CN110249921B - 提高大吴风草中类黄酮和酚类物质含量的培育方法 - Google Patents
提高大吴风草中类黄酮和酚类物质含量的培育方法 Download PDFInfo
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- CN110249921B CN110249921B CN201910569167.2A CN201910569167A CN110249921B CN 110249921 B CN110249921 B CN 110249921B CN 201910569167 A CN201910569167 A CN 201910569167A CN 110249921 B CN110249921 B CN 110249921B
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Abstract
本发明提供提高大吴风草中类黄酮和酚类物质含量的培育方法,属于生物技术领域,包括向大吴风草植株或植株部分经叶面或根部施用促生长组合物,以及,利用紫外线阶段性照射大吴风草植株;上述促生长组合物中包括一种或多种类黄酮化合物和羧酸基团修饰的壳聚糖盐。本发明提供的培育方法能干扰气孔细胞闭合以使得CO2进入细胞不受阻,增加根部环境湿润度以增强植株抗干旱能力,增加光合产物多糖生成量,为类黄酮物质等次生代谢物提供糖苷,生物相容度高,抑制或清除活性氧自由基以保护植株免于氧化损伤,增强苯丙氨酸解氨酶和查尔酮合成酶活性,提高初代代谢向次生代谢的转变效率,增强植株抗氧化、抗辐射及抗非生物逆境胁迫的能力。
Description
技术领域
本发明属于生物技术领域,具体涉及提高大吴风草中类黄酮和酚类物质含量的培育方法。
背景技术
大吴风草(Farfujium japonicum Lindl.),又名莲蓬草,属菊科管状花亚科千里光族款冬亚族大吴风草属植物,多年生草本,分布区域从中国东南部经琉球群岛和台湾地区一直到日本,全株入药。大吴风草作为药食两用的资源植物具有较高的开发价值,其根状茎、叶片、叶柄均可入药或作为蔬菜食用,“辛、温、无毒,性凉、味苦”,并具有退热、解毒、活血之功效,民间广泛用于治疗湿疹、咳嗽、支气管炎、淋巴腺炎、痢疾等疾病;也是重要的园林绿化植物,宜植于大树下、高大建筑物背阴处、林地,也可布置荫蔽处的花坛、花境,常用做路旁的花境做背景或点缀,十分美丽。其生境范围相当广泛,常生于低海拔的林下、山地、山谷、海滨草丛;其野生环境多为林下,郁闭度在80%以上,能成片生长,而且长势较好,土壤基质多为土质疏松、排水良好、富含有机质的土壤。随着野生资源不断被挖掘利用,人们开始人工栽培大吴风草,由于其适应性较强,即使在向阳处、土质瘠薄甚至盐碱地均能生长,所以繁殖比较容易。
大吴风草的化学成分主要为挥发油、萜类(倍半萜、二萜、三萜)、酚类、生物碱类、甾体类以及脂肪酸类等;类黄酮和酚类化合物是植物在长期的生态适应过程中为抵御恶劣生态条件、动物、微生物等攻击而形成的一大类次生代谢产物,调节植物生长,保护植物免受紫外线,抵御致病菌侵袭,影响植物的生殖力和花粉的萌发;类黄酮及黄酮类物质还具有抗癌、抗病毒、抗心脑血管疾病、抗糖尿病并发症、治疗骨质疏松等多种药用作用,还具有很强的抗氧化作用,能清除人体中有害的超氧自由基团,具有抗衰老、增强机体免疫力等生理活性。虽然类黄酮类和酚类化合物具有重要的药理和生理活性,但是在大吴风草体内含量低,不易提取,所以提高大吴风草类黄酮和酚类物质的含量,从而提高大吴风草的开发价值成为研究重点。
发明内容
本发明的目的在于提供一种能干扰气孔细胞闭合以使得CO2进入细胞不受阻,增加根部环境湿润度以增强植株抗干旱能力,增加光合产物多糖生成量,生物相容度高,抑制或清除活性氧自由基,增强苯丙氨酸解氨酶和查尔酮合成酶活性,提高次生代谢转变效率的提高大吴风草中类黄酮和酚类物质含量的培育方法。
本发明为实现上述目的所采取的技术方案为:
提高大吴风草中类黄酮和酚类物质含量的培育方法,包括,向大吴风草植株或植株部分经叶面或根部施用促生长组合物,以及,利用紫外线阶段性照射大吴风草植株;上述促生长组合物中包括一种或多种类黄酮化合物和羧酸基团修饰的壳聚糖盐。该方法能利用促生长组合物和紫外线照射改变大吴风草体内生理代谢水平,促进其光合作用,诱导并促进类黄酮和酚类物质的合成和积累,提高其药用和经济价值,且大大提高了植物对活性氧自由基的清除或抑制能力以保护细胞免于氧化损伤,以及对非生物逆境胁迫的抗性,提高植物产量和品质。
对本发明而言,紫外线阶段性照射操作条件为:紫外灯功率30-45W,紫外线波长为270-320nm,照射周期为30-40d,单次照射时间为15-20min,照射间隔时间为10-12h。更优选地,紫外线阶段性照射于大吴风草花期前30-40d开始进行。少量或适量的紫外照射植物能诱导植物中次生代谢产物的大量生成,进而保护细胞或植株免受辐射伤害,因而使得类黄酮及酚类物质合成途径中相关的基因表达量上升,相关的酶在紫外诱导下被激活或活性上调,从而增加类黄酮及酚类物质的含量,增强植株抗氧化、抗逆境胁迫的能力。
对本发明而言,促生长组合物还包括厚朴酚类似物和异山梨醇二甲醚;厚朴酚类似物选自丙基厚朴酚、异丙基厚朴酚、丁基厚朴酚和异丁基厚朴酚中的至少一种。厚朴酚类似物和异山梨醇二甲醚作为组合物施用于植株,于叶面渗透后,两者能破坏气孔细胞质膜上糖蛋白所含寡糖肽与多肽链之间的共价结构,使气孔细胞间胞间连丝松弛,从而干扰气孔细胞闭合,使得CO2进入细胞不受阻,于根部渗透后进入植株内,两者能利用亲水性能增加根部所处环境的湿润度,参与并促进生物质运输,增强植株抗干旱能力,从不同方面以促进植株光合作用,进而增加光合产物糖的生成量,为类黄酮物质等次生代谢物提供糖苷,达到提高次生代谢产物积累量的目的。
对本发明而言,促生长组合物中的一种或多种类黄酮化合物为大豆黄酮、黄酮醇、二氢黄酮醇、黄烷酮或它们的混合物。进一步优选地,类黄酮化合物之间形成混合物的重量比例为任意比。更优选地,类黄酮化合物中含有黄酮醇化合物槲皮素,且槲皮素重量占比为40-50%。类黄酮化合物施用前期可以吸收UV以减少器官受辐射伤害,但被植株吸收后,能作为植物信号分子,直接或间接的诱导或激活植株中类黄酮合成途径的生物化学通路,并影响上下游各种次生代谢产物的合成和产量。
对本发明而言,黄酮醇包括但不限于槲皮素、山奈酚、山柰苷、黄芪甙、杨梅黄素、异鼠李素、霍香黄酮醇;黄烷酮包括但不限于橙皮素、橙皮苷、柚皮素;二氢黄烷醇包括但不限于二氢槲皮素、二氢山奈酚等。
对本发明而言,羧酸基团修饰的壳聚糖盐中的羧酸基团由氯乙酸或丙烯酸提供,且羧酸基团的取代度为80-95%;羧酸基团修饰的壳聚糖盐选自镁盐、锌盐或它们的混合物。更优选地,羧酸基团修饰的壳聚糖盐为镁盐和锌盐以1:0.5-5 的重量比形成的混合物。在碱性条件下壳聚糖6位上的羟基优先参与反应,生成的壳聚糖衍生物具有很好的水溶性,增加了其被植株吸收和利用的几率,且生成的壳聚糖与金属离子配合物对金属离子具有缓释性,能有利于提高叶绿素活性,提升光合作用效率,或能抑制或清除活性氧自由基,阻止低密度蛋白氧化,避免如盐胁迫或光胁迫等逆境的不良影响。
对本发明而言,羧基基团修饰的壳聚糖盐通过以下步骤获得:将可溶性镁盐或锌盐的水合物溶解后,于70-80℃下震荡搅拌,再配制含有羧酸基团修饰的壳聚糖的反应溶剂并以重量比1:1-2的比例加入盐溶液中,于70-80℃保温反应2-3h 后,加入2-3倍体积的丙酮进行沉淀,减压浓缩,用体积浓度为70%的乙醇溶液洗涤,干燥,即得。壳聚糖接枝羧基基团后,更容易与金属离子形成配合物,分子中的氨基、仲羟基、乙酰氨基等都能与金属离子发生配位反应,且能通过催化活性氧的歧化反应,达到减少活性氧自由基的目的,抑制或避免了氧化损伤。
更优选地,反应溶剂中含有重量浓度分别为2-5wt%的羧酸基团修饰的壳聚糖、0.1-0.5wt%的3,5-二羟基苯甲酸和0.05-0.25wt%的4-羟基苯甲腈。3,5-二羟基苯甲酸和4-羟基苯甲腈加入反应体系中,一方面体系中羟基间具有互斥性,且存在较大的空间位阻,使得金属离子处于在配位时将两者与壳聚糖分子链接起来,避免壳聚糖分子链卷曲团聚而形成更松散的空间结构,其中未配位的羟基能与植株中众多生物质之间形成氢键,并携带生物质运输,提高了产物生物相容度,另一方面两者能促进开放电导钙离子活化的钾离子通道,使得苯丙氨酸解氨酶和查尔酮合成酶活性显著增强,加速酶促反应,提高初代代谢向次生代谢的转变效率,进而增加各种类黄酮及酚类化合物的生成量。
更优选地,羧酸基团修饰的壳聚糖通过以下步骤制备:取壳聚糖分散于5-10 倍量的异丙醇中,搅拌溶胀20-30min后,加入料液比为1:3-4、浓度为35-45%的氢氧化钠溶液,于45-55℃下碱化2-3h后,加入氯乙酸或丙烯酸,转入60-70℃水浴中回流2.5-3.5h,反应完成后,调节体系pH为中性,抽滤,并用浓度为70-75%的乙醇溶液洗涤,再于60-80℃真空干燥即得。
对本发明而言,促生长组合物为液体或可湿性粉剂形式;促生长组合物的施用量为20-100mg/株。该组合物中还含有农业上有益的成分,包括但不限于茉莉酸、茉莉酸甲酯、亚油酸、亚麻酸、有益微生物、微量营养素、肥料。该组合物中的载体包括但不限于水、盐溶液、醇、碳水化合物(如乳糖、直链淀粉或淀粉)、透明质酸。该组合物于大吴风草发芽一个月后开始施用。
对本发明而言,促生长组合物中类黄酮化合物、羧基基团修饰的壳聚糖盐、厚朴酚类似物和异山梨醇二甲醚的重量百分比分别为10-15%、8-15%、3-5%和 1-5%。
本发明的有益效果为:
1)本发明采用促生长组合物与紫外照射相配合的方法,促进植株光合作用,免受辐射伤害,诱导并促进类黄酮和酚类物质的合成和积累,同时提高了植物对活性氧自由基的清除或抑制能力以保护细胞免于氧化损伤,以及提升对如盐胁迫或光胁迫等非生物逆境胁迫的抗性;
2)本发明中促生长组合物能干扰气孔细胞闭合,使得CO2进入细胞不受阻,增加根部所处环境的湿润度,增强植株抗干旱能力,进而增加光合产物糖的生成量,为类黄酮物质等次生代谢物提供糖苷,达到提高次生代谢产物积累量的目的;
3)本发明羧酸基团修饰的壳聚糖盐分子链的制备能避免壳聚糖分子链卷曲团聚,形成分子链更松散的空间结构,具有良好的水溶性和生物相容度,能抑制或清除活性氧自由基,阻止低密度蛋白氧化,且能使得苯丙氨酸解氨酶和查尔酮合成酶活性显著增强,提高初代代谢向次生代谢的转变效率。
本发明采用了上述技术方案提供提高大吴风草中类黄酮和酚类物质含量的培育方法,弥补了现有技术的不足,设计合理,操作方便。
附图说明
图1为促生长组合物不同浓度下的活性氧自由基抑制率变化曲线图;
图2为促生长组合物对大吴风草叶片中可溶性糖含量的影响结果示意图;
图3为促生长组合物对大吴风草叶片中苯丙氨酸解氨酶和查尔酮合成酶活性的影响结果示意图;
图4为大吴风草叶片中总类黄酮含量的变化曲线图;
图5为大吴风草叶片中总酚类物质含量的变化曲线图。
具体实施方式
以下结合具体实施方式和附图对本发明的技术方案作进一步详细描述:
实施例1:
提高大吴风草中类黄酮和酚类物质含量的培育方法,包括,向大吴风草植株或植株部分经叶面或根部施用促生长组合物,以及,利用紫外线阶段性照射大吴风草植株;上述促生长组合物中包括一种或多种类黄酮化合物和羧酸基团修饰的壳聚糖盐。该方法能利用促生长组合物和紫外线照射改变大吴风草体内生理代谢水平,促进其光合作用,诱导并促进类黄酮和酚类物质的合成和积累,提高其药用和经济价值,且大大提高了植物对活性氧自由基的清除或抑制能力以保护细胞免于氧化损伤,以及对非生物逆境胁迫的抗性,提高植物产量和品质。
上述紫外线阶段性照射操作条件为:紫外灯功率35W,紫外线波长为280nm,照射周期为30d,单次照射时间为20min,照射间隔时间为10h,上述紫外线阶段性照射于大吴风草花期前30d开始进行。少量或适量的紫外照射植物能诱导植物中次生代谢产物的大量生成,进而保护细胞或植株免受辐射伤害,因而使得类黄酮及酚类物质合成途径中相关的基因表达量上升,相关的酶在紫外诱导下被激活或活性上调,从而增加类黄酮及酚类物质的含量,增强植株抗氧化、抗逆境胁迫的能力。
上述促生长组合物还包括厚朴酚类似物和异山梨醇二甲醚;厚朴酚类似物为丙基厚朴酚和异丙基厚朴酚等比例混合物。厚朴酚类似物和异山梨醇二甲醚作为组合物施用于植株,于叶面渗透后,两者能破坏气孔细胞质膜上糖蛋白所含寡糖肽与多肽链之间的共价结构,使气孔细胞间胞间连丝松弛,从而干扰气孔细胞闭合,使得CO2进入细胞不受阻,于根部渗透后进入植株内,两者能利用亲水性能增加根部所处环境的湿润度,参与并促进生物质运输,增强植株抗干旱能力,从不同方面以促进植株光合作用,进而增加光合产物糖的生成量,为类黄酮物质等次生代谢物提供糖苷,达到提高次生代谢产物积累量的目的。
促生长组合物中的一种或多种类黄酮化合物为大豆黄酮、槲皮素和柚皮素混合物,其中槲皮素重量占比为45%,大豆黄酮重量占比为25%,柚皮素重量占比为30%。类黄酮化合物施用前期可以吸收UV以减少器官受辐射伤害,但被植株吸收后,能作为植物信号分子,直接或间接的诱导或激活植株中类黄酮合成途径的生物化学通路,并影响上下游各种次生代谢产物的合成和产量。
上述羧酸基团修饰的壳聚糖盐中的羧酸基团由氯乙酸或丙烯酸提供,且羧酸基团的取代度为85%;羧酸基团修饰的壳聚糖盐为镁盐和锌盐混合物,其中镁盐和锌盐以1:1.5的重量比混合。在碱性条件下壳聚糖6位上的羟基优先参与反应,生成的壳聚糖衍生物具有很好的水溶性,增加了其被植株吸收和利用的几率,且生成的壳聚糖与金属离子配合物对金属离子具有缓释性,能有利于提高叶绿素活性,提升光合作用效率,或能抑制或清除活性氧自由基,阻止低密度蛋白氧化,避免如盐胁迫或光胁迫等逆境的不良影响。
上述羧基基团修饰的壳聚糖盐通过以下步骤获得:将可溶性镁盐或锌盐的水合物溶解后,于70℃下震荡搅拌,再配制含有羧酸基团修饰的壳聚糖的反应溶剂并以重量比1:1.5的比例加入盐溶液中,于70℃保温反应3h后,加入2倍体积的丙酮进行沉淀,减压浓缩,用体积浓度为70%的乙醇溶液洗涤,干燥,即得。壳聚糖接枝羧基基团后,更容易与金属离子形成配合物,分子中的氨基、仲羟基、乙酰氨基等都能与金属离子发生配位反应,且能通过催化活性氧的歧化反应,达到减少活性氧自由基的目的,抑制或避免了氧化损伤。
上述反应溶剂中含有重量浓度分别为2.5wt%的羧酸基团修饰的壳聚糖、0.15wt%的3,5-二羟基苯甲酸和0.2wt%的4-羟基苯甲腈。3,5-二羟基苯甲酸和4- 羟基苯甲腈加入反应体系中,一方面体系中羟基间具有互斥性,且存在较大的空间位阻,使得金属离子处于在配位时将两者与壳聚糖分子链接起来,避免壳聚糖分子链卷曲团聚而形成更松散的空间结构,其中未配位的羟基能与植株中众多生物质之间形成氢键,并携带生物质运输,提高了产物生物相容度,另一方面两者能促进开放电导钙离子活化的钾离子通道,使得苯丙氨酸解氨酶和查尔酮合成酶活性显著增强,加速酶促反应,提高初代代谢向次生代谢的转变效率,进而增加各种类黄酮及酚类化合物的生成量。
上述羧酸基团修饰的壳聚糖通过以下步骤制备:取壳聚糖分散于8倍量的异丙醇中,搅拌溶胀20min后,加入料液比为1:3、浓度为45%的氢氧化钠溶液,于45℃下碱化3h后,加入氯乙酸或丙烯酸,转入70℃水浴中回流2.5h,反应完成后,调节体系pH为中性,抽滤,并用浓度为70%的乙醇溶液洗涤,再于70℃真空干燥即得。
上述促生长组合物为可湿性粉剂形式;该组合物于大吴风草发芽一个月后开始施用,施用量为40mg/株,于大吴风草花期前30d开始施用量调整为80mg/株。该组合物中还含有重量占比为10%的农业上有益的成分,包括亚油酸、微量营养素和肥料。
上述促生长组合物中类黄酮化合物、羧基基团修饰的壳聚糖盐、厚朴酚类似物和异山梨醇二甲醚的重量百分比分别为10%、15%、3%和2%。
实施例2:
提高大吴风草中类黄酮和酚类物质含量的培育方法,具体措施如下:
1)取壳聚糖分散于6倍量的异丙醇中,搅拌溶胀30min后,加入料液比为 1:3.5、浓度为40%的氢氧化钠溶液,于50℃下碱化2.5h后,加入氯乙酸,转入 70℃水浴中回流3h,反应完成后,调节体系pH至中性,抽滤,并用浓度为75%的乙醇溶液洗涤,再于80℃真空干燥,即得取代度为90%的羧甲基壳聚糖;
2)将可溶性镁盐MgCl2·6H2O或锌盐ZnCl2溶解后,于80℃下震荡搅拌,再配制含有羧酸基团修饰的壳聚糖的反应溶剂并以重量比1:2的比例加入盐溶液中,保温反应2h后,加入3倍体积的丙酮进行沉淀,减压浓缩,用体积浓度为 70%的乙醇溶液洗涤,干燥,即得羧甲基壳聚糖镁盐和锌盐,上述反应溶剂中含有重量浓度分别为2wt%的羧酸基团修饰的壳聚糖、0.2wt%的3,5-二羟基苯甲酸和0.1wt%的4-羟基苯甲腈;
3)按重量占比分别为45%、15%、25%、15%取槲皮素、大豆黄酮、橙皮素和二氢山奈酚,将上述各组分混合即得类黄酮化合物;
4)按重量百分比分别为15%、12%、5%和5%取类黄酮化合物、羧甲基壳聚糖盐、厚朴酚类似物和异山梨醇二甲醚,混合后,向其中加入水配制成浓度为 50g/L的液体,即为促生长组合物,上述羧甲基壳聚糖盐为镁盐和锌盐按1:2.5 的重量比混合得到,厚朴酚类似物为丙基厚朴酚、异丙基厚朴酚和丁基厚朴酚等比例混合物;
5)将上述促生长组合物于大吴风草发芽一个月后开始施用,喷洒叶片或灌溉根部,施用量为50mg/株,于大吴风草花期前40d开始调整施用量为100mg/ 株;
6)于大吴风草花期前40d开始进行紫外线阶段性照射,紫外灯功率45W,紫外线波长为320nm,单次照射时间为15min,照射间隔时间为12h,照射周期为40d,继续培育30d后,大吴风草器官中类黄酮和酚类物质含量达到最高峰,其中以叶片中含量最高,叶柄次之,根状茎最小。
实施例3:
本实施例与实施例2不同之处在于:
步骤4)所制促生长组合物中未添加厚朴酚类似物和异山梨醇二甲醚;
其他步骤与实施例2中一致,完成大吴风草培育。
实施例4:
本实施例与实施例2不同之处在于:
本实施例所制促生长组合物中未添加羧酸基团修饰的壳聚糖盐;
其他步骤与实施例2中一致,完成大吴风草培育。
实施例5:
本实施例与实施例2不同之处在于:
步骤2)制备羧甲基壳聚糖盐时所用反应溶剂体系中未添加3,5-二羟基苯甲酸和4-羟基苯甲腈;
其他步骤与实施例2中一致,完成大吴风草培育。
实施例6:
本实施例与实施例2不同之处在于:
本实施例中在大吴风草培育期间仅采用施用促生长组合物一种措施,在花期前不配合采用紫外线阶段性照射培育;
其他步骤与实施例2中一致,完成大吴风草培育。
实施例7:
本实施例的培育方法为:于大吴风草花期前40d开始进行紫外线阶段性照射,紫外灯功率45W,紫外线波长为320nm,单次照射时间为15min,照射间隔时间为12h,照射周期为40d,继续培育30d后,大吴风草器官中类黄酮和酚类物质含量达到最高峰,其中以叶片中含量最高,叶柄次之,根状茎最小;
即本实施例中在大吴风草培育期间未施用促生长组合物,完成大吴风草培育。
试验例1:
促生长组合物清除活性氧自由基试验
试验样品:实施例1、2、4所制促生长组合物。
试验方法:采用硝基四氮唑蓝光照还原法,分别配制浓度为3.3×10-6mol·L-1、0.01mol·L-1、4.6×10-5mol·L-1的核黄素、蛋氨酸、硝基四氮唑蓝溶液,于30℃避光恒温20min,于日光灯(92W)下进行光照5min后,每隔3min移取部分溶液进比色皿中于560nm波长下测定其吸光度值,绘制吸光度值随光照时间的变化线,拟合求出直线的斜率K0;分别取各组合物样品,配制成浓度依次为0.05g/L、 0.1g/L、0.15g/L、0.2g/L、0.25g/L、0.3g/L的溶液,分别移取以上三种溶液后,再加入不同浓度的组合物溶液,并测定吸光度值随光照时间的变化,拟合求出直线的斜率Km,由公式抑制率%=(K0-Km)/K0×100%,求得组合物样品对超氧离子的抑制率,作各浓度组合物的抑制率随浓度变化的曲线图,处理及统计结果如附图1所示。
图1为促生长组合物不同浓度下的活性氧自由基抑制率变化曲线图。由图可知,促生长组合物对活性氧自由基均具有抑制作用,且抑制率与浓度均表现为正相关关系;其中实施例1和2催化活性氧歧化的活性差异不明显,且显著高于实施例4,说明组合物能通过羧酸基团修饰的壳聚糖盐来完成清除或减少活性氧自由基,以达到抑制或避免氧化损伤的效果,且效果显著。
试验例2:
促生长组合物对可溶性糖含量的影响
试验样品:实施例2、3及实施例7培育所得大吴风草植株。
试验方法:采用蒽酮比色法测定可溶性糖,三组处理分别称量10g大吴风草叶片烘干,取1g烘干后的叶片用10ml乙醇溶液研磨成匀浆,静置30min后, 4500rpm离心15min,取清液3ml到25ml容量瓶加水定容。然后取1ml稀释液加入5ml蒽酮试剂,煮沸10min,冷却后在620nm测定吸光值。将结果处理并分析,结果如附图2所示。
图2为促生长组合物对大吴风草叶片中可溶性糖含量的影响结果示意图。由图可知,实施例2和3较实施例7中可溶性糖含量有显著提升,而实施例2和3 之间也存在一定差异,说明组合物能促进光合作用进行,进而增加光合产物糖的生成量,其中实施例2较实施例3效果更加显著,能为类黄酮物质等次生代谢物提供更多的糖苷,以达到提高次生代谢产物积累量的目的。
试验例3:
促生长组合物对苯丙氨酸解氨酶和查尔酮合成酶活性的影响
试验样品:实施例2、4、5培育所得大吴风草植株。
试验方法:用紫外分光光度法测定酶活性,取2.0g新鲜大吴风草叶片,加入3ml硼酸缓冲液和0.5g PVP,研磨,10000rpm离心15min,取上清。取0.5mL 苯丙氨酸溶液加入0.5ml上清液。然后加入10ml蒸馏水,在40℃的水浴中加热 30min,再在沸水中煮5min终止反应,在290nm下测定分光光度值,以吸收变化0.01所需酶量作为一个酶活性单位(U)。进行3次重复测定,取平均值。将结果处理并分析,结果如附图3所示。
图3为促生长组合物对大吴风草叶片中苯丙氨酸解氨酶和查尔酮合成酶活性的影响结果示意图。由图可知,实施例4和5酶活性之间差异不大,两者都显著低于实施例2中酶活性,说明组合物能提升苯丙氨酸解氨酶和查尔酮合成酶活性以增加类黄酮化合物的生成和积累量,同时也说明实施例2较实施例5所制组合物能显著增强苯丙氨酸解氨酶和查尔酮合成酶活性,加速类黄酮物质合成的酶促反应。
试验例4:
不同培育方法对大吴风草中类黄酮及酚类物质含量的影响
试验样品:实施例2、6、7培育所得大吴风草植株。
试验方法:以芦丁和白藜芦醇为标准品,并作标准曲线。设定开始施用促生长组合物至花期前调整组合物用量,为第一生长阶段,花期调整组合物施用量的同时开始紫外阶段性照射至紫外照射结束,为第二生长阶段,紫外照射结束至继续培育30d,为第三生长阶段,每一生长阶段结束时,收集大吴风草叶片为试验样品器官。设未施用组合物也未紫外照射的自由生长植株为对比组,精确称定样品粉末0.5g,置于50ml容量瓶中,加95%乙醇40ml,在超声波发生器提取45min,静置冷却,加95%乙醇至刻度,摇匀,静止3-4h后,吸取样液2.0ml,分别置于 25ml容量瓶中,加水至5ml,再加5%亚硝酸钠1.0ml,摇匀放置6min,加入10%的硝酸铝1.0ml,摇匀放置5min,加入5%氢氧化钠10.0ml,混匀后用水定容至刻度,摇匀,放置15min以上,以相应试剂做空白,于510nm处测定吸光度。从标准曲线中得到提取液中类黄酮和酚类的含量。将结果处理并分析,结果如附图4和5所示。
图4为大吴风草叶片中总类黄酮含量的变化曲线图,图5为大吴风草叶片中总酚类物质含量的变化曲线图。
由图4可知,实施例2、6、7的培育方法相较自然生长的植株中总黄酮物质含量高,实施例中实施例2总黄酮含量最高,实施例6次之,实施例7再次,说明促生长组合物和紫外线照射都有利于植株中类黄酮物质含量的积累和增加,且相较而言,促生长组合物提高类黄酮物质含量的效果较紫外线照射更显著。
由图5可知,实施例2、6、7的培育方法相较自然生长的植株中总酚类物质含量高,实施例中实施例2总酚类含量最高,实施例6次之,实施例7再次,说明促生长组合物和紫外线照射都有利于植株中类黄酮物质含量的积累和增加,且相较而言,促生长组合物提高类黄酮物质含量的效果较紫外线照射更显著。
上述实施例中的常规技术为本领域技术人员所知晓的现有技术,故在此不再详细赘述。
以上实施方式仅用于说明本发明,而并非对本发明的限制,本领域的普通技术人员,在不脱离本发明的精神和范围的情况下,还可以做出各种变化和变型。因此,所有等同的技术方案也属于本发明的范畴,本发明的专利保护范围应由权利要求限定。
Claims (7)
1.提高大吴风草中类黄酮和酚类物质含量的培育方法,其特征在于:包括,向所述大吴风草植株或植株部分经叶面或根部施用促生长组合物,以及,利用紫外线阶段性照射大吴风草植株;
所述促生长组合物中包括一种或多种类黄酮化合物和羧酸基团修饰的壳聚糖盐;
所述促生长组合物还包括厚朴酚类似物和异山梨醇二甲醚;所述厚朴酚类似物选自丙基厚朴酚、异丙基厚朴酚、丁基厚朴酚和异丁基厚朴酚中的至少一种;
所述羧酸基团修饰的壳聚糖盐中的羧酸基团由氯乙酸或丙烯酸提供,且羧酸基团的取代度为80-95%;所述羧酸基团修饰的壳聚糖盐选自镁盐、锌盐或它们的混合物;
所述促生长组合物中类黄酮化合物、羧基基团修饰的壳聚糖盐、厚朴酚类似物和异山梨醇二甲醚的重量百分比分别为10-15%、8-15%、3-5%和1-5%。
2.根据权利要求1所述的提高大吴风草中类黄酮和酚类物质含量的培育方法,其特征在于:所述紫外线阶段性照射操作条件为:紫外灯功率30-45W,紫外线波长为270-320nm,照射周期为30-40d,单次照射时间为15-20min,照射间隔时间为10-12h。
3.根据权利要求1所述的提高大吴风草中类黄酮和酚类物质含量的培育方法,其特征在于:所述促生长组合物中的一种或多种类黄酮化合物为大豆黄酮、黄酮醇、二氢黄酮醇、黄烷酮或它们的混合物。
4.根据权利要求1所述的提高大吴风草中类黄酮和酚类物质含量的培育方法,其特征在于:所述羧酸基团修饰的壳聚糖盐通过以下步骤获得:将可溶性镁盐或锌盐的水合物溶解后,再配制含有羧酸基团修饰的壳聚糖的反应溶剂并以重量比1:1-2的比例加入盐溶液中,于70-80℃保温反应2-3h后,加入丙酮进行沉淀,减压浓缩,用乙醇溶液洗涤,干燥,即得。
5.根据权利要求4所述的提高大吴风草中类黄酮和酚类物质含量的培育方法,其特征在于:所述反应溶剂中含有重量浓度分别为2-5wt%的羧酸基团修饰的壳聚糖、0.1-0.5wt%的3,5-二羟基苯甲酸和0.05-0.25wt%的4-羟基苯甲腈。
6.根据权利要求4所述的提高大吴风草中类黄酮和酚类物质含量的培育方法,其特征在于:所述羧酸基团修饰的壳聚糖通过以下步骤制备:取壳聚糖分散于异丙醇中溶胀20-30min后,加入氢氧化钠溶液于45-55℃下碱化2-3h后,加入氯乙酸或丙烯酸,转入60-70℃水浴中回流2.5-3.5h,反应完成后,调节体系pH为中性,抽滤,并用乙醇溶液洗涤,真空干燥即得。
7.根据权利要求1所述的提高大吴风草中类黄酮和酚类物质含量的培育方法,其特征在于:所述促生长组合物为液体或可湿性粉剂形式;所述促生长组合物的施用量为20-100mg/株。
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