沙棘叶黄酮提取物体内外抗氧化活性、应用与护肝作用的研究
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摘要
目前,食品中广泛应用的抗氧化剂多以人工合成抗氧化剂为主,但由于合成抗氧化剂在毒理学上存在安全问题,其广泛应用反而成为了一种食品安全的威胁。因此,如何寻找开发利用自然界存在的天然的植物来源抗氧化剂成为研究的热点。沙棘叶中富含多种生物活性成分,是天然抗氧化剂的良好来源,其不仅能够应用于食品中抑制脂肪和蛋白的氧化,同时还具有有益人体健康的潜在功效,比如清除体内自由基、抑制肝损伤、提高肠道免疫能力等等。本课题以蒙古大果沙棘叶为提纯对象,提纯了具有高度非特异性抗氧化活力和高浓度黄酮类含量的,沙棘叶黄酮提取物,研究了超声波辅助乙醇-盐酸溶液提取最佳工艺,提取物中主要成分分析、提取物的抗氧化稳定性,提取物在生、熟肉糜中对脂肪氧化的抑制作用,提取物对人胚肺成纤维细胞免于受到H2O2损伤的保护作用,提取物对半乳糖胺(Gal-N)诱导肝损伤大鼠的保护作用以及在对小鼠肠道黏膜免疫能力的影响。主要研究结果如下：
1、以超声波辅助乙醇-盐酸溶液对沙棘叶中的总黄酮进行提取,优化提取条件并对其抗氧化能力进行了判定。沙棘叶总黄酮提取物制备最佳加工条件为乙醇体积比50%,料液比1：20,盐酸浓度3mol/L,提取时间30min,超声波功率80W,此工艺条件下的提取率达到6.34%。沙棘叶总黄酮具有较强的还原能力和2,2’-氨基-二(3-乙基-苯并噻唑啉磺酸-6)铵盐(ABTS+)自由基清除能力。
2、比较了沙棘叶黄酮提取物的抗氧化能力,将2%沙棘叶黄酮提取物与0.01%BHA和0.01%Vc的抗氧化活性分别进行对比,沙棘叶黄酮提取物的还原能力、ABTS+自由基和超氧自由基清除率均显著高于BHA和Vc(P<0.05)；提取物的2,2-二苯基-1-间三硝基苯基联肼(DPPH)自由基和羟基自由基清除率基本和BHA的效果相当(P>0.05),但是始终高于Vc的DPPH自由基和羟基自由基清除率(P<0.05)。
3、利用优化过条件后的高效液相色谱法(HPLC)分析了沙棘叶黄酮提物中的主要活性物质。提取物中主要成分有芦丁、杨梅素、槲皮素和异鼠李素,其中芦丁、杨梅素、槲皮素和异鼠李素的总含量分别达到了30.32、20.05、29.01和23.08mg/g。
4、不同的pH值、避光保存、金属离子以及加热对沙棘叶提取物的抗氧化活性(还原能力和ABTS自由基清除活性)影响不显著(P>0.05),但是在阳光和日光灯照射条件下,其抗氧化活性显著降低(P<0.05),为沙棘叶提取物作为天然抗氧化剂在食品领域的稳定应用提供理论依据。
5、将沙棘叶黄酮提取物以0.02%、0.05%、0.10%、0.20%的添加量应用于生肉糜中,在冷藏过程中测定肉糜的色差值、pH值、羰基含量、硫代巴比妥酸值(TBARS值),安排了专业感官评价组并对肉糜的感官质量变化进行了评定。与不添加沙棘叶黄酮提取物的对阴性照组比较,提取物处理组在7天内能显著抑制猪肉蛋白和脂肪氧化过程,增加肉糜颜色的红度值,且随着添加量加大效果增加明显,与BHA效果相当。感官评价也是一样。
6、同样将0.02%、0.05%、0.10%、0.20%的沙棘叶黄酮提取物添加到熟肉糜中,在冷藏过程中测定肉糜的色差值、pH值、羰基含量、硫代巴比妥酸值(TBARS值),并对包括颜色、异味、可接受性、酸败4个感官指标做了比较评定。结果表明,添加提取物处理组对油炸后熟肉饼脂肪氧化抑制作用效果明显,在抑制熟肉糜蛋白和脂肪氧化过程效果明显,与BHA效果相当。感官评定也一样。
7、沙棘叶黄酮提取物可以对细胞起到保护作用,对试验的人胚肺成纤维细胞MRC-5,在试验中减轻了细胞遭受过氧化氢的损伤。沙棘叶黄酮提取物能够增加细胞存活几率,并且可显著提高细胞内超氧化物歧化酶(SOD酶)、过氧化氢酶(CAT酶)和谷胱甘肽过氧化物酶(GSH-Px酶)的活力；同时,对细胞内的丙二醛(MDA)有显著清除作用。
8、沙棘叶黄酮提取物对半乳糖胺(Gal-N)诱导的肝损伤大鼠具有明显的保护作用。首先,沙棘叶黄酮提取物能够显著的降低Gal-N诱导的肝损伤大鼠血清中的谷草转氨酶(AST)和谷丙转氨酶(ALT)水平(P<0.05),从生理角度考虑,其对大鼠的肝脏具有很好的保护以及毒物保护作用,同时对健康具有很大益处；其次,沙棘叶黄酮提取物可显著提高大鼠肝脏组织中的SOD酶、CAT酶和GSH-Px酶的活力(P<0.05),提高肝脏组织的总抗氧化能力(T-AOC)(P<0.05),并且降低肝脏组织中的MDA含量(P<0.05),减轻Gal-N对肝脏的损伤。
9、中剂量沙棘叶黄酮提取物灌胃可以促进小鼠肠道黏膜免疫水平,能够显著提高肠道黏膜中的分泌性免疫球蛋白A (SIgA)、白细胞介素2(IL-2)和白细胞介素4(IL-4)的含量(P<0.05),促进机体肠道T淋巴细胞连续分化,使机体免疫水平得到提高；同时,从干扰素γ (IFN-γ)的含量以及IFN-γ与IL-4的比值可以看出中剂量沙棘叶黄酮提取物灌胃能够提高体液免疫力,细胞免疫能力正常,对抵抗外界异物感染、保证机体肠道健康最有利。另外,高剂量组造成对异物识别敏感性减低,不利于试验动物机体肠道健康。另外由于IFN-γ与IL-4比值低于对照组,有潜在的免疫失调的可能,需要进行进一步深入研究。
None-natural and chemical resourse antioxidants are commercial and wide used in morden food production, to make foods less lipid oxidation. Meanwhile synthetic antioxdants are well-known for their potential health risks, for this reason, they are under stem regulations in food industry. With the development of natural-resourses plant antioxdants is regarded as a effective and good alternatives for the food oxidant deterioration. The result of the research of active component in the seabuckthorn leaves shows the flavonoid of the seabuckthorn leaves is a promising resourse to yielding natural antioxidants. The fuction of the seabuchthorn leaves flavonoid has the capability to savenge free radicals, inhibiting liver damage and improving intestinal immune ability. The objectives of this research were to prepare Seabuckthorn leaves extract with high flavonoid content and antioxidant activity. The optimal extraction condition of total flavonoids of Seabuckthorn leaves, using ethanol-hydrochloric acid solution assisted by ultrasonic was determined, as well as main component analysis using HPLC and antioxidant stability. The antioxidant effects of Seabuckthorn leaves extract in raw and cooked pork patties were evaluated. In addition,it do good to alleviate the damaged of human embryonic lung fibroblast cells MRC-5attacked by H2O2, hepatoprotective effect on Gal-N-induced liver damage in rats and the influence of the intestinal mucosal immunity in mice were also investigated. The main results were as follows:
1. The optimum conditions for extracting total flavonoids from the Seabuckthorn leaves were found to be:ethanol concentration50%, material-to-liquid ratio1:20(g/mL), hydrochloric acid concentration3mol/L, extraction time30min and ultrasonic power80W. Under these optimum conditions, the total yield of flavonoid reached as high as6.34%, and the total flavonoids extract possessed marked antioxidant activities. This study opens a new route for the extraction of total flavonoids from the Seabuckthorn leaves.
2.20mg/kg Seabuckthorn leaves extract has higher reducing power, in comparison to0.1mg/kg BHA and0.1mg/kg ascorbic acid, as same as ABTS+and superoxide radicals scavenging activity shows nice result (P<0.05), but less abilty to BHA in DPPH and hydroxyl radical scavenging activity (P<0.05).
3. The main components of Seabuckthorn leaves extract was analysed using high performance liquid chromatograph (HPLC). The four components were rutin, myricetin, quercetin and isorhamnetin. The total content of each of them was30.32,20.05,29.01and23.08mg/g, respectively.
4. The different pH value, avoid light preservation, metal ions and heating conditions on the antioxidant activities of Seabuckthorn leaves extract (reducing power and ABTS radical scavenging activity) effect was not significant (P>0.05), but in the sunlight and fluorescent light, its antioxidant activity decreased significantly (P<0.05). In addition, this research provided theoretical basis of Seabuckthorn leaves extract for application in food industry as natural antioxidants.
5. The Seabuckthorn leaves extract at a0.02%,0.05%,0.10%and0.20%addition of meat patties (weight/weight). During7days of storage, red color index, pH, carbonyl groups content and TBARS experiment was measured and organized a sensory evaluation experiment to judge the quality of raw pork patties. The results showed that the treatments of the seabuckthorn extract had significant inhibitory effects on lipid oxidation and kept the a*-value of patties in7days, in comparison to the controls, and shows the obviously dose effect ration, be equivalent to BHA, sensory evaluation has the same result.
6. Seabuckthorn leaves extract at a0.02%,0.05%,0.10%and0.20%concentration level(w/w) were added to fried meat patties, during the storage period, red color index, pH, carbonyl groups content and TBARS+value were measured and organized a sensory evaluation experiment to appraise the quality of fried meat patties.. The results shows the addition of Seabuckthorn leaves extract had obviously inhibitory effects on free radical reaction induced lipid oxidation, equivalent to BHA, sensory evaluation has the same result.
7. The Seabuckthorn leaves extract could protect the MRC-5in alleviating the degree of H2O2induced cytotoxicity. The extract treated cells had higher cell viability than the control. Additionally, the cytoprotective effect of Seabuckthorn leaves extract was in conneted with the decline in the MDA level and increases in cell enzyme of the SOD, CAT and GSH-Px activities in treated MRC-5cells.
8. The Seabuckthorn leaves extract processed significant hepatoprotective effect on Gal-N-induced liver damage in rats. First of all, Seabuckthorn leaves extract could significant reduced the ALT and AST level of serum of the rat (P<0.05), indicated that it has good hepatoprotective effect, and were benefit to health. Secondly, compared with negative control group, SOD, CAT and GSH-Px activities and T-AOC of liver of the rats were increased significantly (P<0.05) and the MDA content of liver of the rats was sharply decreased (P<0.05) treated by all dose of Seabuckthorn leaves extract. Our study showed that the Seabuckthorn leaves extract could obviously reduce the liver damage which induced by Gal-N.
9. Medium dose of Seabuckthom leaves extract could promote the intestinal mucosal immunity in mice. Secretory immunoglobulin A (SIgA), interleukin2(IL-2) and interleukin4(IL-4) levels were significant increased (P<0.05), which promoted continuous body intestinal T cell differentiation, as well as the body's immune level was improved. At the same time, from the content of interferon γ (IFN-γ) and the ratio of IFN-y and IL-4, the medium dose of Seabuckthorn leaves extract could promote intestinal mucosal immunity levels, and enhance humoral immunity and cellular immunity ability is normal, to resist outside eyewinker infection to ensure the body most good intestinal health.In addition, high dose group caused the intestinal cellular immunity was inhibited, the body is not sensitive to outside eyewinker, bad for the body intestinal health.
1、以超声波辅助乙醇-盐酸溶液对沙棘叶中的总黄酮进行提取,优化提取条件并对其抗氧化能力进行了判定。沙棘叶总黄酮提取物制备最佳加工条件为乙醇体积比50%,料液比1：20,盐酸浓度3mol/L,提取时间30min,超声波功率80W,此工艺条件下的提取率达到6.34%。沙棘叶总黄酮具有较强的还原能力和2,2’-氨基-二(3-乙基-苯并噻唑啉磺酸-6)铵盐(ABTS+)自由基清除能力。
2、比较了沙棘叶黄酮提取物的抗氧化能力,将2%沙棘叶黄酮提取物与0.01%BHA和0.01%Vc的抗氧化活性分别进行对比,沙棘叶黄酮提取物的还原能力、ABTS+自由基和超氧自由基清除率均显著高于BHA和Vc(P<0.05)；提取物的2,2-二苯基-1-间三硝基苯基联肼(DPPH)自由基和羟基自由基清除率基本和BHA的效果相当(P>0.05),但是始终高于Vc的DPPH自由基和羟基自由基清除率(P<0.05)。
3、利用优化过条件后的高效液相色谱法(HPLC)分析了沙棘叶黄酮提物中的主要活性物质。提取物中主要成分有芦丁、杨梅素、槲皮素和异鼠李素,其中芦丁、杨梅素、槲皮素和异鼠李素的总含量分别达到了30.32、20.05、29.01和23.08mg/g。
4、不同的pH值、避光保存、金属离子以及加热对沙棘叶提取物的抗氧化活性(还原能力和ABTS自由基清除活性)影响不显著(P>0.05),但是在阳光和日光灯照射条件下,其抗氧化活性显著降低(P<0.05),为沙棘叶提取物作为天然抗氧化剂在食品领域的稳定应用提供理论依据。
5、将沙棘叶黄酮提取物以0.02%、0.05%、0.10%、0.20%的添加量应用于生肉糜中,在冷藏过程中测定肉糜的色差值、pH值、羰基含量、硫代巴比妥酸值(TBARS值),安排了专业感官评价组并对肉糜的感官质量变化进行了评定。与不添加沙棘叶黄酮提取物的对阴性照组比较,提取物处理组在7天内能显著抑制猪肉蛋白和脂肪氧化过程,增加肉糜颜色的红度值,且随着添加量加大效果增加明显,与BHA效果相当。感官评价也是一样。
6、同样将0.02%、0.05%、0.10%、0.20%的沙棘叶黄酮提取物添加到熟肉糜中,在冷藏过程中测定肉糜的色差值、pH值、羰基含量、硫代巴比妥酸值(TBARS值),并对包括颜色、异味、可接受性、酸败4个感官指标做了比较评定。结果表明,添加提取物处理组对油炸后熟肉饼脂肪氧化抑制作用效果明显,在抑制熟肉糜蛋白和脂肪氧化过程效果明显,与BHA效果相当。感官评定也一样。
7、沙棘叶黄酮提取物可以对细胞起到保护作用,对试验的人胚肺成纤维细胞MRC-5,在试验中减轻了细胞遭受过氧化氢的损伤。沙棘叶黄酮提取物能够增加细胞存活几率,并且可显著提高细胞内超氧化物歧化酶(SOD酶)、过氧化氢酶(CAT酶)和谷胱甘肽过氧化物酶(GSH-Px酶)的活力；同时,对细胞内的丙二醛(MDA)有显著清除作用。
8、沙棘叶黄酮提取物对半乳糖胺(Gal-N)诱导的肝损伤大鼠具有明显的保护作用。首先,沙棘叶黄酮提取物能够显著的降低Gal-N诱导的肝损伤大鼠血清中的谷草转氨酶(AST)和谷丙转氨酶(ALT)水平(P<0.05),从生理角度考虑,其对大鼠的肝脏具有很好的保护以及毒物保护作用,同时对健康具有很大益处；其次,沙棘叶黄酮提取物可显著提高大鼠肝脏组织中的SOD酶、CAT酶和GSH-Px酶的活力(P<0.05),提高肝脏组织的总抗氧化能力(T-AOC)(P<0.05),并且降低肝脏组织中的MDA含量(P<0.05),减轻Gal-N对肝脏的损伤。
9、中剂量沙棘叶黄酮提取物灌胃可以促进小鼠肠道黏膜免疫水平,能够显著提高肠道黏膜中的分泌性免疫球蛋白A (SIgA)、白细胞介素2(IL-2)和白细胞介素4(IL-4)的含量(P<0.05),促进机体肠道T淋巴细胞连续分化,使机体免疫水平得到提高；同时,从干扰素γ (IFN-γ)的含量以及IFN-γ与IL-4的比值可以看出中剂量沙棘叶黄酮提取物灌胃能够提高体液免疫力,细胞免疫能力正常,对抵抗外界异物感染、保证机体肠道健康最有利。另外,高剂量组造成对异物识别敏感性减低,不利于试验动物机体肠道健康。另外由于IFN-γ与IL-4比值低于对照组,有潜在的免疫失调的可能,需要进行进一步深入研究。
None-natural and chemical resourse antioxidants are commercial and wide used in morden food production, to make foods less lipid oxidation. Meanwhile synthetic antioxdants are well-known for their potential health risks, for this reason, they are under stem regulations in food industry. With the development of natural-resourses plant antioxdants is regarded as a effective and good alternatives for the food oxidant deterioration. The result of the research of active component in the seabuckthorn leaves shows the flavonoid of the seabuckthorn leaves is a promising resourse to yielding natural antioxidants. The fuction of the seabuchthorn leaves flavonoid has the capability to savenge free radicals, inhibiting liver damage and improving intestinal immune ability. The objectives of this research were to prepare Seabuckthorn leaves extract with high flavonoid content and antioxidant activity. The optimal extraction condition of total flavonoids of Seabuckthorn leaves, using ethanol-hydrochloric acid solution assisted by ultrasonic was determined, as well as main component analysis using HPLC and antioxidant stability. The antioxidant effects of Seabuckthorn leaves extract in raw and cooked pork patties were evaluated. In addition,it do good to alleviate the damaged of human embryonic lung fibroblast cells MRC-5attacked by H2O2, hepatoprotective effect on Gal-N-induced liver damage in rats and the influence of the intestinal mucosal immunity in mice were also investigated. The main results were as follows:
1. The optimum conditions for extracting total flavonoids from the Seabuckthorn leaves were found to be:ethanol concentration50%, material-to-liquid ratio1:20(g/mL), hydrochloric acid concentration3mol/L, extraction time30min and ultrasonic power80W. Under these optimum conditions, the total yield of flavonoid reached as high as6.34%, and the total flavonoids extract possessed marked antioxidant activities. This study opens a new route for the extraction of total flavonoids from the Seabuckthorn leaves.
2.20mg/kg Seabuckthorn leaves extract has higher reducing power, in comparison to0.1mg/kg BHA and0.1mg/kg ascorbic acid, as same as ABTS+and superoxide radicals scavenging activity shows nice result (P<0.05), but less abilty to BHA in DPPH and hydroxyl radical scavenging activity (P<0.05).
3. The main components of Seabuckthorn leaves extract was analysed using high performance liquid chromatograph (HPLC). The four components were rutin, myricetin, quercetin and isorhamnetin. The total content of each of them was30.32,20.05,29.01and23.08mg/g, respectively.
4. The different pH value, avoid light preservation, metal ions and heating conditions on the antioxidant activities of Seabuckthorn leaves extract (reducing power and ABTS radical scavenging activity) effect was not significant (P>0.05), but in the sunlight and fluorescent light, its antioxidant activity decreased significantly (P<0.05). In addition, this research provided theoretical basis of Seabuckthorn leaves extract for application in food industry as natural antioxidants.
5. The Seabuckthorn leaves extract at a0.02%,0.05%,0.10%and0.20%addition of meat patties (weight/weight). During7days of storage, red color index, pH, carbonyl groups content and TBARS experiment was measured and organized a sensory evaluation experiment to judge the quality of raw pork patties. The results showed that the treatments of the seabuckthorn extract had significant inhibitory effects on lipid oxidation and kept the a*-value of patties in7days, in comparison to the controls, and shows the obviously dose effect ration, be equivalent to BHA, sensory evaluation has the same result.
6. Seabuckthorn leaves extract at a0.02%,0.05%,0.10%and0.20%concentration level(w/w) were added to fried meat patties, during the storage period, red color index, pH, carbonyl groups content and TBARS+value were measured and organized a sensory evaluation experiment to appraise the quality of fried meat patties.. The results shows the addition of Seabuckthorn leaves extract had obviously inhibitory effects on free radical reaction induced lipid oxidation, equivalent to BHA, sensory evaluation has the same result.
7. The Seabuckthorn leaves extract could protect the MRC-5in alleviating the degree of H2O2induced cytotoxicity. The extract treated cells had higher cell viability than the control. Additionally, the cytoprotective effect of Seabuckthorn leaves extract was in conneted with the decline in the MDA level and increases in cell enzyme of the SOD, CAT and GSH-Px activities in treated MRC-5cells.
8. The Seabuckthorn leaves extract processed significant hepatoprotective effect on Gal-N-induced liver damage in rats. First of all, Seabuckthorn leaves extract could significant reduced the ALT and AST level of serum of the rat (P<0.05), indicated that it has good hepatoprotective effect, and were benefit to health. Secondly, compared with negative control group, SOD, CAT and GSH-Px activities and T-AOC of liver of the rats were increased significantly (P<0.05) and the MDA content of liver of the rats was sharply decreased (P<0.05) treated by all dose of Seabuckthorn leaves extract. Our study showed that the Seabuckthorn leaves extract could obviously reduce the liver damage which induced by Gal-N.
9. Medium dose of Seabuckthom leaves extract could promote the intestinal mucosal immunity in mice. Secretory immunoglobulin A (SIgA), interleukin2(IL-2) and interleukin4(IL-4) levels were significant increased (P<0.05), which promoted continuous body intestinal T cell differentiation, as well as the body's immune level was improved. At the same time, from the content of interferon γ (IFN-γ) and the ratio of IFN-y and IL-4, the medium dose of Seabuckthorn leaves extract could promote intestinal mucosal immunity levels, and enhance humoral immunity and cellular immunity ability is normal, to resist outside eyewinker infection to ensure the body most good intestinal health.In addition, high dose group caused the intestinal cellular immunity was inhibited, the body is not sensitive to outside eyewinker, bad for the body intestinal health.
引文
[1]包文芳,孙一楠.沙棘属植物化学成分研究进展[J].沙棘,1999,12(2)：39.-42.
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