CN105820201A - 从五指毛桃果中提取乔松素-7-O-β-D-葡萄糖苷的方法及其用途 - Google Patents
从五指毛桃果中提取乔松素-7-O-β-D-葡萄糖苷的方法及其用途 Download PDFInfo
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- CN105820201A CN105820201A CN201610322974.0A CN201610322974A CN105820201A CN 105820201 A CN105820201 A CN 105820201A CN 201610322974 A CN201610322974 A CN 201610322974A CN 105820201 A CN105820201 A CN 105820201A
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- pinocembrin
- ethanol
- glucose glycosides
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- methanol
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
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- C07H17/07—Benzo[b]pyran-4-ones
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
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- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
本发明公开了一种从五指毛桃果中提取、分离一种黄酮类化合物—乔松素‑7‑O‑β‑D‑葡萄糖苷的方法及其用途,属于植物源农药技术领域。具体是通过将五指毛桃果粗提物经过大孔树脂柱层析,Sephadex LH‑20凝胶柱层析和重结晶的方法分离得到化合物,该化合物对柑橘采后病原菌—意大利青霉具有较好的抑菌活性,对柑橘青霉病有较好的抗病性,可用于开发成一种新型的植物源柑橘防腐保鲜剂。
Description
技术领域
本发明属于植物源农药技术领域,具体涉及到一种从五指毛桃果中提取、分离一种黄酮类化合物—乔松素-7-O-β-D-葡萄糖苷的方法及其在柑橘采后保鲜中的用途。
背景技术
五指毛桃果(Ficus simplicissima Lour.)为桑科无花果属植物裂掌榕的果实,收载于历年《中国药典》和多种中药学古书籍中。五指毛桃是广东地区的道地中药材,具有很好的益气健脾功效,有“广东人参”之美誉。五指毛桃性平,味甘、辛,有健脾补肺、利湿舒筋之功,可用于脾虚浮肿、食少无力、肺痨咳嗽、盗汗、风湿痹痛、产后无乳等症,在广东民间使用历史悠久。现代药理学研究表明五指毛桃可以保护胃黏膜、减轻肝损伤、提高机体免疫力、减轻脑缺血缺氧的损害。动物急性毒性实验研究表明小鼠灌服五指毛桃水提物后14d内,动物均无死亡,各脏器均无异常。表明五指毛桃毒性极低,生物安全性好。关于五指毛桃的抗菌活性研究,仅见五指毛桃水煎煮提取液对大肠杆菌、枯草芽胞杆菌、金黄色葡萄球菌有抑制作用。
由意大利青霉(Penicillium italicum)引起的青霉病和指状青霉(Penicilliumdigitatum)引起的绿霉病,是造成柑橘采后腐烂发生的最主要的两种病害,腐烂率一般为10%~30%,严重时可高达40%~50%以上,造成巨大的经济损失。长期以来,主要采用化学合成药剂作为保鲜剂对柑橘进行保鲜,这些化学杀菌剂能杀死或抑制病原微生物的生长,有较好的防腐保鲜效果,但同样会造成药剂残留、污染环境,抗药性等弊端。另外,一些化学防腐剂有潜在的致癌、致突变、致畸的可能性。
近年来,人们开始转向从天然植物中寻找安全环保的柑橘植物源保鲜剂。天然植物源保鲜剂具有高效、低毒、低残留、无污染、使用安全等优点,在应用上易受消费者欢迎。另外,植物源药剂不易产生抗药性和不会造成环境污染问题。因此,有必要从天然植物中寻找、开发天然植物源防腐剂对柑橘进行保鲜以减少对化学药剂的依赖。
乔松素-7-O-β-D-葡萄糖苷是一种黄酮类化合物,目前从植物中分离到此化合物的报道较少,主要存在于羽叶金合欢、扯根菜、扁枝槲寄生、南酸枣、老鹰茶、玫瑰花、青皮木、荔枝核、光果甘草和青钱柳等植物中。尚未见五指毛桃果中乔松素-7-O-β-D-葡萄糖苷的报道,本发明为首次从五指毛桃果中分离得到乔松素-7-O-β-D-葡萄糖苷。
发明内容
本发明的目的是提供一种从五指毛桃果中分离得到一种黄酮类化合物—乔松素-7-O-β-D-葡萄糖苷的方法。
本发明的另一个目的是提供乔松素-7-O-β-D-葡萄糖苷在柑橘采后保鲜剂中的应用。
本发明提供的黄酮化合物的结构如下:
本发明的上述目的是通过下面的技术方案得以实现的:
(1)五指毛桃果于鼓风干燥箱中40-50℃烘干2h,粉碎机粉碎后过40目筛,取筛下药材粉末,以体积分数为90%的乙醇溶液为提取溶剂,按照料液比(kg∶L)=1:(10-40)超声波辅助提取30-90min,超声波辅助提取的温度为50-80℃,重复提取3次,合并提取液并过滤,回收乙醇,滤液浓缩至无醇味,即得五指毛桃果提取物浸膏;
(2)将步骤(1)中的五指毛桃果提取物浸膏悬浮于10-20倍浸膏重量的蒸馏水中,经D101大孔树脂柱层析,先用纯水洗脱至流出液无颜色、再分别依次用5倍柱体积30%乙醇、5倍柱体积50%乙醇和5倍柱体积95%乙醇梯度洗脱,收集95%乙醇洗脱液,浓缩至无醇味,即得95%乙醇洗脱浓缩物;
(3)将步骤(2)中95%乙醇洗脱浓缩物溶解于甲醇中,95%乙醇洗脱浓缩物与甲醇的质量体积比(g:mL)为(1.0-5.0):10,将该溶液于离心机上5000-8000r/min离心3-10min,取上清液用Sephadex LH-20凝胶柱层析分离,用甲醇洗脱,收集8-10个柱体积洗脱液,薄层层析合并目标化合物流分,减压浓缩,向得到的浓缩物中加入甲醇并加热至浓缩物刚好能全部溶解为止,放入4℃冰箱中,析出淡黄色沉淀物即为目标化合物。
其中步骤(2)采用的大孔树脂还可为除D101型之外的其他弱极性和低极性大孔树脂。
本发明制得的目标化合物乔松素-7-O-β-D-葡萄糖苷可添加常规辅料制成防治柑橘采后病原菌的药物组合物,用于柑橘采后防腐保鲜,防治由意大利青霉(Penicillium italicum)引起的青霉病和指状青霉(Penicillium digitatum)引起的绿霉病。
王静等通过***溶剂萃取老鹰茶提取物,依次用石油醚、二氯甲烷、乙酸乙酯、水饱和正丁醇溶剂萃取,再通过大孔树脂柱分离正丁醇萃取部位,用乙醇-水***梯度洗脱,分为10%、30%、50%、95%4个部分,对95%部分用硅胶柱进行反复分离,并结合ODS、Sephadex LH-20和制备型HPLC分离纯化出乔松素-7-O-β-D-葡萄糖苷(王静,陆维丽,张义龙,唐敏芳,汤文建,李俊.老鹰茶的化学成分研究[J].安徽医科大学学报,2014,49(4):479-483.)。付明等通过***溶剂萃取扯根菜提取物,依次用石油醚、醋酸乙酯、正丁醇萃取,醋酸乙酯部位反复经硅胶柱色谱、Sephadex LH-20柱色谱和薄层色谱等,最终从醋酸乙酯部分分离得到乔松素-7-O-β-D-葡萄糖苷(付明,魏麟,余娟,胡朝暾.扯根菜的化学成分研究[J].中国药学杂志,2013,48(22):1911-1914)。刘红燕通过***溶剂萃取玫瑰花提取物,依次用石油醚、氯仿、乙酸乙酯、正丁醇萃取,乙酸乙酯部位反复经硅胶柱色谱、Sephadex LH-20柱色谱,最终从醋酸乙酯部分分离得到乔松素-7-O-β-D-葡萄糖苷(刘红燕.玫瑰花的化学成分研究[J].天然产物研究与开发,2013,25(1):47-49.)。上述方法都使用了大量的有机溶剂,硅胶柱层析,甚至是制备高效液相色谱技术,需要的成本高,不利于大样品量的制备。
本发明的优点在于有机溶剂使用量少,操作步骤简单,适合规模化生产制备乔松素-7-O-β-D-葡萄糖苷。
附图说明
图1为实施例1制得的乔松素-7-O-β-D-葡萄糖苷的结构式;
图2为实施例1制得的乔松素-7-O-β-D-葡萄糖苷的核磁共振氢谱;
图3为实施例1制得的乔松素-7-O-β-D-葡萄糖苷的核磁共振碳谱;
图4为乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌的抑制效果图;
图5为乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝显微形态的影响图;
图6为乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝生长量的影响图;
图7为乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝相对电导率的影响图;
图8为乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌几丁质酶活性的影响图;
图9为乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌β-1,3-葡聚糖酶活性的影响。
具体实施方式
实施例1:化合物的制备方法
五指毛桃果于鼓风干燥箱中45℃烘干2h,粉碎机粉碎后过40目筛,取筛下的五指毛桃果粉末5.0Kg,用50L体积分数为90%的乙醇溶液浸泡,超声波辅助提取60min,提取温度为60℃,重复三次至提取液颜色变淡为止,合并提取液并过滤,滤液在45℃条件下浓缩至无醇味,共得浸膏350g。将浸膏悬浮于4L蒸馏水中,经D101大孔树脂柱层析,先用纯水洗脱至流出液无颜色、再分别依次用5倍柱体积30%乙醇、5倍柱体积50%乙醇和5倍柱体积95%乙醇梯度洗脱,水洗脱组分弃用,其他洗脱组分浓缩干燥得干膏,备用。95%乙醇洗脱物经干燥得到7.5g粉末。将7.5g 95%乙醇洗脱部位干燥粉末溶解于20mL甲醇中,5000-8000r/min离心6min,取上清液经Sephadex LH-20凝胶柱层析,甲醇洗脱,收集8-10个柱体积洗脱液,薄层层析检测合并相同流分,得到A~I共8个流分。经检测流分E为目标化合物流分,取250mg流分E溶解于3.0—5.0mL甲醇中并加热使其全部溶解,然后放入4℃冰箱中,经甲醇重结晶析出淡黄色沉淀物21.5mg。
化合物的结构确证:1H-NMR(600MHz,DMSO-d6)显示一个1-取代苯环信号δH 7.55(2H,d,J=7.6Hz,H-2',6'),7.44(3H,m,H-3',4',5'),以及一对间位耦合质子信号6.21(1H,d,J=1.5Hz,H-8),6.16(1H,d,J=1.5Hz,H-6),还有一个酚羟基质子信号12.05(5-OH),5.66(1H,d,J=12.9,H-2),4.99(1H,d,J=7.4Hz,H-1”)为糖的端基质子,确定其为β-构型,13C-NMR(150MHz,DMSO-d6)数据显示该化合物有21个C,结合H谱确定化合物为一个黄酮糖苷类化合物,ESI-MS给出准分子离子峰m/z 417.00[M-H]-,其他H信号如下:3.66(1H,d,J=9.4Hz,H-6”a),3.15~3.45(6H,H-3a,2”,3”,4”,5”,6”b),2.85(1H,d,J=16.7Hz,H-3b)。13C-NMR(150MHz,DMSO-d6),79.1(C-2),42.6(C-3),197.3(C-4),163.4(C-5),97.1(C-6),165.8(C-7),96.0(C-8),163.0(C-9),103.7(C-10),138.9(C-1'),127.2(C-2',6'),129.1(C-3',4',5'),100.0(C-1”),73.5(C-2”),76.8(C-3”),69.9(C-4”),77.6(C-5”),61.0(C-6”)。鉴定该黄色沉淀物为乔松素-7-O-β-D-葡萄糖苷。
实施例2:化合物抑制柑橘采后意大利青霉菌活性
1材料 意大利青霉(Penicillium italicum,ACCC 30399)购于中国农业微生物菌种保藏中心。培养基:PDA和PDB(不含琼脂粉的PDA)培养基;细胞壁降解酶诱导培养基(KNO3 2.0g,K2HPO4 1.0g,MgSO4·7H2O 0.5g,KCl 0.5g,FeSO4 0.01g,维生素B1 0.1g,L-天门冬酰胺0.5g,几丁质或葡聚糖、CMC 10.0g,H2O 1000mL),pH调至5.0,分装后121℃灭菌20min。
2实验方法
①乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌的抑制效果
采用生长速率法测定乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝生长的抑制毒力。从已活化的意大利青霉菌落边缘用灭菌的打孔器打取菌饼(Ф=5mm),菌丝面朝下放置于已经凝固的含药培养基中央,27℃培养。每个处理4次重复,试验重复2次。培养7d后十字交叉法测量菌落直径,与空白对照比较,采用下面公式计算各浓度药物处理对菌丝生长的抑制率。根据生物统计几率值换算表,将抑制百分率换算成几率值,采用DPS 7.05软件以试验中设定的浓度为横坐标,抑制几率值为纵坐标,得到药剂的毒力回归方程和有效浓度EC50和EC95。
②乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝形态的影响
将乔松素溶液加入50mL PDB培养基的三角瓶中,使其终浓度达到0.2528mg·mL-1(EC50与EC95之和的一半),不加乔松素-7-O-β-D-葡萄糖苷溶液作为对照组,向每个三角瓶中加入200μL意大利青霉菌悬液,置于27℃,180rpm振荡培养24和48h后,在10x40倍电子显微镜下观察。
③乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌孢子萌发的影响
无菌条件下,配制意大利青霉孢子悬浮液,用移液器按9:1比例将菌悬液与不同浓度的乔松素-7-O-β-D-葡萄糖苷溶液滴入凹玻片凹槽中,使乔松素-7-O-β-D-葡萄糖苷作用的终浓度为0.8、0.4、0.2、0.1、0.05、0.025mg·mL-1,每个处理重复3次。将玻片置于27℃培养,24h后进行镜检,观察各处理和对照组的意大利青霉孢子的萌发情况,在显微镜下统计100-150个孢子的萌发数,计算孢子萌发率和抑制率。
④乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝生长量的影响
在无菌的条件下,取1.0mL菌悬液加入装有100mL PDB液体培养基的250mL三角瓶中,27℃、180rpm振荡培养48h,待菌丝球生长良好时加入乔松素-7-O-β-D-葡萄糖苷处理液,使终浓度为0.2528mg·mL-1,对照加入等量的无菌水。分别在继续培养的0、3、6、12、24h取样,用布氏漏斗抽滤收集菌丝,用蒸馏水对菌丝冲洗3次后,在真空冷冻干燥机中将菌丝冻干至恒重,即为菌丝体的重量。每个处理重复5次,试验重复2次。
⑤乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌细胞膜透性影响
电导率法测定:取1.0mL菌悬液加入装有100mL PDB液体培养基的250mL三角瓶中,27℃、180rpm振荡培养48h。称取等量的菌丝分别放入装有100mL0.2528mg·mL-1乔松素-7-O-β-D-葡萄糖苷溶液和100mL无菌水的250mL三角瓶中,在27℃、180rpm的摇床上继续培养,在处理0、1、2、4、6h取样,在转速为3000rpm离心10min,采用DDS-11A数显电导率仪测定对照组和乔松素-7-O-β-D-葡萄糖苷处理的溶剂空白电导率(C0)和上清液的电导率(Ct),最后将菌丝沸水浴保温10min,待冷却至室温后,测定其电导率(Cd)。每个处理重复三次。用相对电导率(relative electric conductivity,REC)来表示细胞膜透性,相对电导率(%)的计算公式如下:
⑥几丁质酶(Chitinase,CHI)活性的测定
几丁质酶液的提取:取1.0g菌丝,加入5mL乙酸-乙酸钠缓冲液(含0.1M、pH 5.2,含5mMβ-巯基乙醇;1mM聚乙二醇6000;1mM EDTA;4%PVP和0.05%Triton X-100),冰浴下研磨呈匀浆后离心(4℃、12 000rpm,20min),收集上清液备用。
几丁质酶活性的测定参考Boller的方法并稍作改进,以每毫克蛋白酶解胶状几丁质产生1×10-9moL N-乙酰葡萄糖胺为一个CHI活性单位(U·mg-1protein)。
⑦β-1,3-葡聚糖酶(β-1,3-glucanase,GLU)活性的测定
β-1,3-葡聚糖酶液的提取方法与几丁质酶液的提取方法相同。β-1,3-葡聚糖酶活性测定参考Abeles和Forrence的方法并稍作改进,以菌丝中每毫克蛋白分解昆布多糖产生1μg葡萄糖为1个GLU活性单位(U·mg-1protein)。
⑧数据统计分析采用SPSS 19.0和DPS 7.05数据统计软件对试验数据进行统计分析,用Duncan新复极差法进行显著性差异分析,显著性差异水平:显著(P<0.05);极显著(P<0.01);采用Excel 2003软件进行数据处理及图形制作。
3实验结果
①乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌的抑制效果
试验结果如说明书附图中图4所示,随着化合物处理浓度的逐渐升高,其对意大利青霉的抑菌效果也逐渐增强;当处理浓度为0.8mg·mL-1时,对意大利青霉的抑菌率达到100%,完全抑制了意大利青霉的生长。采用DPS v8.05软件进行统计分析,得到以抑菌率几率值为Y,药剂浓度(0.025~0.4mg·mL-1)对数值为X的毒力回归方程:Y=7.8219+3.1150X,相关系数r为0.9836,说明乔松素-7-O-β-D-葡萄糖苷的处理浓度与抑菌率呈显著的线性关系;同时也计算出EC50和EC95分别为0.0846和0.4209mg·mL-1。
②乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝形态的影响
试验结果如说明书附图中图5所示,对照组的意大利青霉菌丝细胞壁光滑、粗细一致、分枝正常、内部原生质分布均匀(图5A);乔松素-7-O-β-D-葡萄糖苷处理12h后,意大利青霉菌丝顶端膨大,菌丝缠绕扭结、粗细不均(图5B);处理24h后的菌丝细长、皱缩、分枝增多,甚至出现内含物消解、菌丝空腔和断裂现象(图5C)。
③乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌孢子萌发的影响
由表1可看出,不同浓度的乔松素-7-O-β-D-葡萄糖苷处理对意大利青霉的孢子萌发有显著的抑制作用。随着处理浓度的增加,意大利青霉菌的孢子萌发率均逐渐减少,抑制效果增强,且各处理与对照组之间的差异极显著(P<0.01)。当乔松素-7-O-β-D-葡萄糖苷处理浓度为0.4mg·mL-1时,该处理的孢子萌发率仅为3.5%,抑制率高达96.3%。综上所述,乔松素-7-O-β-D-葡萄糖苷对意大利青霉的孢子萌发具有显著的抑制效果。
表1 乔松素-7-O-β-D-葡萄糖苷对意大利青霉孢子萌发的抑制作用
注:表中数据为三次重复的平均值±标准方差,同列有不同大小写字母的表示经邓肯氏多重检验分别在0.01及0.05水平差异显著。
④乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌丝生长量的影响
由说明书附图中图6可知,在整个培养期间,对照组的菌丝生长量呈上升趋势,至24h时,生长量达到最大值,菌丝干重为1.22±0.032g·100mL-1;而加入乔松素-7-O-β-D-葡萄糖苷(0.2528mg·mL-1)处理的培养基中菌丝生长明显受到抑制,培养6h内,菌丝的生长量急剧下降,随后下降较为平缓,至24h时菌丝的生长量达到最小值,干重为0.53±0.011g·100mL-1,仅为同期对照组菌丝干重的43.4%。由此可见,乔松素对意大利青霉的菌丝生长具有显著的抑制效果。
⑤乔松素-7-O-β-D-葡萄糖苷对意大利青霉菌细胞膜透性的影响
细胞膜遭到破坏时,菌体内部电解质会泄漏外渗到菌液中,从而使菌液的电导率升高,菌丝相对电导率的大小可以间接反映细胞膜透性的完整性。由说明书附图中图7可知,对照组的相对电导率呈缓慢上升的变化趋势;乔松素-7-O-β-D-葡萄糖苷处理组相对电导率随处理时间而逐渐升高,其上升幅度显著高于对照组(P<0.05),说明随着时间的延长,意大利青霉的细胞膜透性逐渐增加,细胞中大量的内含物向外渗漏,使得菌液电导率快速上升,破坏细胞膜透性的完整性。
⑥乔松素-7-O-β-D-葡萄糖苷对意大利青霉细胞壁降解酶活性的影响
乔松素-7-O-β-D-葡萄糖苷对意大利青霉细胞壁降解酶活性的影响结果见说明书附图中的图8。从图8可以看出,对照组的菌丝几丁质酶(CHI)活性呈缓慢下降趋势,在整个培养期间没有显著差异;而乔松素-7-O-β-D-葡萄糖苷处理的菌丝CHI活性先显著上升,在处理2h时达到最大值,随后缓慢下降。在处理2、4、6h时的CHI活性分别为55.6、50.7和52.2U·mg-1protein,均比同期对照组的CHI活性高,由此可见乔松素显著提高意大利青霉细胞降解酶CHI活性。
乔松素-7-O-β-D-葡萄糖苷同样也会增加意大利青霉菌中另外一个细胞壁降解相关酶β-1,3-葡聚糖酶(GLU)活性的变化,它是分解真菌细胞壁的一种溶菌酶。由说明书附图中图9可知,对照组的菌丝β-1,3-葡聚糖酶(GLU)活性呈缓慢上升的变化趋势;而乔松素-7-O-β-D-葡萄糖苷处理的菌丝β-1,3-葡聚糖酶(GLU)活性呈急剧上升趋势,且上升幅度显著大于对照组。在处理2、4、6h时的GLU活性分别为5.59、5.17和6.23U·mg-1protein,均比同期对照组的GLU活性高,可见GLU活性的增高会引起意大利青霉菌细胞壁发生自溶现象。
Claims (5)
1.一种从五指毛桃果中提取乔松素-7-O-β-D-葡萄糖苷的方法,其特征在于:所述乔松素-7-O-β-D-葡萄糖苷的结构如下:
2.如权利要求1所述的一种从五指毛桃果中提取乔松素-7-O-β-D-葡萄糖苷的方法,其特征在于:包括以下步骤:
(1)五指毛桃果于鼓风干燥箱中40-50℃烘干2h,粉碎机粉碎后过40目筛,取筛下药材粉末,以体积分数为90%的乙醇溶液为提取溶剂,按照料液比(kg∶L)=1:(10-40)超声波辅助提取30-90min,超声波辅助提取的温度为50-80℃,重复提取3次,合并提取液并过滤,回收乙醇,滤液浓缩至无醇味,即得五指毛桃果提取物浸膏;
(2)将步骤(1)中的五指毛桃果提取物浸膏悬浮于10-20倍浸膏重量的蒸馏水中,经D101大孔树脂柱层析,先用纯水洗脱至流出液无颜色、再分别依次用5倍柱体积30%乙醇、5倍柱体积50%乙醇和5倍柱体积95%乙醇梯度洗脱,收集95%乙醇洗脱液,浓缩至无醇味,即得95%乙醇洗脱浓缩物;
(3)将步骤(2)中95%乙醇洗脱浓缩物溶解于甲醇中,95%乙醇洗脱浓缩物与甲醇的质量体积比(g:mL)为(1.0-5.0):10,将该溶液于离心机上5000-8000r/min离心3-10min,取上清液用Sephadex LH-20凝胶柱层析分离,用甲醇洗脱,收集8-10个柱体积洗脱液,薄层层析合并目标化合物流分,减压浓缩,向得到的浓缩物中加入甲醇并加热至浓缩物刚好能全部溶解为止,放入4℃冰箱中,析出淡黄色沉淀物即为目标化合物。
3.如权利要求2所述的一种从五指毛桃果中提取乔松素-7-O-β-D-葡萄糖苷的方法,其特征在于:包括以下步骤:五指毛桃果于鼓风干燥箱中45℃烘干2h,粉碎机粉碎后过40目筛,取筛下的五指毛桃果粉末5.0Kg,用50L 90%乙醇浸泡,超声波辅助提取60min,提取温度为60℃,重复三次至提取液颜色变淡为止,合并提取液并过滤,滤液在45℃条件下浓缩至无醇味,共得浸膏350g;将浸膏悬浮于4L蒸馏水中,经D101大孔树脂柱层析,先用纯水洗脱至流出液无颜色、再分别依次用5倍柱体积30%乙醇、5倍柱体积50%乙醇和5倍柱体积95%乙醇梯度洗脱,水洗脱组分弃用,其他洗脱组分浓缩干燥得干膏,备用;95%乙醇洗脱物经干燥得到7.5g粉末,将7.5g 95%乙醇洗脱部位干燥粉末溶解于20mL甲醇中,5000-8000r/min离心6min,取上清液经Sephadex LH-20凝胶柱层析,甲醇洗脱,收集8-10个柱体积洗脱液,薄层层析检测合并相同流分,得到A~I共8个流分;经检测流分E为目标化合物流分,取250mg流分E溶解于3.0—5.0mL甲醇中并加热使其全部溶解,然后放入4℃冰箱中,经甲醇重结晶析出淡黄色沉淀物21.5mg。
4.权利要求1所述化合物在制备柑橘采后防腐保鲜剂中的应用,其特征在于:所述柑橘采后病原菌选自意大利青霉和指状青霉。
5.一种防治柑橘采后病原菌的药物组合物,其特征在于:包括权利要求1所述的化合物。
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