蒜头果有效成分及其生物活性研究
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摘要
本论文以广西特色林产资源蒜头果为原料,系统进行了蒜头果果皮果肉、果壳、种仁和枝叶多个部位提取脂肪酸、挥发油、多糖、黄酮、木质素、蛋白质及抗氧化成分,考察了有效成分的抗肿瘤、抗氧化和抑菌多种生物活性,研究结果对广西蒜头果种植和综合利用具有重要指导意义。主要研究内容和取得的成果如下：
(1)分别采用溶剂法索氏提取蒜头果脂肪酸,水蒸气蒸馏法提取蒜头果挥发油,GC-MS联用技术分析了甲脂化后脂肪酸和挥发油其化学成分组分,结果表明：种仁中脂肪酸得率为60.5%±2.5%(质量分数),鉴定出10种化合物,占总量的98.98%,主要化合物和相对含量分别为神经酸(二十四碳烯-15-酸)55.70%、油酸(十八碳烯酸)23.81%、莽酸(二十二碳烯酸)13.13%；新鲜种仁、枝叶和果皮果肉中挥发油得率分别为0.1±0.02%、0.2士0.03%和1.1±0.12%。种仁挥发油中有15种成分被鉴定,占总量的99.90%,其中苯甲醇(58.42%)相对含量较高,苯甲醛(29.66%)次之。枝叶挥发油中有8种成分被鉴定,占总量的99.87%,主要化学成分及相对含量分别为苯甲醛90.68%、苯甲醇2.95%、苯甲酸4.17%、扁桃腈0.07%。果皮果肉挥发油中有7种成分被鉴定,占总量的99.83%,主要化学成分及相对含量分别为苯甲醛87.15%、苯甲醇6.51%、扁桃腈5.66%,结果表明蒜头果各部位均含苯甲醛和苯甲醇。
(2)建立了苯酚-硫酸法测定蒜头果渣多糖的分析方法。在单因素实验基础上,采用响应面法对蒜头果渣多糖超声波提取工艺进行优化,在提取功率490W、提取时间45min、料液比1：25(g·mL-1)的最佳工艺条件下,蒜头果渣多糖的得率为5.02%。利用水提醇沉、Sevag法脱蛋白、大孔树脂-聚酰胺脱色、DEAE-52纤维素离子交换柱、透析、凝胶Sephadex G-100柱分离纯化了蒜头果粗多糖,制得两个组份多糖GP-1和GP-2,HPGPC分析得到其分子量分别为42.95、2597kDa。HPLC分析蒜头果多糖GP-1中葡萄糖、鼠李糖、树胶醛糖的摩尔比为：5.8：3.2：1,而GP-2中葡萄糖、鼠李糖、树胶醛糖、甘露糖、果糖的摩尔比为13：3.4：1.6：1：1。体外抗肿瘤试验结果显示,GP-1和GP-2对鼻咽癌(CNE-1)和肺癌(H460)细胞均有一定的抑制作用,而对人正常肝细胞(HL-7702)基本无杀伤能力,GP-2对两种癌细胞的抑制作用强于GP-1,对CNE-1最大抑制率为64-49%。
(3)采用溶剂浸提法对蒜头果叶和蒜头果叶渣总黄酮提取工艺进行研究。结果表明,在最佳工艺条件下,蒜头果叶和残渣总黄酮得率分别为16.78%、12.31%。蒜头果叶(渣)黄酮经树脂HPD400纯化后总黄酮的含量分别达到79.80%、78.62%,纯度提高了39.19%、43.97%。首次采用制备液相色谱技术从蒜头果叶中分离得到化合物Ⅰ,由紫外光谱、红外光谱、质谱和核磁共振确定其化合物Ⅰ为芦丁,高效液相色谱外标法测得该化合物的纯度达95.59%。活性试验结果表明：纯化的叶总黄酮与残渣总黄酮对DPPH·的清除率均比纯化的芦丁化合物、单宁酸对照品和未纯化的总黄酮的清除率要高。当浓度为1000μg·mL-1时,纯化叶总黄酮和渣总黄酮清除率分别为91.69%、89.78%。抗油脂氧化试验结果表明蒜头果叶纯化总黄酮对猪油具有较好的抗氧化能力。蒜头果叶粗提取和纯化总黄酮对金黄色葡萄球菌、白色念珠菌和沙门氏菌均有不同程度的抑制作用,其中对金黄色葡萄球菌抑菌圈大于12.0mm。蒜头果具有较强的抗氧化作用和抑菌能力,其活性主要源于芦丁化合物。
(4)以蒜头果壳和树枝为原料,采用一般碱法、超声波辅助和微波辅助提取木质素,分别测定了蒜头果壳和枝中木质素含量及木质素的提取率,并对所提取的木质素进行红外和紫外光谱分析。克拉森木素定量法测得蒜头果壳和枝中木质素含量别为44.41(士0.65)%和34.57(±0.52)%,在碱浓度0.5mol·L-1,碱液量30mL·g-1、超声波处理时间1h、水浴温度40℃提取工艺条件下,果壳和枝木质素提取率分别为45.21(±0.59)%、63.78(±0.73)%。紫外和红外图谱显示超声波辅助法提取的木质素保持了木质素原有结构,存在明显的愈疮木基和紫丁香基苯环结构。并通过粘度法比较了三种不同提取方法所得到的木质素相对分子质量。
(5)从蒜头果的果皮果肉中提取抗氧化剂,并采用两种体系评价其抗氧化性能。结果表明：以pH4的水为溶剂、料液比1：30(g·mL-1)、70℃水浴1h条件下提取2次,蒜头果皮果肉多酚抗氧化成分得率为3.29%。其多酚化合物对羟基自由基(·OH)和二苯基苦基肼自由基(DPPH·)均有较强的清除作用,最大清除率分别为74.00%、76.54%,其中对DPPH·清除率强弱依次为蒜头果皮果肉水提取物>抗坏血酸>单宁酸。
(6)以脱脂后的蒜头果渣为原料,测定了蒜头果渣中蛋白质含量。通过单因素实验和正交试验优化蛋白质提取的工艺条件。结果表明,蒜头果渣的蛋白质含量为31.07%,蛋白质提取的优化工艺条件为：提取温度为室温,提取溶剂为磷酸盐缓冲液(pH=7.5),提取时间为3h,料液比(W/V为1：25。在此优化条件下,蛋白质提取率为86.01%。
(7)通过对蒜头果脂肪酸、挥发油、多糖、黄酮、蛋白质、木质素、多酚抗氧化成分提取及其生物活性能力的考察,证明蒜头果中隐藏丰富的资源,开展对其种植和研究有着重要意义。
Malania oleifera Chun et S. Lee is an important and special oil tree cultivated in Guangxi, China. In this thesis, systematic research was carried out on different parts of Malania oleifera, such as seeds, shell, branches with leaves and outer part of pericarp (exocarp and mesocarp). Chemical component of fatty acids, essential oils, polysaccharides, flavonoids, lignins, protein and antioxidant were studied. The antitumor, antioxidation, antilipooxidant and antimicrobial activities of effective constituent were investigated. Research results obtained in this study will greatly enhance the comprehensive utilization rate of Malania oleifera and provide basic data for further development of Malania oleifera. The main research contents and results were showed as follows:
(1) The extraction of fatty acids from the seeds was conducted using petroleum ether (90-120℃) in a soxhlet extractor and essential oils were extracted by hydrodistillation. Total fatty acids (TFAs) of lipids were transformed into their corresponding methyl esters and analyzed by gas chromatography-mass spectrometry (GC-MS). The yield of fatty acids extracted from the seeds was60.5±2.5%, which contains fourteen chromatographic components, of which10compounds were identified, representing98.98%of the lipids, the major components were nervonic acid (55.70%), octadecenoic acid (23.81%), docosenoic acid (13.13%); The yields of essential oils were0.1±0.02%,0.2±0.03%and1.1±0.12%, respectively from fresh seeds, branches with leaves and outer part of pericarp. Seventeen chromatographic components and fifteen compounds (99.90%) were identified in the essential oils of seeds with benzyl alcohol (58.42%) and benzaldehyde (29.66%) as main constituents. Ten components and eight compounds were identified, representing99.87%of the essential oils of branches with leaves, the major componts were benzaldehyde (90.68%), benzyl alcohol (2.95%), benzoic acid (4.17%) and mandelonitrile (0.07%). Seven compounds were identified, representing99.83%of the essential oils of outer part of pericarp, the major constituents include benzaldehyde (87.15%), benzyl alcohol (6.51%) and mandelonitrile (5.66%).
(2) The analysis method of polysaccharides with pertinence to the residue of Malania oleifera was established. Based on the results of single factor experiment, response surface method (RSM) was used for optimizing ultrasound-assisted extraction of malania polysaccharides. Results showed that the optimized conditions of ultrasonic power490W, extraction time45min and ratio of solid to liquid as1:25, under such circumstances, the yield of polysaccharide was5.02%. The crude polysaccharides were purified by alcohol sedimenting technique, sevag method (deproteinization), macroporous-polyamide resin (decoloration), DEAE cellulose-52chromatography and sephadex G-100column chromatography in that order. Two main fractions, GP-1and GP-2, were obtained through the extraction and purification steps. High performance gel permeation chromatography (HPGPC) of the individual fractions showed that each fraction was represented by a narrow peak on the chromatograms. The average molecular weights of the two fractions were determined to be42.95kDa and2597kDa for fractions GP-1and GP-2, respectively. The GP-1was composed of glucose, rhamnose, and arabinose in a molar ratio of5.8:3.2:1while GP-2was composed of glucose, rhamnose, arabinose, mannose and fructose in a molar ratio of13:3.4:1.6:1:1. Furthermore, the influence of fractions GP-1and GP-2upon antitumor activities to the human nasopharyngeal carcinoma (CNE-1) cells and lung cancer (H460) cells in vitro were evaluated. The results indicated that GP-1and GP-2played an inhibited effect on the cancer cells and didn't kill human normal liver cells (HL-7702). Growth inhibition rate of GP-2against CNE-1cells was higher than that of GP-1and the maximum inhibition rate reached64.49%.
(3) The extraction of total flavonoids from Malania oleifera leaves and its residue after volatile oil extraction were studied with the methods of solvent extraction. Results showed that the yields of total flavonoids from M. oleifera leaves and its residue were16.78%and12.31%under the optimized conditions, respectively. The purity of flavones were79.80%and78.62%increased by39.19%and43.97%respectively from crude extract flavones of Malania oleifera leaves and its residue after purification by HPD400resin. Rutin has been firstly purified from its crude extracts from Malania oleifera by HPLC. The confirmation of chemical structure was performed by MP, IR, MS,'HNMR and UV spectroscopy. The purity of rutin reached95.59%by analytical HPLC. The antioxidant, antilipooxidant and antimicrobial activities of Malania oleifera flavonoids were investigated. The result showed that the scavenging activity of total flavonoid separated by HPD400on DPPH·were better than that of crude extract, rutin and tannic acid, the highest scavenging rates of purification of total flavonoids from leaves (residues) reached89.78%and91.69%in the cocentration of1000μg·mL-1, respectively. The purification of total flavonoids from malania leaves had very strong antioxidation on lard. Bacteriostasic experiments showed that purification of total flavonoids have bacteriostatic bioactivities against Monilia albican, Salmonella, Staphyloccocus aureus to some extent, the bacterial inhibition diameters of samples on Staphylococcus aureus were greater than12.0mm. Rutin palyed a significant role in antioxidation and antimicrobial activity.
(4) Lignins were extracted from Malania oleifera shell and branch by traditional alkaline method, ultrasound-assisted method and microwave-assisted method. The yields and the contents of lignins in Malania oleifera shell and branch were determined. Meanwhile, the lignins obtained were structurally analyzed by infrared and (IR) and UV spectroscopy. The contents of lignin in M. oleifera shell and branch were44.41(±0.65)%and34.57(±0.52)%respectively by Klason method, the lignins extraction rates reached45.21(±0.59)%and63.78(±0.73)%respectively from Malania oleifera shell and branch under the conditions of ultrasound-assisted extraction at40℃water bath for1hour with 0.5mol·L-1KOH water as the solvent and the solvent/material ratio of30:1(mL·L-1). UV and IR spectral analyses showed that the lignin extracted by ultrasound-assisted method remained its original structure, there were many syringyl and guaiacyl groups in Malania oleifera shell and branch. And compared the relative molecular weight of lignins extracted by three different extraction methods with viscosity method.
(5) The antioxidant components were extracted by reflux extraction from peel and flesh of Malania oleifera and its antioxidation were studied under two different situations. The results showed that the polyphenols from Malania oleifera flesh extraction rate reaches3.29%under the optimized conditions of extraction at70℃water bath for1hour twice with water (pH4) as the solvent and the material/solvent ratio of1:30(g·mL-1). The clearing capacity of polyphenols for·OH and DPPH·were very significant by scavenging test, were74.00%and76.54%respectively, in the order of clearing capacity for DPPH·were water extraction> ascorbic acid> tannic acid.
(6) The content of protein was measured, and single factor experiment and orthogonal factor experiment were carried out to optimize the technology of extraction, which was used to extract the protein from Malania oleifera residue. Results showed that the content of protein in Malania oleifera residue was31.07%, the optimum conditions were as follows:The extraction temperature is room-temperature, the extraction solvent is phosphate buffer solution, and pH=7.5, the extraction time is3hours and the ratio of material to liquid is1:25. Under this circumstance, the extraction rate reaches86.01%.
(7) It was revealed that abundant resources were hidden in Malania oleifera by the means of components analysis of fatty acids, essential oils, polysaccharides, flavonoids, proteins, lignin and antioxidant of polyphenols. Therefore, research carried on Malania oleifera has great significance.
(1)分别采用溶剂法索氏提取蒜头果脂肪酸,水蒸气蒸馏法提取蒜头果挥发油,GC-MS联用技术分析了甲脂化后脂肪酸和挥发油其化学成分组分,结果表明：种仁中脂肪酸得率为60.5%±2.5%(质量分数),鉴定出10种化合物,占总量的98.98%,主要化合物和相对含量分别为神经酸(二十四碳烯-15-酸)55.70%、油酸(十八碳烯酸)23.81%、莽酸(二十二碳烯酸)13.13%；新鲜种仁、枝叶和果皮果肉中挥发油得率分别为0.1±0.02%、0.2士0.03%和1.1±0.12%。种仁挥发油中有15种成分被鉴定,占总量的99.90%,其中苯甲醇(58.42%)相对含量较高,苯甲醛(29.66%)次之。枝叶挥发油中有8种成分被鉴定,占总量的99.87%,主要化学成分及相对含量分别为苯甲醛90.68%、苯甲醇2.95%、苯甲酸4.17%、扁桃腈0.07%。果皮果肉挥发油中有7种成分被鉴定,占总量的99.83%,主要化学成分及相对含量分别为苯甲醛87.15%、苯甲醇6.51%、扁桃腈5.66%,结果表明蒜头果各部位均含苯甲醛和苯甲醇。
(2)建立了苯酚-硫酸法测定蒜头果渣多糖的分析方法。在单因素实验基础上,采用响应面法对蒜头果渣多糖超声波提取工艺进行优化,在提取功率490W、提取时间45min、料液比1：25(g·mL-1)的最佳工艺条件下,蒜头果渣多糖的得率为5.02%。利用水提醇沉、Sevag法脱蛋白、大孔树脂-聚酰胺脱色、DEAE-52纤维素离子交换柱、透析、凝胶Sephadex G-100柱分离纯化了蒜头果粗多糖,制得两个组份多糖GP-1和GP-2,HPGPC分析得到其分子量分别为42.95、2597kDa。HPLC分析蒜头果多糖GP-1中葡萄糖、鼠李糖、树胶醛糖的摩尔比为：5.8：3.2：1,而GP-2中葡萄糖、鼠李糖、树胶醛糖、甘露糖、果糖的摩尔比为13：3.4：1.6：1：1。体外抗肿瘤试验结果显示,GP-1和GP-2对鼻咽癌(CNE-1)和肺癌(H460)细胞均有一定的抑制作用,而对人正常肝细胞(HL-7702)基本无杀伤能力,GP-2对两种癌细胞的抑制作用强于GP-1,对CNE-1最大抑制率为64-49%。
(3)采用溶剂浸提法对蒜头果叶和蒜头果叶渣总黄酮提取工艺进行研究。结果表明,在最佳工艺条件下,蒜头果叶和残渣总黄酮得率分别为16.78%、12.31%。蒜头果叶(渣)黄酮经树脂HPD400纯化后总黄酮的含量分别达到79.80%、78.62%,纯度提高了39.19%、43.97%。首次采用制备液相色谱技术从蒜头果叶中分离得到化合物Ⅰ,由紫外光谱、红外光谱、质谱和核磁共振确定其化合物Ⅰ为芦丁,高效液相色谱外标法测得该化合物的纯度达95.59%。活性试验结果表明：纯化的叶总黄酮与残渣总黄酮对DPPH·的清除率均比纯化的芦丁化合物、单宁酸对照品和未纯化的总黄酮的清除率要高。当浓度为1000μg·mL-1时,纯化叶总黄酮和渣总黄酮清除率分别为91.69%、89.78%。抗油脂氧化试验结果表明蒜头果叶纯化总黄酮对猪油具有较好的抗氧化能力。蒜头果叶粗提取和纯化总黄酮对金黄色葡萄球菌、白色念珠菌和沙门氏菌均有不同程度的抑制作用,其中对金黄色葡萄球菌抑菌圈大于12.0mm。蒜头果具有较强的抗氧化作用和抑菌能力,其活性主要源于芦丁化合物。
(4)以蒜头果壳和树枝为原料,采用一般碱法、超声波辅助和微波辅助提取木质素,分别测定了蒜头果壳和枝中木质素含量及木质素的提取率,并对所提取的木质素进行红外和紫外光谱分析。克拉森木素定量法测得蒜头果壳和枝中木质素含量别为44.41(士0.65)%和34.57(±0.52)%,在碱浓度0.5mol·L-1,碱液量30mL·g-1、超声波处理时间1h、水浴温度40℃提取工艺条件下,果壳和枝木质素提取率分别为45.21(±0.59)%、63.78(±0.73)%。紫外和红外图谱显示超声波辅助法提取的木质素保持了木质素原有结构,存在明显的愈疮木基和紫丁香基苯环结构。并通过粘度法比较了三种不同提取方法所得到的木质素相对分子质量。
(5)从蒜头果的果皮果肉中提取抗氧化剂,并采用两种体系评价其抗氧化性能。结果表明：以pH4的水为溶剂、料液比1：30(g·mL-1)、70℃水浴1h条件下提取2次,蒜头果皮果肉多酚抗氧化成分得率为3.29%。其多酚化合物对羟基自由基(·OH)和二苯基苦基肼自由基(DPPH·)均有较强的清除作用,最大清除率分别为74.00%、76.54%,其中对DPPH·清除率强弱依次为蒜头果皮果肉水提取物>抗坏血酸>单宁酸。
(6)以脱脂后的蒜头果渣为原料,测定了蒜头果渣中蛋白质含量。通过单因素实验和正交试验优化蛋白质提取的工艺条件。结果表明,蒜头果渣的蛋白质含量为31.07%,蛋白质提取的优化工艺条件为：提取温度为室温,提取溶剂为磷酸盐缓冲液(pH=7.5),提取时间为3h,料液比(W/V为1：25。在此优化条件下,蛋白质提取率为86.01%。
(7)通过对蒜头果脂肪酸、挥发油、多糖、黄酮、蛋白质、木质素、多酚抗氧化成分提取及其生物活性能力的考察,证明蒜头果中隐藏丰富的资源,开展对其种植和研究有着重要意义。
Malania oleifera Chun et S. Lee is an important and special oil tree cultivated in Guangxi, China. In this thesis, systematic research was carried out on different parts of Malania oleifera, such as seeds, shell, branches with leaves and outer part of pericarp (exocarp and mesocarp). Chemical component of fatty acids, essential oils, polysaccharides, flavonoids, lignins, protein and antioxidant were studied. The antitumor, antioxidation, antilipooxidant and antimicrobial activities of effective constituent were investigated. Research results obtained in this study will greatly enhance the comprehensive utilization rate of Malania oleifera and provide basic data for further development of Malania oleifera. The main research contents and results were showed as follows:
(1) The extraction of fatty acids from the seeds was conducted using petroleum ether (90-120℃) in a soxhlet extractor and essential oils were extracted by hydrodistillation. Total fatty acids (TFAs) of lipids were transformed into their corresponding methyl esters and analyzed by gas chromatography-mass spectrometry (GC-MS). The yield of fatty acids extracted from the seeds was60.5±2.5%, which contains fourteen chromatographic components, of which10compounds were identified, representing98.98%of the lipids, the major components were nervonic acid (55.70%), octadecenoic acid (23.81%), docosenoic acid (13.13%); The yields of essential oils were0.1±0.02%,0.2±0.03%and1.1±0.12%, respectively from fresh seeds, branches with leaves and outer part of pericarp. Seventeen chromatographic components and fifteen compounds (99.90%) were identified in the essential oils of seeds with benzyl alcohol (58.42%) and benzaldehyde (29.66%) as main constituents. Ten components and eight compounds were identified, representing99.87%of the essential oils of branches with leaves, the major componts were benzaldehyde (90.68%), benzyl alcohol (2.95%), benzoic acid (4.17%) and mandelonitrile (0.07%). Seven compounds were identified, representing99.83%of the essential oils of outer part of pericarp, the major constituents include benzaldehyde (87.15%), benzyl alcohol (6.51%) and mandelonitrile (5.66%).
(2) The analysis method of polysaccharides with pertinence to the residue of Malania oleifera was established. Based on the results of single factor experiment, response surface method (RSM) was used for optimizing ultrasound-assisted extraction of malania polysaccharides. Results showed that the optimized conditions of ultrasonic power490W, extraction time45min and ratio of solid to liquid as1:25, under such circumstances, the yield of polysaccharide was5.02%. The crude polysaccharides were purified by alcohol sedimenting technique, sevag method (deproteinization), macroporous-polyamide resin (decoloration), DEAE cellulose-52chromatography and sephadex G-100column chromatography in that order. Two main fractions, GP-1and GP-2, were obtained through the extraction and purification steps. High performance gel permeation chromatography (HPGPC) of the individual fractions showed that each fraction was represented by a narrow peak on the chromatograms. The average molecular weights of the two fractions were determined to be42.95kDa and2597kDa for fractions GP-1and GP-2, respectively. The GP-1was composed of glucose, rhamnose, and arabinose in a molar ratio of5.8:3.2:1while GP-2was composed of glucose, rhamnose, arabinose, mannose and fructose in a molar ratio of13:3.4:1.6:1:1. Furthermore, the influence of fractions GP-1and GP-2upon antitumor activities to the human nasopharyngeal carcinoma (CNE-1) cells and lung cancer (H460) cells in vitro were evaluated. The results indicated that GP-1and GP-2played an inhibited effect on the cancer cells and didn't kill human normal liver cells (HL-7702). Growth inhibition rate of GP-2against CNE-1cells was higher than that of GP-1and the maximum inhibition rate reached64.49%.
(3) The extraction of total flavonoids from Malania oleifera leaves and its residue after volatile oil extraction were studied with the methods of solvent extraction. Results showed that the yields of total flavonoids from M. oleifera leaves and its residue were16.78%and12.31%under the optimized conditions, respectively. The purity of flavones were79.80%and78.62%increased by39.19%and43.97%respectively from crude extract flavones of Malania oleifera leaves and its residue after purification by HPD400resin. Rutin has been firstly purified from its crude extracts from Malania oleifera by HPLC. The confirmation of chemical structure was performed by MP, IR, MS,'HNMR and UV spectroscopy. The purity of rutin reached95.59%by analytical HPLC. The antioxidant, antilipooxidant and antimicrobial activities of Malania oleifera flavonoids were investigated. The result showed that the scavenging activity of total flavonoid separated by HPD400on DPPH·were better than that of crude extract, rutin and tannic acid, the highest scavenging rates of purification of total flavonoids from leaves (residues) reached89.78%and91.69%in the cocentration of1000μg·mL-1, respectively. The purification of total flavonoids from malania leaves had very strong antioxidation on lard. Bacteriostasic experiments showed that purification of total flavonoids have bacteriostatic bioactivities against Monilia albican, Salmonella, Staphyloccocus aureus to some extent, the bacterial inhibition diameters of samples on Staphylococcus aureus were greater than12.0mm. Rutin palyed a significant role in antioxidation and antimicrobial activity.
(4) Lignins were extracted from Malania oleifera shell and branch by traditional alkaline method, ultrasound-assisted method and microwave-assisted method. The yields and the contents of lignins in Malania oleifera shell and branch were determined. Meanwhile, the lignins obtained were structurally analyzed by infrared and (IR) and UV spectroscopy. The contents of lignin in M. oleifera shell and branch were44.41(±0.65)%and34.57(±0.52)%respectively by Klason method, the lignins extraction rates reached45.21(±0.59)%and63.78(±0.73)%respectively from Malania oleifera shell and branch under the conditions of ultrasound-assisted extraction at40℃water bath for1hour with 0.5mol·L-1KOH water as the solvent and the solvent/material ratio of30:1(mL·L-1). UV and IR spectral analyses showed that the lignin extracted by ultrasound-assisted method remained its original structure, there were many syringyl and guaiacyl groups in Malania oleifera shell and branch. And compared the relative molecular weight of lignins extracted by three different extraction methods with viscosity method.
(5) The antioxidant components were extracted by reflux extraction from peel and flesh of Malania oleifera and its antioxidation were studied under two different situations. The results showed that the polyphenols from Malania oleifera flesh extraction rate reaches3.29%under the optimized conditions of extraction at70℃water bath for1hour twice with water (pH4) as the solvent and the material/solvent ratio of1:30(g·mL-1). The clearing capacity of polyphenols for·OH and DPPH·were very significant by scavenging test, were74.00%and76.54%respectively, in the order of clearing capacity for DPPH·were water extraction> ascorbic acid> tannic acid.
(6) The content of protein was measured, and single factor experiment and orthogonal factor experiment were carried out to optimize the technology of extraction, which was used to extract the protein from Malania oleifera residue. Results showed that the content of protein in Malania oleifera residue was31.07%, the optimum conditions were as follows:The extraction temperature is room-temperature, the extraction solvent is phosphate buffer solution, and pH=7.5, the extraction time is3hours and the ratio of material to liquid is1:25. Under this circumstance, the extraction rate reaches86.01%.
(7) It was revealed that abundant resources were hidden in Malania oleifera by the means of components analysis of fatty acids, essential oils, polysaccharides, flavonoids, proteins, lignin and antioxidant of polyphenols. Therefore, research carried on Malania oleifera has great significance.
引文
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