黑木耳分级多糖制备及抗氧化研究
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摘要
黑木耳(A uricularia auricula),是一种真菌,又名木耳或光木耳。黑木耳是我国重要的药食同源的食用菌。其富含丰富的氨基酸、铁及多糖。其主要的活性成分为黑木耳多糖,具有很高的生物活性如：抗癌、降血脂和抗血凝功效。在生产利用与科研上,越来越受重视。
本文以黑木耳子实体为原料,用pH6、7、8磷酸盐缓冲溶液浸提黑木耳多糖,用乙醇浓度为100%分级黑木耳多糖。用ABTS+·、DPPH·、超氧自由基、羟基自由基、过氧化氢、金属离子螯合、还原力和脂质过氧化为指标测定多糖的体外抗氧化性。用8周昆明小鼠分成10组雌雄各半,每组10只,灌胃APG6、APG7、APG8多糖100mg/kg、150mg/kg、200 mg/kg,测试其体内SOD、CAT、GSH-PX、GSH. MDA含量,研究黑木分级耳多糖对小鼠体内抗氧化作用的影响。将330只小鼠随机分成11组,辐射模型组,对照组、给药组分别灌胃APG6、APG7、APG8多糖100 mg/kg、150mg/kg、200 mg/kg,14d时照射4.0Gy的60Coγ-射线建立辐射氧化损伤模型,14d后测定小鼠存活率,对免疫系统及抗氧化的影响。研究不同剂量黑木耳分级多糖对小鼠辐射、免疫、氧化系统作用的影响及对小鼠辐射损伤的保护作用,为以后抗氧化及抗辐射生物源药物开发与利用黑木耳多糖提供科学参考。主要结果如下：
1用pH6、7、8磷酸盐缓冲溶液提取黑木耳多糖,用100%乙醇分级,研究黑木耳分级多糖的分子量分布。APG6、APG7、APG8的多糖得率分别为：11.57%、19.46%、12.72%。多糖含量分别为：93.12%、89.43%、96.93%。高效液相色谱法对APG6、APG7、APG8进行纯度检测,APG6、APG7单一组分。质均分子量Mw分别为：4.3840×104、9.4335×103、4.4775×104。
2用制备的APG6、APG7、APG8多糖测定体外抗氧化。研究结果表明酸溶性黑木耳分级多糖APG6具有较强的清除ABTS、DPPH、金属离子螯合能力、还原能力,并且清除ABTS和DPPH自由基能力高于阳性对照Vc。碱溶性黑木耳分级多糖APG8具有较强的清除超氧自由基和羟基自由基能力。而中性黑木耳分级多糖APG7抗脂质过氧化抑制力高于其他多糖。
3 APG6、APG7、APG8做体内抗氧化小鼠试验。结果表明各剂量组多糖均能提高小鼠血清和肝组织中SOD、GSH-PX、GSH、CAT酶,降低MDA含量,随着剂量的增加效果越显著,其中APG8组效果最强。
4小鼠受照4.0Gy的60Coγ-射线,建立辐射损伤模型,分别从辐射指标、免疫功能、抗氧化功能等研究APG6、APG7、APG8对小鼠的辐射保护作用。各组多糖都能显著的提高小鼠存活率；减少由于辐射损伤引起的体液免疫、细胞免疫和非特异性免疫功能下降,促进机体免疫功能恢复,缩短免疫功能恢复时间；增强辐射损伤小鼠SOD、GSH-Px抗氧化酶的活力,降低MDA含量,减少自由基对机体的损伤,增强机体清除自由基的能力。APG8组辐射小鼠恢复效果最优。
Auricularia auricula is a mushroom, commonly called Judas's ear fungus or Jew's Ear. It is the Chinese traditional non-toxic edible mushroom, which has been widely used in Chinese cuisines and is known for their pharmaceutical effects in Chinese herbal medicine.The edible fungus is rich in amino acids, protein,iron,acalcium and polysaccharides.Of particular significance among these function components is polysaccharides.A. auricular polysaccharide have many biological activities, including antitumor activity, preventive hypocholesterolemia and anticoagulant activity.
In this paper,A.auricular fruiting bodies as raw material,pH 6,7,8 phosphate buffer extracted with polysaccharides,and than 100% ethanol concentration of purified polysaccharide.As ABTS,DPPH,hydroxyl free radical,hydroxyl radicals, hydrogen peroxide, metal ion chelating ability,reduce power and induced lipid peroxidation determination of polysaccharide in vitro antioxidant activity.8-week-old Kunming mice,half male and female,10 in each group.Oral administration in mice fed with APG6,APG7 and APG8, the oral dose 100 mg/kg,150mg/kg,200 mg/kg according to the weight.At the 28d,tested in SOD,CAT,GSH-PX,GSH,MDA for the determination of the indicators.Further study of the relationship between antioxidant and A. auricular polysaccharides.The preliminary study on the role of APG6,APG7 and APG8 on the y-ray radiation protection in mice.To 330 mices half male and female,10 in each group.Mice were randomly divided into modle control group,irradiated control group,dose irradiation group.Oral administration in mice fed with APG6,APG7 and APG8 the oral dose 100 mg/kg,150mg/kg,200 mg/kg according to the weight.Mice of the oral group of APG6,APG7,APG8 and,irradiated control group before irradiation be administrated for 14 days,for the next day,4.0Gy he packet 60Co y-ray single irradiation is required.Mice of oral group APG6,APG7 and APG8 after irradiation take administration of 14 days from the current day.On the 28th day after irradiation,all mice were killed by cervical dislocation.For a preliminary study of the A.auricular polysaccharide protective effects in mice radiation, immunity and antioxidant activities.For the future biological antioxidant and anti-radiation drug development and utilization of the source A. auricular polysaccharides provide scientific reference.The main results are as follows:
1 With pH 6,7,8 phosphate buffer extraction of polysaccharides,100% ethanoland purified pH 6,7,8.Studyon APG6,APG7,APG8 polysaccharides molecular weight distribution.The extraction rate of APG6,APG7,APG8 polysaccharides were 11.57%,19.46%,2.72%.Content of APG6,APG7,APG8 polysaccharides were 93.12%,89.43%,96.93%.Measured by Ubbelohde viscometer polysaccharide APG6,APG7 and APG8 viscosity average molecular weight 7.458×105.128×105 and 7.082×105.High efficiency liquid chromatography testing APG6, APG7, APG8 for purity, APG6, APG7 single component, and MW:4.3840×104,9.4335×103,4.4775×104.
2 APG6,APG7,APG8 polysaccharides were evaluated on their antioxidant activities in vitro.The results of extracorporeal antioxidation experiment showed that APG6 had very good potential for scavenging ABTS,DPPH,metal ion chelating ability,reduce power.APG6 had clearer ABTS and DPPH radical scavenging than positive control Vc.APG8 could clean up hydroxyl free radical and hydroxyl radicals.And APG7 was higher inhibiting lipid peroxidation than the other polysaccharides.
3 To study the effects of APG6,APG7,APG8 on in vivo antioxidant activities of normal mice.The results show that APG6,APG7,APG8 also could improve the activity of SOD,GSH-PX,GSH,CAT, reduce the content of MDA in their serum and liver.We also can see if we increasing the dose,the in vivo antioxidant activities of normal mice also increases gradually.APG8 have higher than the other polysaccharides.
4 To establish a mouse model of 60Coγ-injury in 4.0Gy,oral gavage of APG6,APG7 and APG8 detection of survival rate,immune-function and anti-oxidation.For a preliminary study of the y-radiation on APG6,APG7 and APG8 protective effect in mice.All polysaccharides could improved survival rate,reduce the radiation damage caused by humoralimmunity,cellular immunity and non-specific immune function,promote recovery of immune function,reducing immune function recovery time. As to simple irradiated mice,the serum,liver and brain SOD,GSH-Px,TChE were significantly increased,the content of MDA significantly decreased.APG6,APG7 and APG8 can directly scavenge free radicals,at the same time,mobilize the whole body irradiated mice scavenging system.The APG8 group had the optimal recovery effect of radiation in mice.
本文以黑木耳子实体为原料,用pH6、7、8磷酸盐缓冲溶液浸提黑木耳多糖,用乙醇浓度为100%分级黑木耳多糖。用ABTS+·、DPPH·、超氧自由基、羟基自由基、过氧化氢、金属离子螯合、还原力和脂质过氧化为指标测定多糖的体外抗氧化性。用8周昆明小鼠分成10组雌雄各半,每组10只,灌胃APG6、APG7、APG8多糖100mg/kg、150mg/kg、200 mg/kg,测试其体内SOD、CAT、GSH-PX、GSH. MDA含量,研究黑木分级耳多糖对小鼠体内抗氧化作用的影响。将330只小鼠随机分成11组,辐射模型组,对照组、给药组分别灌胃APG6、APG7、APG8多糖100 mg/kg、150mg/kg、200 mg/kg,14d时照射4.0Gy的60Coγ-射线建立辐射氧化损伤模型,14d后测定小鼠存活率,对免疫系统及抗氧化的影响。研究不同剂量黑木耳分级多糖对小鼠辐射、免疫、氧化系统作用的影响及对小鼠辐射损伤的保护作用,为以后抗氧化及抗辐射生物源药物开发与利用黑木耳多糖提供科学参考。主要结果如下：
1用pH6、7、8磷酸盐缓冲溶液提取黑木耳多糖,用100%乙醇分级,研究黑木耳分级多糖的分子量分布。APG6、APG7、APG8的多糖得率分别为：11.57%、19.46%、12.72%。多糖含量分别为：93.12%、89.43%、96.93%。高效液相色谱法对APG6、APG7、APG8进行纯度检测,APG6、APG7单一组分。质均分子量Mw分别为：4.3840×104、9.4335×103、4.4775×104。
2用制备的APG6、APG7、APG8多糖测定体外抗氧化。研究结果表明酸溶性黑木耳分级多糖APG6具有较强的清除ABTS、DPPH、金属离子螯合能力、还原能力,并且清除ABTS和DPPH自由基能力高于阳性对照Vc。碱溶性黑木耳分级多糖APG8具有较强的清除超氧自由基和羟基自由基能力。而中性黑木耳分级多糖APG7抗脂质过氧化抑制力高于其他多糖。
3 APG6、APG7、APG8做体内抗氧化小鼠试验。结果表明各剂量组多糖均能提高小鼠血清和肝组织中SOD、GSH-PX、GSH、CAT酶,降低MDA含量,随着剂量的增加效果越显著,其中APG8组效果最强。
4小鼠受照4.0Gy的60Coγ-射线,建立辐射损伤模型,分别从辐射指标、免疫功能、抗氧化功能等研究APG6、APG7、APG8对小鼠的辐射保护作用。各组多糖都能显著的提高小鼠存活率；减少由于辐射损伤引起的体液免疫、细胞免疫和非特异性免疫功能下降,促进机体免疫功能恢复,缩短免疫功能恢复时间；增强辐射损伤小鼠SOD、GSH-Px抗氧化酶的活力,降低MDA含量,减少自由基对机体的损伤,增强机体清除自由基的能力。APG8组辐射小鼠恢复效果最优。
Auricularia auricula is a mushroom, commonly called Judas's ear fungus or Jew's Ear. It is the Chinese traditional non-toxic edible mushroom, which has been widely used in Chinese cuisines and is known for their pharmaceutical effects in Chinese herbal medicine.The edible fungus is rich in amino acids, protein,iron,acalcium and polysaccharides.Of particular significance among these function components is polysaccharides.A. auricular polysaccharide have many biological activities, including antitumor activity, preventive hypocholesterolemia and anticoagulant activity.
In this paper,A.auricular fruiting bodies as raw material,pH 6,7,8 phosphate buffer extracted with polysaccharides,and than 100% ethanol concentration of purified polysaccharide.As ABTS,DPPH,hydroxyl free radical,hydroxyl radicals, hydrogen peroxide, metal ion chelating ability,reduce power and induced lipid peroxidation determination of polysaccharide in vitro antioxidant activity.8-week-old Kunming mice,half male and female,10 in each group.Oral administration in mice fed with APG6,APG7 and APG8, the oral dose 100 mg/kg,150mg/kg,200 mg/kg according to the weight.At the 28d,tested in SOD,CAT,GSH-PX,GSH,MDA for the determination of the indicators.Further study of the relationship between antioxidant and A. auricular polysaccharides.The preliminary study on the role of APG6,APG7 and APG8 on the y-ray radiation protection in mice.To 330 mices half male and female,10 in each group.Mice were randomly divided into modle control group,irradiated control group,dose irradiation group.Oral administration in mice fed with APG6,APG7 and APG8 the oral dose 100 mg/kg,150mg/kg,200 mg/kg according to the weight.Mice of the oral group of APG6,APG7,APG8 and,irradiated control group before irradiation be administrated for 14 days,for the next day,4.0Gy he packet 60Co y-ray single irradiation is required.Mice of oral group APG6,APG7 and APG8 after irradiation take administration of 14 days from the current day.On the 28th day after irradiation,all mice were killed by cervical dislocation.For a preliminary study of the A.auricular polysaccharide protective effects in mice radiation, immunity and antioxidant activities.For the future biological antioxidant and anti-radiation drug development and utilization of the source A. auricular polysaccharides provide scientific reference.The main results are as follows:
1 With pH 6,7,8 phosphate buffer extraction of polysaccharides,100% ethanoland purified pH 6,7,8.Studyon APG6,APG7,APG8 polysaccharides molecular weight distribution.The extraction rate of APG6,APG7,APG8 polysaccharides were 11.57%,19.46%,2.72%.Content of APG6,APG7,APG8 polysaccharides were 93.12%,89.43%,96.93%.Measured by Ubbelohde viscometer polysaccharide APG6,APG7 and APG8 viscosity average molecular weight 7.458×105.128×105 and 7.082×105.High efficiency liquid chromatography testing APG6, APG7, APG8 for purity, APG6, APG7 single component, and MW:4.3840×104,9.4335×103,4.4775×104.
2 APG6,APG7,APG8 polysaccharides were evaluated on their antioxidant activities in vitro.The results of extracorporeal antioxidation experiment showed that APG6 had very good potential for scavenging ABTS,DPPH,metal ion chelating ability,reduce power.APG6 had clearer ABTS and DPPH radical scavenging than positive control Vc.APG8 could clean up hydroxyl free radical and hydroxyl radicals.And APG7 was higher inhibiting lipid peroxidation than the other polysaccharides.
3 To study the effects of APG6,APG7,APG8 on in vivo antioxidant activities of normal mice.The results show that APG6,APG7,APG8 also could improve the activity of SOD,GSH-PX,GSH,CAT, reduce the content of MDA in their serum and liver.We also can see if we increasing the dose,the in vivo antioxidant activities of normal mice also increases gradually.APG8 have higher than the other polysaccharides.
4 To establish a mouse model of 60Coγ-injury in 4.0Gy,oral gavage of APG6,APG7 and APG8 detection of survival rate,immune-function and anti-oxidation.For a preliminary study of the y-radiation on APG6,APG7 and APG8 protective effect in mice.All polysaccharides could improved survival rate,reduce the radiation damage caused by humoralimmunity,cellular immunity and non-specific immune function,promote recovery of immune function,reducing immune function recovery time. As to simple irradiated mice,the serum,liver and brain SOD,GSH-Px,TChE were significantly increased,the content of MDA significantly decreased.APG6,APG7 and APG8 can directly scavenge free radicals,at the same time,mobilize the whole body irradiated mice scavenging system.The APG8 group had the optimal recovery effect of radiation in mice.
引文
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