蔷薇红景天原花青素的分离纯化及抗氧化、抗肿瘤活性研究
|
摘要
本文以张家口产的蔷薇红景天根茎为原料,研究蔷薇红景天原花青素分离纯化工艺,并对其抗氧化和抗肿瘤活性进行了初步研究,为进一步开发该新食物资源提供依据。研究方法:
(1)蔷薇红景天原花青素纯化工艺研究。首先,采用醇提法获得蔷薇红景天乙醇提取物,再通过乙酸乙酯萃取的方法分离蔷薇红景天原花青素低聚物与高聚物,最后,采用AB-8大孔吸附树脂吸附的方法,对上述两种原花青素粗品进一步分离纯化。在前期考察了上样液浓度、上样流速、解析液浓度等条件的基础上,本研究对AB-8大孔吸附树脂的上样液pH、解析液pH和径高比进行筛选,优化AB-8大孔吸附树脂的纯化工艺,筛选最佳吸附、解析条件,制备较高纯度的蔷薇红景天原花青素低聚物和高聚物。
(2)蔷薇红景天原花青素的抗氧化性研究。通过体外实验研究蔷薇红景天原花青素对羟自由基、DPPH自由基、邻苯三酚自氧化产生的超氧阴离子、亚硝酸根离子的清除作用;利用整体动物实验观察蔷薇红景天原花青素低聚物对小鼠外周血、肝脏、心脏和脑组织中抗氧化酶(SOD,GSH-Px)和脂质过氧化物(MDA)的影响。
(3)蔷薇红景天原花青素低聚物抗肿瘤活性研究。建立肉瘤-180(S180)荷瘤动物模型,观察S180在动物体内的生长情况,根据平均瘤重计算抑瘤率。
研究结果:
(1)乙酸乙酯萃取最佳工艺为萃取时间25 min、体积比(乙酸乙酯︰提取液)1.5︰1、萃取次数4次;AB-8大孔吸附树脂的最佳上样液pH为4.5,解析液pH为5,径高比为1︰40(cm)。由此分离纯化得到蔷薇红景天原花青素低聚物的纯度为88. 3%,而蔷薇红景天原花青素高聚物的纯度为98%。
(2)蔷薇红景天原花青素具有较强的清除DPPH自由基、羟自由基和亚硝酸根离子的能力,且清除效果均优于抗坏血酸。经蔷薇红景天原花青素低聚物处理的小鼠外周血、心脏、肝脏、脑组织中SOD、GSH-Px活性有所增加,MDA含量显著下降; 200、400 mg/kgbw剂量组可明显提高上述组织中GSH-Px活性,降低MDA含量,与对照组相比均有显著性差异(P<0.05),三个剂组的外周血、心脏、脑组织中SOD活性明显高于对照组(P<0.05),肝脏SOD活性与对照组比较差异不显著(P>0.05)。
(3)蔷薇红景天原花青素低聚物具有一定的抗肿瘤活性,且雌雄小鼠之间存在差异。低、中、高三个剂量组雄性动物的平均瘤重与模型对照组比较均明显降低,且有显著性差异(P<0.05),其抑瘤率分别为47.5%、51.4%、56.9%;而雌性动物的平均瘤重较模型对照组有所降低,但统计学处理无显著性差异,抑瘤率分别为24.4%、40%、41.1%。
结论:本研究采用乙酸乙酯萃取与AB-8大孔吸附树脂吸附相结合分离纯化蔷薇红景天原花青素,得到原花青素产品纯度较高,效果良好;蔷薇红景天原花青素具有良好的自由基清除作用,其低聚物能有效对抗动物机体的脂质过氧化,并且具有一定的抗肿瘤活性。
In order to provide theoretical foundation for developing new food resource, the present paper used rhizome of Rhodiola rosea as raw material to study separation and purification techniques of proanthocyanidins, and also conducted assays investigating its anti-oxidant functions and anti-tumor effects.
The main researches are as follows:
(1) Study of purification of Rhodiola rosea proanthocyanidins. Firstly, Rhodiola rosea was extracted by ethanol, before the extract was purified by acetic ether leaching and AB-8 macro porous resin absorption. Based on former researches about sample concentration, flow velocity of sample loading and concentration of desorption solution, the present study optimized purification technique of AB-8 macro porous resin absorption as the sample liquid pH、desorption solution pH and diameter to height ratio. The optimized conditions of leaching, absorption and desorption were explored, and samples of oligomer and polymer with high purity were achieved.
(2) Researches on anti-oxidative effects of Rhodiola rosea proanthocyanidins. The eliminating effects on·OH,·DPPH,·O2 produced by Pyrogallol auto-oxidation and NO2- of Rhodiola rosea proanthocyanidins were evaluated by tests in vitro. And integral animal experiments were employed to observe Rhodiola rosea Proanthocyanidin oligomers’influence on SOD, GSH-Px,and MDA levels of peripheral blood, liver, heart and brain.
(3) Study on anti-tumor property of Rhodiola rosea proanthocyanidins. The S180 tumor–bearing animal model was established, and we monitored the development state of sarcoma in animal body; According to the average tumor heavy,the inhibitory rate (refers to Rhodiola rosea proanthocyanidins’function on inhibiting tumor growth) was calculated.
The main results are along these lines:
(1) The optimized conditions of acetic ether leaching were: extracting time of 25 min, volume rate (acetic ether to extractant) of 1.5:1, operation times of 4. The optimized conditions of AB-8 macro porous resin absorption were: sample liquid pH of 4.5, desorption solution pH of 5, and diameter to height ratio of 1:40 (cm). Under the above optimized conditions, Rhodiola rosea Proanthocyanidin was separated and refined, with product purity reaching 88. 3%, and polymer product purity was 98%.
(2) Rhodiola rosea proanthocyanidin had strong ability of diminishing·OH, DPPH·,·O2 and NO2-, and the outcome was better than ascorbic acid. After administration of Rhodiola rosea proanthocyanidins, activities of SOD and GSH-Px in mouse blood, heart, liver and brain improved, while MDA content declined; 200 and 400 mg/kgbw dose groups notably increased GSH-Px activity and decreased MDA content in the mentioned 4 tissue samples, as significant differences were observed in comparison to the control group(P<0.05); however, SOD activity in liver showed no significant difference between dose groups and control group(P>0.05), while in the other 3 tissue samples, SOD activities in dose groups were significantly different from the control group(P<0.05).
(3)Rhodiola rosea proanthocyanidin oligomer unfolded some degree of anti-tumor activity, which was dissimilar among mice of different genders. The average tumor weights of male mice from low, middle and high dose groups obviously reduced, and displayed significant differences in contrast to the control group, the inhibitory rates (refer to Rhodiola rosea proanthocyanidins’function on inhibiting tumor growth) were 47.5%, 51.4% and 56.9% separately. However, though average tumor weights of female mice from dose groups were less than the control group, no significant difference was revealed in the data, and inhibitory rates were 24.4%, 40% and 41.1% separately.
The results indicated that the separation method of acetic ether leaching along with AB-8 macro porous resin absorption had admirable effects, and Rhodiola rosea proanthocyanidin products were with high purity. Through the anti-oxidative experiments and anti-tumor explorations, Rhodiola rosea proanthocyanidin oligomer was proved to be an excellent natural antioxidant with some degree of anti-tumor activity.
(1)蔷薇红景天原花青素纯化工艺研究。首先,采用醇提法获得蔷薇红景天乙醇提取物,再通过乙酸乙酯萃取的方法分离蔷薇红景天原花青素低聚物与高聚物,最后,采用AB-8大孔吸附树脂吸附的方法,对上述两种原花青素粗品进一步分离纯化。在前期考察了上样液浓度、上样流速、解析液浓度等条件的基础上,本研究对AB-8大孔吸附树脂的上样液pH、解析液pH和径高比进行筛选,优化AB-8大孔吸附树脂的纯化工艺,筛选最佳吸附、解析条件,制备较高纯度的蔷薇红景天原花青素低聚物和高聚物。
(2)蔷薇红景天原花青素的抗氧化性研究。通过体外实验研究蔷薇红景天原花青素对羟自由基、DPPH自由基、邻苯三酚自氧化产生的超氧阴离子、亚硝酸根离子的清除作用;利用整体动物实验观察蔷薇红景天原花青素低聚物对小鼠外周血、肝脏、心脏和脑组织中抗氧化酶(SOD,GSH-Px)和脂质过氧化物(MDA)的影响。
(3)蔷薇红景天原花青素低聚物抗肿瘤活性研究。建立肉瘤-180(S180)荷瘤动物模型,观察S180在动物体内的生长情况,根据平均瘤重计算抑瘤率。
研究结果:
(1)乙酸乙酯萃取最佳工艺为萃取时间25 min、体积比(乙酸乙酯︰提取液)1.5︰1、萃取次数4次;AB-8大孔吸附树脂的最佳上样液pH为4.5,解析液pH为5,径高比为1︰40(cm)。由此分离纯化得到蔷薇红景天原花青素低聚物的纯度为88. 3%,而蔷薇红景天原花青素高聚物的纯度为98%。
(2)蔷薇红景天原花青素具有较强的清除DPPH自由基、羟自由基和亚硝酸根离子的能力,且清除效果均优于抗坏血酸。经蔷薇红景天原花青素低聚物处理的小鼠外周血、心脏、肝脏、脑组织中SOD、GSH-Px活性有所增加,MDA含量显著下降; 200、400 mg/kgbw剂量组可明显提高上述组织中GSH-Px活性,降低MDA含量,与对照组相比均有显著性差异(P<0.05),三个剂组的外周血、心脏、脑组织中SOD活性明显高于对照组(P<0.05),肝脏SOD活性与对照组比较差异不显著(P>0.05)。
(3)蔷薇红景天原花青素低聚物具有一定的抗肿瘤活性,且雌雄小鼠之间存在差异。低、中、高三个剂量组雄性动物的平均瘤重与模型对照组比较均明显降低,且有显著性差异(P<0.05),其抑瘤率分别为47.5%、51.4%、56.9%;而雌性动物的平均瘤重较模型对照组有所降低,但统计学处理无显著性差异,抑瘤率分别为24.4%、40%、41.1%。
结论:本研究采用乙酸乙酯萃取与AB-8大孔吸附树脂吸附相结合分离纯化蔷薇红景天原花青素,得到原花青素产品纯度较高,效果良好;蔷薇红景天原花青素具有良好的自由基清除作用,其低聚物能有效对抗动物机体的脂质过氧化,并且具有一定的抗肿瘤活性。
In order to provide theoretical foundation for developing new food resource, the present paper used rhizome of Rhodiola rosea as raw material to study separation and purification techniques of proanthocyanidins, and also conducted assays investigating its anti-oxidant functions and anti-tumor effects.
The main researches are as follows:
(1) Study of purification of Rhodiola rosea proanthocyanidins. Firstly, Rhodiola rosea was extracted by ethanol, before the extract was purified by acetic ether leaching and AB-8 macro porous resin absorption. Based on former researches about sample concentration, flow velocity of sample loading and concentration of desorption solution, the present study optimized purification technique of AB-8 macro porous resin absorption as the sample liquid pH、desorption solution pH and diameter to height ratio. The optimized conditions of leaching, absorption and desorption were explored, and samples of oligomer and polymer with high purity were achieved.
(2) Researches on anti-oxidative effects of Rhodiola rosea proanthocyanidins. The eliminating effects on·OH,·DPPH,·O2 produced by Pyrogallol auto-oxidation and NO2- of Rhodiola rosea proanthocyanidins were evaluated by tests in vitro. And integral animal experiments were employed to observe Rhodiola rosea Proanthocyanidin oligomers’influence on SOD, GSH-Px,and MDA levels of peripheral blood, liver, heart and brain.
(3) Study on anti-tumor property of Rhodiola rosea proanthocyanidins. The S180 tumor–bearing animal model was established, and we monitored the development state of sarcoma in animal body; According to the average tumor heavy,the inhibitory rate (refers to Rhodiola rosea proanthocyanidins’function on inhibiting tumor growth) was calculated.
The main results are along these lines:
(1) The optimized conditions of acetic ether leaching were: extracting time of 25 min, volume rate (acetic ether to extractant) of 1.5:1, operation times of 4. The optimized conditions of AB-8 macro porous resin absorption were: sample liquid pH of 4.5, desorption solution pH of 5, and diameter to height ratio of 1:40 (cm). Under the above optimized conditions, Rhodiola rosea Proanthocyanidin was separated and refined, with product purity reaching 88. 3%, and polymer product purity was 98%.
(2) Rhodiola rosea proanthocyanidin had strong ability of diminishing·OH, DPPH·,·O2 and NO2-, and the outcome was better than ascorbic acid. After administration of Rhodiola rosea proanthocyanidins, activities of SOD and GSH-Px in mouse blood, heart, liver and brain improved, while MDA content declined; 200 and 400 mg/kgbw dose groups notably increased GSH-Px activity and decreased MDA content in the mentioned 4 tissue samples, as significant differences were observed in comparison to the control group(P<0.05); however, SOD activity in liver showed no significant difference between dose groups and control group(P>0.05), while in the other 3 tissue samples, SOD activities in dose groups were significantly different from the control group(P<0.05).
(3)Rhodiola rosea proanthocyanidin oligomer unfolded some degree of anti-tumor activity, which was dissimilar among mice of different genders. The average tumor weights of male mice from low, middle and high dose groups obviously reduced, and displayed significant differences in contrast to the control group, the inhibitory rates (refer to Rhodiola rosea proanthocyanidins’function on inhibiting tumor growth) were 47.5%, 51.4% and 56.9% separately. However, though average tumor weights of female mice from dose groups were less than the control group, no significant difference was revealed in the data, and inhibitory rates were 24.4%, 40% and 41.1% separately.
The results indicated that the separation method of acetic ether leaching along with AB-8 macro porous resin absorption had admirable effects, and Rhodiola rosea proanthocyanidin products were with high purity. Through the anti-oxidative experiments and anti-tumor explorations, Rhodiola rosea proanthocyanidin oligomer was proved to be an excellent natural antioxidant with some degree of anti-tumor activity.
引文
[1] Yousef GG,Grace MH,Cheng DM,et al.Comparative phytochemical characterizeation of three Rhodiola species [J].Phytochemistry,2006,67:2380-2391.
[2] Panossian A,Wikman G,Sarris J.Rosenroot(Rhodiola rosea):Traditional use,chemical composition,pharmacology and clinical efficacy[J].Phytomedicine,2010,17:481-493.
[3]孟玉彩.蔷薇红景天中原花青素提取、精制及稳定性研究[D].保定:河北农业大学,2009.
[4]边庆荣,赵文,蒋东升,等.红景天毒理学评价[J].卫生毒理学杂志,1991,(5):40-41.
[5] Darbinyan V,Kteyan A,Panossian A,et al. Rhodiola rosea in stress induced fatigue-a double blind cross-over of a standardized extract SHR-5 with a repeated low-dose regimen on the mental performance of healthy physicians during night duty. Phytomedicine,2000,7(5):365-371.
[6]赵文,王庭欣,齐小菊,等.蔷薇红景天提取液抗疲劳作用研究[J].营养学报,2006,28:180-181.
[7]赵文,蒋东升,边庆荣,等.蔷薇红景天对小鼠免疫功能的影响及抑瘤作用[J].营养学报,2000,22:102-104.
[8] Min BS,Bae KH,Kim YH,et al.Screening of korean plants against human immunodeficiency virus type1 protease.Phytotherapy research,1999,13:680-682.
[9]姜文华,孟晓婷.红景天素抗老化和抗痴呆效应的实验研究[J].白求恩医科大学学报,2001,27:127-129.
[10]常洁琴,崔广智,薛贵平,等.玫瑰红景天对小鼠学习记忆的影响(简报).中国中药杂志,1994,19(6):370.
[11] Joslyn MA,Dittmar HFK. The proanthocyanidins of pinot blanc grape[J].American Journal of Enology and Viticulture,1967,18(1):1-10.
[12] Masquelier J,Michaud J,Laparra J,et al. Flavonoids and pycnogenols[J].International Journal of Vitamin and Nutrition Researeh,1970,49:307-311.
[13]石碧,狄莹.植物多酚[M].北京:科学出版社,2000,10:137.
[14] Freitas VAP,Glorie Y, Bourgeois G,et al. Characterisation of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry[J].Phytoehemistry,1998,49(5):1435.
[15]杨其蒀.天然药物化学[M].北京:中国医药科技出版社,1996,6.
[16]何钊,任其龙.层析法分离葡萄籽中原花青素的研究[J].食品科学,2004,25(8):70-73.
[17]孙达旺.植物单宁化学[M].北京:中国林业出版社,1992:130-142.
[18]韩菊,魏福祥,瞿丽萍,等.葡萄籽中低聚原花色素的性能研究[J].食品科学,2003,24(2):36-38.
[19]国植,徐莉.原花青素:具有广阔发展前景的植物药[J].国外医药一植物药分册,1996,11(5):196-204.
[20] Smith AK, June H, Noble AC.Effect of viscosity on the bittemess and astringency of grapeseed tannin [M].Food quality and preference,1996,7:161-166.
[21]吕丽爽.葡萄籽低聚原花青素的研制[D].无锡:江南大学,2000.
[22]魏孝义,谢海辉,周文华等.原花青素的制备工艺[P].CN1273985A.
[23]樊丽华.葡萄籽中原花青素提取方法的进一步研究[J].新疆大学学报(自然科学版),2004,21(l):102-104.
[24]李莹,赵谋明,于立梅,等.采用大孔吸附树脂纯化马尾松树皮原花青素及其抗氧化性研究[J].食品与发酵工业,2006,32(12):145-149.
[25]徐晓云,潘思轶,谢笔钧,等.树脂法精制沙棘籽原花色素的研究[J].农业工程学报,2005,21(1):152-154.
[26]张泽生,赵春艳,曹力心.大孔吸附树脂分离纯化山楂果中原花青素的研究[J].现代食品科技,2006,22(2):16.
[27] Karim NM. Method for extraction of proanthocyaidins from plant material[P].US 5912363.
[28] Ariga Toshiaki. Extraction of proanthocyaidins[P].JP 5623415.
[29] Zdunczyk Z,Frejnagel S,Wroblewska M,et al. Biologicalactivity of polyphenol extracts from different plant sources[J].Food Research International,2002,35:183- 186.
[30] Toshiaki A.The antioxidative function,preventive actionon disease and utilization of proanthocyanidins[J].Bio Factors,2004,21:197-201.
[31] Yamakoshi J,Kataoka S,Koga T,et al. Proanthocyanidins rich extract from grape seeds attenuates the development of aortic atherrosclerosis in cholesterol fed rabbits[J].Athero sclerosis,1999,142:139-149.
[32] Jacqueline EW,Senthilmohan ST,Peskin AV.Antioxidant activity of procyanidin-containing plant extracts at different phs[J].Food Chemistry,2005,77:155-161.
[33] Bagchi D,Garg A,Krohn RL,et al.Oxygen free radical scavenging abilities of vitamins C and E,and a grape seed proanthocyanidin extract in vitro[J].Res Commun Mol Pathol Pharmacol,1997,95(2):179-189.
[34] Lu Y,ZhaoWZ,Chen Z,et al.Procyanidins from grape seeds protect against phorbol ester-induced oxidative cellular and genotoxic damage[J].Acta Pharmacol Sin,2004,25 (8):1083-1089.
[35]钟进义,栗世如,张燕滨.葡多酚对小鼠亚慢性辐射损伤的防护作用研究[J].中国公共卫生,1999,15(11):1012- 1013.
[36]段玉清,谢笔钧.莲房原花青素体内抗氧化研究[J].Acta Nurtrinenta Sinica,2003,25 (3):306-308.
[37] Katia Tebib,Lotfi Bitri,Pierre Besancon,et al.Polymeric grape seed tannins prevent plasma cholesterol changes in high-cholesterol-fed rats[J].Food Chemistry,1994,49(4):403-406.
[38] Bas JMD,Femandez-larrea J,Blay M,et al.Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats[J].Faseb J Express Article,2005,10:1096-3095.
[39] Huynh TH,Teel RW.Selective induction of apoptosis in human mam-mary cancer cells(MCF-7 ) by pycnogenol[J].Anticancer Res,2000,20 (4):24 l7-2420.
[40] Vayalil P K,Mittal A.Proanthocyanidins from grape seeds inhibit expression of matrix metalloproteinases in human prostate carcinoma cells,which is associated with the inhibition of activation of MAPK and NFκB[J].Carcinogen,2004,25 (6):987-995.
[41]李莹.白藜芦醇和原花青素诱导胃癌细胞凋亡分子机理的初步研究[D].西安:第四军医大学,2003.
[42] Pinent M,Blay M,Blade M C,et al.Grape seed derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines[J].Endocrinol,2004,145 (11):4985-4990.
[43]徐桂花,徐惠娟.葡萄籽中低聚原花青素调节大鼠血脂的作用[J].农业科学研究,2006,3:44-45.
[44] Subathira S,Molan AL,Meagher LP,et al.Variation in antimicrobial action of proanthocyanidins from doryecnium rectum against rumen baeteria [J].Phytoehemistry,2004,65:2485-2497.
[45] Browlnee HE,Hedger J,Scott IM.Effects of a range of procyanidins on the coeoa pathogen crinipellis perniciosa[J].Physiological and MolecularPlantpathology,1992,40(4):227-232.
[46] Salawu M B,Acamovic T,Stewart CS.Callianidra calothyrsus leaf extrzcts effects on microbial growth and enzye activities [M].Toxin Plants Other Nat Toxicatints,1997:509- 513.
[47]段玉清,张海晖,邓乾春,等.莲房原花青素对免疫功能的影响[J].食品研究与开发,2007,28(12):1-5.
[48] Sharma S D,Katiyar S K.Dietary grape-seed proanthocyanidins inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12[J].Carcinogenesis,2006,2(1):95-102.
[49] Liu FJ,Zhang YX,Lau BH.Procyanidins from the bark of Ha ma melis virginiana exhibit antimutagenic properties against nitroaromatic compounds[J].Cell Mol life sci,1998,54(10): 1168.
[50]凌智群,张晓辉,谢笔钧,等.原花青素的药理学研究进展[J].中国药理学通报,2002,18(l):9-12.
[51]周素娟.葡萄籽提取物原花青素的研究概况及其在我国保健食品中的应用[J].中国食品卫生杂志,2007,19(3):284-286.
[52]赵超英,姚小曼.葡萄籽提取物原花青素的营养保健功能(综述)[J].中国食品卫生杂志, 2000(6):38-41.
[53]段玉清,谢笔钧,等.原花青素在化妆品领域的研究与开发现状[J].香料香精化妆品, 2002,12(6):23.
[54]周忻.大孔吸附树脂分离纯化中草药有效成分的影响因素[J].中国药物应用与监测,2006(2):50-53.
[55]吕洁丽,杨中汉,袁珂.新型凝胶树脂及大孔吸附树脂在中草药成分分离纯化中的应用[J].中药材,2005,28(3):239-242.
[56]孟玉彩,赵文,李慧玲,张会彦.蔷薇红景天中原花青素含量的测定方法[J].食品研究与开发,2009,30(4):133-136.
[57]范明远,叶青.体内自由基清除剂及抗氧化剂-原花青素的研究进展[J].中国预防医学杂志,2001,2(4):303-305.
[58] Concepcion SM,Jose AL,Fulgencio SC.A Procedure to Measure the Antiradcal Efficiency of Polyphenols[J].J Sci Food Agric,1998,76:270-276.
[59]姚育翠.玫瑰花中原花青素的分离分析及抗氧化活性研究[D].浙江:浙江大学,2008.
[60]翁瑞光.萝葡婴萃取物于模式系统之抗氧化性[J].食品科学(台),1998,25(3):268.
[61]王继锋,王石泉,汤国枝.山植原花色素的抗氧化作用研究[J].天然产物研究与开发,2001(2):46-48.
[62]贾之慎,邬建敏,唐孟成.比色法测定Fenton反应产生的羟自由基[J].生物化学与生物物理进展,1996:23(2),184-185.
[63]钱丽燕,郭淑春.水果中亚硝酸盐的测定[J].吉林粮食高等专科学校学报,1997,12(2):11-13.
[64]李洪军,建全,解纯刚,等.野生马齿觅抑制亚硝胺合成的体外试验[J].营养学报,1998,20(3):361-362.
[65]邹洪,袁悼斌.邻苯三酚自氧化的电化学研究[J].分析试验室,1997,16 (4) :36-39.
[66]赵文恩,韩雅珊,乔旭光.类胡萝卜素清除活性氧自由基的机理[J].化学通报,1999(4): 18-22.
[67]刘杰超,焦中高,周红平,等.甜樱桃红色素的体外抗氧化活性[J].果树学报,2006,23(5):756-759.
[68]张长贵.蚕豆中原花青素提取、纯化及抗氧化性质研究[D].重庆:西南大学,2007.
[69]李姣,王珂,王瑞坡,等.芦笋多糖提取纯化工艺及体外抗氧化研究[J].食品科学,2011,32(8):65-69.
[70]许申鸿,杭瑚,李运平.超氧化物歧化酶邻苯三酚测活法的研究及改进[J].化学通报,2001,8:516-519.
[71]邓修惠,黄学梅.改良DTNB比色法测定血清GSH-Px活力[J].重庆医学,2000,9(5):445.
[72]刘小杰,何国庆,熊正英.沙棘油对小鼠心肌自由基代谢和超微结构的影响[J].营养学报,2002,24(2):126-129.
[73]刘小杰,何国庆,熊正英.口服沙棘油对游泳小鼠肝、心、脑自由基代谢影响的实验研究[J].食品科学,2001,22(10):71-73.
[74]潘绥,郭维新,李跃平.甲醛对人体超氧化物岐化酶和神经行为功能的影响[J].中国职业医学,2001(1):55-56.
[75]方允中,李文杰.自由基与酶[J].北京:北京科学出版社,1989:261-263.
[76]程文凯.脂质过氧化与抗氧化酶[J].工业卫生与职业病,1993,19(4):254-256.
[77]黄玲,望宝贵,刘娅.翅果油抗氧化作用的研究[J].卫生研究,2002,31(3):172-174.
[78]李湘鸣,罗方妮,方企圣.臭氧对大鼠肺灌洗液某些生化指标及肺形态的影响[J].中国公共卫生学报,1996,15(4):234-235.
[79]潘洪志.DNA氧化损伤标志物8-羟基脱氧鸟苷其检测[J].中国卫生检验杂志,2003,13(4):404-405.
[80]赵伟,童丽丽,乔永康.邻苯二甲酸二乙基己酯对小鼠血液中的SOD及MDA的影响[J].公共卫生与预防医学,2007,18(1):12-15.
[81]杨婷,曽璨，彭琳．食品中亚硝酸盐与人类肿瘤关系的研究进展[J].医学信息,2011,7:3115-3116.
[82]丁之恩.亚硝酸盐和亚硝胺在食品中的作用及其机理[J].安徽农业大学学报,1994,21(2):199-205.
[83]李延红,周占业,史齐,等.低浓度亚硝酸钠预处理对乙醇损伤PC12细胞的保护作用[J].中国药理学通报,2011,27(7):952-956.
[84]王彦玲,王蓓,程建新,等.牛黄天龙胶囊的抗肿瘤作用及其毒性[J].实用癌症杂志,2005,20(3):253-255.
[85]路景涛,魏伟,陈敏珠.青篙素抗肿瘤作用的初步研究[J].安徽医药,2007,11(1):14-15.
[86] Itch H,Shimura K,Kawade M,et al.Inhibitory action of a (1-6)-beat-D-glucan-protein complex(F-III-2-b)isolated from Agaricus blazei Murill on Meth A fibrosarcoma-bearing mice and its antitumor mechanism[J].Japanese Jounal of Pharmacology,1994,66(2):265-271.
[87] Itch H,Shimura K,Kawade M,et al.Antitumor effects of a new polysaceharide-protein complex (ATOM)prepared from Agaricus blazei(Iwadestrain101)‘Himematsutake’and its mechanisms in tumor-bearing mice[J].Anticancer Researeh,1997,17(l):277-284.
[88]宋炳生,杨玉龙.枸杞多糖的免疫调节作用[J].药学实践杂志,1997,15(2):69-72.
[89] Foley GE,Drolet BP,et al.Isolation and serial Propagation of malignant and normal cells in semi-defined media origins of CCRF cell lines[J].Cancer Research,1960,20:930-938.
[90]郑国强,刘安军,滕安国,等.雌雄小鼠胸腺和脾脏的免疫比较[J].安徽农业科学,2011,39(16):9743-9745.
[91] Cavallott D,Artico M,Lannetti G,et al.Occurrence of adrenergic nerve fibers in human thymus during immune reponse[J].Neurochem Int,2002,40(3):211-221.
[92]刘仰斌,李启华.小鼠胸腺形态结构的增龄性变化[J].中国组织工程研究与临床康复,2010,14(31):5805-5808.
[2] Panossian A,Wikman G,Sarris J.Rosenroot(Rhodiola rosea):Traditional use,chemical composition,pharmacology and clinical efficacy[J].Phytomedicine,2010,17:481-493.
[3]孟玉彩.蔷薇红景天中原花青素提取、精制及稳定性研究[D].保定:河北农业大学,2009.
[4]边庆荣,赵文,蒋东升,等.红景天毒理学评价[J].卫生毒理学杂志,1991,(5):40-41.
[5] Darbinyan V,Kteyan A,Panossian A,et al. Rhodiola rosea in stress induced fatigue-a double blind cross-over of a standardized extract SHR-5 with a repeated low-dose regimen on the mental performance of healthy physicians during night duty. Phytomedicine,2000,7(5):365-371.
[6]赵文,王庭欣,齐小菊,等.蔷薇红景天提取液抗疲劳作用研究[J].营养学报,2006,28:180-181.
[7]赵文,蒋东升,边庆荣,等.蔷薇红景天对小鼠免疫功能的影响及抑瘤作用[J].营养学报,2000,22:102-104.
[8] Min BS,Bae KH,Kim YH,et al.Screening of korean plants against human immunodeficiency virus type1 protease.Phytotherapy research,1999,13:680-682.
[9]姜文华,孟晓婷.红景天素抗老化和抗痴呆效应的实验研究[J].白求恩医科大学学报,2001,27:127-129.
[10]常洁琴,崔广智,薛贵平,等.玫瑰红景天对小鼠学习记忆的影响(简报).中国中药杂志,1994,19(6):370.
[11] Joslyn MA,Dittmar HFK. The proanthocyanidins of pinot blanc grape[J].American Journal of Enology and Viticulture,1967,18(1):1-10.
[12] Masquelier J,Michaud J,Laparra J,et al. Flavonoids and pycnogenols[J].International Journal of Vitamin and Nutrition Researeh,1970,49:307-311.
[13]石碧,狄莹.植物多酚[M].北京:科学出版社,2000,10:137.
[14] Freitas VAP,Glorie Y, Bourgeois G,et al. Characterisation of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry[J].Phytoehemistry,1998,49(5):1435.
[15]杨其蒀.天然药物化学[M].北京:中国医药科技出版社,1996,6.
[16]何钊,任其龙.层析法分离葡萄籽中原花青素的研究[J].食品科学,2004,25(8):70-73.
[17]孙达旺.植物单宁化学[M].北京:中国林业出版社,1992:130-142.
[18]韩菊,魏福祥,瞿丽萍,等.葡萄籽中低聚原花色素的性能研究[J].食品科学,2003,24(2):36-38.
[19]国植,徐莉.原花青素:具有广阔发展前景的植物药[J].国外医药一植物药分册,1996,11(5):196-204.
[20] Smith AK, June H, Noble AC.Effect of viscosity on the bittemess and astringency of grapeseed tannin [M].Food quality and preference,1996,7:161-166.
[21]吕丽爽.葡萄籽低聚原花青素的研制[D].无锡:江南大学,2000.
[22]魏孝义,谢海辉,周文华等.原花青素的制备工艺[P].CN1273985A.
[23]樊丽华.葡萄籽中原花青素提取方法的进一步研究[J].新疆大学学报(自然科学版),2004,21(l):102-104.
[24]李莹,赵谋明,于立梅,等.采用大孔吸附树脂纯化马尾松树皮原花青素及其抗氧化性研究[J].食品与发酵工业,2006,32(12):145-149.
[25]徐晓云,潘思轶,谢笔钧,等.树脂法精制沙棘籽原花色素的研究[J].农业工程学报,2005,21(1):152-154.
[26]张泽生,赵春艳,曹力心.大孔吸附树脂分离纯化山楂果中原花青素的研究[J].现代食品科技,2006,22(2):16.
[27] Karim NM. Method for extraction of proanthocyaidins from plant material[P].US 5912363.
[28] Ariga Toshiaki. Extraction of proanthocyaidins[P].JP 5623415.
[29] Zdunczyk Z,Frejnagel S,Wroblewska M,et al. Biologicalactivity of polyphenol extracts from different plant sources[J].Food Research International,2002,35:183- 186.
[30] Toshiaki A.The antioxidative function,preventive actionon disease and utilization of proanthocyanidins[J].Bio Factors,2004,21:197-201.
[31] Yamakoshi J,Kataoka S,Koga T,et al. Proanthocyanidins rich extract from grape seeds attenuates the development of aortic atherrosclerosis in cholesterol fed rabbits[J].Athero sclerosis,1999,142:139-149.
[32] Jacqueline EW,Senthilmohan ST,Peskin AV.Antioxidant activity of procyanidin-containing plant extracts at different phs[J].Food Chemistry,2005,77:155-161.
[33] Bagchi D,Garg A,Krohn RL,et al.Oxygen free radical scavenging abilities of vitamins C and E,and a grape seed proanthocyanidin extract in vitro[J].Res Commun Mol Pathol Pharmacol,1997,95(2):179-189.
[34] Lu Y,ZhaoWZ,Chen Z,et al.Procyanidins from grape seeds protect against phorbol ester-induced oxidative cellular and genotoxic damage[J].Acta Pharmacol Sin,2004,25 (8):1083-1089.
[35]钟进义,栗世如,张燕滨.葡多酚对小鼠亚慢性辐射损伤的防护作用研究[J].中国公共卫生,1999,15(11):1012- 1013.
[36]段玉清,谢笔钧.莲房原花青素体内抗氧化研究[J].Acta Nurtrinenta Sinica,2003,25 (3):306-308.
[37] Katia Tebib,Lotfi Bitri,Pierre Besancon,et al.Polymeric grape seed tannins prevent plasma cholesterol changes in high-cholesterol-fed rats[J].Food Chemistry,1994,49(4):403-406.
[38] Bas JMD,Femandez-larrea J,Blay M,et al.Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats[J].Faseb J Express Article,2005,10:1096-3095.
[39] Huynh TH,Teel RW.Selective induction of apoptosis in human mam-mary cancer cells(MCF-7 ) by pycnogenol[J].Anticancer Res,2000,20 (4):24 l7-2420.
[40] Vayalil P K,Mittal A.Proanthocyanidins from grape seeds inhibit expression of matrix metalloproteinases in human prostate carcinoma cells,which is associated with the inhibition of activation of MAPK and NFκB[J].Carcinogen,2004,25 (6):987-995.
[41]李莹.白藜芦醇和原花青素诱导胃癌细胞凋亡分子机理的初步研究[D].西安:第四军医大学,2003.
[42] Pinent M,Blay M,Blade M C,et al.Grape seed derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines[J].Endocrinol,2004,145 (11):4985-4990.
[43]徐桂花,徐惠娟.葡萄籽中低聚原花青素调节大鼠血脂的作用[J].农业科学研究,2006,3:44-45.
[44] Subathira S,Molan AL,Meagher LP,et al.Variation in antimicrobial action of proanthocyanidins from doryecnium rectum against rumen baeteria [J].Phytoehemistry,2004,65:2485-2497.
[45] Browlnee HE,Hedger J,Scott IM.Effects of a range of procyanidins on the coeoa pathogen crinipellis perniciosa[J].Physiological and MolecularPlantpathology,1992,40(4):227-232.
[46] Salawu M B,Acamovic T,Stewart CS.Callianidra calothyrsus leaf extrzcts effects on microbial growth and enzye activities [M].Toxin Plants Other Nat Toxicatints,1997:509- 513.
[47]段玉清,张海晖,邓乾春,等.莲房原花青素对免疫功能的影响[J].食品研究与开发,2007,28(12):1-5.
[48] Sharma S D,Katiyar S K.Dietary grape-seed proanthocyanidins inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12[J].Carcinogenesis,2006,2(1):95-102.
[49] Liu FJ,Zhang YX,Lau BH.Procyanidins from the bark of Ha ma melis virginiana exhibit antimutagenic properties against nitroaromatic compounds[J].Cell Mol life sci,1998,54(10): 1168.
[50]凌智群,张晓辉,谢笔钧,等.原花青素的药理学研究进展[J].中国药理学通报,2002,18(l):9-12.
[51]周素娟.葡萄籽提取物原花青素的研究概况及其在我国保健食品中的应用[J].中国食品卫生杂志,2007,19(3):284-286.
[52]赵超英,姚小曼.葡萄籽提取物原花青素的营养保健功能(综述)[J].中国食品卫生杂志, 2000(6):38-41.
[53]段玉清,谢笔钧,等.原花青素在化妆品领域的研究与开发现状[J].香料香精化妆品, 2002,12(6):23.
[54]周忻.大孔吸附树脂分离纯化中草药有效成分的影响因素[J].中国药物应用与监测,2006(2):50-53.
[55]吕洁丽,杨中汉,袁珂.新型凝胶树脂及大孔吸附树脂在中草药成分分离纯化中的应用[J].中药材,2005,28(3):239-242.
[56]孟玉彩,赵文,李慧玲,张会彦.蔷薇红景天中原花青素含量的测定方法[J].食品研究与开发,2009,30(4):133-136.
[57]范明远,叶青.体内自由基清除剂及抗氧化剂-原花青素的研究进展[J].中国预防医学杂志,2001,2(4):303-305.
[58] Concepcion SM,Jose AL,Fulgencio SC.A Procedure to Measure the Antiradcal Efficiency of Polyphenols[J].J Sci Food Agric,1998,76:270-276.
[59]姚育翠.玫瑰花中原花青素的分离分析及抗氧化活性研究[D].浙江:浙江大学,2008.
[60]翁瑞光.萝葡婴萃取物于模式系统之抗氧化性[J].食品科学(台),1998,25(3):268.
[61]王继锋,王石泉,汤国枝.山植原花色素的抗氧化作用研究[J].天然产物研究与开发,2001(2):46-48.
[62]贾之慎,邬建敏,唐孟成.比色法测定Fenton反应产生的羟自由基[J].生物化学与生物物理进展,1996:23(2),184-185.
[63]钱丽燕,郭淑春.水果中亚硝酸盐的测定[J].吉林粮食高等专科学校学报,1997,12(2):11-13.
[64]李洪军,建全,解纯刚,等.野生马齿觅抑制亚硝胺合成的体外试验[J].营养学报,1998,20(3):361-362.
[65]邹洪,袁悼斌.邻苯三酚自氧化的电化学研究[J].分析试验室,1997,16 (4) :36-39.
[66]赵文恩,韩雅珊,乔旭光.类胡萝卜素清除活性氧自由基的机理[J].化学通报,1999(4): 18-22.
[67]刘杰超,焦中高,周红平,等.甜樱桃红色素的体外抗氧化活性[J].果树学报,2006,23(5):756-759.
[68]张长贵.蚕豆中原花青素提取、纯化及抗氧化性质研究[D].重庆:西南大学,2007.
[69]李姣,王珂,王瑞坡,等.芦笋多糖提取纯化工艺及体外抗氧化研究[J].食品科学,2011,32(8):65-69.
[70]许申鸿,杭瑚,李运平.超氧化物歧化酶邻苯三酚测活法的研究及改进[J].化学通报,2001,8:516-519.
[71]邓修惠,黄学梅.改良DTNB比色法测定血清GSH-Px活力[J].重庆医学,2000,9(5):445.
[72]刘小杰,何国庆,熊正英.沙棘油对小鼠心肌自由基代谢和超微结构的影响[J].营养学报,2002,24(2):126-129.
[73]刘小杰,何国庆,熊正英.口服沙棘油对游泳小鼠肝、心、脑自由基代谢影响的实验研究[J].食品科学,2001,22(10):71-73.
[74]潘绥,郭维新,李跃平.甲醛对人体超氧化物岐化酶和神经行为功能的影响[J].中国职业医学,2001(1):55-56.
[75]方允中,李文杰.自由基与酶[J].北京:北京科学出版社,1989:261-263.
[76]程文凯.脂质过氧化与抗氧化酶[J].工业卫生与职业病,1993,19(4):254-256.
[77]黄玲,望宝贵,刘娅.翅果油抗氧化作用的研究[J].卫生研究,2002,31(3):172-174.
[78]李湘鸣,罗方妮,方企圣.臭氧对大鼠肺灌洗液某些生化指标及肺形态的影响[J].中国公共卫生学报,1996,15(4):234-235.
[79]潘洪志.DNA氧化损伤标志物8-羟基脱氧鸟苷其检测[J].中国卫生检验杂志,2003,13(4):404-405.
[80]赵伟,童丽丽,乔永康.邻苯二甲酸二乙基己酯对小鼠血液中的SOD及MDA的影响[J].公共卫生与预防医学,2007,18(1):12-15.
[81]杨婷,曽璨，彭琳．食品中亚硝酸盐与人类肿瘤关系的研究进展[J].医学信息,2011,7:3115-3116.
[82]丁之恩.亚硝酸盐和亚硝胺在食品中的作用及其机理[J].安徽农业大学学报,1994,21(2):199-205.
[83]李延红,周占业,史齐,等.低浓度亚硝酸钠预处理对乙醇损伤PC12细胞的保护作用[J].中国药理学通报,2011,27(7):952-956.
[84]王彦玲,王蓓,程建新,等.牛黄天龙胶囊的抗肿瘤作用及其毒性[J].实用癌症杂志,2005,20(3):253-255.
[85]路景涛,魏伟,陈敏珠.青篙素抗肿瘤作用的初步研究[J].安徽医药,2007,11(1):14-15.
[86] Itch H,Shimura K,Kawade M,et al.Inhibitory action of a (1-6)-beat-D-glucan-protein complex(F-III-2-b)isolated from Agaricus blazei Murill on Meth A fibrosarcoma-bearing mice and its antitumor mechanism[J].Japanese Jounal of Pharmacology,1994,66(2):265-271.
[87] Itch H,Shimura K,Kawade M,et al.Antitumor effects of a new polysaceharide-protein complex (ATOM)prepared from Agaricus blazei(Iwadestrain101)‘Himematsutake’and its mechanisms in tumor-bearing mice[J].Anticancer Researeh,1997,17(l):277-284.
[88]宋炳生,杨玉龙.枸杞多糖的免疫调节作用[J].药学实践杂志,1997,15(2):69-72.
[89] Foley GE,Drolet BP,et al.Isolation and serial Propagation of malignant and normal cells in semi-defined media origins of CCRF cell lines[J].Cancer Research,1960,20:930-938.
[90]郑国强,刘安军,滕安国,等.雌雄小鼠胸腺和脾脏的免疫比较[J].安徽农业科学,2011,39(16):9743-9745.
[91] Cavallott D,Artico M,Lannetti G,et al.Occurrence of adrenergic nerve fibers in human thymus during immune reponse[J].Neurochem Int,2002,40(3):211-221.
[92]刘仰斌,李启华.小鼠胸腺形态结构的增龄性变化[J].中国组织工程研究与临床康复,2010,14(31):5805-5808.