CN115177648A - 一种快速促进采摘菊叶黄酮累积和富硒转化的方法及应用 - Google Patents
一种快速促进采摘菊叶黄酮累积和富硒转化的方法及应用 Download PDFInfo
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- CN115177648A CN115177648A CN202211019445.5A CN202211019445A CN115177648A CN 115177648 A CN115177648 A CN 115177648A CN 202211019445 A CN202211019445 A CN 202211019445A CN 115177648 A CN115177648 A CN 115177648A
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Abstract
本发明提供了一种快速促进采摘菊叶黄酮累积和富硒转化的方法及应用。利用离体叶片激发菊叶自身抗氧化能力和抗病性的防御系统,通过纳米硒喷洒、UV‑A紫外光激发、蓝光照射、通风干燥的胁迫诱导组合,实现快速促进采摘菊叶黄酮累积和富硒转化,同时实现查尔酮、花青素等物质的富集。本方法在菊科物种中黄酮累积含量提高超过4倍,达到极显著水平;有机硒含量提高达37倍以上,以活性硒胱氨酸形态为主,没有无机硒残留。本发明主要适用于以大品种生物医药用植物叶片为反应器,进行黄酮提取物生产和富硒活性物质转化利用,有效提高产量和原材料利用率,降低植物栽种和富硒培养成本,有利于实现目标活性物质的稳定可控生产和标准化管理。
Description
技术领域
本发明属于生物技术领域,特别涉及一种快速促进采摘菊叶黄酮累积和富硒转化的方法及应用。
背景技术
生物黄酮,即众多黄酮类化合物,包括黄酮、黄酮醇、黄酮、黄烷醇、异黄酮和花青素,是人们日常饮食中最丰富的多酚,具有广泛的生物学效应,主要包括有抑制HL-60白血病细胞生长和溶解癌细胞的作用,清除体内的自由基及毒素,消炎、抗过敏、广谱抗菌、抗病毒;同时还可以调节血脂,降低血液粘稠度,改善血清脂质,延长红血球寿命并增强造血功能,能够有效预防、减少心脑血管等疾病的发生。
有研究表明,植物中类黄酮存在差异代谢产物,参与花青素和苯丙烷的生物合成;在众多类黄酮化合物中,花青素是一种重要的抗氧化剂,是强效的自由基清除剂,具有抗氧化、抗突变、预防心脑血管疾病、保护肝脏和抑制肿瘤细胞发生等多种保健功能。槲皮素除了具有强抗氧化作用外,还具有扩冠作用,同时对由ADP、胶原或凝血酶引起的血小板聚集和血栓具有抑制作用,并已应用于临床,如心血管系统疾病、活血化瘀、降脂降压等。
菊科植物(Asteraceae)约一千属,是双子叶植物纲中第一大科,也是种子植物中种类最多的一科。其广布全世界,富有经济价值。我国境内,包括归化及栽培年代已久的菊科植物约有240属,2300种之多;其中,菊科药用植物约有120属,500多种,如茵陈(Artemisia)、红花(Carthamus)、旋覆花(Inula)、蒲公英(Tataxacum)、苍耳(Xanthium)等均已广泛入药。菊科植物含有复杂多样的活性成分,使得菊科植物资源在医药、农药、保健品、化妆品、添加剂等方面的研究和开发利用取得了可喜的进展。
诱导抗性是近年来发展的果蔬采后病害控制的新技术之一,主要是通过激发果蔬自身防御系统,来增强抗氧化能力和抗病性。而生物黄酮作为植物重要的防御物质,可以在外界诱导下达到富集作用,从而应用在天然活性物生产领域。
植株中的生物黄酮含量受外界诱导影响,黄酮水平可以通过外界因素刺激诱导。有研究表明,LED灯诱导刺激可以提高相关基因的表达水平,刺激植物的类黄酮含量的增加,紫外线光辐射诱导可以通过诱导植物结构基因和调节基因的表达调控花青素的合成。另有研究表明,干旱胁迫会导致控制查尔酮合酶合成的基因表达上调,将苯丙代谢引入黄酮代谢,从而进入不同的代谢通路,形成不同的类黄酮物质。
发明内容
为了克服现有技术的不足,应对中药种植面积缺乏、产品化程度低而植物提取行业发展迅速的情况,本发明的首要目的就是利用非生物胁迫,对离体的菊叶片进行加工处理,诱导黄酮富集。本发明提出了一种快速促进采摘菊叶黄酮累积和富硒转化的方法,即通过叶片离株、硒溶液处理、光照干燥的条件胁迫,促使黄酮类物质的富集,总含量提高超过400%,同时还引起花青素、黄酮醇、黄烷醇类的大量物质累积。
本发明的另一目的在于提供一种上述快速促进采摘菊叶黄酮累积和富硒转化的方法的应用;通过开展对菊科大样本的广泛应用,通过高丰度的天然活性黄酮提取,实现在复方药物或候选药物、功能食品、保健食品等方面的转化应用。
本发明目的通过下述技术方案实现:
一种快速促进采摘菊叶黄酮累积和富硒转化的方法,包括如下步骤:
(1)采集新鲜菊叶,清洗晾干;
(2)在叶片上喷洒纳米硒溶液;
(3)将叶片放置处理后,得到黄酮累积和富硒转化的叶片。
步骤(1)所述的菊叶为菊科植物的叶片,优选为包括翼茎阔苞菊、南美蟛蜞菊、艾蒿中的至少一种,更优选为翼茎阔苞菊(Pluchea sagittalis(Lam.)Cabera)。
步骤(1)所述的清洗为使用水进行清洗。
步骤(2)所述的纳米硒溶液的浓度为10~20mg/L;优选为12.5mg/L。
步骤(2)所述的纳米硒为多糖化纳米硒;优选为绿藻多糖化纳米硒,制备方法参照中国发明专利CN201610893963.8一种绿藻多糖化纳米硒及其制备方法与应用。
步骤(3)所述的放置处理的时间为24~72h,优选为48h。
步骤(3)所述的放置处理为包括光诱导和干燥胁迫中的至少一种,优选为光诱导和干燥胁迫。
上述的光诱导的具体步骤为:
将叶片放置于紫外光下激发后,用蓝光照射。
上述的紫外光的波长为315~400nm,辐射照度为10~20W/m2。优选为波长352nm,辐射照度15W/m2。
上述的激发的时间为4~8h,优选为6h。
上述的蓝光的波长为400~500nm,优选为470nm。
上述的蓝光照射的时间为12~36h,优选为24h。
上述的干燥胁迫的具体步骤为:
将叶片放置于20~30℃条件下通风干燥12~36h,优选为放置于25℃条件下通风干燥24h。
上述的富硒转化为有机硒代谢转化和富集,将无机硒转化为活性硒胱氨酸。
上述的黄酮为包括黄酮类化合物,优选为包括花青素(Anthocyanins)、黄酮(Flavonoid)、黄酮醇(Flavonols)、黄烷醇(Flavanols)、异黄酮(Isoflavones)中的至少一种。
上述快速促进采摘菊叶黄酮累积和富硒转化的方法在菊科深加工中的应用。
本发明所提供的富集方式得到的菊叶片提取物富集有大量类黄酮物质,可以应用于生物功能制品,起到抗氧化、抗炎症,预防心血管疾病的作用。
本发明所述方法能够有效提高菊叶黄酮的提取率,已完成三种菊科样品的实验,增长率分别达到490%、716%、1436%。同时通过UPLC-MS/MS检测,对选择富集率最高的品种——翼茎阔苞菊(Pluchea sagittalis(Lam.)Cabera)进行了组间差异代谢物分析,筛选出主要差异代谢物,证明本方法对黄酮代谢的黄酮代谢通路和花青素合成通路的刺激作用。
本发明相对于现有技术,具有如下的优点及效果:
第一,相对于正常的生长富集,离株叶片在进行光照干燥后,总黄酮含量发生了4倍以上的提高,指导种植业合理选取采摘周期和频率,关注后期加工和保存的方式,有效解决植提物市场成本高、产量低的问题。
第二,离体阔苞菊叶片进行光照干燥后,类黄酮成分中花青素种类、槲皮素-葡糖苷物质显著富集。对于植提物的抗氧化、抗突变、预防心脑血管疾病、保护肝脏和抑制肿瘤细胞发生等多种药用保健功能进行了补充。
第三、经本方法处理后,三种菊叶水提取物中总硒含量提高分别达70倍、42倍和37倍,均未检出无机硒残留,其有机硒以活性硒胱氨酸形态为主,分别占70%、75%和68%。
附图说明
图1是叶片黄酮含量累积测试结果图。
图2是叶片硒含量累积测试结果图。
图3是组间PCA主成分分析结果图。
图4是实施例4与对照实施例4-1的差异代谢物统计结果图。
图5是实施例4与对照实施例4-1的KEGG富集统计结果图。
图6是实施例4与对照实施例4-2的差异代谢物统计结果图。
图7是实施例4与对照实施例4-2的KEGG富集统计结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下面实施方案中若未注明具体试验条件,则通常按照常规试验条件或按照试剂公司所建议的试验条件。所使用的材料、试剂等,若无特殊说明,均为从商业途径得到的试剂和材料。
实施例1
(1)采摘:选取同一株翼茎阔苞菊(Pluchea sagittalis(Lam.)Cabera)植株上生长成熟度类似的叶片,流水清洗后自然晾干,每组取1g左右叶片进行实验,称量叶片鲜重;
(2)硒处理:将叶片铺开,两面均匀喷洒12.5mg/L纳米硒溶液。(即绿藻多糖化纳米硒,绿藻多糖化纳米硒的制备方法参照中国发明专利CN201610893963.8一种绿藻多糖化纳米硒及其制备方法与应用);
(3)光诱导:将叶片放置于UV-A紫外光(352nm,15W/m2)下激发6小时后,用蓝光(470nm)照射24小时;
(4)干燥胁迫:将叶片放置于25℃条件下通风干燥24小时;
(5)提取:取处理后的叶片,研磨至粉末状,溶解于50%乙醇,匀浆后并用超声波提取10次(每次提取10秒,之后暂停10秒),之后将匀浆液置于翻转台翻转5小时,60℃热浴2小时,14000rpm离心10分钟,收集上清液。
对照实施例1
(1)采摘:选取同一株翼茎阔苞菊植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重;
(2)密封冷冻保鲜;将采摘清洗后的叶片晾干,密封放置于-80℃低温冷冻保鲜;
(3)提取:取处理后的叶片,研磨至粉末状,溶解于50%乙醇,匀浆后并用超声波提取10次(每次提取10秒,之后暂停10秒),匀浆液置于翻转台翻转5小时,60℃热浴2小时,14000rpm离心10分钟,收集上清液。
实施例2
与实施例1基本相同,区别仅在于:所述的步骤(1)为:
采摘:选取同一株艾蒿植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重。
对照实施例2
与对照实施例1基本相同,区别仅在于:所述的步骤(1)为:
采摘:选取同一株艾蒿植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重。
实施例3
与实施例1基本相同,区别仅在于:所述的步骤(1)为:
采摘:选取同一株南美蟛蜞菊植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重。
对照实施例3
与对照实施例1基本相同,区别仅在于:所述的步骤(1)为:
采摘:选取同一株南美蟛蜞菊植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重。
实施例4
(1)采摘:选取同一株翼茎阔苞菊植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重;
(2)光诱导:将叶片放置于UV-A紫外光(352nm,15W/m2)下激发6小时后,用蓝光(470nm)照射24小时;
(3)干燥胁迫:将叶片放置于25℃条件下通风干燥24小时;
(4)提取:将样品放置于冻干机中真空冷冻干燥;利用研磨仪研磨(30Hz,1.5分钟)至粉末状;称取100mg的粉末,溶解于1.2mL 70%甲醇提取液中,每30分钟涡旋一次,每次持续30秒,共涡旋6次,样本置于4℃冰箱过夜;离心(转速12000rpm,10分钟)后,吸取上清,用微孔滤膜过滤样品。
对照实施例4-1
(1)采摘:选取同一株翼茎阔苞菊植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重;
(2)干燥胁迫:将叶片放置于25℃条件下通风干燥24小时;
(3)密封冷冻保鲜;将采摘清洗后的叶片晾干,密封放置于-80℃低温冷冻保鲜;
(4)提取:将样品放置于冻干机中真空冷冻干燥;利用研磨仪研磨(30Hz,1.5分钟)至粉末状;称取100mg的粉末,溶解于1.2mL 70%甲醇提取液中,每30分钟涡旋一次,每次持续30秒,共涡旋6次,样本置于4℃冰箱过夜;离心(转速12000rpm,10分钟)后,吸取上清,用微孔滤膜过滤样品。
对照实施例4-2
(1)采摘:选取同一株翼茎阔苞菊植株上生长成熟度类似的叶片,流水清洗后自然晾干,称量叶片鲜重;
(2)密封冷冻保鲜;将采摘清洗后的叶片密封,并放置于-80℃低温冷冻保鲜;
(3)提取:将样品放置于冻干机中真空冷冻干燥;利用研磨仪研磨(30Hz,1.5分钟)至粉末状;称取100mg的粉末,溶解于1.2mL 70%甲醇提取液中,每30分钟涡旋一次,每次持续30秒,共涡旋6次,样本置于4℃冰箱过夜;离心(转速12000rpm,10分钟)后,吸取上清,用微孔滤膜过滤样品。
测试例1
对实施例1~4、对照实施例1~4采用NaNO2-Al(NO3)3-NaOH方法进行黄酮含量检测。采用高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)法-检测总硒、无机硒和硒胱氨酸含量。结果如下:
表1-1叶片黄酮含量累积及硒含量测试结果表
所述NaNO2-Al(NO3)3-NaOH方法步骤如下:称取芦丁标准品,配制不同浓度的标准品溶液,分别取100μL待测液加入10μL 5%亚硝酸钠、10μL 10%硝酸铝、80μL 4%氢氧化钠溶液显色,放置10分钟后,在510nm处测量吸光度,绘制芦丁标准曲线,得回归方程;对翼茎阔苞菊叶片黄酮提取液进行检测,吸取提取液,用上述方法显色,在510nm处测量吸光度,根据吸光度对照回归方程得提取液总黄酮质量浓度,按以下公式求得提取液中总黄酮含量(mg/g):
式中:C为样品提取液中总黄酮的质量浓度,V为提取液体积,N为稀释倍数,M为样品质量。根据标准曲线回归方程计算出不同处理的叶片在不同时期所含总黄酮含量,结果如图1所示。
所述HPLC-ICP-MS检测总硒、无机硒和硒胱氨酸方法步骤如下:
(1)将菊叶片提取液加入超滤管(0.5mL~3kDa)中,14000rpm,4℃离心30min,收集滤液。
(2)标准液配制:硒酸盐(GBW 10033)、亚硒酸盐(GBW 10032)和硒胱氨酸(GBW10087)标准物质的质量摩尔浓度分别为0.525、0.543、0.560μmol/g。用超纯水配制硒混合标准溶液(各含1.00μg/mL),用超纯水稀释成0.0、1.00、5.00、10.0、25.0、50.0、100.0ng/mL的系列混合标准工作溶液。标准品及溶液标准物质于4℃下冷藏保存,所有工作溶液均现配现用。
(3)样本测定采用HPLC(安捷伦1260Infinity型)与ICP-MS(8900型)联用。HPLC工作条件为:色谱柱(Zorbax SB-Aq型反相色谱柱[150mm×4.6mm,5μm,安捷伦]),柱温25℃,流动相为1%甲醇(pH值分别为2.7、7.0的10mM柠檬酸溶液含5mM己烷磺酸钠),梯度洗脱,进样体积10μL,流速1mL/min。ICP-MS工作条件为:射频功率1550W,采样深度8.0mm,蠕动泵转速0.3r/s,高纯氩气载气流量0.90L/min,高纯氩气补偿气流量为0.25L/min,雾化室温度2℃,积分时间:0.5s,采集时间10min。
(4)根据标准曲线计算总硒、无机硒和硒胱氨酸含量,结果如图2所示。
由结果得到以下结论:
(1)实施例1~3中叶片黄酮含量明显高于相应对照实施例1~3,这是由于翼茎阔苞菊、艾蒿和南美蟛蜞菊叶采摘后进行本组合方法的处理,促黄酮含量显著提升,总黄酮累积分别达14倍、4.9倍和7.4倍。说明不同种菊科植物经本方法处理诱导后,叶片黄酮含量都发生明显代谢合成与富集。
(2)实施例4中翼茎阔苞菊叶片黄酮含量明显高于对照实施例1,总黄酮累积达2.13倍。说明单纯干燥处理及光诱导也可促发菊叶黄酮发生富集;而实施例1中叶片黄酮含量明显高于对照实施例4,这是由于翼茎阔苞菊采摘后使用了纳米硒处理,黄酮含量显著提升。但提升效率明显低于实施例1~3中三因素优化组合处理。
(3)实施例1~3与对照实施例1~3相比,经本方法处理后,三种菊叶水提取物中总硒含量提高分别达70倍、42倍和37倍,均未检出无机硒残留,其有机硒以活性硒胱氨酸形态为主,分别占70%、75%和68%。对比实施例4与对照实施例4-1和4-2,菊叶总硒含量和硒胱氨酸含量无明显提升。说明本优化组合处理方法可显著促进菊叶有机硒代谢转化和富集。
组间PCA主成分分析结果如图3所示,实验结果证明,不同种菊科植物经本方法处理诱导后,叶片黄酮含量都发生明显富集。
测试例2
对实施例4、对照实施例4-1、对照实施例4-2采用UPLC-MS/MS方法进行代谢组分析,结果如下:
表2-1实施例4与对照实施例4-1差异代谢物筛选
注:差异代谢物筛选条件:重要性投影(VIP)≥0.8且差异倍数值(fold change)≥1.6和≤0.62
表2-2实施例4与对照实施例4-2差异代谢物筛选
注:差异代谢物筛选条件:重要性投影(VIP)≥0.8且差异倍数值(FC)≥1.6和≤0.62
UPLC-MS/MS方法步骤如下:数据采集仪器系统主要包括超高效液相色谱(UPLC)(SHIMADZU Nexera X2)和串联质谱(MS/MS)(Applied Biosystems4500QTRAP)。具体条件为:色谱柱:Agilent SB-C18 1.8μm,2.1mm*100mm;流动相:A相为超纯水(加入0.1%的甲酸),B相为乙腈(加入0.1%的甲酸);洗脱梯度:0.00min B相比例为5%,9.00min内B相比例线性增加到95%,并维持在95%1min,10.00-11.10min,B相比例降为5%,并以5%平衡至14min;流速0.35ml/min;柱温40℃;进样量4μl。
由实验结果可知,实施例4与对照实施例4-1,实施例4对于类黄酮代谢物,如花青素(Anthocyanins)、黄酮(Flavonoid)、黄酮醇(Flavonols)、黄烷醇(Flavanols)、异黄酮(Isoflavones)产生明显的富集作用;根据KEGG注释和富集结果(表1及图4、图5),实施例4的差异代谢物主要富集花青素代谢和异黄酮代谢。结果说明,本方法光照诱导对于花青素代谢和异黄酮代谢通路的刺激,具体生物学代谢机制仍待进一步确认。
实施例4与对照实施例4-2对照,实施例4中,花青素(Anthocyanins)、查尔酮(Chalcones)类物质发生显著富集,而二氢黄酮(Dihydroflavone)和二氢黄酮醇(Dihydroflavonol)类物质都呈现减少趋势;根据KEGG注释和富集结果(表2及图6、图7),实施例4的差异代谢物主要富集在黄酮代谢。结果说明,本方法的干燥胁迫对于黄酮代谢通路的刺激,具体生物学代谢机制仍待进一步确认。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于包括如下步骤:
(1)采集新鲜叶片,清洗晾干;
(2)将叶片铺开,在叶片上喷洒纳米硒溶液;
(3)将叶片放置处理后,得到富集黄酮类物质的叶片。
2.根据权利要求1所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于:
步骤(1)所述的新鲜叶片为菊科植物的叶片。
3.根据权利要求1所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于:
步骤(2)所述的纳米硒溶液的浓度为10~20mg/L;
步骤(2)所述的纳米硒为多糖化纳米硒。
4.根据权利要求1所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于:
步骤(3)所述的放置处理的时间为24~72h。
5.根据权利要求1所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于:
步骤(3)所述的放置处理为包括光诱导和干燥胁迫中的至少一种。
6.根据权利要求5所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于:
所述的光诱导的具体步骤为:
将叶片放置于紫外光下激发后,用蓝光照射。
7.根据权利要求6所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于:
所述的紫外光的波长为315~400nm,辐射照度为10~20W/m2;
所述的激发的时间为4~8h。
8.根据权利要求6所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于:
所述的蓝光的波长为400~500nm;
所述的蓝光照射的时间为12~36h。
9.根据权利要求5所述的快速促进采摘菊叶黄酮累积和富硒转化的方法,其特征在于所述的干燥胁迫的具体步骤为:
将叶片放置于20~30℃条件下通风干燥12~36h。
10.权利要求1~9任一所述的快速促进采摘菊叶黄酮累积和富硒转化的方法在菊科深加工中的应用。
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