鹰嘴豆异黄酮抑制酪氨酸酶及其包合性能研究
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摘要
鹰嘴豆(Cicer arietinum L)豆科鹰嘴豆属,因其面形奇特,尖如鹰嘴而得此名。别名桃豆、鸡碗豆,新疆俗称诺胡提,又称桃尔豆,耐干旱、耐贫瘠,是目前世界上栽培面积较广的食用豆类作物之一,为世界第二大消费豆类,在我国新疆已有2500年的栽培历史。研究表明鹰嘴豆具有广泛的生理活性,如防癌、降血脂、保护心血管功能等,其化合物的提取及其保健食品的开发具有广阔的前景,其中重要的营养成分鹰嘴豆异黄酮(CAI)含量达到0.33%。鹰嘴豆异黄酮能有效防止癌细胞增殖,具有调节血脂预防心血管疾病、改善骨质疏松等功效。目前,无论是国内还是国外关于鹰嘴豆的研究很少,而对鹰嘴豆异黄酮的包合和应用性能方面的研究更是少之又少。
由于异黄酮含有酚羟基,化学反应性强,稳定性差,光和热足以促使其分解,失去抗氧化活性和其他生物学功能,使其在贮存和应用中也受到一定的限制。与环糊精形成包合物后,将显著改善药物的理化性质。本课题研究了从鹰嘴豆中提取异黄酮的具体工艺,取发芽后的鹰嘴豆用石油醚脱脂,然后选用65%乙醇为提取剂,在85。C下,提取2次(每次2小时),乙酸乙酯萃取后用大孔吸附树脂进行精制,得到较纯的鹰嘴豆异黄酮精品,并对照鹰嘴豆芽素A进行紫外扫描,确定260nm为其最大吸收波长。
用研磨法制备β-环糊精及羟丙基节-环糊精与鹰嘴豆异黄酮的包合物,由红外吸收光谱对包合物进行表征。运用饱和溶液法和紫外分光光度法测试包合体系的溶解度,并测定鹰嘴豆异黄酮在不同浓度的β-环糊精及羟丙基-β-环糊精溶液下吸光度的变化,根据Benesi-Hildebrand方程确定了包合反应的包合比为1:1、表观包合稳定常数Kfθ和包合反应的热力学参数ΔGθ,ΔHθ和ΔSθ,并由此得出包合反应主要由焓变控制,推动力为范德华力。
在30℃,pH=6.8的Na2HPO4-NaH2PO4缓冲体系中,分别以L-酪氨酸和L-3,4-二羟基本氨酸(L-DOPA)为底物,通过测定不同浓度提取物加入量的条件下吸光度随时间的变化,来研究鹰嘴豆异黄酮提取物对酪氨酸酶单酚酶及二酚酶活性的抑制动力学效应。实验结果表明,鹰嘴豆异黄酮对酪氨酸酶单酚酶和二酚酶都有良好抑制作用,对单酚酶和二酚酶活力的相对抑制率达到50%的鹰嘴豆异黄酮浓度(IC50)约分别为0.07mg/mL和0.03mg/mL;在降低酪氨酸酶酶活的同时,鹰嘴豆异黄酮能明显延长单酚酶的迟滞时间,0.08mg/mL鹰嘴豆异.黄酮能使其迟滞时间由1.26 min延长至2.62 min。通过Lineweaver-Burk图显示鹰嘴豆异黄酮对二酚酶的抑制作用表现为竞争性抑制,通过做Dixon曲线得出抑制常数(KI)为0.0193mg/mL。
Chickpea(Cicer arietinum) belongs to the Family Leguminosae, whose name is indicative of its shape. It is an annual or perennial herb growing in arid and semi-arid area of the world, it is also the second largest consumption legume and widely cultivated. It has been grown in XinJiang for 2500 years. Research shows that chickpea has several physiological effects,such as anticancer,antilipemic,andanti-angiocarpy properties.Extraction of the compounds and the development of health food have broad prospects.The resource of isoflavones is contained in chickpea, which is up to 0.33% in its germinated seeds. Cicer arietinum isoflavones(CAI) are effective in preventing the proliferation of cancer cells, decreasing blood lipids to prevent cardiovascular disease, curing osteoporosis.However,the researchs about Chickpea are limited not only in domestic but also in foreign, especially in inclusion and application.
Due to the phenolic hydroxyl, isoflavones has strong chemical reactivity, poor stability, accelerated decomposition under conditions of light and heat, also easy to lose antioxidant activity and other biological functions,so its application and storage also subject to certain restrictions. Using Cyclodextrins to form inclusion compounds will significantly improve the physical and chemical properties of Cicer arietinum isoflavones.
In this paper, the specific extraction methods of total isoflavones from chickpea was studied. Choose the germinant chickpea powder, degreasing by petroleun ether, using 65% ethanol and extracting the chickpea powder for two times (2 hours per time)under 85℃,then refined by macroporous adsorption resins after extracted by ethyl acetate to obtain the refinement. Confirm the maximum absorption wavelength is 260nm by scanning UV absorption spectrum compared with Biochanin A.
The inclusion complexes ofβ-cyclodextrin(β-CD) and hydroxypropyl-β-cyclodextrin (Hp-β-CD) with cicer arietinum isoflavones(CAI) were prepared by the grinding method and characterized by infrared absorption spectrum. UV spectrophotometry was used to measure the solubility of inclusion complex as well as the changes of absorbance of Cicer arietinum isoflavones under different concentration ofβ-cyclodextrin(β-CD) and hydroxypropyl-β-cyclodextrin (Hp-β-CD), The inclusion equilibrium constant(Kfθ)of inclusion complex was determined by Benesi-Hildebrand equation and the thermodynamic parameters of the inclusion reaction,such as△Gθ△Hθand△Sθwere obtained from the inclusion constants at different temperatures.The whole inclusion process was controlled mainly by enthalpy and driven by Van Der Waals Forces.
The inhibitory kinetics of cicer arietinum isoflavones on the activity of monophenolase and diphenolase contained in tyrosinase is studied via enzymological kinetic method. The changes of absorbance in time under different concentration of the extract are measured when take L-Tyrosine and L-DOPA(L-3,4-dihydroxyphenylalanine) as substrates respectively with Na2HPO4-NaH2PO4 as buffer solution(pH=6.8) at 30℃. It was found that Cicer arietinum isoflavones can inhibit both the monophenolase and diphenolase activities of tyrosinase well. The Cicer arietinum isoflavones concentrations corresponding to 50%inhibitory rate(IC50)are 0.07 mg/mL for monophenolase and 0.03 mg/mL for diphenolase,respectively.Moreover,cicer arietinum isoflavones can also extent the lag time of catalytic oxidation of L-Tyrosine by monophenolase,for example,0.08 mg/mL of Cicer arietinum isoflavones can extend the lag time from 1.26 min to 2.62 min. The inhibition kinetics of Cicer arietinum isoflavones analyzed by Lineweaver-Burk plots demonstrate that the inhibition effect of Cicer arietinum isoflavones for the oxidation of L-DOPA expresses as what a competitive inhibitor does.The inhibition constant of competitive reversible inhibitor KI were determined to be 0.0193 mg/mL.
由于异黄酮含有酚羟基,化学反应性强,稳定性差,光和热足以促使其分解,失去抗氧化活性和其他生物学功能,使其在贮存和应用中也受到一定的限制。与环糊精形成包合物后,将显著改善药物的理化性质。本课题研究了从鹰嘴豆中提取异黄酮的具体工艺,取发芽后的鹰嘴豆用石油醚脱脂,然后选用65%乙醇为提取剂,在85。C下,提取2次(每次2小时),乙酸乙酯萃取后用大孔吸附树脂进行精制,得到较纯的鹰嘴豆异黄酮精品,并对照鹰嘴豆芽素A进行紫外扫描,确定260nm为其最大吸收波长。
用研磨法制备β-环糊精及羟丙基节-环糊精与鹰嘴豆异黄酮的包合物,由红外吸收光谱对包合物进行表征。运用饱和溶液法和紫外分光光度法测试包合体系的溶解度,并测定鹰嘴豆异黄酮在不同浓度的β-环糊精及羟丙基-β-环糊精溶液下吸光度的变化,根据Benesi-Hildebrand方程确定了包合反应的包合比为1:1、表观包合稳定常数Kfθ和包合反应的热力学参数ΔGθ,ΔHθ和ΔSθ,并由此得出包合反应主要由焓变控制,推动力为范德华力。
在30℃,pH=6.8的Na2HPO4-NaH2PO4缓冲体系中,分别以L-酪氨酸和L-3,4-二羟基本氨酸(L-DOPA)为底物,通过测定不同浓度提取物加入量的条件下吸光度随时间的变化,来研究鹰嘴豆异黄酮提取物对酪氨酸酶单酚酶及二酚酶活性的抑制动力学效应。实验结果表明,鹰嘴豆异黄酮对酪氨酸酶单酚酶和二酚酶都有良好抑制作用,对单酚酶和二酚酶活力的相对抑制率达到50%的鹰嘴豆异黄酮浓度(IC50)约分别为0.07mg/mL和0.03mg/mL;在降低酪氨酸酶酶活的同时,鹰嘴豆异黄酮能明显延长单酚酶的迟滞时间,0.08mg/mL鹰嘴豆异.黄酮能使其迟滞时间由1.26 min延长至2.62 min。通过Lineweaver-Burk图显示鹰嘴豆异黄酮对二酚酶的抑制作用表现为竞争性抑制,通过做Dixon曲线得出抑制常数(KI)为0.0193mg/mL。
Chickpea(Cicer arietinum) belongs to the Family Leguminosae, whose name is indicative of its shape. It is an annual or perennial herb growing in arid and semi-arid area of the world, it is also the second largest consumption legume and widely cultivated. It has been grown in XinJiang for 2500 years. Research shows that chickpea has several physiological effects,such as anticancer,antilipemic,andanti-angiocarpy properties.Extraction of the compounds and the development of health food have broad prospects.The resource of isoflavones is contained in chickpea, which is up to 0.33% in its germinated seeds. Cicer arietinum isoflavones(CAI) are effective in preventing the proliferation of cancer cells, decreasing blood lipids to prevent cardiovascular disease, curing osteoporosis.However,the researchs about Chickpea are limited not only in domestic but also in foreign, especially in inclusion and application.
Due to the phenolic hydroxyl, isoflavones has strong chemical reactivity, poor stability, accelerated decomposition under conditions of light and heat, also easy to lose antioxidant activity and other biological functions,so its application and storage also subject to certain restrictions. Using Cyclodextrins to form inclusion compounds will significantly improve the physical and chemical properties of Cicer arietinum isoflavones.
In this paper, the specific extraction methods of total isoflavones from chickpea was studied. Choose the germinant chickpea powder, degreasing by petroleun ether, using 65% ethanol and extracting the chickpea powder for two times (2 hours per time)under 85℃,then refined by macroporous adsorption resins after extracted by ethyl acetate to obtain the refinement. Confirm the maximum absorption wavelength is 260nm by scanning UV absorption spectrum compared with Biochanin A.
The inclusion complexes ofβ-cyclodextrin(β-CD) and hydroxypropyl-β-cyclodextrin (Hp-β-CD) with cicer arietinum isoflavones(CAI) were prepared by the grinding method and characterized by infrared absorption spectrum. UV spectrophotometry was used to measure the solubility of inclusion complex as well as the changes of absorbance of Cicer arietinum isoflavones under different concentration ofβ-cyclodextrin(β-CD) and hydroxypropyl-β-cyclodextrin (Hp-β-CD), The inclusion equilibrium constant(Kfθ)of inclusion complex was determined by Benesi-Hildebrand equation and the thermodynamic parameters of the inclusion reaction,such as△Gθ△Hθand△Sθwere obtained from the inclusion constants at different temperatures.The whole inclusion process was controlled mainly by enthalpy and driven by Van Der Waals Forces.
The inhibitory kinetics of cicer arietinum isoflavones on the activity of monophenolase and diphenolase contained in tyrosinase is studied via enzymological kinetic method. The changes of absorbance in time under different concentration of the extract are measured when take L-Tyrosine and L-DOPA(L-3,4-dihydroxyphenylalanine) as substrates respectively with Na2HPO4-NaH2PO4 as buffer solution(pH=6.8) at 30℃. It was found that Cicer arietinum isoflavones can inhibit both the monophenolase and diphenolase activities of tyrosinase well. The Cicer arietinum isoflavones concentrations corresponding to 50%inhibitory rate(IC50)are 0.07 mg/mL for monophenolase and 0.03 mg/mL for diphenolase,respectively.Moreover,cicer arietinum isoflavones can also extent the lag time of catalytic oxidation of L-Tyrosine by monophenolase,for example,0.08 mg/mL of Cicer arietinum isoflavones can extend the lag time from 1.26 min to 2.62 min. The inhibition kinetics of Cicer arietinum isoflavones analyzed by Lineweaver-Burk plots demonstrate that the inhibition effect of Cicer arietinum isoflavones for the oxidation of L-DOPA expresses as what a competitive inhibitor does.The inhibition constant of competitive reversible inhibitor KI were determined to be 0.0193 mg/mL.
引文
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