CN114766519B - 黄芩提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途 - Google Patents
黄芩提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/22—Lamiaceae or Labiatae [Mint family], e.g. thyme, rosemary, skullcap, selfheal, lavender, perilla, pennyroyal, peppermint or spearmint
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
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- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
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- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
本发明公开了一种黄芩甲醇提取物,以及该黄芪甲醇提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途。本发明中的黄芩为常见大宗中药材,在我国来源广,成本低,用量少,具有清热燥湿、泻火解毒、止血、安胎的功效。黄芩甲醇提取物可以有效抑制PDA培养基、玉米培养基和小麦培养基中脱氧雪腐镰刀菌烯醇的生物合成,效果好,安全无毒副作用。
Description
技术领域
本发明涉及农产品质量安全领域,具体的说涉及黄芩提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途。
背景技术
黄芩,别名山茶根、土金茶根,是唇形科黄芩属多年生草本植物。黄芩是我国传统中药材之一,其主要活性成分为黄芩苷、汉黄芩苷、黄芩素、汉黄芩素等黄酮类化合物,具有清热燥湿、泻火解毒、止血、安胎的功效。现代药学研究表明,黄芩在抗炎、抗肿瘤、降血压等药理方面发挥了良好的功效。
脱氧雪腐镰刀菌烯醇(DON),又名呕吐毒素,主要由禾谷镰刀菌(Fusariumgraminearum)和黄色镰刀菌(Fusarium culmorum)产生的单端孢霉烯族类毒素,广泛存在于小麦、玉米等粮食作物中,具有很高的细胞毒性和免疫毒性,会导致厌食、呕吐、腹泻、发烧、站立不稳、反应迟钝等急性中毒症状,严重时损害造血系统造成死亡。
鉴于DON的强烈毒性和广泛分布性,国内外已经制定了不同农产品、食品和饲料中DON的限量标准。为更好的防控DON污染,减少财产损失,保障人民健康和生命安全,开发能够高效抑制DON产生、安全、无毒的抑菌剂具有非常重要的经济价值和社会意义。
发明内容
本发明首先提供了一种黄芩甲醇提取物,该黄芩甲醇提取物是通过如下方法制备得到的:
将黄芩粉碎,过0.42mm试验筛;
加入甲醇混匀,黄芩粉末与甲醇的质量体积比(g:mL)为1:1-100,36-44℃超声10-100min,用纱布过滤,4500rpm离心15min后取上清;
可重复上述步骤,合并取得的上清液;
用旋转蒸发仪减压浓缩,回收甲醇,得到黄芩甲醇提取物。
其中,黄芩粉末与甲醇的质量体积比(g:mL)优选为1:4-100。
本发明还提供了上述黄芩甲醇提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途。
黄芩甲醇提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂时,加水配制成浓度为1-100g/L的溶液;
优选的浓度为2-50g/L。
本发明提供的黄芩甲醇提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途,其具备以下优点:
(1)本发明首次发现了黄芩甲醇提取物在不同基质中可以有效抑制DON的生成;
(2)本发明的黄芩为常见大宗中药材,来源广、价格低廉,形成的抑制剂生成工艺简易、成本低,能够大规模应用;
(3)本发明的黄芩具有清热燥湿、泻火解毒、止血、安胎的功效,在抗炎、抗肿瘤、降血压等药理方面发挥了良好的作用,形成的抑制剂对人畜安全性好,对环境无污染;
(4)本发明的黄芩甲醇提取物为复合提取物,与现有的其它单种成分抑制剂相比,抑制效果更为显著、成本更低、且不易导致产毒真菌产生抗药性,适合长期使用;
(5)本发明的抑制剂使用简单,普通人员简单培训即可使用,有益于大规模推广。
附图说明
图1不同浓度黄芩甲醇提取物对PDA培养基中DON生成的影响
图2不同浓度黄芩甲醇提取物对小麦培养基中DON生成的影响
具体实施方式
下列实施例中所用到的材料及其来源或制备方法。
(1)试剂
葡萄糖、琼脂(上海源叶生物科技有限公司);
乙腈、甲醇、乙酸铵(美国Merck公司);
黄芩(安徽道源堂中药饮片有限公司);
DON标准品(纯度大于99%,美国Romer公司)
Agilent Poroshell 120EC-C18色谱柱(美国安捷伦科技有限公司)
玉米、小麦(普通市售产品)
(2)仪器
超高效液相色谱仪(美国Waters公司);
TRIPLE QUADTM 5500三重四级杆质谱仪(美国AB SCIEX公司);
HSC-24B氮吹仪(上海楚定分析仪器有限公司);
Milli-Q超纯水仪(美国Millipore公司);
AL104分析天平(瑞士梅特勒-托利多仪器有限公司);
SK8210LHC超声波清洗机(上海科导超声仪器有限公司);
BJ-800A食品粉碎机(杭州德清拜杰电器有限公司);
SX-500高压灭菌锅(日本TOMY公司);
MGC-300H人工气候箱(上海一恒科学仪器有限公司);
GZX-CF101-2-BS电热恒温鼓风干燥箱(上海跃进医疗器械有限公司);
Heraeus Multifuge X3高速离心机(美国Thermo Fisher scientific公司);
冷冻干燥机(宁波新芝生物科技股份有限公司)。
(3)菌株培养基的制备
PDA培养基:去皮马铃薯200g煮沸30min后取滤液,加入葡萄糖20g、琼脂16g,用蒸馏水定容至1000mL,115℃高压灭菌30min,降温到55℃左右倒平板,每平板20mL。
PDB液体培养基:去皮马铃薯200g煮沸30min后取滤液,加入葡萄糖20g,用蒸馏水定容至1000mL,115℃高压灭菌30min。
玉米培养基:称取50g优质玉米至250mL三角瓶,加入20mL蒸馏水混匀,用透气封口膜密封瓶口,浸泡过夜,高压灭菌(120℃,30min),冷却后将玉米摇散待用。
小麦培养基:称取50g优质小麦至250mL三角瓶,加入20mL蒸馏水混匀,用透气封口膜密封瓶口,浸泡过夜,高压灭菌(120℃,30min),冷却后将小麦摇散待用。
实施例1黄芩甲醇提取物的制备
将黄芩用食品粉粹机粉碎,过0.42mm试验筛,混合均匀。
称取100g黄芩粉末至2000mL三角瓶,加入800mL甲醇混匀,40℃超声60min,用纱布过滤至新的2000mL三角瓶中,4500rpm离心15min后取上清;
残渣重复加入800mL甲醇混匀,超声,离心后取上清;
合并取得的上清液,用旋转蒸发仪减压浓缩,回收甲醇,得到黄芩甲醇提取物。
实施例2不同样品中DON的精准测定及方法学验证
(1)产毒菌株活化及孢子液的制备
将禾谷镰刀菌菌株F4582(购至德国微生物菌种保藏中心DSMZ)接种于PDA培养基,28℃黑暗培养7天后,接种至PDB液体培养基中,25℃下150r/min震荡继续培养5天。取孢子液,显微镜观察孢子浓度,用无菌水调整至105个/mL,用于后续接种。
(2)菌株接种
①接种于PDA培养基:将100μL培养好的孢子液接种至PDA培养基中心,28℃黑暗培养9天后观察到培养基菌丝生已长满整个培养皿,禾谷镰刀菌菌株F4582整体呈现出黄色,中间稍有些砖红色,中心的老菌丝有些塌陷,边缘的菌丝紧贴在培养皿上。
②接种于小麦培养基或玉米培养基:将100μL培养好的孢子液接种至50g灭菌小麦培养基或玉米培养基的三角瓶中,接种第1周,每天振荡三角瓶1次,使孢子液与培养基充分接触。28℃黑暗中培养28天后观察到三角瓶中长有黄色的菌丝。
(3)样品测定
①DON提取方法
将PDA培养基、小麦培养基或玉米培养基于50℃烘箱干燥,粉碎混匀后,准确称取2g粉碎样品于50mL离心管,加入10mL乙腈/水(84/16,v/v),旋涡震荡1min,浸泡5min后,超声提取1小时。4000r/min离心10min后,取5mL上清液,在40℃下氮气吹干,1mL的5mmol/L乙酸铵水溶液/甲醇(80/20,v/v)溶解残渣,涡旋30s,超声1min,涡旋30s,充分溶解后,过0.22μm滤膜,UPLC-MS/MS测定。
②超高效液相色谱-串联质谱(UPLC-MS/MS)检测条件
色谱柱:Agilent Poroshell 120EC-C18色谱柱(100mm×3.0mm,2.7mm);流动相:流动相A为5mmol/L乙酸铵溶液,流动相B为甲醇;梯度洗脱程序:0~0.5mim,10% A;4min,90% A;4.5min,90% A;4.7min,10% A;6min,10% A;流速0.4mL/min;进样量3μL;柱温40℃。
DON的具体质谱参数如下表1所示
表1DON的质谱参数
(4)方法学验证
通过考察线性、检出限(limit of detection,LOD)和定量限(limit ofquantitation,LOQ)、回收率和精密度来评价所建立PDA培养基、小麦培养基或玉米培养基中DON分析方法的灵敏度、准确性和重复性。
分别取不含DON的空白PDA培养基、小麦培养基或玉米培养基于50℃烘箱干燥,粉碎混匀后,准确称取2g粉碎样品于50mL离心管,加入10mL乙腈/水(84/16,v/v),旋涡震荡1min,浸泡5min后,超声提取1h。4000r/min离心10min后,取5mL上清液,在40℃下氮气吹干,1mL的5mmol/L乙酸铵水溶液/甲醇(80/20,v/v)溶解残渣,涡旋30s,超声1min,涡旋30s,分别获得三种空白基质溶液。多次制备,将多份同种同种空白基质溶液混合后待用。
取适量DON标准品粉末,用乙腈溶解稀释成质量浓度为1mg/L的标准工作液。用空白基质溶液稀释标准工作液得到1、2、5、10、20、50、100和200μg/L浓度的基质标准溶液,以DON浓度为横坐标、峰面积为纵坐标,建立DON的基质标准曲线并用于样品浓度测定。以定性离子通道的3倍信噪比(signal-to-noise ratio,S/N)确定目标毒素的检测限(LOD)、定量离子通道的10倍信噪比确定目标毒素的定量限(LOQ)。采用加标回收试验法考察回收率和精密度:选取空白PDA培养基、小麦培养基或玉米培养基样品分别按照添加浓度5、50和100μg/kg加入适量的标准工作液,每个浓度选取5个平行样。回收率为测定值和理论值的百分比,日内精密度和日间精密度分别为同一天和连续5天测定结果的相对标准偏差(relativestandard deviation,RSD)。
实验结果显示PDA培养基、小麦培养基和玉米培养基中DON在各自范围内线性关系良好,相关系数(R2)均大于0.990。PDA中DON的定量限为1μg/kg,检出限为0.4μg/kg;小麦中DON的定量限为2μg/kg,检出限为1μg/kg;玉米中DON的定量限为5μg/kg,检出限为2μg/kg。加标回收试验结果表明,PDA中DON回收率范围为88.7%~102.9%(n=5),精密度(RSD)范围为5.6%~11.5%(n=5);小麦中DON的回收率范围为82.3%~98.3%(n=5),精密度(RSD)范围为6.9%~11.0%(n=5);玉米中DON的回收率范围为84.6%~101.4%(n=5),精密度(RSD)范围为5.6%~11.5%(n=5)。
以上数据表明,采用的分析方法灵敏、准确、可靠,满足PDA培养基、小麦培养基和玉米培养基中DON的准确定量,可以采用该方法验证黄芩甲醇提取物对PDA培养基、小麦培养基或玉米培养基中DON生物合成的抑制作用进行研究。
实施例3PDA培养基中黄芩甲醇提取物对DON合成的抑制作用
量取适量实施例1中制备的黄芩甲醇提取物溶于10mL无菌超纯水,过滤除菌后加入90mL灭菌后的PDA培养基,使得最终添加浓度分别达到0、0.5、2、5和20g/L。混匀倒板后,接种100μL实施例1中制备的菌种孢子液,于28℃恒温恒湿培养箱黑暗培养培养9天,UPLC-MS/MS检测DON的产量。每个浓度设置平行5份。
结果表明,与对照组相比(黄芩甲醇提取物浓度0g/L),当黄芩甲醇提取物的浓度达到2g/L以上时,对DON的生物合成有明显的抑制效果(P<0.05),抑制率达到72.1%;抑制效果随着黄芩甲醇提取物浓度的升高而增强,当黄芩甲醇提取物的浓度为5g/L时,DON产量下降88.7%;当黄芩甲醇提取物浓度达到20g/L时几乎完全抑制DON的产生(图1)。
实施例4小麦培养基中黄芩甲醇提取物对DON合成的抑制作用
量取适量黄芩甲醇提取物溶于无菌超纯水,分别配制浓度分别为1、5、10和50g/L的黄芩甲醇提取物溶液,过滤除菌。取小麦培养基,120℃高压灭菌30分钟后分别加入不同浓度的20mL黄芩甲醇提取物溶液,对照组加入20mL无菌超纯水。用无菌透气封口膜密封锥形瓶,摇匀后加入100μL实施例1制备得到的菌种孢子液于28℃恒温恒湿培养箱黑暗培养培养28天,检测DON的产量。每个浓度设置平行5份。
结果显示,与对照组相比,当黄芩甲醇提取物的浓度为50g/L时可显著抑制DON的合成(P<0.01),DON的产量下降77.0%(图2)。
实施例5玉米培养基中黄芩甲醇提取物对DON合成的抑制作用
量取适量黄芩甲醇提取物溶于无菌超纯水,分别配制浓度分别为1、5、10、50和100g/L的黄芩甲醇提取物溶液,过滤除菌。取玉米培养基,120℃高压灭菌30分钟后分别加入不同浓度的20mL黄芩甲醇提取物溶液,对照组加入20mL无菌超纯水。用无菌透气封口膜密封锥形瓶,摇匀后加入100μL实施例1制备得到的菌种孢子液于28℃恒温恒湿培养箱黑暗培养培养28天,检测DON的产量。每个浓度设置平行5份。
结果与小麦培养基类似,与对照组相比,当黄芩甲醇提取物的浓度为50g/L时即可显著抑制DON的合成(P<0.01),DON的产量下降率超过50%。随着黄芩甲醇提取物的浓度的升高,其对DON的产量下降率逐渐提高。
Claims (3)
1.黄芩甲醇提取物作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途;
其中所述的黄芩甲醇提取物是通过如下方法制备得到的:
将黄芩粉碎,过0.42mm试验筛;
加入甲醇混匀,黄芩粉末与甲醇的质量体积比g:mL为1:1-100,36-44℃超声10-100min,用纱布过滤,4500rpm离心15min后取上清;
用旋转蒸发仪减压浓缩,回收甲醇,得到黄芩甲醇提取物。
2.根据权利要求1所述的用途,其中黄芩甲醇提取物在使用时加水配制成浓度为1-100g/L的溶液。
3.根据权利要求2所述的用途,其中黄芩甲醇提取物在使用时加水配制成浓度为2-50g/L的溶液。
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