CN112535226A - 朴树及其提取物在银杏相关产品中的应用 - Google Patents
朴树及其提取物在银杏相关产品中的应用 Download PDFInfo
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- CN112535226A CN112535226A CN202011217613.2A CN202011217613A CN112535226A CN 112535226 A CN112535226 A CN 112535226A CN 202011217613 A CN202011217613 A CN 202011217613A CN 112535226 A CN112535226 A CN 112535226A
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- hackberry
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- extract
- ginkgo
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Abstract
朴树(Celtis sinensis Pers.)及其提取物在银杏相关产品中的应用,本发明分离得到的朴树系列提取物能够减轻银杏酸对HepG2细胞的增殖抑制作用,可将细胞存活率从26.7%提高到63.0%;对T细胞介导炎症反应有较好的抑制作用,对Con A活化的T细胞增殖抑制率最高可达95.4%;朴树叶乙醇提取物能够有效抑制银杏酸导致的过敏反应。利用朴树提取物或直接利用朴树叶加入到银杏茶等制品能有效起到减毒增效作用。本发明为银杏茶、白果等含有银杏酸产品的安全应用提供了新方法,为低毒银杏茶产品的开发提供了一种新的解决方案。
Description
技术领域
本发明涉及保健食品领域,尤其涉及朴树及其提取物在银杏相关产品中的应用。
背景技术
银杏叶富含黄酮类化合物、萜内酯类化合物、聚戊烯醇等活性物质,目前市场上有很多以银杏叶为原料制成的茶叶或茶饮品,对人体心脑血管和神经系统等方面有一定保健功效,且抗疲劳,抗衰老,广受追求健康的现代消费者欢迎。银杏茶中除了含有活性物质、蛋白质、氨基酸、矿物质、维生素等营养成分,还含有一定量的银杏酸。银杏酸可引起严重的炎症反应,并表现在肝损伤和致敏等方面。所以,在国内外的银杏提取物(EGB)质量标准中,主要质控指标除规定了活性成分的含量外(银杏总黄酮≥24%,银杏总内酯≥6%),还对银杏叶中银杏酸的含量进行了严格的限制。例如,2015版《中国药典》对银杏叶提取物中总银杏酸的要求是不得超过10mg/kg。因此,在制备EGB的过程中,需要通过分离、纯化等工艺将产品中残留的银杏酸控制在质量标准范围,但这些方法难以应用到银杏茶、白果等银杏制品的加工工艺中,需要研发其他方法保证其食用的安全性。到目前为止,直接将银杏叶制备成茶,在冲泡过程中,银杏酚酸能溶出多少、有无危害、如何防控等问题尚未得到系统阐述和有效解决。
为了实现银杏茶等银杏食用产品的减毒增效,通过大量筛选和深入研究,本发明对朴树 (Celtis sinensis Pers.)叶、枝和皮中活性成分进行了提取和分离,筛选出能够减弱银杏酸的肝细胞毒性和抑制T细胞介导的炎症反应的活性组分,为银杏茶等银杏产品的合理应用提供了技术支撑。
发明内容
解决的技术问题:本发明针对银杏茶在冲泡过程中浸出的银杏酸的量与潜在的安全隐患,提供了一种朴树及其提取物在银杏相关产品中的应用,该提取物可与银杏茶配伍减毒,减少银杏酸带来的潜在肝损伤和致敏性风险。
技术方案:朴树(Celtis sinensis Pers.)及其提取物在制备银杏产品中的应用。
朴树提取物在制备银杏茶中的应用。
上述朴树提取物为朴树叶、朴树枝或朴树皮的提取物。
朴树叶成分的提取方法为:朴树叶磨成粉,用70wt.%乙醇热回流提取3次,减压浓缩除去乙醇后,依次用二氯甲烷、乙酸乙酯和正丁醇萃取,分别筛选得到正丁醇萃取组分、水溶性组分2个有效组分。
朴树枝成分的提取方法为:朴树枝磨成粉,分别用30wt.%、60wt.%和90wt.%的乙醇热回流提取,提取液经减压浓缩出去乙醇,将所得水溶液用20%HCl调至pH 3~4,随后用石油醚萃取,剩下的酸水液用20wt.%NaOH调至pH 9.2,再用二氯甲烷萃取,在此分离过程中,共收集、干燥、筛选得到90%醇提碱化后得到的沉淀、30%醇提上层的水溶性组分、60%醇提CH2Cl2萃取后水溶性成分、90%醇提碱溶后萃取得到的生物碱、90%醇提CH2Cl2萃取后水溶性成分。
朴树皮成分的提取方法为:朴树枝磨成粉,用70wt.%的乙醇热回流提取,提取液经减压浓缩出去乙醇,将所得水溶液用20wt.%HCl调至pH 3~4,随后用石油醚萃取,剩下的酸水液用20wt.%NaOH调至pH 9.2,再用二氯甲烷萃取,在此分离过程中,共收集、干燥、筛选得到树皮提取液与石油醚萃取的脂溶性混合成分、树皮CH2Cl2萃取后水溶性成分、树皮提取得到的生物碱组分。
用HPLC法测定银杏茶浸出的银杏酸种类及含量;用人HepG2肝细胞增殖测定银杏酸对肝细胞的毒性作用,小鼠炎症模型考察银杏酸的致敏性;利用植物化学研究手段,对朴树叶、枝和皮中的活性成分进行提取和分级分离,比较收集到的各组分对银杏酸的减毒效果。
银杏茶冲泡过程中浸出银杏酸的测定:按照银杏茶商品说明,用200ml开水泡茶并保温 15min,过滤收集茶水,向茶叶渣中再次加入200ml开水保温15min,过滤收集茶水,如此反复冲泡3次,将3次茶水减压浓缩至干燥,加适量甲醇溶解后经HPLC检测分析其中的银杏酸种类及含量。
银杏酸的毒性测试:用MTT法检测HepG2细胞的增殖,分析银杏酸的肝细胞毒性;建立小鼠接触性皮炎模型,考察银杏酸的接触性致敏性;建立足垫注射模型,考察银杏酸对近端淋巴结(免疫器官)的影响。
有益效果:本发明分离得到的朴树系列提取物能够减轻银杏酸对HepG2细胞的增殖抑制作用;对T细胞介导炎症反应有较好的抑制作用;朴树叶乙醇提取物能够有效抑制银杏酸导致的过敏反应。利用朴树提取物或直接利用朴树叶加入到银杏茶等制品能有效起到减毒增效作用。本发明为银杏茶、白果等含有银杏酸产品的安全应用提供了新方法,为低毒银杏茶产品的开发提供了一种新的解决方案。
附图说明
图1:4种银杏茶冲泡过程中浸出银杏酸的含量;
图2:银杏茶中银杏酸的组成;
图3:银杏酸(左:总银杏酸;右:银杏酸单体)的肝细胞毒性;
图4:银杏酸对免疫系统的影响;A:注射了银杏酸的实验组小鼠的腘窝淋巴结重量显著升高;B:淋巴结中淋巴细胞数目显著增多;C:结果表明银杏酸可导致小鼠耳肿胀;
图5:朴树提取物对银杏酸导致的肝损伤的抑制作用;其中A结果表明,朴树叶的BA相高剂量、Water相高剂量、朴树枝2#高剂量、6#低剂量和朴树皮的14#提取物低剂量可以显著减轻银杏酸C13:0对HepG2肝细胞增殖的抑制作用;B结果表明,朴树叶的BA相高剂量、Water相高剂量、朴树枝的2#低剂量、10#和朴树皮的14#提取物高剂量可以显著减轻银杏酸C15:1对HepG2肝细胞增殖的抑制作用。
图6:朴树提取物对T细胞介导的炎症反应的抑制作用;
图7:朴树叶的提取分离路线图。
具体实施方式
下面结合附图及具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明,而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
本发明的目的在于提供一种解决银杏酸导致的毒副作用问题的方法,旨在提升银杏茶、白果等难以脱除银杏酸的产品的使用安全性。
在本发明实施例中,银杏茶在冲泡过程中浸出的银杏酸含量较高(超过10mg/kg),具有一定的安全隐患,银杏酸可以到导致不同程度的肝损伤和免疫系统紊乱。朴树提取物可以减轻银杏酸导致的上述毒副作用。
实施例1
1.实验目的
比较不同品牌银杏茶在冲泡过程中浸出的银杏酸含量。
2.实验材料
银杏茶分别从湖北、江苏、浙江和山东四处产地购买,其中湖北和江苏的银杏茶为叶片状,浙江和山东的银杏茶为粉末状茶包;总银杏酸和白果新酸标准品购于南京景竹生物科技有限公司。
3.实验方案
银杏茶冲泡时总银杏酸的提取方法:分别取四种不同产地的银杏茶各15g,精密称量,装入保温杯,加入100℃开水200mL泡茶,于恒温水浴锅中保温15min,过滤,分开收集茶水和茶叶渣。向茶叶渣中继续加入100℃开水200mL,处理方法与第一次泡茶相同,重复泡三次,共得12份茶叶水,用旋转蒸发仪将水蒸干,残渣加甲醇适量使其充分溶解,放置冷却,摇匀即得。
HPLC色谱条件:安捷伦XDB-C18色谱柱(4.6×250mm,5μm),柱温30℃,梯度洗脱(以甲醇为流动相A,水为流动相B,0~30min,50%-80%A;30~33min,80%-50%A),紫外检测器,检测波长为360nm,流速0.8mL/min,进样量10μL,运行时间37min。
总银杏酸含量的计算方法:参照2015版《中国药典》第一部中银杏叶提取物的总银杏酸测定和计算方法,以白果新酸对照品外标法计算总银杏酸含量。
4.实验结果
开水泡银杏茶后,分别取3次茶水进行蒸干、醇溶、HPLC检测分析。结果如图1所示,每次泡茶都会有一定量的银杏酸浸出。产地不同、形态不同的四种银杏茶泡出的银杏酸量存在很大差异,其中粉末状茶包(产地为浙江和山东)泡出的银杏酸要高于叶片银杏茶(产地为湖北和江苏)。通过与标准品比较发现,银杏茶泡出的银杏酸包括C13:0、C15:1、C17:2、C15:0和C17:1(图2)。以1g银杏茶为例,经三次开水冲泡后,不同产地茶水中总银杏酸含量分别为:湖北2.8019mg,江苏1.5967mg,浙江5.1552mg,山东8.3514mg。由此可见,银杏茶在冲泡过程中,不仅溶出了银杏黄酮和银杏内酯等活性物质,同时也会溶出毒性物质 --银杏酸,且含量较高。
实施例2:
1.实验目的
阐明银杏酸的肝细胞毒性和免疫毒性。
2.实验材料
DMEM培养基、RPMI 1640培养基、PBS磷酸缓冲液(Gibco);胎牛血清(Fetal BovineSerum,FBS,Hyclone);胰酶消化液(碧云天生物技术);台盼蓝(阿拉丁试剂);噻唑蓝(MTT,Sigma);人肝癌细胞HepG2购于中国科学院典型培养物保藏委员会细胞库。
清洁级Balb/c小鼠,雌性,6周龄,体重20±2g,由扬州大学比较医学中心提供。随机分成2组,每组8只。分为对照组(Sham.)、银杏酸实验组(S.c.),在21±2℃、自由取食、自由饮水和12h昼夜交替的条件下饲养。动物的实验操作严格按照善待实验动物的指导性意见(中华人民共和国国家科学技术委员会,2006),实验过程中所有的操作,均尽可能减轻动物的痛苦,损伤和使用数量。
3.实施方案
3.1 MTT法检测HepG2细胞增殖
HepG2细胞培养条件为DMEM(含10%FBS),37℃,5%CO2的无菌条件下培养。在 96孔板中每孔接种4×103个HepG2细胞100μL,DMEM完全培养基,置于37℃培养(5%CO2),待细胞贴壁6h后,每孔加不同浓度药物再培养72h。终止培养前4h加入20μL MTT(4mg/mL) 溶液。4h后吸去上清,每孔加入200μL DMSO,振荡,540nm处检测吸光度,并记录读数。存活率(%)=(ODcontrol-ODsample)/ODcontrol×100
3.2小鼠腘窝淋巴结模型
两组分别于D0在小鼠右下足垫进行皮下注射,银杏酸组给以1mg总银杏酸20μL,对照组给以等体积溶剂。七天后,以颈椎脱臼法处死小鼠,迅速取出注射近端的腘窝淋巴结(PLN),去除脂肪组织,立刻称重。
3.3小鼠接触性皮炎模型
动物分组:对照组(Sham.)、银杏酸实验组(S.c.)。
致敏:银杏酸组于D0、D7和D14采用背部三点注射法给每只小鼠注射0.15mg总银杏酸(300μL,溶剂PBS:FCA=1:1),对照组采用同样的方法注射等量溶剂。
激发:银杏酸组于最后一次致敏后的第7日即D21在小鼠右耳耳廓内外两侧涂3%总银杏酸(50μL/只,丙酮溶解)进行攻击,对照组涂等量溶剂。攻击后24h,用螺旋测微仪量小鼠左右耳厚,根据右耳减去左耳的厚度差判断小鼠的皮炎程度。
4.实验结果
银杏茶中银杏酸主要有5种C13:0、C15:0、C15:1、C17:1和C17:2,其中以C13:0、C15:1 和C17:1的含量最丰富。银杏类产品被报道的副作用主要为肝损伤和过敏反应,因此我们首先考察了银杏酸对HepG2肝细胞增殖的影响。由图3(左)的结果可见,总银杏酸在100μg/mL 的浓度下对HepG2细胞的体外增殖有显著抑制作用;其中主要的细胞毒性成分可能为银杏酸 C13:0和C15:1,银杏酸C17:1对HepG2细胞的增殖几乎没有影响(图3,右)。我们将总银杏酸注射到小鼠足垫,7天后取足垫附近的腘窝淋巴结观察,发现银杏酸可导致淋巴结肿大。与注射同体积溶剂的对照组比较,注射了银杏酸的实验组小鼠的腘窝淋巴结重量显著升高(图4:A),淋巴结中淋巴细胞数目显著增多(图4:B)。采用小鼠耳肿胀模型,考察银杏酸是否能导致小鼠接触性皮炎。先通过背部多点注射银杏酸的方式诱导,7天后将银杏酸涂耳朵攻击,于攻击10h后量小鼠耳厚。图4:C结果表明银杏酸可导致小鼠耳肿胀。综上结果表明,银杏酸不仅会导致肝损伤,还会对免疫系统产生刺激,引起炎症细胞过度增殖,导致外周免疫器官——淋巴结肿大,并在接触部位表现出炎症反应。
实施例3:
1.实验目的
获得一系列朴树提取物。
2.实验材料
朴树叶从贵州采摘,经鉴定为野生品种;甲醇、乙酸乙酯、二氯甲烷、正丁醇等试剂(分析纯,购自南京姜华化玻仪器有限公司)。
3.实验方案
3.1朴树叶提取物的制备
朴树中的活性成分的提取分离如图7所示。向100g朴树叶粉末加入100mL乙醇,静置过夜,次日补加70%乙醇,按料液比1:4(g:mL)于50℃回流提取3次,每次提取2h。合并提取液经旋转蒸发除去乙醇,用体积比1:1的二氯甲烷萃取3次,得到二氯甲烷萃取组分(CH2Cl2);然后,水溶液用乙酸乙酯萃取3次,得到乙酸乙酯萃取组分(EA);最后,水溶液用正丁醇萃取3次,得到正丁醇萃取组分(BA);剩下的为水溶性组分(Water)。
3.2朴树枝提取物的制备
将朴树枝进行除尘、晾干并烘干后,置于粉碎机中进行粉碎,粉碎成40目。称重,将朴树枝粉末均分成3份,分别用30%、60%、90%的乙醇提取,料液比为1:20,回流提取4h,过滤,得提取液。浓缩蒸至无醇味,将所得水溶液用20%HCl调至pH 3~4,得到的酸水液用等体积石油醚萃取,然后用20%NaOH将酸水液调至pH 9.2,再用等体积二氯甲烷萃取,收集二氯甲烷层。通过上述提取,共收集、干燥得到以下10个组分:
01#:30%醇提(未蒸干醇)得到的生物碱;
02#:90%醇提碱化后得到的沉淀;
03#:30%醇提上层的水溶性组分;
04#:60%醇提CH2Cl2萃取后水溶性成分;
05#:30%醇提(蒸干至无醇)得到的生物碱;
06#:60%醇提CH2Cl2与水溶的混合性成分;
07#:60%醇提正常得到的生物碱;
08#:90%醇提碱溶后萃取得到的生物碱;
09#:90%醇提CH2Cl2与水的混合性成分;
10#:90%醇提CH2Cl2萃取后水溶性成分。
3.3朴树皮提取物的制备
将朴树外皮进行除尘、晾干并烘干后,置于粉碎机中进行粉碎,粉碎成40目。称重,用 70%的乙醇提取朴树皮粉末,后续处理同3.2。通过以上提取,共收集、干燥得到以下6个组分:
11#:树皮碱化后的沉淀;
12#:树皮脂溶性成分;
13#:树皮提取液水与脂溶性混合成分(石油醚萃取);
14#:树皮CH2Cl2萃取后水溶性成分;
15#:树皮提取液水与脂溶性混合成分(CH2Cl2萃取);
16#:树皮提取得到的生物碱。
4.实验结果
大量研究表明,朴树中含有黄酮、皂苷、生物碱等成分。因此,本发明分别按照黄酮、皂苷和生物碱的提取方法对朴树叶、枝和皮中的化学成分进行提取和分离。通过颜色反应,鉴定发现朴树叶中含有黄酮、皂苷类成分,朴树皮和枝中含有生物碱类成分。用芦丁法计算得到,朴树叶中总黄酮质量分数约为10.76%;用齐墩果酸法计算得到,朴树叶中总三萜质量分数为2.38%。用不同极性的溶剂对朴树叶醇提物进行萃取,分别得到二氯甲烷萃取组分 (CH2Cl2)、乙酸乙酯萃取组分(EA)、正丁醇萃取组分(BA)和水溶性(Water)组分;其中,乙酸乙酯可将朴树叶提取物中的黄酮苷元、萜类等成分萃取出来,正丁醇可将皂苷、黄酮糖苷等成分萃取出来。同时参照生物碱的提取步骤,对朴树枝和朴树皮中的物质进行提取和分离,得到16个组分(1#~16#)。
实施例4:
1.实验目的
研究朴树提取物对银杏酸的减毒作用。
2.实验材料
同实施例2;朴树提取物为实施例3中实验获得。
3.实验方案
3.1朴树提取物对银杏酸导致的HepG2细胞毒性影响的测定
HepG2细胞培养条件为DMEM(含10%FBS),37℃,5%CO2的无菌条件下培养。在 96孔板中每孔接种4×103个HepG2细胞100μL,DMEM完全培养基,置于37℃培养(5%CO2),待细胞贴壁6h后,每孔依次加入100μM的银杏酸和100μg/mL的朴树提取物再培养72h,对照孔仅补加100μL的培养基。终止培养前4h加入20μL MTT(4mg/mL)溶液。4h后吸去上清,每孔加入200μL DMSO,振荡,540nm处检测吸光度,并记录读数。存活率(%) =(ODcontrol-ODsample)/ODcontrol×100
3.2朴树提取物体外抗炎活性测定
从小鼠淋巴结分离得到T淋巴细胞,以5×105个细胞、50μL/孔种96孔板,1640完全培养基,置于37℃培养(5%CO2),随后每孔加入5μg/mL、50μL Con A溶液,对照孔补加50 μL培养基;将朴树提取物配置成所需浓度,向Con A活化的T细胞各孔分别加入不同浓度的朴树提取物100μL,对照孔补加100μL培养基。培养箱培养48h后,加入20μL MTT(4mg/mL) 溶液。4h后吸去上清,每孔加入200μL DMSO,振荡,540nm处检测吸光度,并记录读数。存活率(%)=(ODcontrol-ODsample)/ODcontrol×100
4.实验结果
图5:A结果表明,HepG2细胞与100μmol/L的银杏酸C13:0共培养72h后存活率仅有23.4%,与100μmol/L的银杏酸C15:1共培养72h后存活率仅有26.7%,表明HepG2细胞增殖被显著抑制;将朴树叶的BA相高剂量、Water相高剂量、朴树枝2#高剂量、6#低剂量和朴树皮的14#提取物低剂量加入培养体系后,可以将银杏酸C13:0损伤的HepG2细胞存活率分别提高到28.8%、30.2%、63.0%、29.1%和33.0%,显著减轻银杏酸C13:0对HepG2肝细胞增殖的抑制作用;将朴树叶的BA相高剂量、Water相高剂量、朴树枝的2#低剂量、10#和朴树皮的14#提取物高剂量加入培养体系后,可以将银杏酸C15:1损伤的HepG2细胞存活率分别提高到32.5%、35.5%、58.4%、56.9%和34.8%,显著减轻银杏酸C15:1对HepG2肝细胞增殖的抑制作用。
图6结果表明,将Con A活化48h后T细胞存活率计为100%时,添加朴树枝的3#、4#、8#和朴树皮的13#、16#提取物后,Con A活化的T细胞存活率可分别降低至6.76%、6.80%、 4.58%、56.9%和5.30%,表明朴树提取物对炎症性T细胞增殖有显著的抑制作用。其中,以 8#和16#的抑制作用最好且呈现剂量依赖性,提示朴树中存在抗炎活性物质,有望用于抗银杏酸过敏的治疗。
Claims (6)
1.朴树(Celtis sinensis Pers.)及其提取物在制备银杏产品中的应用。
2.朴树提取物在制备银杏茶中的应用。
3.根据权利要求1或2所述的应用,其特征在于所述朴树提取物为朴树叶、朴树枝或朴树皮的提取物。
4.根据权利要求3所述的应用,其特征在于朴树叶成分的提取方法为:朴树叶磨成粉,用70wt.%乙醇热回流提取3次,减压浓缩除去乙醇后,依次用二氯甲烷、乙酸乙酯和正丁醇萃取,分别筛选得到正丁醇萃取组分、水溶性组分2个有效组分。
5.根据权利要求3所述的应用,其特征在于朴树枝成分的提取方法为:朴树枝磨成粉,分别用30wt.%、60wt.%和90wt.%的乙醇热回流提取,提取液经减压浓缩出去乙醇,将所得水溶液用20% HCl调至pH 3~4,随后用石油醚萃取,剩下的酸水液用20wt.% NaOH调至pH 9.2,再用二氯甲烷萃取,在此分离过程中,共收集、干燥、筛选得到90%醇提碱化后得到的沉淀、30%醇提上层的水溶性组分、60%醇提CH2Cl2萃取后水溶性成分、90%醇提碱溶后萃取得到的生物碱、90%醇提CH2Cl2萃取后水溶性成分。
6.根据权利要求3所述的应用,其特征在于朴树皮成分的提取方法为:朴树枝磨成粉,用70wt.%的乙醇热回流提取,提取液经减压浓缩出去乙醇,将所得水溶液用20wt.% HCl调至pH 3~4,随后用石油醚萃取,剩下的酸水液用20wt.% NaOH调至pH 9.2,再用二氯甲烷萃取,在此分离过程中,共收集、干燥、筛选得到树皮提取液与石油醚萃取的脂溶性混合成分、树皮CH2Cl2萃取后水溶性成分、树皮提取得到的生物碱组分。
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