冬枣黄酮的提取及其抗氧化作用的研究
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摘要
目的:本研究通过单因素及正交试验,研究超声波法提取冬枣提取物(DongzaoJujube Extract,DJE)黄酮的最佳提取条件,探讨加工储存条件对冬枣黄酮稳定性和抗氧化能力的影响,并评价DJE对S_(180)荷瘤小鼠的抗氧化作用。
方法:1、超声波提取工艺的研究:在单因素试验的基础上进行L_9(3~4)正交试验,通过极差分析和方差分析探讨超声波法提取冬枣黄酮的最佳提取条件。
2、冬枣黄酮的稳定性研究:采用比色法分别测定DJE中总黄酮的含量和对二苯代苦味酰基自由基(1,1-diphenyl-2-picrylhydazyl,DHHP)的清除能力,比较研究各种外界因素(温度、光照、酸碱度、金属离子以及常用食品添加剂等)对DJE中黄酮的含量及其抗氧化稳定性的影响。
3、DJE半数致死量(LD_(50))的测定:通过Horn法测定DJE的半数致死量(LD_(50))。
4、动物分组及模型建立:50只健康昆明小鼠,雌雄各半,随机分为5组。肿瘤模型组小鼠左前肢腋窝皮下接种0.2mLS_(180)荷瘤小鼠腹腔积液混悬液瘤细胞,浓度为1.0×10~7cfu/mL;EED及EAED组分别给予EED及EAED100mg/(kg·d)灌胃;CY对照组给予环磷酰胺(Cyclophosphamide(CY))20mg/(kg·2d);CY对照组、肿瘤对照组及空白对照组,均给予0.15mL/10g生理盐水灌胃,实验期为10d。
5、抗氧化系统功能评价:分别测定血浆及肝匀浆中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活力及丙二醛(MDA)的含量,红细胞溶血度,单细胞琼脂电泳(single-cell gelelectrophoresis,SCGE)测定。
结果:1、各因素对DJE黄酮的影响程度由大到小依次为:提取时间＞料液比＞乙醇浓度。超声波法提取DJE黄酮的最佳工艺条件是:料液比为1:25时,用70%的乙醇溶液在超声波中提取30min。
2、稳定性的实验结果表明,冬枣提取物的热稳定性、光稳定性均较好,pH在5.0～8.0范围内稳定性较好;浓度为0.025%～4.0%的NaCl、1%～20%的蔗糖对冬枣黄酮的稳定性无明显影响;金属离子K~+、Mg~(2+)、Zn~(2+)对DJE的稳定性影响较小,但Cu~(2+)、Fe~(3+)可明显降低冬枣黄酮的稳定性。
3、LD_(50)＞5000mg/kg,属实际无毒级。
4、DJE对S_(180)荷瘤小鼠血浆中的SOD活力有升高作用,EED组及EAED组的SOD活力均高于CY对照组(P＜0.05)及肿瘤对照组(P＜0.05)。EED组和EAED组间无统计学差异(P＞0.05)。
DJE对MDA的含量有降低作用,EED组及EAED组的MDA含量均低于肿瘤对照组(P＜0.05),但均高于空白对照组(P＜0.05)。EED组和EAED组间无统计学差异(P＞0.05)。
肝匀浆各组结果与肿瘤对照组比较均无统计学差异(P＞0.05)。
彗星电泳实验显示较高剂量的DJE在体外对血淋巴细胞的氧化损伤有一定的保护作用。
结论:1、超声波提取DJE黄酮的最佳工艺为:料液比为1:25,乙醇浓度70%,提取时间30min。
2、冬枣提取物中的黄酮稳定性较好。
3、DJE可提高S_(180)荷瘤小鼠的机体抗氧化能力。
Objective:To study the excellent extracting condition for falconoids in the Dongzao Jujube Extract(DJE) by the method of ultrasonic extracting,through monofactorial experiments and orthogonal experiments and investigate the DJE falconoids effects on the stability and oxidization in different processing and saving conditions and evaluate the DJE effects of antioxidative activity by establishing the S_(180)-tumor bearing mice model.
Methods:1.Studied the extracting method by ultrasonic:Based on the monofactorial experiments to study the L_9(3~4) orthogonal experiments,the excellent falconoids extracting condition could be sure through analysis of range and variance methods.
2.Stability of flavones in DJE:To determine the content of total flavonoids in DJE and the cleaning capability of DHHP,comparative studied the influences in every external factor(time,illumination,power of hydrogen,metal ion,commonly used food additive and so on) for the content of falconoids and antioxidative stabilizer.
3.Determination of the DJE's half lethal dose(LD_(50)):Determined the DJE's half lethal dose(LD_(50)) with Horn's method.
4.Animals grouping and modeling:50 healthy Kunming mice,half male and half female,were randomly divided into five groups with 10 mice in each group.0.2mL S_(180) mouse sarcoma cells suspension(concentration of 1.0×10~7cfu/mL) was inoculated subcutaneously into the left armpit region of each tumor model mouse.Mice in the EED group and EAED group were gavaged by100mg/(kg·d) EED and100mg/(kg·d) EAED respectively.Mice in the CY control group were given 20mg/(kg·2d) CY (Cyclophosphamide injection(CY)).Mice in the CY control group,tumor control group and blank control group were gavaged 0.15mL/10g normal saline once a day.The experimental period was 10 days.
5.Functional evaluation of antioxidative system:The activities of superoxide dismutase(SOD),glutathione-peroxidase(GSH-Px),the content of malanydiadehyde (MDA) in plasma and liver homogenate,hemolytic degree of RBC and single-cell gelelectrophoresis(SCGE) were measured respectively.
Results:1.Every factor could affect the falconoids in DJE,the order was:extracting times＞ratio of dosage liquor＞concentration of ethanol.The excellent falconoids extracting conditions for ultrasonic extracting were as follows:The ration of dosage liquor was 1:25;the concentration of ethanol is 70%;the extracting time is 30 min.
2.It could be seen that thermostability,light stability and pH limited in 5.0-8.0 were stationary in the DJE's steady experiment.There were no significant effects by the NaCl (0.025%-4.0%) or cane sugar(1%-20%) for the stability of flavones in DJE.There were little effect on DJE stability by the metal ion such as K~+,Mg~(2+),Zn~(2+),but Cu~(2+),Fe~(3+) could cut down the stability of flavones in DJE.
3.The dosage of LD_(50) was above 5000mg/kg,which actually belongs to the non-toxic level.
4.DJE had the effect of increasing the SOD activity in plasma.The SOD activities in EED and EADE groups were both significantly higher than that in the CY control group (P＜0.05) and the tumor control group(P＜0.05).And there were no significant difference between EED and EAED groups(P＞0.05).
DJE had the decreasing effect on the level of MDA.The MDA level in the two DJE group were both significantly lower than that in the tumor group(P＜0.05) but both higher than that in the blank control group(P＜0.05).There were no significant difference between EED and EAED groups(P＞0.05).
There was no significant difference(P＞0.05) on the every results of liver homogenate between the two DJE groups and tumor group.
There was significant protection effect of oxidative damage on the blood-lympho-cytes in vitro in SCGE experimentation.
Conclusion:1.The excellent falconoids extracting conditions for ultrasonic extracting were as follows:The ration of dosage liquor is 1:25;the concentration of ethanol is 70%;the extracting time is 30 min.
2.DJE's flavones are stationary.
3.DJE can raise the S_(180)-tumor bearing mice's antioxidation activity.
方法:1、超声波提取工艺的研究:在单因素试验的基础上进行L_9(3~4)正交试验,通过极差分析和方差分析探讨超声波法提取冬枣黄酮的最佳提取条件。
2、冬枣黄酮的稳定性研究:采用比色法分别测定DJE中总黄酮的含量和对二苯代苦味酰基自由基(1,1-diphenyl-2-picrylhydazyl,DHHP)的清除能力,比较研究各种外界因素(温度、光照、酸碱度、金属离子以及常用食品添加剂等)对DJE中黄酮的含量及其抗氧化稳定性的影响。
3、DJE半数致死量(LD_(50))的测定:通过Horn法测定DJE的半数致死量(LD_(50))。
4、动物分组及模型建立:50只健康昆明小鼠,雌雄各半,随机分为5组。肿瘤模型组小鼠左前肢腋窝皮下接种0.2mLS_(180)荷瘤小鼠腹腔积液混悬液瘤细胞,浓度为1.0×10~7cfu/mL;EED及EAED组分别给予EED及EAED100mg/(kg·d)灌胃;CY对照组给予环磷酰胺(Cyclophosphamide(CY))20mg/(kg·2d);CY对照组、肿瘤对照组及空白对照组,均给予0.15mL/10g生理盐水灌胃,实验期为10d。
5、抗氧化系统功能评价:分别测定血浆及肝匀浆中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活力及丙二醛(MDA)的含量,红细胞溶血度,单细胞琼脂电泳(single-cell gelelectrophoresis,SCGE)测定。
结果:1、各因素对DJE黄酮的影响程度由大到小依次为:提取时间＞料液比＞乙醇浓度。超声波法提取DJE黄酮的最佳工艺条件是:料液比为1:25时,用70%的乙醇溶液在超声波中提取30min。
2、稳定性的实验结果表明,冬枣提取物的热稳定性、光稳定性均较好,pH在5.0～8.0范围内稳定性较好;浓度为0.025%～4.0%的NaCl、1%～20%的蔗糖对冬枣黄酮的稳定性无明显影响;金属离子K~+、Mg~(2+)、Zn~(2+)对DJE的稳定性影响较小,但Cu~(2+)、Fe~(3+)可明显降低冬枣黄酮的稳定性。
3、LD_(50)＞5000mg/kg,属实际无毒级。
4、DJE对S_(180)荷瘤小鼠血浆中的SOD活力有升高作用,EED组及EAED组的SOD活力均高于CY对照组(P＜0.05)及肿瘤对照组(P＜0.05)。EED组和EAED组间无统计学差异(P＞0.05)。
DJE对MDA的含量有降低作用,EED组及EAED组的MDA含量均低于肿瘤对照组(P＜0.05),但均高于空白对照组(P＜0.05)。EED组和EAED组间无统计学差异(P＞0.05)。
肝匀浆各组结果与肿瘤对照组比较均无统计学差异(P＞0.05)。
彗星电泳实验显示较高剂量的DJE在体外对血淋巴细胞的氧化损伤有一定的保护作用。
结论:1、超声波提取DJE黄酮的最佳工艺为:料液比为1:25,乙醇浓度70%,提取时间30min。
2、冬枣提取物中的黄酮稳定性较好。
3、DJE可提高S_(180)荷瘤小鼠的机体抗氧化能力。
Objective:To study the excellent extracting condition for falconoids in the Dongzao Jujube Extract(DJE) by the method of ultrasonic extracting,through monofactorial experiments and orthogonal experiments and investigate the DJE falconoids effects on the stability and oxidization in different processing and saving conditions and evaluate the DJE effects of antioxidative activity by establishing the S_(180)-tumor bearing mice model.
Methods:1.Studied the extracting method by ultrasonic:Based on the monofactorial experiments to study the L_9(3~4) orthogonal experiments,the excellent falconoids extracting condition could be sure through analysis of range and variance methods.
2.Stability of flavones in DJE:To determine the content of total flavonoids in DJE and the cleaning capability of DHHP,comparative studied the influences in every external factor(time,illumination,power of hydrogen,metal ion,commonly used food additive and so on) for the content of falconoids and antioxidative stabilizer.
3.Determination of the DJE's half lethal dose(LD_(50)):Determined the DJE's half lethal dose(LD_(50)) with Horn's method.
4.Animals grouping and modeling:50 healthy Kunming mice,half male and half female,were randomly divided into five groups with 10 mice in each group.0.2mL S_(180) mouse sarcoma cells suspension(concentration of 1.0×10~7cfu/mL) was inoculated subcutaneously into the left armpit region of each tumor model mouse.Mice in the EED group and EAED group were gavaged by100mg/(kg·d) EED and100mg/(kg·d) EAED respectively.Mice in the CY control group were given 20mg/(kg·2d) CY (Cyclophosphamide injection(CY)).Mice in the CY control group,tumor control group and blank control group were gavaged 0.15mL/10g normal saline once a day.The experimental period was 10 days.
5.Functional evaluation of antioxidative system:The activities of superoxide dismutase(SOD),glutathione-peroxidase(GSH-Px),the content of malanydiadehyde (MDA) in plasma and liver homogenate,hemolytic degree of RBC and single-cell gelelectrophoresis(SCGE) were measured respectively.
Results:1.Every factor could affect the falconoids in DJE,the order was:extracting times＞ratio of dosage liquor＞concentration of ethanol.The excellent falconoids extracting conditions for ultrasonic extracting were as follows:The ration of dosage liquor was 1:25;the concentration of ethanol is 70%;the extracting time is 30 min.
2.It could be seen that thermostability,light stability and pH limited in 5.0-8.0 were stationary in the DJE's steady experiment.There were no significant effects by the NaCl (0.025%-4.0%) or cane sugar(1%-20%) for the stability of flavones in DJE.There were little effect on DJE stability by the metal ion such as K~+,Mg~(2+),Zn~(2+),but Cu~(2+),Fe~(3+) could cut down the stability of flavones in DJE.
3.The dosage of LD_(50) was above 5000mg/kg,which actually belongs to the non-toxic level.
4.DJE had the effect of increasing the SOD activity in plasma.The SOD activities in EED and EADE groups were both significantly higher than that in the CY control group (P＜0.05) and the tumor control group(P＜0.05).And there were no significant difference between EED and EAED groups(P＞0.05).
DJE had the decreasing effect on the level of MDA.The MDA level in the two DJE group were both significantly lower than that in the tumor group(P＜0.05) but both higher than that in the blank control group(P＜0.05).There were no significant difference between EED and EAED groups(P＞0.05).
There was no significant difference(P＞0.05) on the every results of liver homogenate between the two DJE groups and tumor group.
There was significant protection effect of oxidative damage on the blood-lympho-cytes in vitro in SCGE experimentation.
Conclusion:1.The excellent falconoids extracting conditions for ultrasonic extracting were as follows:The ration of dosage liquor is 1:25;the concentration of ethanol is 70%;the extracting time is 30 min.
2.DJE's flavones are stationary.
3.DJE can raise the S_(180)-tumor bearing mice's antioxidation activity.
引文
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1 石丽霞,张振家.复方大枣口服液对小鼠免疫机能的影响[J].第四军医大学吉林军医学院(现吉林医药学院)学报,2005,26(1):41-42.
2 焦艳,闻杰,于晓红,等.膜荚黄芪茎叶总黄酮对小鼠细胞免疫功能的影响[J].中国中西医结合杂志,1999,19(6):356-358.
3 孙奕,王景明,骆永珍.淫羊藿总黄酮促进免疫功能低下小鼠IL-2和NK活性的实验研究[J].中草药,2002,33(7):635-636.
4 Christian B.Frank L.Glucoconicoids enhance OX-idative stress-induced cell death in hipocampal neuronsinvitro[J].Endocrinology,1997,138:101.
5 Kanowski S,Herrmann W M,Stephan K,et al.Proof of eficacy of the Ginkgo biloba special extract EGB761 in outpatients suffering from mild to moderateprimary degenerative dementia of the Alzheimer type ormulti-infant dementia[J].Pharmacopsychiatry,1996,29(2):47-50.
6 Oishi M,Mochizuki Y,Takasu T,et al.Eflee-tiveness of traditional Chinese medicine in Alzheimer disease[J].Alzheimer Dis Assoc Disord,1998,12(3):247-250.
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