食用菌抗氧化活性研究及竹荪抗氧化物质提取工艺优化
摘要
许多食用菌都含有多种活性物质,具有提高人体免疫力、抗肿癌、抗氧化等功效。食用菌在我国栽培历史悠久,资源丰富,是新药物的宝贵资源库。本文主要对食用菌抗氧化活性、竹荪抗氧化物质提取工艺优化及抗氧化特性进行研究,研究结果如下:
(1)通过总还原能力,羟自由基清除能力和超氧阴离子自由基抑制能力三种测定方法对45种食用菌液体发酵产物体外抗氧化活性进行测定,并进行抗氧化活性模糊综合评价。结果表明:45种食用菌发酵产物中,多数发酵液、菌丝水提液和菌丝醇提液具有抗氧化活性,模糊综合评价法分析表明发酵液抗氧化活性最强的是桦褐孔菌,菌丝醇提液抗氧化活性最强的是赤芝,菌丝水提液抗氧化活性最强的是榆耳。
(2)通过总还原能力,羟自由基清除能力,超氧阴离子自由基抑制能力和DPPH·自由基的清除能力四种测定方法对26种食用菌子实体干品水提液和醇提液体外抗氧化活性进行测定,并进行抗氧化活性模糊综合评价。结果表明:26种食用菌子实体干品水、醇提取液均具有抗氧化活性,模糊综合评价法分析表明抗氧化活性最强的水、醇提取液分别为竹荪的水提液和醇提液。说明竹荪抗氧化活性强,具有较高的研究和应用价值。
(3)采用L_9(3~4)正交设计法优化竹荪抗氧化物质提取条件。正交因素为乙醇浓度(A)、回流温度(B)、料液比(C)和提取时间(D) 4个因素,以DPPH·清除率为评价指标。结果表明,影响抗氧化物质提取效果的主要因素为回流温度,最佳的提取工艺为:乙醇浓度90%,回流温度90℃,料液比1︰35,回流时间2.5 h,提取次数1次。
(4)利用D-半乳糖制造衰老模型小鼠,研究竹荪提取物在衰老模型小鼠体内的抗氧化作用。结果表明:竹荪提取物低剂量(50 mg/kg·d),中剂量(100 mg/kg·d)和高剂量(200 mg/kg·d)对衰老模型小鼠的体重增加量不显著(P>0.05),说明竹荪提取物在实验剂量下没有明显的毒、副作用;中剂量和高剂量下,小鼠肝重、脾重与空白组差异均不显著(P>0.05),与模型组差异显著(P<0.05),说明竹荪提取物能修复和保护小鼠受损肝脏、脾脏;竹荪提取物可明显提高衰老模型小鼠血清中SOD酶活力,降低衰老模型小鼠血清中MDA含量,并且竹荪提取物与小鼠血清中SOD酶活力和MDA含量呈明显的剂量效应关系。
(5)对竹荪抗氧化物质的成分特性进行研究。结果表明:竹荪抗氧化物质提取得率为15.33%;1.00 g竹荪对DPPH·清除率相当于397.18μg V_C或相当于557.47 mg V_E对DPPH·清除率;依次用石油醚、乙酸乙酯、正丁醇萃取粗提物水溶液得到四个分离相物质,利用分光光度法对成分进行初步鉴定。结果表明粗提物中含有大量多糖类物质,少量多酚类物质和微量黄酮类物质;水相主要含多糖类物质物质,固形物占总提取物的83.77%;石油醚相和正丁醇相含有微量多酚类物质,固形物含量极少;乙酸乙酯相中含有较多多酚类物质,不含有多糖类物质和黄酮类物质,固形物只占总提取物的10.72%,但其抗氧化能力却与含有水相仅差7.06%,说明乙酸乙酯相中物质抗氧化能力明显强于水相中抗氧化物质。乙酸乙酯相近红外光谱分析表明含有酚类,酯类和缔合态羧酸类物质。薄层层析分析表明至少含有7种物质。
It has been investigated that most mushrooms contain several kinds of active components which can improve human immunity, and have antitumor and antioxidant activity and so on. As there are so many species of mushrooms in China,it is a valuable resource pool of new medicine and health food.this paper mainly studies antioxidant activities species of mushrooms and extraction process optimization for Dictyophora indusiata, and the research results are as follows:
(1) Antioxidant activities of liquid fermented products of 45 species of mushrooms were compared by using the reducing power measurement, the salicylic acid method and the self-oxidation method of pyrogallol.The results showed that most liquid fermenting products of 45 species of mushrooms had antioxidant properties in vitro. In addition, to evaluate the overall antioxidant capacities of them, a fuzzy appraisal mathematical model was employed. It had been found that the fermented filtrate of Inonotus obliquus, the methanol extract of Ganoderma lucidum’hypha and the water extract of Gloeostereum incarnatum’hypha had the highest antioxidant activity among all tested mushrooms respectively.
(2) Antioxidant activities of extracts from the dried fruiting bodies of 26 species of mushrooms were firstly compared by using the reducing power measurement, the salicylic acid method, the self-oxidation method of pyrogallol and the scavenging ability on DPPH·(1,1-Diphenyl-2-picrylhydrazyl). The results showed that the extracts of all 26 species had antioxidant properties in vitro. To evaluate the overall antioxidant capacities of them, a fuzzy appraisal mathematical model was employed. The result showed that the water and ethanol extracts of D. indusiata had the highest antioxidant activity among all tested mushrooms.
(3) Thus, D. indusiata was further studied for a potential antioxidant source. The extract conditions including ethanol concentration, extract temperature, material and solvent ratio and extract time were investigated by L_9(3~4)orthogonal design with the scavenging quality of DPPH·as the antioxidation activity index. The result showed that extract temperature was the major factor affecting the extract rate, and that the optimal extract process was to use 90% ethanol with a material/solvent ratio of 1/35 at 90℃for 2.5 h.
(4) The antioxidant activity in vivo of D. indusiata extract had been investigated,the result showed that none of the low dose(50 mg/kg·d),middle dose(100 mg/kg·d),high dose(200 mg/kg·d) had significant influence on body weight(P>0.05), which indicated that the D. indusiata extract haven't poisonous or side effect. In the respect of liver and spleen weight, middle dose and high dose reached the level of control, which indicated that the D. indusiata extract could protect impaired apparatus; The extract of D. indusiata could significantly increase SOD vitality and reduce MDA content of mice which injected in D-galactose,and the dose and antioxidant activity had dose-response relationship.
(5) Antioxidant characteristics of D.indusiata extract had been studied.The results showed that the yield of D. indusiata extract was 15.33%, and the antioxidant activity of 1.00 g D. indusiata extract equaled to that of 397.18μg VC or 557.47 mg VE. The crude extraction contained plenty of polysaccharide, a little polyphenol, and traces of flavone. Petroleumether, ethylacetate, isobutanol were utilized successively to separation the crude extract. It could be found that the petroleumether, ethylacetate, isobutanol extractions all mainly contained polyphenol, and the water extraction contained plenty of polysaccharide. Moreover, antioxidative substance in ethylacetate had been further investigated for its lower content (10.72%) but higher antioxidant activity, which was just 7.06% lower than the extraction in water. According to the infrared spectroscopy absorption curve of material dissolved in ethylacetate, it could been derived that there are phenolic, esters and associate carboxylic acid in extraction of ethylacetate.Based on the thin-layer chromatogram, it could been derived that there are at least seven kinds of substances in extraction of ethylacetate.
(1)通过总还原能力,羟自由基清除能力和超氧阴离子自由基抑制能力三种测定方法对45种食用菌液体发酵产物体外抗氧化活性进行测定,并进行抗氧化活性模糊综合评价。结果表明:45种食用菌发酵产物中,多数发酵液、菌丝水提液和菌丝醇提液具有抗氧化活性,模糊综合评价法分析表明发酵液抗氧化活性最强的是桦褐孔菌,菌丝醇提液抗氧化活性最强的是赤芝,菌丝水提液抗氧化活性最强的是榆耳。
(2)通过总还原能力,羟自由基清除能力,超氧阴离子自由基抑制能力和DPPH·自由基的清除能力四种测定方法对26种食用菌子实体干品水提液和醇提液体外抗氧化活性进行测定,并进行抗氧化活性模糊综合评价。结果表明:26种食用菌子实体干品水、醇提取液均具有抗氧化活性,模糊综合评价法分析表明抗氧化活性最强的水、醇提取液分别为竹荪的水提液和醇提液。说明竹荪抗氧化活性强,具有较高的研究和应用价值。
(3)采用L_9(3~4)正交设计法优化竹荪抗氧化物质提取条件。正交因素为乙醇浓度(A)、回流温度(B)、料液比(C)和提取时间(D) 4个因素,以DPPH·清除率为评价指标。结果表明,影响抗氧化物质提取效果的主要因素为回流温度,最佳的提取工艺为:乙醇浓度90%,回流温度90℃,料液比1︰35,回流时间2.5 h,提取次数1次。
(4)利用D-半乳糖制造衰老模型小鼠,研究竹荪提取物在衰老模型小鼠体内的抗氧化作用。结果表明:竹荪提取物低剂量(50 mg/kg·d),中剂量(100 mg/kg·d)和高剂量(200 mg/kg·d)对衰老模型小鼠的体重增加量不显著(P>0.05),说明竹荪提取物在实验剂量下没有明显的毒、副作用;中剂量和高剂量下,小鼠肝重、脾重与空白组差异均不显著(P>0.05),与模型组差异显著(P<0.05),说明竹荪提取物能修复和保护小鼠受损肝脏、脾脏;竹荪提取物可明显提高衰老模型小鼠血清中SOD酶活力,降低衰老模型小鼠血清中MDA含量,并且竹荪提取物与小鼠血清中SOD酶活力和MDA含量呈明显的剂量效应关系。
(5)对竹荪抗氧化物质的成分特性进行研究。结果表明:竹荪抗氧化物质提取得率为15.33%;1.00 g竹荪对DPPH·清除率相当于397.18μg V_C或相当于557.47 mg V_E对DPPH·清除率;依次用石油醚、乙酸乙酯、正丁醇萃取粗提物水溶液得到四个分离相物质,利用分光光度法对成分进行初步鉴定。结果表明粗提物中含有大量多糖类物质,少量多酚类物质和微量黄酮类物质;水相主要含多糖类物质物质,固形物占总提取物的83.77%;石油醚相和正丁醇相含有微量多酚类物质,固形物含量极少;乙酸乙酯相中含有较多多酚类物质,不含有多糖类物质和黄酮类物质,固形物只占总提取物的10.72%,但其抗氧化能力却与含有水相仅差7.06%,说明乙酸乙酯相中物质抗氧化能力明显强于水相中抗氧化物质。乙酸乙酯相近红外光谱分析表明含有酚类,酯类和缔合态羧酸类物质。薄层层析分析表明至少含有7种物质。
It has been investigated that most mushrooms contain several kinds of active components which can improve human immunity, and have antitumor and antioxidant activity and so on. As there are so many species of mushrooms in China,it is a valuable resource pool of new medicine and health food.this paper mainly studies antioxidant activities species of mushrooms and extraction process optimization for Dictyophora indusiata, and the research results are as follows:
(1) Antioxidant activities of liquid fermented products of 45 species of mushrooms were compared by using the reducing power measurement, the salicylic acid method and the self-oxidation method of pyrogallol.The results showed that most liquid fermenting products of 45 species of mushrooms had antioxidant properties in vitro. In addition, to evaluate the overall antioxidant capacities of them, a fuzzy appraisal mathematical model was employed. It had been found that the fermented filtrate of Inonotus obliquus, the methanol extract of Ganoderma lucidum’hypha and the water extract of Gloeostereum incarnatum’hypha had the highest antioxidant activity among all tested mushrooms respectively.
(2) Antioxidant activities of extracts from the dried fruiting bodies of 26 species of mushrooms were firstly compared by using the reducing power measurement, the salicylic acid method, the self-oxidation method of pyrogallol and the scavenging ability on DPPH·(1,1-Diphenyl-2-picrylhydrazyl). The results showed that the extracts of all 26 species had antioxidant properties in vitro. To evaluate the overall antioxidant capacities of them, a fuzzy appraisal mathematical model was employed. The result showed that the water and ethanol extracts of D. indusiata had the highest antioxidant activity among all tested mushrooms.
(3) Thus, D. indusiata was further studied for a potential antioxidant source. The extract conditions including ethanol concentration, extract temperature, material and solvent ratio and extract time were investigated by L_9(3~4)orthogonal design with the scavenging quality of DPPH·as the antioxidation activity index. The result showed that extract temperature was the major factor affecting the extract rate, and that the optimal extract process was to use 90% ethanol with a material/solvent ratio of 1/35 at 90℃for 2.5 h.
(4) The antioxidant activity in vivo of D. indusiata extract had been investigated,the result showed that none of the low dose(50 mg/kg·d),middle dose(100 mg/kg·d),high dose(200 mg/kg·d) had significant influence on body weight(P>0.05), which indicated that the D. indusiata extract haven't poisonous or side effect. In the respect of liver and spleen weight, middle dose and high dose reached the level of control, which indicated that the D. indusiata extract could protect impaired apparatus; The extract of D. indusiata could significantly increase SOD vitality and reduce MDA content of mice which injected in D-galactose,and the dose and antioxidant activity had dose-response relationship.
(5) Antioxidant characteristics of D.indusiata extract had been studied.The results showed that the yield of D. indusiata extract was 15.33%, and the antioxidant activity of 1.00 g D. indusiata extract equaled to that of 397.18μg VC or 557.47 mg VE. The crude extraction contained plenty of polysaccharide, a little polyphenol, and traces of flavone. Petroleumether, ethylacetate, isobutanol were utilized successively to separation the crude extract. It could be found that the petroleumether, ethylacetate, isobutanol extractions all mainly contained polyphenol, and the water extraction contained plenty of polysaccharide. Moreover, antioxidative substance in ethylacetate had been further investigated for its lower content (10.72%) but higher antioxidant activity, which was just 7.06% lower than the extraction in water. According to the infrared spectroscopy absorption curve of material dissolved in ethylacetate, it could been derived that there are phenolic, esters and associate carboxylic acid in extraction of ethylacetate.Based on the thin-layer chromatogram, it could been derived that there are at least seven kinds of substances in extraction of ethylacetate.
引文
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[1]李红,张元湖.应用DPPH法测定苹果提取物的抗氧化能力[J].山东农业大学学报(自然科学版),2005,36(1):35-38.
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[1]赵艳红,李建科,李国秀.天然抗氧化物体外活性评价方法的优选与优化[J].食品科学,2008,29(6):64-69.
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[1]李红,张元湖.应用DPPH法测定苹果提取物的抗氧化能力[J].山东农业大学学报(自然科学版),2005,36(1):35-38.
[2] Aruoma,O. I.Free radicals,oxidative stress,and antioxidants in human health and disease[J].Journal of the American Oil Chemists’Society,1998,75:199-212.
[3] Parejoa I,Viladomata F,Bastida J, et al.Investigation of Bolivian plant extracts for their radical scavenging activity and antioxidant activity[J].Life Science,2003,73:1667-1681.
[4] Lee Y.-L.Taste quality,antioxidant and antimutagenic properties of Hypsizigus marmoreus fruit bodies[D].Master’s Thesis,Taiwan,Taichung:National Chung-Hsing University,2003.
[5] Mizuno.T,Sakai.T,Chihara. G.Health foods and medicinal usages of mushrooms[J].Food Review International,1995,11,69-81.
[6] Banerjee A.,Cumin N,Dasgupta,et al. In vitro study of antioxidant activity of Syzygium fruit [J].Food Chemistry,2005,90:727-733.
[7]梁华,陆芽春,李雪林.几种食用菌清除氧自由基的研究[J].食品科技,2005(7):94-97.
[8]陈瑗,周玫.药用化学[M].北京:人民卫生出版社,2002:8-20.
[9] Wei Yi,Xiaoqin Wu,Rihui Cao,et al.Biological evaluations of novel vitamin C esters as mushroom tyrosinase inhibitors and antioxidants[J].Food Chemistry,2009 (117):381–386.
[1]卯晓岚.中国大型真菌[M].郑州:河南科学技术出版社,2000.
[2]黄年来.中国大型真菌原色图鉴[M].北京:中国农业出版社,1998.
[3]朱利泉,邓艳霞.竹荪的研究与利用[J].中国野生植物资源,19(3):21-23.
[4]郭渝南,刘晓玲,范娟.竹荪的营养与药用功效[J].食用菌,2004(4):44-45.
[5]吕作舟.食用菌栽培学[M].北京:高等教育出版社,2006.
[6]屠六帮.竹荪菌柄、菌托、菌盖的营养价值及作用[J].江苏食用菌,1990,2:13.
[7] Ukai S,Kiho T,HaraC,et al.Antitumor activity of various polysaccharids isolated from Dictyophora indusiata,Ganoderma japonicum,Cordyceps cicadae,Auricularia auricular-judae,and Auricularia species[J].Chen Pharm Bull,1983,31(2):741-744.
[8]谭敬军.竹荪抑菌特性研究[J].食品科学,2001,22(9):73-75.
[9]王钦德,杨坚.食品试验设计与统计分析[M].北京:中国农业大学出版社,2003.
[10]吕峰.我国薏苡仁资源主要品质及薏苡仁活性多糖的研究[D].福州:福建农林大学博士学位论文,2008.
[11] E L柯斯乐.扩散—流体系统中的传质[M].王宇新,姜忠义,译.北京:化学工业出版社,2002:1-30.
[12]王唯勇,韩鲁佳,王振,等.植物功能成分浸提过程动力学研究进展[J].中国农业大学学报,2006,11(1):100-104.
[1]刘昌孝.药物评价实验设计与统计学基础[M].北京:军事医学科学出版社,1999.
[2]凌关庭.抗氧化食品与健康[M].北京:化学工业出版社,2004.
[3] Pablo M.Nitric oxide protection of rat liver from lipid peroxidation, collagen accumulation,and liver damage induced by carbon tetrachloride [J].Biochemical Pharmacol,1998,56(6):773-779.
[4] M. Lazar,Rychly,V. Klim,et al.Free Radicals in Chemistry and Biology, Boca Raton[M].Florida:CRC Press Inc,1989:5-37.
[5]丁秀红,高荫榆,晁红娟,等.毛竹叶多糖体内抗氧化作用研究[J].食品科学,2008,29(5):427-430.
[6]李磊.几种天然抗氧化物质的抗氧化活性比较及茶多酚的抗氧化保健功效研究[D].硕士学位论文,杭州:浙江大学,2008.
[7]曾红,周忠波,张利莉,等.裂盖马鞍菌提取物体内抗氧化活性研究[J].天然产物研究与开发,2009,21:667-671.
[8]浅田浩二.生物体内的活性氧清除系统—抗氧化酶[J].日本医学介绍,林洁译,1994,15(7).
[9]卢新华,谷彬,刘思妤,等.苦菜体内外抗氧化作用的实验研究[J].右江医学,2007,35(2):120-121.
[1]吴谋成.食品分析与感官评定[M].北京:中国农业出版社,2002.
[2]杨普香,黎小平.桑叶茶黄酮化合物的测定方法研究[J].食品科学,2001,22(10):81.
[3]吕峰.我国薏苡仁资源主要品质及薏苡仁活性多糖的研究[D].博士学位论文,福州:福建农林大学,2008.
[4]李发美,胡育筑.分析化学[M].北京:中国医药科技出版社,2006.
[5]宦双燕.波谱分析[M].北京:中国纺织出版社,2008.
[6]檀东飞,梁明,吴若菁,等.棘托竹荪乙酸乙酯提取物的化学成分研究[J].天然产物研究与开发,2003,15(1):34-37.