut[J]. Journal of Chinese Materia Medica,2000,25 (1):7-10.
[15]WAN L, CHEN X F, DU J. Studies on the anti inflammatory and analgesic Effect of Hu Tao Qing Pi[J]. pharmacological and Clinical of Chinese Medicines,1999,15 (2):29-30.
[16]宋君.植物间的他感作用[J].生态学杂志,1990,9(6)：43-47.
[17]翟明普,贾黎明.森林植物间的他感作用[J].北京林业大学学报,1993,15(3)：138-147.
[18]别智鑫,翟梅枝,周博,等.核桃青皮鲜样水提液对盆栽苗生长的影响[J].陕西农业科学,2007,2：59-60.
[19]翟梅枝,李鹏影,黄海玲.核桃青皮活性物质P4-2对烟草体内生理生化指标的影响[J].西北林学院学报,2009,24(4)：143-146.
[20]别智鑫,张国贞,蔡永社,等.核桃青皮化感物质初提溶剂的选择[J].杨凌职业技术学院学报, 2007,6(2)：5-8.
[21]朴仁哲,赵洪颜,朴京一,等.核桃楸青果皮提取液对植物生长的影响[J].天然产物研究与开发,2006,18：11-14.
[22]何衍彪,詹儒林,赵艳龙.植物源农药的研究和应用[J].热带农业科学,2004,24(3)：58-62.
[23]张兴.植物性农药在生防中的地位和作用[J].世界农业,2003,2(1)：31-32.
[24]翟梅枝,张凤云,魏花,等.核桃青皮中次生代谢物质的生物活性研究-杀虫活性[J].西北林学院学报,2006,21(1)：122-125.
[25]孙墨珑,袁海舰,宋湛谦,等.核桃楸树皮乙醇提取物的杀虫活性[J].东北林业大学学报,2007,35(1)：60-62.
[26]梁永峰.核桃青皮提取物对小麦蚜虫的毒杀和拒食活性研究[J].安徽农业科学,2010,38(27)：15020-15021.
[27]曹雁平.我国天然食用色素研究现状[J].食品与发酵工业,2007,33(1)：80-84.
[28]吕俊芳,张美丽,刘启瑞,等.核桃外果皮中棕褐色色素的提取及性质测试[J].化学研究与应用,2001,13(4)：387-390.
[29]李庆勇,王丽敏,祖元刚,等.抗肿瘤植物原料药的现状与发展趋势[J].中国药房,2009,20(1)：228-230.
[30]韩旭东,张皓楠,等.流式细胞仪工作原理及在中医药研究中的应用[J].实验室科学,2010,13(3)： 158-160.
[31]周金黄,刘干中.免疫药理学进展—基础与临床[M].北京：中国科学技术出版社,1993：367.
[32]Waggoner S E. Cervical cancer[J]. Lancet,2003,361 (9376):2217-2220.
[33]Dunne A L, Price M E, Mothersill C, et al. Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells[J]. Br J Cancer, 2003,89 (12):2277-2280.
[34]胡野,凌志强,单小云,等.细胞凋亡的分子医学[M].北京：军事医学科学出版社,2002：2
[35]彭黎明,姚震.妇科恶性肿瘤与细胞凋亡[J].医学综述,2002,8(1)：5-7.
[36]Tishler R B, Lampu D M, Park S, et al. Microtubule-active drug taxol, vinblastine, and nocodazole increase the levels of transcrptionally active P53[J]. Cancer Res,1995,55 (3):602-605.
[37]Samel D, Deana D A, De Witte P. The effect of purified extract of Fagopyrum esculent um(buckwheat)on protein kinases involved in signal transduction pathways[J]. Planta Med,1996,62 (2):106-110.
[38]程春旭,高颜茹.抗肿瘤药物作用机制的研究进展[J].吉林医学,2009,30(22)：3080-3083.
[39]张莉萍,蒋纪恺,谭荣安.苦参碱对K562细胞株端粒酶活性和细胞周期的影响[J].中华肿瘤杂志,1998,20(5)：328-330.
[40]徐秀,月杨戚.活化型高分子量激肽原潜在的抗肿瘤作用及其分子机制[J].中国生物化学与分子生物学报,2006,22(9)：694-697.
[41]王广秀.白黎芦醇抗肿瘤作用的研究进展[J].国外医学·肿瘤学分册,2006,32(12)：894.
[42]Takara K, Sakaeda T, Okumura K. An update on overcoming (MDR)mediated multidrug resistance in cancer chemotherapy [J]. Curr Pharm Des,2006,12(3):273-286.
[43]石宇雄,黄永明,许少键,等.参麦注射液对人成骨肉瘤多药耐药细胞株R-OS-732逆转作用[J].广东医学,2006,27(8)：1200-1201.
[44]李雪梅,吴运光,潘达鑫.新型抗肿瘤药槐定碱[J].中国新药杂志,2006,15(8)：655-658.
[45]Lin J, Dong H F, Oppenhein J J, et al. Effects of astragli radix on the growth of different cancer cell lines[J]. World J Gastroenterol,2003,9(3):670-673.
[46]黄自丽,黄修燕,郑起.中药抗肿瘤作用及其作用机制研究进展[J].医学综述,2010,16(3)：386-389.
[47]Mans D R, Rocha A B, Schwartsmann G, et al. Anti-cancer drug discovery and development in Brazil:targeted plant collection as a rational strategy to acquire candidate anti-cancer compounds[J]. Oncologist,2000,5:185-198.
[48]Duffin J. Poisoning the spindle:serendipity and discovery of the anti-tumor properties of the vinca alkaloids[J]. Canc Bull Med Hist,2000,17:155-192.
[49]赵锐,赵玮玮.抗癌植物药紫杉醇研究与动态[J].中草药,2009,40(7)：1172-附2.
[50]徐艳红.宫颈癌的致病因素及预防的研究综述[J].中国实用医药,2009,25(4)：252-253.
[51]李华,高国兰.HPV 与宫劲癌的研究进展[J].实用癌症杂志,2007,22(4)：420-422.
[52]De Villiers EM, Gunst K, Stein H, et al. Esophageal squamous cell cancer in patients with head and neck cancer prevalence of human papillomavirus DNA sequences[J]. Int J Cancer,2004,109 (2): 253.
[53]Rion G, Faver M, Jeannel D, et al. Association between poor prognosis in early stage invasion cervical carcinomas and non-detection of HPV DNA[J]. Lancet,1990,335:1171.
[54]乔玉环,张梦真,史记蓉.子宫颈癌及其癌前病变组织端粒酶活性的研究[J].中华妇产科杂志,2001,36(8)：483.
[55]Fu J, Jin Y, Arend LJ. Smac3, a novel smac/DIABLO splicing variant, attenuates the stability and apoptosis inhibiting activity of X-linked inhibitor of apoptosis protein[J]. J Biol Chem,2003,278 (52):52660.
[56]贾艳菊,代玲,张灿.龙葵生物碱诱导HeLa细胞凋亡的研究[J].动物医学进展,2010,31(8)：51-54.
[57]崔晓燕,罗琼,杨明亮,等.枸杞多糖对人宫颈爱细胞生长及细胞凋亡影响[J].中国公共卫生,2006,22(12)：1411-1412.
[58]赵令武,万福生.白英水提物诱导人宫颈癌HeLa细胞凋亡的实验研究[J].中国临床药理学与治疗学,2007,12(8)：883-886.
[59]李志玖,郑芳,蒋飞,等.自藜芦醇对宫颈癌HeLa细胞凋亡的诱导作用[J].郧阳医学院学报,2009,28(1)：16-17,
[60]陈艳,田莉,盛习峰,等.紫花牡荆素诱导人宫颈癌HeLa细胞凋亡[J].湖南师范大学学报(医学版),2010,7(2)：11-13.
[61]JI Y B, MA H T, YANG B, et al. Antitumor effects of different extract fractions from Qinglongyi[J]. China Tradit Herb Drugs,2004,35 (10):1145-1147.
[62]JI Y B, JI C F, MA H T. Study on the effects of cool and hot ethanol extracts from Qinglongyi on the biochemical function of tumor membrane in H22 mice[J]. China J Chin Mater Med,2005,30 (7): 531-534.
[63]LIU W, JI Y B. Study on acute toxicity experiment of mice and anti-tumor function in vivo of Chinese traditional medicine Qinglongyi[J]. J Harbin Univ Com(Nat Sci Ed),2004,20(1):4-10.
[64]LIU W, LIN W H, JI Y B. Study on the acute toxicity experiment of mice and anti-tumor function in vitro of the Qinglongyi[J]. China J Chin Mater Med,2004,29(9):887-890.
[65]张野平,杨志博,景永奎,等.胡桃醌对肿瘤细胞的增殖抑制作用和抗菌作用[J].沈阳药学院学报,1993,10(4)：271-274.
[66]孔陵生,邹翔,季宇彬.青龙衣中胡桃醌对A-549细胞周期及凋亡影响[J].哈尔滨商业大学学报(自然科学版),2008,24(6)：641-644.
[67]季宇彬,曲中原,邹翔,等.青龙衣中胡桃醌对S180肉瘤小鼠的抑瘤作用研究[J].中国药学杂志,2009,44(2)：195-199.
[68]周媛媛,王栋.青龙衣中二芳基庚烷类化学成分的研究[J].时珍国医国药,2009,20(8)：1936-1937.
[69]刘红兵.中草药东京枫杨Pterocarya tonkinesis (Franch.)Dode抗肿瘤活性成分的研究[D].青岛：中国海洋大学,2004,42：88.
[70]Liu J X, Di D L, Huang X Y, Li C. Two new diarylheptanoids from the pericarps of Juglans regia L.[J]. China Chem Lett,2007,18 (8):943-946.
[71]Liu J X, Meng M, Di D L, et al. Simultaneous determination of three diaryhptanoids and an a-tetralone derivative in the green walnut husks(Juglans regia L.)by high-performance liquid chromatography with photodiode array detector[J]. J Chrom A,2008,1190 (1):80-85.
[72]周媛媛,王栋,牛峰.抗肿瘤中药青龙衣化学成分的研究[J].中草药,2010,41(1)：11-14.
[73]卫小红,邵杰,王军辉,等.齐墩果酸诱导人肺腺癌细胞A549凋亡及其与细胞内钙离子的关系[J].同济大学学报(医学版),2009,30(5)：19-23.
[74]灵林蔚,蒲蔷,陈晓珍,等.齐墩果酸对卵巢癌细胞IGROV1和乳腺癌细胞MDA-MB-231生长的抑制作用[J].应用与环境生物学报,2010,16(2)：202-204.
[75]徐新伟,郭玲玲,顾振纶,等.熊果酸对乳腺癌SK-BR-3细胞增殖和凋亡的影响[J].苏州大学学报(医学版),2009,29(1)：68-70.
[76]卢丹,冯常炜,马彦娟.熊果酸对食管癌KYSE105细胞增殖、凋亡的影响[J].胃肠病学和肝病学杂志,2010,19(2)：130-133.
[77]王辉,方全华,李海军.熊果酸诱导人肝癌BEL-7404细胞凋亡的作用及其机制[J].肿瘤基础与临床,2010,23(2)：93-95.
[78]夏阳,苟欣.熊果酸诱导前列腺癌细胞株PC-3凋亡的实验研究[J].重庆医科大学学报,2009,34(12)：1661-1665.
[79]中国科学院上海药物研究所.中草药有效成分提取与分离[M].上海：上海科学技术出版社,1983.
[80]袁珂,刘延泽,冀春茹.中草药化学成分研究中几种不同提取方法的比较研究[J].天然产物研究与开发,1997,9(4)：57-61.
[81]向智敏,毕丽君,张骊,等.超临界二氧化碳萃取墨红花香气成分的研究[J].天然产物研究与开发,1996,8(4)：42-46.
[82]吴文君,刘惠霞,朱靖博,等.天然产物杀虫剂—原理·方法·实践[M].西安：陕西科学技术出版社,1998.
[83]储茂泉,古宏晨,刘国杰.中草药浸提过程的动力学模型[J].中草药,2000,31(7)：504-506.
[84]李才国,刘睿.木瓜中齐墩果酸的提取、分离及鉴定[J].现代商贸工业,2007,19(3)：159-162.
[85]韦国锋,程金生,黄祖良,等.大孔吸附树脂纯化女贞子中齐墩果酸的研究[J].中国现代应用药学杂志,2008,25(2)：90-92.
[86]李海鹏,徐怀德等.光皮木瓜齐墩果酸超声波辅助提取及纯化工艺[J].农业工程学报,2008,24(10)：222-226.
[87]黄晓君,殷志琦,叶文才,等.女贞子的化学成分研究[J].中国中药杂志,2010,35(7)：861-864.
[88]朱春林,杨洪元.亚微肝泰片中齐墩果酸的含量测定[J].中国中医药信息杂志,2010,17(11)：47-48.
[89]沈晓丹,袁杰,徐乃玉,等HPLC测定蓝萼香茶菜中齐墩果酸和熊果酸的含量[J].中成药,2010,32(12)： 2182-2183.
[90]陈亮,甘辉,刘洪.反相高效液相色谱法测定木瓜中齐墩果酸和乌索酸的含量[J].安徽农业科学,2007,35(32)：10183-10184.
[91]邓世星,孙志勇,周卿,等.紫外光谱法测定齐墩果酸含量[J].遵义医学院学报,2003,26(6)：556-557.
[92]张彦功,杨玉琴,李建,等.不同产地牛膝药材中齐墩果酸的含量测定[J]贵阳中医学院学报, 2010,32(1)：20-21.
[93]胡野,凌志强,单小云.细胞凋亡的分子医学[M].北京：军事医学科学出版社,2002：2210.
[94]中草药诱导人宫颈癌HeLa细胞凋亡及其分子机制[J].江西医学院学报,2006,46(5)：176-177.
[95]陈奇.中药药理研究方法学[M].北京：人民卫生出版社,1994：112.
[96]Zhu Y M. Synthesis and anti-HIV activity of oleanolic acid derivatives. Bioorganic and Medicinal Chemistry Letters,2001,11:3115-3118.
[97]张建平,包海花,刘海峰,等.薄层色谱法鉴别蛇莓中齐墩果酸[J].牡丹江医学院学报,2010,31 (1)： 57-58.
[98]闫花丽,赵陆华,朱丹妮,等.衍生化气相色谱法测定夏枯草中齐墩果酸和熊果酸的含量[J].中国中药杂志,1999,24(12)：744-745.
[99]袁晓环,王伟光,李春彦,等HPLC法检测蛇莓中齐墩果酸的含量[J].中国生化药物杂志,2010,31 (3)： 195-197.
[100]张兴旺,梅丽娟,邵赟,等HPLC法测定甘青铁线莲中的齐墩果酸[J].分析试验室,2009,28.Suppl：238-240.
[101]谭朝阳,袁宏佳HPLC法测定龙牙楤木药材中齐墩果酸的含量[J].中国中医药信息杂志2010,17(1)：46-47.
[102]陈婷婷,热娜·卡斯木HPLC法测定新疆鼠尾草三萜有效部位中熊果酸的含量[J].新疆医科大学学报,2008,31(4)：394-395.