CN105906638B - 一种快速制备叶绿素和叶绿素降解产物的方法 - Google Patents
一种快速制备叶绿素和叶绿素降解产物的方法 Download PDFInfo
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 34
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- QPDWBRHRBKXUNS-IEEIVXFASA-L chlorophyllide b Chemical compound C1([C@H](C2=O)C(=O)OC)=C(N3[Mg]N45)C2=C(C)\C3=C\C(=N2)C(CC)=C(C=O)\C2=C\C4=C(C=C)C(C)=C5\C=C/2[C@@H](C)[C@H](CCC(O)=O)C1=N\2 QPDWBRHRBKXUNS-IEEIVXFASA-L 0.000 claims description 13
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- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 claims description 3
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- RKEBXTALJSALNU-LDCXZXNSSA-N 3-[(3R,21S,22S)-16-ethenyl-11-ethyl-4-hydroxy-3-methoxycarbonyl-12,17,21,26-tetramethyl-7,23,24,25-tetrazahexacyclo[18.2.1.15,8.110,13.115,18.02,6]hexacosa-1,4,6,8(26),9,11,13(25),14,16,18(24),19-undecaen-22-yl]propanoic acid Chemical compound CCC1=C(C2=NC1=CC3=C(C4=C([C@@H](C(=C5[C@H]([C@@H](C(=CC6=NC(=C2)C(=C6C)C=C)N5)C)CCC(=O)O)C4=N3)C(=O)OC)O)C)C RKEBXTALJSALNU-LDCXZXNSSA-N 0.000 description 2
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- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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Abstract
本发明公开了一种快速制备高纯度叶绿素和叶绿素降解产物的方法。本发明首次采用温州蜜柑果皮和甘薯叶片为原料来制备叶绿素及其衍生物,提供的方法不仅取材方便、快速、成本低,而且易操作,可以在三天内完成全部8种产物的制备,所得产品纯度可以达到90%‑95%,成本远远低于同种进口标准品的价格。本方法可以高效快速制备实验所需毫克级的叶绿素及其衍生物的标准对照品,适合在商业实验室及企业推广使用,具有广泛的应用前景。
Description
技术领域
本发明属于化学和酶学的交叉领域,具体涉及一种快速制备高纯度叶绿素和叶绿素降解产物的方法。
背景技术
目前实验室分析叶绿素及其降解产物时,通常使用进口品牌的标准对照品作为参照。这些标准对照品通常由制备型薄层色谱(TLC)和制备型HPLC提纯自菠菜叶片和绿藻等材料,纯度常为90%-98%,通常纯度越高,价格越贵。进口叶绿素标准品常存在供货期长(一周至一个月),价格昂贵(以进口品牌sigma的叶绿素a为例,每毫克价格在6000-8000RMB),分装及运输过程易见光降解,低温保存造成运输不便等问题。由于进口标准品购买过程的上述不便,且分析样品中叶绿素时标准品用量较大,实验成本很高,因此实验室亟需一种叶绿素及其降解产物标准品的快速制备方法,然而,目前并无这方面的报道。本方法不仅取材方便、快速、成本低,而且易操作,可以在三天内完成全部实验,所得产品纯度可以达到90%-95%,成本远远低于同种进口标准品的价格。本方法可以高效快速制备实验所需毫克级的叶绿素及其衍生物的标准对照品,适合在商业实验室及企业推广使用,具有广泛的应用前景。
发明内容
本发明的目的在于提供一种快速制备高纯度叶绿素和叶绿素降解产物的方法,该方法可快速分离制备叶绿素a和叶绿素b及其降解产物,方法快速简单、易操作、成本低。通过该方法获得的叶绿素及其降解产物纯度达到90%-95%,可以作为标准品使用,或其他商品化用途,为叶绿素相关的生理生化提供可靠的标准品做参照。
为了达到上述目的,本发明采取以下技术措施:
一种快速制备高纯度叶绿素和叶绿素降解产物的方法,包括:
(1)材料准备:新鲜蔬菜叶片和新鲜柑橘果实洗净晾干,将新鲜柑橘自果皮表层削取的0.2~0.3mm厚度的表皮,作为新鲜柑橘果皮,备用;
优选的,蔬菜叶片为将市场上购买来的新鲜蔬菜(菠菜、甘薯叶等)摘选叶片,挑除老叶和黄叶并去掉叶柄,洗净后阴暗处晾干。新鲜柑橘,果皮带绿,成熟度以色差指数CI为-2.0~2.0,清水洗净,晾干;
(2)总叶绿素的提取,以下步骤在避光条件下进行:
a.将(1)中洗净晾干的蔬菜叶片称取100g,用液氮冷冻,在研钵或样品破碎仪中磨成细粉;
b.将样品粉末与提取液(N,N-二甲基甲酰胺,含0.1%碳酸镁,w/v)按1g:2mL的比例混合。超声清洗仪100%功率(100W),冰浴超声20min~30min,期间上下混匀样品数次。4000g~5000g离心5min~10min,取上清。沉淀按上述样品粉末与提取液的比例重新加入提取液抽提。重复冰浴超声、离心和取上清过程,直至残渣无色,将收集的上清合并;
c.合并的上清分装至250mL分液漏斗中,每个分液漏斗100mL,按1:1的体积比加入正己烷混合,充分振荡后静置至完全分层。弃上层正己烷相(含类胡萝卜素),下层放入新的分液漏斗中,按上述上清与正己烷的体积比重新加入正己烷,继续振荡后静置分层,弃上层正己烷相,重复该步骤至上层无色,合并下层液;
d.下层液与正己烷按4:1的体积比混合,剧烈振荡后-20℃冰箱中放置2~3h。4000g~5000g离心10min~15min,移除最上层白色乳油状物及中间层正己烷相。按下层液和正己烷1:1的体积比继续加入正己烷,剧烈振荡后4000g~5000g离心10min,移除上层,重复该步骤至上层无色,移除上层保留下层液;
e.下层液与10%氯化钠水溶液按体积比1:5~6混合,振荡1min,4000g~5000g离心5min,收集上清。剩余下层继续与无水乙醚按10:1的体积比混合,振荡1min,静置至分层,收集上清,重复该步骤一遍,将收集的上清合并。加入等体积的1:1(v/v)的正己烷:无水乙醚溶液混合后,再加入5倍体积的10%氯化钠水溶液,振荡1min,静置至分层,收集合并上层液。合并的上层液加入等体积的正己烷,振荡后加入无水硫酸钠至无结块。上清在避光条件下30℃真空浓缩仪中干燥或者氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存,得叶绿素粗提干燥物;
(3)叶绿素a和叶绿素b(chlorophyll a和chlorophyll b)的分离:称取步骤(2)中所得总叶绿素粗提干燥物10mg,用1:1(v/v)的甲醇:丙酮溶解,配成终浓度2mg/mL,8000g~12000g离心10min~15min,上清经0.22μm微孔滤膜过滤后分装至2mL上样瓶中。可按常规方式的液相色谱分离法进行叶绿素a和叶绿素b的分离。
优选的:实验室常见的分析型、半制备型或者制备型的液相色谱仪(配备PDA(二极管阵列检测器)或紫外可见光检测器)使用常用的C18色谱柱,均可用于叶绿素a和叶绿素b的分离。流动相A为甲醇,流动相B为甲醇:丙酮=1:1;用50%流动相A+50%流动相B等度洗脱,分别收集chlorophyll a色谱峰和chlorophyll b色谱峰的馏分,避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(4)脱镁叶绿素a和脱镁叶绿素b(pheophytin a和pheophytin b)的制备
(a)脱镁叶绿素a(pheophytin a)的制备:称取2mg步骤(3)中分离干燥所得的叶绿素a,于10mL离心管中用4mL无水乙醚溶解配制成500μg/mL。逐滴滴入13%的盐酸进行酸化,每加一滴盐酸充分振荡,至溶液变成黑色后立即停止。离心管中加入4mL双蒸水,振荡10秒,静置分层后吸走下层水相。继续加入4mL双蒸水,振荡静置分层后吸走下层水相,重复该步骤数次,用PH试纸测定下层水相的酸碱度在6.8-7.2时停止加入双蒸水。4000g~5000g离心10min,收集上清。避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(b)脱镁叶绿素b(pheophytin b)的制备:称取2mg步骤(3)中分离干燥所得的叶绿素b,于10mL离心管中用4mL无水乙醚溶解配制成500μg/mL。逐滴滴入30%的盐酸进行酸化,每加一滴盐酸充分振荡,至溶液变成黑色后立即停止。后续操作同(a)中所述步骤,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(5)脱植基叶绿素a和脱植基叶绿素b(chlorophyllide a和chlorophyllide b)的制备
(a)粗酶提取液的准备:称取步骤(1)中的新鲜柑橘果皮100g,液氮冷冻后,在研钵或样品破碎仪中磨成细粉末。每g样品粉末中加入1mL-20℃预冷的丙酮,混匀后,超声清洗仪100%功率(100W),冰浴超声10min~15min。4000g~5000g离心5min~10min,弃上清,沉淀继续加入100mL-20℃预冷的丙酮,重复上述冰浴超声、离心、弃上清的过程2~3遍。弃上清后,沉淀在牛皮纸上摊开,通风橱中吹5min至干后倒入研钵,加入液氮,研磨成细粉。沉淀继续加入50mL-20℃预冷的丙酮,超声清洗仪100%功率(100W),冰浴超声10min~15min,4000g~5000g离心5min~10min,弃上清,重复上述冰浴超声、离心和弃上清的过程3~4遍至沉淀完全无色。每100g新鲜柑橘果皮得到的无色沉淀加入50mL缓冲液(丙酮:0.2M Tris-HCl(PH=8.0)=1:1),充分搅匀所得悬浊液即为1份粗酶提取液;
(b)脱植基叶绿素a的制备:称取5mg步骤(3)中分离干燥所得的叶绿素a,无水乙醚溶解配制成500μg/mL,加入1份粗酶提取液混合,用缓冲液补足至100mL。水浴锅中42℃反应2h,期间振荡样品数次或者磁力搅拌器上42℃恒温搅拌反应2h;4000g~5000g离心10min,收集上清。避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(c)脱植基叶绿素b的制备:称取5mg步骤(3)中分离干燥所得的叶绿素b,后续操作同(b)中所述步骤,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(6)脱镁叶绿酸甲酯a和脱镁叶绿酸甲酯b(pheophorbide a和pheophorbide b)的制备
(a)脱镁叶绿酸甲酯a的制备:称取2mg步骤(5)中干燥所得脱植基叶绿素a,10mL离心管中无水乙醚溶解配制成500μg/mL。逐滴滴入13%的盐酸进行酸化,每加一滴充分振荡,至溶液变成黑色后立即停止。离心管中加入4mL双蒸水,振荡10秒,静置分层后吸走下层水相。继续加入4mL双蒸水,振荡静置至分层后吸走下层水相,重复该步骤数次,用PH试纸测定下层水相的pH在6.8-7.8时停止加入双蒸水。4000g~5000g离心10min,收集上清,避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(b)脱镁叶绿酸甲酯b的制备:称取2mg步骤(5)中干燥所得脱植基叶绿素b,后续操作同(a)中所述步骤,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存。
本发明与现有技术相比,具有以下优点和显著的效果:
本发明提供的方法,不仅快速、低成本,而且简单、易操作。本方法所用高效液相色谱(HPLC)为实验室较为常见的仪器,半制备及分析型HPLC均可满足小量分离制备叶绿素a和叶绿素b及其衍生物的需求。这是首次采用柑橘果皮和蔬菜叶片为原料来制备叶绿素及其衍生物。本方法可以在三天内单人次完成全部实验,所得产品纯度可以达到90%-95%,成本远远低于同种进口标准品的价格。可以高效快速制备实验所需毫克级的叶绿素及其衍生物的标准对照品,适合在商业实验室及企业推广使用,具有广泛的应用前景。
具体实施方式
本发明实施例中所述技术方案如未特别说明,均为本领域常规技术。
实施例1:
叶绿素a和叶绿素b的提纯制备
(1)材料准备:将市场上购买来的新鲜甘薯叶摘选叶片,挑除老叶和黄叶并去掉叶柄,洗净后阴暗处晾干备用。将市场上购买来的新鲜温州蜜柑(色差指数CI为-2.0~0.0)清水洗净,晾干,备用;
(2)总叶绿素的提取(以下步骤在避光条件下进行):
a.将(1)中洗净晾干的甘薯叶片称取100g,用液氮冷冻,在研钵或样品破碎仪(IKA,德国)中磨成细粉;
b.将样品粉末倒入500mL离心瓶中(Beckman),加入200mL提取液(N,N-二甲基甲酰胺,含0.1%碳酸镁,w/v)混匀。超声清洗仪100%功率(100W),冰浴超声20min,期间上下混匀样品数次。5000g离心5min,取上清。沉淀重新加入200mL提取液抽提。重复冰浴超声、离心和取上清过程,直至残渣无色,将收集的上清合并(约1000mL);
c.合并的上清分装至250mL分液漏斗中,每个分液漏斗100mL,按1:1的体积比加入正己烷混合,充分振荡后静置1min至完全分层。弃上层正己烷相(含类胡萝卜素),下层放入新的分液漏斗中,上清重新加入100mL正己烷,继续振荡后静置分层,弃上层正己烷相,重复该步骤至上层无色,合并下层液;
d.下层液与正己烷按4:1的体积比混合,剧烈振荡后冰箱中-20℃放置2h。分装至50mL螺口离心管中,5000g离心10min,移除最上层白色乳油状物及中间层正己烷相。按下层液和正己烷1:1的体积比继续加入正己烷,剧烈振荡后5000g离心10min,移除上层,重复该步骤至上层无色,移除上层保留下层液;
e.合并后分装入500mL离心瓶中(Beckman)的下层液,与10%氯化钠水溶液按体积比1:5混合,振荡1min,5000g离心5min,收集上清。剩余下层继续与无水乙醚按10:1的体积比混合,振荡1min,静置至分层,收集上清,重复该步骤一遍,将收集的上清合并。合并的上清分装入250mL分液漏斗中,加入等体积的1:1的正己烷:无水乙醚溶液,混合后,再加入5倍体积的10%氯化钠水溶液,振荡1min,静置至分层,收集合并上层液。合并的上层液加入等体积正己烷,振荡后加入无水硫酸钠至无结块。上清在避光条件下30℃真空浓缩仪中干燥或者氮气吹干,获得叶绿素粗提干燥物约120mg。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(3)叶绿素a和叶绿素b(chlorophyll a和chlorophyll b)的分离:将步骤(2)中所得总叶绿素粗提干燥物称重,用1:1(v:v)的甲醇:丙酮溶液溶解,浓度为2mg/mL,12000g超速离心10min,上清经0.22μm滤膜过滤入上样瓶中。用于分离叶绿素a/b的仪器型号及色谱条件如下:戴安Dionex Ultimate 3000半制备型高效液相色谱,配备Ultimate RSVariable Wavelength Detector检测器;色谱柱:COSMOSIL,5C18-MS-Ⅱ,10mm id.×250mm;流动相A为甲醇,流动相B为甲醇:丙酮=1:1;用50%流动相A+50%流动相B等度洗脱,3.0毫升/min,B等度洗脱,检测波长为654nm和665nm。叶绿素a的出峰时间约为9.3min,叶绿素b的出峰时间约为7.7min,收集chlorophyll a的色谱峰和chlorophyll b的色谱峰的洗脱馏分,上清在避光条件下30℃真空浓缩仪中干燥或者氮气吹干。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存,分离得到的叶绿素a和叶绿素b的指标详见表1。
实施例2:
叶绿素a和叶绿素b的降解产物的制备
(1)脱镁叶绿素a和脱镁叶绿素b(pheophytin a和pheophytin b)的制备
(a)脱镁叶绿素a(pheophytin a)的制备:称取2mg实施例1步骤(3)中分离干燥所得的叶绿素a,于10mL离心管中用4mL无水乙醚溶解配制成500μg/mL。逐滴滴入13%的盐酸进行酸化,每加一滴盐酸充分振荡,至溶液变成黑色后立即停止。离心管中加入4mL双蒸水,振荡10秒,静置分层后吸走下层水相。继续加入4mL双蒸水,振荡静置分层后吸走下层水相,重复该步骤数次,用PH试纸测定下层水相的酸碱度在6.8-7.2时停止加入双蒸水。4000g~5000g离心10min,收集上清。避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,2mg的叶绿素a获得约1.8mg脱镁叶绿素a。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(b)脱镁叶绿素b(pheophytin b)的制备:称取2mg实施例1步骤(3)中分离干燥所得的叶绿素b,于10mL离心管中用4mL无水乙醚溶解配制成500μg/mL。逐滴滴入30%的盐酸进行酸化,每加一滴盐酸充分振荡,至溶液变成黑色后立即停止。后续操作同(a)中所述步骤,2mg的叶绿素b获得约1.8mg脱镁叶绿素b。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
以上分离得到的脱镁叶绿素a和脱镁叶绿素b的指标见表1。
(2)脱植基叶绿素a和脱植基叶绿素b(chlorophyllide a和chlorophyllide b)的制备
(a)粗酶提取液的准备:将实施例1步骤(1)中准备的新鲜温州蜜柑自果皮表层用手术刀削取0.2~0.3mm厚度的表皮。称取100g表皮,用液氮冷冻,放入研钵中继续加入液氮研磨成细粉末,装入500mL离心瓶中(Beckman)中。加入100mL-20℃预冷的丙酮,混匀后,超声清洗仪100%功率(100W),冰浴超声10min。4000g离心5min,弃上清,沉淀继续加入100mL-20℃预冷的丙酮,重复上述冰浴超声、离心、弃上清的过程2遍。弃上清后,沉淀在牛皮纸上摊开,通风橱中吹5min至干后倒入研钵,加入液氮,研磨成细粉(越细越好)。沉淀继续加入50mL-20℃预冷的丙酮,超声清洗仪100%功率(100W),冰浴超声10min,4000g离心5min,弃上清,重复上述冰浴超声、离心和弃上清的过程4遍至沉淀完全无色。每100g新鲜柑橘果皮得到的无色沉淀加入50mL缓冲液(丙酮:0.2M Tirs-HCl(PH=8.0)=1:1),充分搅匀所得悬浊液即为1份粗酶提取液;
(b)脱植基叶绿素a的制备:称取5mg实施1例步骤(3)中分离干燥所得的叶绿素a,无水乙醚溶解配制成500μg/mL,加入1份粗酶提取液混合,用缓冲液补足至100mL。水浴锅中42℃反应2h,期间振荡样品数次或者磁力搅拌器上42℃恒温搅拌反应2h;4000g离心10min,收集上清。避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,5mg的叶绿素a获得约3.4mg脱植基叶绿素a。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(c)脱植基叶绿素b的制备:称取5mg实施1例步骤(3)中分离干燥所得的叶绿素b,后续操作同(b)中所述步骤,5mg的叶绿素b获得约3.2mg脱植基叶绿素b。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
以上分离得到的脱植基叶绿素a和脱植基叶绿素b的指标见表1。
(3)脱镁叶绿酸甲酯a和脱镁叶绿酸甲酯b(pheophorbide a和pheophorbide b)的制备
(a)脱镁叶绿酸甲酯a的制备:称取2mg步骤(2)中干燥所得脱植基叶绿素a,10mL离心管中无水乙醚溶解配制成500μg/mL。逐滴滴入13%的盐酸进行酸化,每加一滴充分振荡,至溶液变成黑色后立即停止。离心管中加入4mL双蒸水,振荡10秒,静置分层1min后吸走下层水相。继续加入4mL双蒸水,振荡静置至分层后吸走下层水相,重复该步骤数次,用PH试纸测定下层水相的酸碱度在6.8-7.2时停止加入双蒸水。4000g离心10min,收集上清,避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,2mg的叶绿素a获得约1.7mg脱镁叶绿酸甲酯a。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(b)脱镁叶绿酸甲酯b的制备:称取2mg步骤(2)中干燥所得脱植基叶绿素b,后续操作同(a)中所述步骤,2mg的叶绿素b获得获得约1.7mg脱镁叶绿酸甲酯b。样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存。
以上分离得到的脱镁叶绿酸甲酯a和脱镁叶绿酸甲酯b的指标见表1。
表1各种制备物纯度检测结果如下:
Claims (3)
1.一种快速制备脱植基叶绿素a和脱植基叶绿素b的方法,包括:
(1)材料准备:新鲜蔬菜叶片和新鲜柑橘果实洗净晾干,将新鲜柑橘自果皮表层削取的0.2~0.3mm厚度的表皮,作为新鲜柑橘果皮,备用;
(2)总叶绿素的提取,以下步骤在避光条件下进行:
a.将(1)中洗净晾干的蔬菜叶片称取100g,用液氮冷冻,在研钵或样品破碎仪中磨成细粉;
b.将样品粉末与提取液按1g:2mL的比例混合;超声清洗仪100%功率,冰浴超声20min~30min,期间上下混匀样品数次;4000g~5000g离心5min~10min,取上清;沉淀按上述样品粉末与提取液的比例重新加入提取液抽提;重复冰浴超声、离心和取上清过程,直至残渣无色,将收集的上清合并;
所述的提取液为:N,N-二甲基甲酰胺溶液,含0.1%碳酸镁,w/v;
c.合并的上清分装至250mL分液漏斗中,每个分液漏斗100mL,按1:1的体积比加入正己烷混合,充分振荡后静置至完全分层;弃上层正己烷相,下层放入新的分液漏斗中,按上述上清与正己烷的体积比重新加入正己烷,继续振荡后静置分层,弃上层正己烷相,重复该步骤至上层无色,合并下层液;
d.下层液与正己烷按4:1的体积比混合,剧烈振荡后-20℃冰箱中放置2~3h;4000g~5000g离心10min~15min,移除最上层白色乳油状物及中间层正己烷相;
按下层液和正己烷1:1的体积比继续加入正己烷,剧烈振荡后4000g~5000g离心10min,移除上层,重复该步骤至上层无色,移除上层保留下层液;
e.下层液与10%氯化钠水溶液按体积比1:5~6混合,振荡1min,4000g~5000g离心5min,收集上清;剩余下层继续与无水乙醚按10:1的体积比混合,振荡1min,静置至分层,收集上清,重复该步骤一遍,将收集的上清合并;加入等体积的1:1,v/v的正己烷:无水乙醚溶液混合后,再加入5倍体积的10%氯化钠水溶液,振荡1min,静置至分层,合并收集的上清;加入与上清等体积的正己烷,振荡后加入无水硫酸钠至无结块;上清在避光条件下30℃真空浓缩仪中干燥或者氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存,得叶绿素粗提干燥物;
(3)叶绿素a和叶绿素b的分离:称取步骤(2)中所得总叶绿素粗提干燥物10mg,用1:1,v/v的甲醇:丙酮溶解,配成终浓度2mg/mL,8000g~12000g离心10min~15min,上清经0.22μm微孔滤膜过滤后分装至2mL上样瓶中;
按常规方式的液相色谱分离法进行叶绿素a和叶绿素b的分离;
(4)脱植基叶绿素a和脱植基叶绿素b的制备
(a)粗酶提取液的准备:称取步骤(1)中的新鲜柑橘果皮100g,液氮冷冻后,在研钵或样品破碎仪中磨成细粉末;
每克样品粉末中加入1mL-20℃预冷的丙酮,混匀后,超声清洗仪100%功率,冰浴超声10min~15min;4000g~5000g离心5min~10min,弃上清,沉淀继续加入100mL-20℃预冷的丙酮,重复上述冰浴超声、离心、弃上清的过程2~3遍;弃上清后,沉淀在牛皮纸上摊开,通风橱中吹5min至干后倒入研钵,加入液氮,研磨成细粉;沉淀继续加入50mL-20℃预冷的丙酮,超声清洗仪100%功率,冰浴超声10min~15min,4000g~5000g离心5min~10min,弃上清,重复上述冰浴超声、离心和弃上清的过程3~4遍至沉淀完全无色;每100g新鲜柑橘果皮得到的无色沉淀加入50mL缓冲液,充分搅匀所得悬浊液即为1份粗酶提取液;
所述的缓冲液为:丙酮:0.2M Tris-HCl=1:1,v/v,其中0.2M Tris-HCl的PH为8.0;
(b)脱植基叶绿素a的制备:称取5mg步骤(3)中分离干燥所得的叶绿素a,无水乙醚溶解配制成500μg/mL,加入1份粗酶提取液混合,用缓冲液补足至100mL;水浴锅中42℃反应2h,期间振荡样品数次或者磁力搅拌器上42℃恒温搅拌反应2h;4000g~5000g离心10min,收集上清;避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存;
(c)脱植基叶绿素b的制备:称取5mg步骤(3)中分离干燥所得的叶绿素b,后续操作同(b)中所述步骤,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存。
2.权利要求1所述的方法,其特征在于:步骤(1)中所述的蔬菜叶片为将市场上购买来的新鲜蔬菜摘选叶片,挑除老叶和黄叶并去掉叶柄,洗净后阴暗处晾干;所述的新鲜柑橘果皮为将市场上购买来的新鲜柑橘清水洗净,晾干,削取表皮,厚度为0.2~0.3mm。
3.权利要求1所述的方法,其特征在于:步骤(3)中进行叶绿素a和叶绿素b的分离时,实验室常见的分析型、半制备型或者制备型的液相色谱仪使用常用的C18色谱柱,均用于叶绿素a和叶绿素b的分离;流动相A为甲醇,流动相B为甲醇:丙酮=1:1;用50%流动相A+50%流动相B等度洗脱,分别收集chlorophyll a色谱峰和chlorophyll b色谱峰的馏分,避光条件下,30℃真空浓缩仪中干燥或者常温下氮气吹干,样品用锡箔纸包裹避光,冰箱中-80℃长期保存或者-20℃短期保存。
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