摘要
人参皂苷Rg3(ginsenoside-Rg3)属于原人参二醇型皂苷,是一种从人参(Panax ginseng C. A. Meyer)中分离提取出来的天然化合物。由于其对肿瘤细胞有很强的抑制作用,所以在人医临床被广泛用于治疗各种肿瘤疾病。Rg3在自然界中存在20-C键的两种差向异构体,即20(R)-Rg3和20(S)-Rg3,近年来研究表明两种Rg3异构体在许多免疫活性研究中存在显著差异。因此,本论文通过研究Rg3两种异构体的免疫佐剂作用、抗氧化作用和对巨噬细胞功能的调节作用,研究Rg3两种异构体的免疫学特性,并比较两者间免疫活性的差异。
1Rg3不同构型免疫佐剂作用的研究
目的：采用OVA作为模式抗原,探讨Rg3两种不同构型的免疫佐剂作用和对小鼠Thl/Th2免疫平衡的调节机制。方法：将32只BALB/c小鼠随机分为4组,分别为生理盐水对照组、OVA对照组、OVA+20(R)-Rg3佐剂组和OVA+20(S)-Rg3佐剂组。第一次免疫后,间隔3周再免疫第二次。分别采集一免和二免后第14天小鼠血液,并检测血清IgM抗体水平、IgG及其亚类抗体水平。二免后第14天,所有供试小鼠处死并分离培养脾脏淋巴细胞,RT-PCR法检测OVA抗原刺激后脾淋巴细胞中相关细胞因子的表达；采用MTT法测脾淋巴细胞增殖试验；Western-blot法检测血清中细胞因子的表达。结果：20(R)-Rg3或20(S)-Rg3做为免疫佐剂与OVA对照组相比,均能显著提高小鼠一免和二免后血清中抗OVA特异性IgG及其亚类(IgG1, IgG2a, IgG2b, IgG3)和IgM水平；促进小鼠脾淋巴体外增殖(P<0.05)；显著增强细胞因子Interleukin-4(IL-4), Interleukin-10(IL-10), Interferon-y (IFN-y), Interleukin-12(IL-12)以及核转录因子T-bet和GATA-3的表达(P<0.05)；显著增强血清中细胞因子Interleukin-5(IL-5)和Interferon-γ (IFN-γ)的表达(P<0.05)。且OVA+20(R)-Rg3组各项指标均优于OVA+20(S)-Rg3组。结果表明,20(R)-Rg3和20(S)-Rg3均具有明显的免疫佐剂效应,并且能调节机体Th1/Th2免疫平衡,20(R)-Rg3免疫佐剂效果优于其异构体20(S)-Rg3。
2Rg3不同构型抗氧化作用的研究
目的：用环磷酰胺(Cyclophosphamide Cy)建立小鼠免疫抑制模型,在此基础上应用20(R)-Rg3和20(S)-Rg3处理小鼠,观察20(R)-Rg3和20(S)-Rg3对免疫抑制小鼠胸腺指数、脾脏指数、酸性磷酸酶(Acid Phosphatase ACP)、超氧化物歧化酶(Superoxide Dismutase SOD)、一氧化氮(Nitric Oxide NO)、溶菌酶(Lysozyme LZM)、总抗氧化能力(Total Antioxidant Capacity T-AOC)、黄嘌呤氧化酶(Xanthine Oxidase XOD)、过氧化氢酶(Catalase CAT)和丙二醛(Malondialdehyde MDA)等免疫学和抗氧化指标的影响,探讨20(R)-Rg3和20(S)-Rg3对小鼠抗氧化能力的调节作用。结果：20(R)-Rg3或20(S)-Rg3均可提高由Cy诱导的免疫抑制导致降低的小鼠胸腺指数、脾脏指数、ACP活性、SOD活性、LZM活性、T-AOC和CAT活性；降低Cy诱导的NO、XOD活性和MDA水平的升高。结果表明,20(R)-Rg3和20(S)-Rg3均能对抗Cy诱导的小鼠免疫抑制,具有明显的抗氧化作用,20(R)-Rg3抗氧化活性优于其异构体20(S)-Rg3。
3Rg3不同构型对巨噬细胞功能调节作用的研究
目的：体外分析20(R)-Rg3和20(S)-Rg3对巨噬细胞系RAW264.7(?)乎吸爆发和吞噬活性的影响及诱导鼠源巨噬细胞的细胞因子(IL-10和TNF-α)和Nuclear factor kappa B (NF-κB)分泌的作用。方法：实验一经20(R)-Rg3或20(S)-Rg3预培养的RAW264.7,用二氢诺丹明(Dihydrorhodaminel23DHR)处理后,与粉红色荧光乳胶微球共同培养,由于细胞的呼吸爆发作用可引起细胞内本身不发光的DHR转化为发绿色荧光的诺丹明(Rhodamine ROD),用流式细胞仪和激光共聚焦显微镜同时测定粉红色荧光(代表细胞吞噬或粘附粉红色荧光微球的数量)和绿色荧光(间接反应了细胞呼吸爆发的力度),以检测20(R)-Rg3和20(S)-Rg3对巨噬细胞Raw2647呼吸爆发和吞噬活性的影响。实验二以Raw-blue cells作为研究对象,分别加入不同浓度的20(R)-Rg3或20(S)-Rg3(1、10、100μg/m1)以刺激细胞,然后从细胞培养上清中检测分泌型碱性磷酸酶(SEAP)的表达。实验三以不同浓度的20(R)-Rg3或20(S)-Rg3(1、10、100μg/ml)刺激RAW264.7,并收集细胞,用RT-PCR法检测细胞分泌的细胞因子Interleukin-10(IL-10)水平和Tumor necrosis factor-α (TNF-α)水平。结果：20(R)-Rg3能显著提高RAW264.7的呼吸爆发作用和吞噬活性(P<0.05),相反,20(S)-Rg3显著抑制了正常RAW264.7的呼吸爆发作用和吞噬活性(P<0.05)；20(R)-Rg3或20(S)-Rg3刺激细胞内NF-κBB的活化随浓度的不断增大而降低,但不受LPS特异性抑制剂PMB抑制,且20(R)-Rg3刺激细胞内NF-κB的活化程度大于20(S)-Rg3(P<0.05)；体外20(R)-Rg3或20(S)-Rg3刺激细胞内TNF-a和IL-10的分泌随浓度的不断增大而降低。与20(S)-Rg3相比,20(R)-Rg3能显著刺激细胞内TNF-a和IL-10的分泌(P<0.05)。结果表明,20(R)-Rg3可能是通过增强细胞呼吸爆发作用和吞噬作用清除机体内的活性氧自由基,发挥抗氧化作用,但20(S)-Rg3抑制细胞呼吸爆发作用和吞噬作用的机制有待进一步研究。
综上所述,20(R)-Rg3的免疫佐剂作用、抗氧化能力和巨噬细胞调节作用都优于20(S)-Rg3,是一种理想的免疫调节剂。
Ginsenoside-Rg3is native compound, extract from Panax ginseng C. A. Mayer, belong to protopanaxadiol type saponin. This saponin exerts many pharmacological activities such as tumor-suppressing and antimetastatic, so is widely used to treat many kinds of tumor diseases. Two optical isomers of20(R)-Rg3and20(S)-Rg3have been found in ginseng products. They are epimers of each other depending on the position of the hydroxyl (OH) group on carbon-20. The space position of chemical group(s) in a molecular structure may significantly influence its pharmacological activities. So, this study was designed to evaluate Rg3and its epimers,20(R)-Rg3and20(S)-Rg3, for their effects on adjuvant, antioxidant and regulatory role in macrophages.
1Adjuvant effects of Rg3and its epimers
Objective To measure the adjuvant effects on the immune response against ovalbumin (OVA) of20(R)-Rg3and20(S)-Rg3, and the effect of immune balance between Thl and Th2.
Methods32mice were randomly divided into4groups of8animals each. At week0and3, mice were subcutaneously immunized twice with10μg of OVA alone or with10μg of OVA mixed in20(R)-Rg3(50μg) or20(S)-Rg3(50μg) on Day1and Day15. Two weeks post the last immunization, blood samples were collected to examine IgG and the IgG subclasses, as well as interferon (IFN)-γ and interleukin (IL)-5; splenocytes were prepared to measure proliferative responses to stimulations with concanavalin A (ConA), lipopolysacharide (LPS) and OVA, and mRNA expressions of cytokines and transcription factors by RT-PCR. Results Both20(R)-Rg3and20(S)-Rg3exhibited the adjuvant effects on OVA-induced immune responses.20(R)-Rg3promoted significantly higher serum specific IgG and the IgG isotype responses in association with highly up-regulated serum IFN-y and IL-5than20(S)-Rg3. In addition,20(R)-Rg3significantly enhanced splenocyte proliferative responses to ConA, LPS and OVA, as well as mRNA expression of IFN-y, IL-12, IL-4and IL-10and transcription factors T-bet and GATA-3by splenocytes when compared with the20(S)-Rg3. Therefore, ginsenoside Rg3is sterospecific in stimulation of the immune response, and20(R)-Rg3has more potent adjuvant activity than20(S)-Rg3.
2Antioxidant effects of Rg3and its epimers
Objective The present study was designed to evaluate Rg3and its epimers,20(R)-Rg3and20(S)-Rg3, for their effects on oxidative stress induced by cyclophosphamide (Cy) in mice. Methods Forty-eight mice were randomly distributed into6groups and The animals were intraperitoneally administered saline solution, Cy,20(R)-Rg3,20(S)-Rg3,20(R)-Rg3+Cy or20(S)-Rg3+Cy respectively. The spleen, thymus and serum were collected to measure the indices of the organs and oxidative parameters. Results The findings showed that Rg3significantly inhibited Cy-induced oxidative stress in mice by increasing the indices of the spleen and thymus and total antioxidant capacity, elevating the activities of catalase, superoxidase dismutase and lysozyme as well as decreasing the activity of xanthine oxidase and the levels of malondialdehyde and nitric oxide. Rg3was stereospecific in antioxidant activities as R form exhibited significantly higher antioxidant effects than S form. Therefore, R form might be a better candidate when Rg3is considered as an antioxidant agent.
3Immunomodulatory effect of Rg3and its epimers on macrophage
Objective To investigate the effect of macrophage stimulated by Rg3on the activation of the phagocytosis rate, oxidative burst activity, the secretion of cytokines and NF-κB. Methods In experiment A, RAW264.7cells were stimulated with20(R)-Rg3or20(S)-Rg3for24h. After incubation of RAW264.7with pink fluorescent beads, intracellular dihydrorhodamine123(DHR) was oxidized into green fluorescent rhodamine123(ROD) by oxidative burst products. The cells with beads and ROD were simultaneously detected by a flow cytometer or laser scanning confocal microscope. In experiment B, RAW-blue cells were stimulated with20(R)-Rg3or20(S)-Rg3for24h, then the SEAP in supernate were detected. RAW-blue can be induced by activated NF-κB to secrete SEAP protein, which is a macrophage cell line (RAW264.7). In experiment C, RAW264.7cells were stimulated with20(R)-Rg3or20(S)-Rg3for24h. The secretions of TNF-a and IL-10were quantified by RT-PCR. Results20(R)-Rg3increased the phagocytosis rate and respiratory burst activity of macrophage, but20(S)-Rg3decreased the phagocytosis rate and respiratory burst activity of macrophage. Both 20(R)-Rg3and20(S)-Rg3significantly promoted the secretion of IL-10and TNF-α(P <0.05) and the activation of NF-κB (P<0.05).
In conclusion, the immune adjuvant effect, antioxidant effect and Immunomodulatory effect on macrophages of20(R)-Rg3are better than20(S)-Rg3.20(R)-Rg3is an ideal adjuvant candidate with antioxidant and immunomodulatory activities.
引文
1. Xie H, Wang Y, Wang G, Sheng I, Liu Z. Content determination of ginsenoside Rg and ginsenoside Re in Shenfu injection. W China J Parm Sci 2006;21:208-209
2. Shinkai K, Akedo H, Mukai M, Imamura F, Isoai A, Kobayashi M, et al. Inhibition of in vitro tumor cell invasion by ginsenoside Rg3. Jpn J Cancer Res 1996;87:357-362
3. Mochizuki M, Yoo YC, Matsuzawa K, Sato K, Saiki I, Tono-oka S, et al. Inhibitory effect of tumor metastasis in mice by saponins, ginsenoside-Rb2,20(R)-and 20(S)-ginsenoside-Rg3, of red ginseng. Biol Pharm Bull 1995;18:1197-1202
4. 程慧,宋新波,张丽娟.人参皂苷Rg3与Rh2的研究进展.药物评价研究2010；33：307-310
5. Kim ND, Kim EM, Kang KW, Cho MK, Choi SY, Kim SG. Ginsenoside Rg3 inhibits phenylephrine-induced vascular contraction through induction of nitric oxide synthase. British Journal of Pharmacology 2003;140:661-670
6. Tian J, Fu F, Geng M, Jiang Y, Yang J, Jiang W, et al. Neuroprotective effect of 20(S)-ginsenoside Rg3 on cerebral ischemia in rats. Neurosci Lett 2005;374:92-97
7. Li YX, Qu XB, Zhao Y, Song Y, Zhang W, Zhao B. Raman spectroscopy study on the structure of ginsenoside Rg3. Spectrosc Spect Anal 2008;28:569-571
8. Mochizuki M, Yoo YC, Matsuzawa K, Sato K, Saiki I, Tonooka S, et al. Inhibitory Effect of Tumor-Metastasis in Mice by Saponins, Ginsenoside-Rb2, 20(R)-Ginsenoside-Rg3 and 20(S)-Ginsenoside-Rg3, of Red-Ginseng. Biol Pharm Bull 1995;18:1197-1202
9. Chung SH, Park MW, Ha J.20(S)-ginsenoside Rg3 enhances glucose-stimulated insulin secretion and activates AMPK. Biol Pharm Bull 2008;31:748-751
10. 李欣,万红贵,卢定强,韦萍.人参皂甙的抗肿瘤研究进展.生物加工过程2003；1：13-17
11. 范文宇.浅谈中药人参的药理作用与应用研究.新疆中医药2010；28：89-92
12. 刘安全.浅谈人参的治疗作用和保健功能.内蒙古中医药2011；24：42
13. Yun TK. Panax ginseng--a non-organ-specific cancer preventive? Lancet Oncol 2001;2:49-55
14. 陈燕.鲜人参、生晒参和红参的比较研究.海峡药学2006；18：137-139
15. Li X, Li L. Pharmacological changes of ginseng resulting from the processing. Zhongguo Zhong Yao Za Zhi 1991;16:3-7
16. Kwon SW, Han SB, Park IH, Kim JM, Park MK, Park JH. Liquid chromatographic determination of less polar ginsenosides in processed ginseng. J Chromatogr A 2001;921:335-339
17. Lau AJ, Seo BH, Woo SO, Koh HL. High-performance liquid chromatographic method with quantitative comparisons of whole chromatograms of raw and steamed Panax notoginseng. J Chromatogr A 2004;1057:141-149
18. Park JH, Kim WY, Kim JM, Han SB, Lee SK, Kim ND, et al. Steaming of ginseng at high temperature enhances biological activity. Journal of Natural Products 2000;63:1702-1704
19. 冯鑫,陈晓林,石磊,田义新.西洋参传统加工及现代加工技术展望.人参研究2010：1：27-28
20. 张崇禧,李向高,郑友兰.西洋参的加工与贮藏研究进展.人参研究1994；1：14-18
21. 安美花,安金花.人参研究进展.宁夏农林科技2010；6
22. Song X, Hu S. Adjuvant activities of saponins from traditional Chinese medicinal herbs. Vaccine 2009;27:4883-4890
23. 许弟群,王人卫,曹芳.人参皂甙Rg1对大负荷运动训练小鼠免疫功能的影响.西南师范大学学报2011；5：192-197
24. 周娅红.人参总皂苷对免疫低下小鼠免疫功能的影响.中国中医急症2010：19：1559-1561
25. 刘伟宏,龚守良,李新民.人参三醇组甙对雄性大鼠免疫器官的辐射防护作用.白求恩医科大学学报1994；20：32-34
26. 骆艳秋,尹柏双,徐文勇,姜成.人参叶皂甙对鸡免疫功能调节的研究.安徽农业科学2008；36：202-204
27. 王海凤,郭兵,许丽,刘荣欣.人参皂苷对免疫抑制小鼠T细胞亚群(CD4+/CD8+)及血清TNF-a含量的影响.中国饲料2011;14：41-43
28. 吕梦捷,曾耀英,宋兵.人参皂苷Rb1对小鼠T淋巴细胞体外活化、增殖及凋亡的影响.中草药2011；42：743-748
29. 胡银英,郭江梅,黄艳琴,袁芳.人参皂甙Rg3刺激淋巴细胞CD4上调以及对胃癌细胞MKN-45的杀伤作用.实验与检验医学2011；29：24-26
30. 陈宇,郑纯威,陈国江,张文娟.人参皂苷Rg1免疫佐剂作用的研究.军事医学科学院院刊2009；33：251-292
31. 张丽媛,吴铁.人参皂苷Rh2抗肿瘤作用机制的研究进展.安徽农业科学2008；36：1467-1468
32. 张克坚,刘耕陶.人参叶总皂苷对老龄大鼠白细胞介导的防御功能的影响.中药药理与临床1998；14：17-19
33. 曹丽,顾学文,张天一,汪骅.人参皂甙对小鼠腹腔巨噬细胞活性的影响及其作用机制初步探讨.中国免疫学杂志2011；27：423-433
34. 吕梦捷,曾耀英,宋兵.人参皂甙Rbl对小鼠腹腔巨噬细胞体外吞噬及细胞因子和NO分泌的影响.细胞与分子免疫学杂志2011；27：242-248
35. 王英,楼建国.人参皂苷Rg3对NK细胞与K562细胞的结合率及毒性作用影响的实验研究.中药材2011：34：1270-1273
36. 葛迎春,李晨燕,任慧君.人参皂甙Rh2注射液对(H22)小鼠免疫功能的影响.特产研究2002；24：4-7
37. 郭志廷,韦旭斌,梁剑平.人参总皂苷及其衍生物对小鼠淋巴细胞和NK细胞免疫活性的影响.中国兽医学报2007；27：715-722
38. 刘红樱,葛均波,马晓娟,史大卓.人参皂苷Rbl对氧化型低密度脂蛋白诱导的人单核细胞源树突状细胞免疫成熟的影响.中国中西医结合杂志2011：31：350-354
39. 于君丽,窦德强,陈晓红,杨红振,胡晓燕.人参皂苷Ro促进小鼠脾细胞增殖及调节小鼠脾细胞Th1/Th2细胞因子的产生.药学学报2005；4：332-336
40. 郭志廷,伊鹏霏,褚秀玲,韦旭斌.人参皂苷Rh2衍生物体外诱生小鼠细胞因子 的作用及对NO水平的影响.畜牧与兽医2007；39：1-3
41. 陈英杰,王红燕,徐馁绪.人参化学成分及其抗肿瘤抗心律失常构效关系的研究.中国科学基金1995；9：46-48
42. 薛东波.人参皂甙Rg3抗肿瘤转移作用的研究进展.中国中西医结合外科杂志2001；7：289-290
43. Zhu JH, Takeshita T, Kitagawa I. Suppression of the formation of sister chromatid exchages by low concentrations of ginsenoside Rh2 in human blood lymphocytes. J Cancer Res 1995;55:221-223
44. 夏丽娟,韩锐.人参皂甙Rh2体外对小鼠黑色素瘤细胞的分化诱导作用.药学学报1996；31：742-745
45. 程晓耕,赵雪俭,迟宝琦.IFN和PDS合剂体内抗流感病毒的作用.微生物学杂志2004；24：64-65
46. 褚秀玲,苏建青,韦旭斌.人参皂苷免疫调节和抗病毒作用研究进展.中兽医医药杂志2008；5：20-23
47. 李亚平,郝秀华,赵春芳.人参皂甙Rg3、Rb3抗病毒作用的研究.中国老年学杂志2001：21：215-216
48. 胡云亮,孔祥峰,李祥瑞.10种中药成分对培养细胞生长和抵抗病毒感染的影响.畜牧与兽医2004；35：4-7
49. 厉曙光,白莉华,王力强,陈科,于晓楠,李欣.人参对果蝇寿命的影响及其抗氧化作用.卫生研究2008；37：104-106
50. 李欣,厉曙光,于晓楠,张晶.人参拮抗DEHP对果蝇寿命及脂质过氧化的影响.环境与职业医学2008；25：148-151
51. 程俊霖,周黎明,朱玲.人参茎叶总皂苷对衰老小鼠的作用研究.四川生理科学杂志2004；26：97-99
52. 睢大员,于晓风,曲绍春.西洋参叶20S-原人参二醇组皂苷对大鼠实验性心室重构的影响.中国药学杂志2007；42：108-110
53. 陈冠敏,陈小萍,何聆,黄宗锈,郑丽红.人参片对大鼠血脂水平及延缓衰老作用的实验研究.实用预防医学2002；9：311-313
54. 何苗.人参总皂苷对四氯化碳诱导的大鼠肝纤维的保护作用.安徽医药2011;15：423-425
55. 沈志强,吴蓝鸥,雷伟亚,陈植和,刘吉开.人参皂苷Rgl对中性粒细胞与血小板之间粘附的影响.中草药2002；33：138-140
56. 于晓风,曲绍春,刘巍.西洋参茎叶20S-原人参二醇组皂苷对实验性脑缺血大鼠血小板功能及血液流变学的影响.中药材2011；34：272-275
57. Sotanimi E, Haapajoski E, Rautio A. Ginseng therapy in noninsulin-dependent diabetic patients. Diabetes Care 1995;18:1373-1375
58. Yang Z, Sun H, Ye Y. Ginsenoside Rd from Panax notoginseng is cytotoxic towards HeLa cancer cells and induces apoptosis. Chem Biodivers 2006;3:187-197
59. 胡松华,林锋强.人参皂甙Rb1的免疫佐剂作用.中国兽医学报2003；23：480-482
60. 储岳峰,颜新敏,胡元亮.几种中药成分的免疫增强活性及其作用效果.中国兽医科技2005：35：67-70
61. O'Hagan DT, MacKichan ML, Singh M. Recent developments in adjuvants for vaccines against infectious diseases. Biomolecular engineering 2001;18:69-85
62. Zhang QY, Kang XM, Zhao WH. Antiangiogenic effect of low-dose cyclophosphamide combined with ginsenoside Rg3 on Lewis lung carcinoma. Biochem Bioph Res Co 2006;342:824-828
63. Yuan CS, Shao ZH, Xie JT, Vanden Hoek TL, Mehendale S, Aung H, et al. Antioxidant effects of American ginseng berry extract in cardiomyocytes exposed to acute oxidant stress. Bba-Gen Subjects 2004;1670:165-171
64. Park EK, Choo MK, Han MJ, Kim DH. Ginsenoside Rhl possesses antiallergic and anti-inflammatory activities. Int Arch Allergy Imm 2004; 133:113-120
65. Rivera E, Daggfeldt A, Hu S. Ginseng extract in aluminium hydroxide adjuvanted vaccines improves the antibody response of pigs to porcine parvovirus and Erysipelothrix rhusiopathiae. Vet Immunol Immunop 2003;91:19-27
66. Rivera E, Hu S, Concha C. Ginseng and aluminium hydroxide act synergistically as vaccine adjuvants. Vaccine 2003;21:1149-1157
67. Waller KP, Hu S, Concha C, Lin F. Adjuvant effect of ginseng extracts on the immune responses to immunisation against Staphylococcus aureus in dairy cattle. Vet Immunol Immunop 2003;91:29-37
68. Hu YL, Kong XF, Rui R, Wang DY, Li XG. Effects of Chinese herbal medicinal ingredients on peripheral lymphocyte proliferation and serum antibody titer after vaccination in chicken. Int Immunopharmacol 2004;4:975-982
69. Rivera E, Pettersson FE, Inganas M, Paulie S, Gronvik KO. The Rbl fraction of ginseng elicits a balanced Thl and Th2 immune response. Vaccine 2005;23:5411-5419
70. Hu S, Sun JH, Song XM. Adjuvant effects of protopanaxadiol and protopanaxatriol saponins from ginseng roots on the immune responses to ovalbumin in mice. Vaccine 2007;25:1114-1120
71. Chang KH, Jee HS, Lee NK, Park SH, Lee NW, Paik HD. Optimization of the enzymatic production of 20(S)-ginsenoside Rg(3) from white ginseng extract using response surface methodology. N Biotechnol 2009;26:181-186
72. Kim ND, Kang SY, Park JH, Schini-Kerth VB. Ginsenoside Rg3 mediates endothelium-dependent relaxation in response to ginsenosides in rat aorta:role of K+ channels. Eur J Pharmacol 1999;367:41-49
73. Chen D, Cui JS, Liu XX. Study on Hela cell apoptosis induced by 20(S)-ginsendoside Rg3 in cervical cancer. Chinese Journal of Laboratory Diagnosis 2008;12:463-466
74. Xin Y, Ni JS, Wang XR, Shi B, Wang YH, Wu JX. Inhibitory effect of 20(S)-ginsenoside Rg3 on B16 melanoma metastasis. Journal of Jilin University (Medicine Edition) 2004;30:540-542
75. Li X, Guan YS, Zhou XP, Sun L, Liu Y, He Q, et al. [Anticarcinogenic effect of 20(R)-ginsenoside Rg3 on induced hepatocellular carcinoma in rats]. Sichuan da xue xue bao Yi xue ban= Journal of Sichuan University 2005;36:217-220
76. Gao S, Tang W, Zhang Y, Gao J, Ding X. The Anti-fatigue Effect of 20(R)-Ginsenoside Rg3 in Mice by Intranasally Administration. Biol Pharm Bull 2008;31:2024-2027
77. Wang H, Lin Y. Advances in anti-tumor effects of ginsenoside Rg3. Med Rec 2009;15:323-326
78. Xie F, Li Y, Su F, Hu S. Adjuvant effect of Atractylodis macrocephalae Koidz. polysaccharides on the immune response to foot-and-mouth disease vaccine. Carbohydrate Polymers 2012;87:1713-1719
79. 李萍,熊凡,富青,杨莉.一种简便的RNA完整性检测方法.湖北中医学院学报2005：7：39-40
80. In:Commission CP ed, Pharmacopoeia of the People's Republic of China, Part Ⅰ: People's Medical Publishing House; 2005:205-206
81. Ding L. Investigation of ginseng markets. Modern Chinese Medicine 2010; 12:44-46
82. Liu CX, Xiao PG. Recent advances on ginseng research in China. Journal of ethnopharmacology 1992;36:27-38
83. Seong Y, Shin C, Kim H, Baba A. Inhibitory effects of ginseng total saponins on glutamate-induced swelling of cultured astrocytes. Biol Pharm Bull Commun 1995;18:1776-1780
84. Kitts DD, Wijewickreme AN, Hu C. Antioxidant properties of a North American ginseng extract. Molecular and cellular biochemistry 2000;203:1-10
85. Joo S, Yoo Y, Ahn B, Nam S. Prevention of inflammation-mediated neurotoxicity by Rg3 and its role in microglial activation. Biol Pharm Bull 2008;31:1392-1397
86. Hu S, Concha C, Lin F, Persson Waller K. Adjuvant effect of ginseng extracts on the immune responses to immunisation against Staphylococcus aureus in dairy cattle. Vet Immunol Immunopathol 2003;91:29-37
87. Song X, Chen J, Sakwiwatkul K, Li R, Hu S. Enhancement of immune responses to influenza vaccine (H3N2) by ginsenoside Re. International immunopharmacology 2010;10:351-356
88. Zhai L, Li Y, Wang W, Wang Y, Hu S. Effect of oral administration of ginseng stem-and-leaf saponins (GSLS) on the immune responses to Newcastle disease vaccine in chickens. Vaccine 2011;29:5007-5014
89. Zhai L, Li Y, Wang W, Hu S. Enhancement of humoral immune responses to inactivated Newcastle disease and avian influenza vaccines by oral administration of ginseng stem-and-leaf saponins in chickens. Poultry science 2011;90:1955-1959
90. Liu Z-H, Li L, Qian F. Process optimization of ginsenoside Rg3 prepared by hydrolysing Panax Notoginseng saponins by acetic acid. Chinese Journal of Hospital Pharmacy 2009;29:881-884
91. Li X, Wan HG, Lu DQ, Wei P. Advance of research on antitumour activity of ginsenosides. Chinese Journal of Bioprocess Engineering 2003;1:13-15
92. Kang DI, Lee JY, Yang JY, Jeong SM, Lee JH, Nah SY, et al. Evidence that the tertiary structure of 20(S)-ginsenoside Rg(3) with tight hydrophobic packing near the chiral center is important for Na(+) channel regulation. Biochem Biophys Res Commun 2005;333:1194-1201
93. Feldman M MJ. Cell cooperation in the antibody response. In:Roitt I, Brostoff J, Male D eds, Immunology.6 ed. London:Mosby Publication; 2001:131-146
94. Marciani DJ, Press JB, Reynolds RC, Pathak AK, Pathak V, Gundy LE, et al. Development of semisynthetic triterpenoid saponin derivatives with immune stimulating activity. Vaccine 2000;18:3141-3151
95. Zima AV, LA B. Redox regulation of cardiac calcium channels and transporters. Cardiovasc Res 2006;71:310-321
96. 刘春英,张勇,高玲.活性氧的信号转导功能.食品与药品2007；9：72-72
97. Fujino G, Noguchi T, Takeda K, Ichijo H. Thioredoxin and protein kinases in redox signaling. Semin Cancer Biol 2006; 16:427-435
98. Zima A, Blatter L. Redox regulation of cardiac calcium channels and transporters. Cardiovasc Res 2006;71:310-321
99. Sauer H, Rahimi G, Hescheler J, Wartenberg M. Role of reactive oxygen species and phosphatidyinositol 3-kinase in cardio myocyte diferentiation of embryonic stem cells. FEBS Lett 2000;476:218-223
100. 秦涛余,陈志伟.机体内活性氧生理功能研究进展.食品与药品2008；6：12-16
101. Sohji N, Keisuke H, Masaaki K. Hepatitis C Virus-Induced Reactive Oxygen Species Raise Hepatic Iron Level in Mice by Reducing Hepcidin Transcription. Gastroenterology 2008; 134:226-238
102. Lina W, Tsaia W-L, Shao R-X. Hepatitis C Virus Regulates Transforming Growth Factor β1 Production Through the Generation of Reactive Oxygen Species in a Nuclear Factor κB-Dependent Manner. Gastroenterology 2010;138:2509-2518
103. Leila G, Muzammil A, Wei Z. Oligonol a low molecular weight polyphenol of lychee fruit extract inhibits proliferation of influenza virus by blocking reactive oxygen species-dependent ERK phosphorylation. Phytomedicine 2010;17:1047-1056
104. Malgorzata G, Jacek B. Increased mitochondrial superoxide dismutase expression and lowered production of reactive oxygen species during rotavirus infection. Virology 2010;404:293-303
105. Tripathi D, Jena G. Intervention of astaxanthin against cyclophos-406 phamide-induced oxidative stress and DNA damage:a study in mice. Chem Biol Interact 2009;180:398-406
106. Oboh G, Akomolafe T, Adefegha S, Adetuyi A. Inhibition of 409 cyclophosphamide-induced oxidative stress in rat brain by polar and 410 non-polar extracts of Annatto (Bixa orellana) seeds. Exp Toxicol Pathol 2010;63:257-262
107. Seifried HE, Anderson DE, Fisher EI, Milner JA. A review of the interaction among dietary antioxidants and reactive oxygen species. J Nutr Biochem 2007;18:567-579
108. Kozarski M, Klaus A, Niksic M, Jakovljevic D, Helsper JPFG, Griensven LJLDV. Antioxidative and immunomodulating activities of polysaccharide extracts of the medicinal mushrooms Agaricus bisporus, Agaricus brasiliensis, Ganoderma lucidum and Phellinus linteus. Food Chem 2011;129:1667-1675
109. Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 2010;42:1634-1650
110. Tripathi DN, Jena GB. Intervention of astaxanthin against cyclophosphamide-induced oxidative stress and DNA damage:a study in mice. Chem Biol Interact 2009;180:398-406
111. Oboh G, Akomolafe TL, Adefegha SA, Adetuyi AO. Inhibition of cyclophosphamide-induced oxidative stress in rat brain by polar and non-polar extracts of Annatto (Bixa orellana) seeds. Exp Toxicol Pathol 2010;63:257-262
112. Barroso AS, Quissell DO, Colepicolo P. Modulation of SOD activity in rat submandibular glands. Arch Oral Biol 2003;48:133-139
113. Ravid A, Rocker D, Machlenkin A, Rotem C, Hochman A, Kessler-Icekson G, et al. 1,25-dihydroxyvitamin D-3 enhances the susceptibility of breast cancer cells to doxorubicin-induced oxidative damage. Cancer Res 1999;59:862-867
114. Lisdat F, Ge B. Superoxide sensor based on cytochrome c immobilized on mixed-thiol SAM with a new calibration method. Anal Chim Acta 2002;454:53-64
115. Tunon-Blanco P, Barroso MF, de-los-Santos-Alvarez N, Lobo-Castanon MJ, Miranda-Ordieres AJ, Delerue-Matos C, et al. Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity assessment. J Electroanal Chem 2011;659:43-49
116. Kaya S, Sutcu R, Cetin ES, Aridogan BC, Delibas N, Demirci M. Lipid peroxidation level and antioxidant enzyme activities in the blood of patients with acute and chronic fascioliasis. Int J Infect Dis 2007;11:251-255
117. Tsuchiya K, Jiang JJ, Yoshizumi M, Tamaki T, Houchi H, Minakuchi K, et al. Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD. Free Radical Bio Med 1999;27:347-355
118. Sueishi Y, Hori M, Kita M, Kotake Y. Nitric oxide (NO) scavenging capacity of natural antioxidants. Food Chem 2011;129:866-870
119. Shuai XH, Hu TJ, Liu HL, Su ZJ, Zeng Y, Li YH. Immunomodulatory effect of a Sophora subprosrate polysaccharide in mice. Int J Biol Macromol;46:79-84
120. Zeng XX, Qiao DL, Luo JG, Ke CL, Sun Y, Ye H. Immunostimulatory activity of the polysaccharides from Hyriopsis cumingii. Int J Biol Macromol 2010;47:676-680
121. Song LS, Cong M, Wang LL, Zhao JM, Qiu LM, Li L, et al. The enhanced immune protection of Zhikong scallop Chlamys farreri on the secondary encounter with Listonella anguillarum. Comp Biochem Phys B 2008; 151:191-196
122. Elangovan N, Chiou TJ, Tzeng WF, Chu ST. Cyclophosphamide treatment causes impairment of sperm and its fertilizing ability in mice. Toxicology 2006;222:60-70
123. Auger M, Ross J. In The Macrophage. Oxford:Oxford Univ. Press; 1992
124. Galloway T, Depledge M. Ecotoxicology; 2001
125. Dahlgren C, Karlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999;232:3-14
126. Neumann N, Stafford J, D Barreda, Ainsworth A, Belosevic M. Antimicrobial mechanisms of fish phagocytes and their role in host defense. Dev Comp Immunol 2001;25:807-825
127. J.R. Fine-scale spatial variation of persistent organic pollutants in bottlenose dolphins (Tursiops truncatus) in Biscayne Bay. Florida:Environ; 2007
128. Gougerot-Pocidalo M, Benna JE, Elbim C, Chollet-Martin S, Dang M. Regulation of human neutrophil oxidative burst by pro-and anti-inflammatory cytokines. J Soc Biol 2002;196:37-46
129. Gauss K, Nelson-Overton L, DW DS, Gao Y, DeLeo F, Quinn M. Role of NF-kappaB in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor-a. J Leukoc Biol 2007;82:729-741
130. McDonald PP, Cassatella MA. Activation of transcription factor NF-KB by phagocytic stimuli inhuman neutrophils FEBS Letters 1997;412:583-586
131. B BB, Greenwald D, Hulmes J, Chang M, Pan Y, Mathison J. Identity of tumor necrosis factor and the macrophage-secreted factor cachectin. Nature 1985;316:552-554
132. Bellavite P. The superoxide-forming enzymatic system of phagocytes. Free Radic Biol Med 1988;4:225-230
133. Malech H, Gallin J. Current concepts:Immunology neutrophils in human diseases. N Engl J Med 1987;317:687-700
134. Shingu M, Todoroki T, Nobunaga M. Generat ion of su-peroxide by immuno logically stimulated normal human neutrophils and possible modulation by intracellular and extracellular SOD and rheumatoid factors. Inflammation; 11:143-150
135. Wang X, Qi Y. The effect of Cistanche deserticola polysaccharides(CDPS) on marcrophages activation. Chinese Pharmacological Bulletin 2009;25:790-793
136. Paulsen B. Plant polysacchafides with immunostimulatory activities. Curr Org Chem 2001;5:939-950
137. Wang H, Chen N. Effects of PDF on Expression of TLR2 and TLR4 mRNA in Kidneys of rats with Endotoxic shock. Chin J Lab Diagn 2008;12:436-440
138. Lee DC, Lau AS. Effects of Panax ginseng on Tumor Necrosis Factor-a-Mediated Inflammation:A Mini-Review. Molecules 2011; 16:2802-2816
139. Ding A, Porteu F, Sanchez E, Nathan C. Shared actions of endotoxin and taxol on TNF receptors and TNF release. Science 1990;248:370
