CN112522033A - 一种具有广谱杀菌作用的植物精油 - Google Patents
一种具有广谱杀菌作用的植物精油 Download PDFInfo
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Classifications
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- 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
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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
- A01N65/40—Liliopsida [monocotyledons]
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
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- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
本发明提供了一种具有抑菌活性的香兰雪精油及其制备方法与应用。所述香兰雪精油是采用水蒸气蒸馏法对盛开期的香兰雪花朵进行蒸馏提取得到的。抗菌抑菌药敏试验及最低抑菌浓度(MIC)和最低杀菌浓度(MBC)表明，这种精油具有对细菌(大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、表皮葡萄球菌等)和真菌(白色念珠菌、申克孢子丝菌、红色毛癣菌、酿酒酵母等)的抑菌作用，且具有对细菌和真菌的广谱性抑制作用。由此发现香雪兰精油可用于消杀品和日用产品的开发应用。
Description
技术领域
本发明属于日用产品制造领域，具体涉及一种具有广谱杀菌作用的植物精油。
背景技术
细菌和真菌在自然界中无处不在，其中大部分对人类直接或间接有益，但也有少数种属对环境、人类和动物有害。由于微生物的变异、人类生活环境的改变以及某些宿主自身变化等因素的影响，细菌、真菌和病毒等病原微生物所致的感染性疾病迄今仍在严重威胁着人类的健康，其中有些难以彻底控制。例如，由于病原微生物耐药性的增高和不良反应等原因，现有的抗生素及化学合成抗菌药物在临床应用上受到诸多限制，后果也越来越严重。所以，从天然化合物中寻找高效低毒的抗菌物质，用来抑制细菌和真菌的感染已形成趋势。细菌对抗生素产生耐药性是由于抗生素的成分较为单一，而单方精油中含有的化学成分少则数十种，多则四百多种，使得细菌难以产生耐药性。因此，植物精油作为新兴的天然抗菌和治疗药物越来越受到人们的广泛关注。
香雪兰(Freesia hybrida)是一种具有较高经济价值的园艺作物，由于香雪兰花期较长，花色丰富，长期以来一直作为观赏花卉而被广泛种植。此外，香雪兰花香清幽，不同品种释放气味不同，且花朵单产量高，是提取植物香精和精油的良好材料。香雪兰植物精油的制备、抑菌方面的研究还鲜见报道。
发明内容
本发明的一个目的是提供一种具有广谱杀菌作用的香兰雪精油及其制备方法。
本发明所提供的香兰雪精油是采用水蒸气蒸馏法对香兰雪(Freesia hybrida)花朵进行蒸馏提取得到的。
所述香兰雪花朵优选为盛开期的香雪兰(Freesia hybrida)花朵。
所述水蒸气蒸馏法中的蒸馏温度为100℃，蒸馏时间为3-4小时。
所述蒸馏提取前将所述香兰雪花朵浸没在水中。
所述蒸馏提取时，油水分离器中加入适量的有机溶剂(如正己烷、丙酮、石油醚等)，蒸馏结束后经静置20分钟，待精油溶于有机溶剂(正己烷、丙酮、石油醚等)后，取油水分离器中上层有机溶剂，放入经过超声清洗的收集瓶中，然后再经过自然挥发去除有机溶剂最终得到香雪兰花朵精油。
本发明的另一个目的是提供上述香兰雪精油的应用。
本发明所提供的应用是所述香兰雪精油在制备抑制和/或杀灭细菌和/或真菌产品中的应用。
所述细菌具体可为：表皮葡萄球菌、枯草芽孢杆菌、金黄色葡萄球菌和大肠杆菌，优选为表皮葡萄球菌和枯草芽孢杆菌，但不局限于此。
所述真菌具体可为：白色念珠菌、酿酒酵母、红色毛癣菌和申克孢子丝菌，优选为白色念珠菌、酿酒酵母和红色毛癣菌，但不局限于此。
本发明确定了香雪兰精油对细菌(表皮葡萄球菌、枯草芽孢杆菌、金黄色葡萄球菌、大肠杆菌)和真菌(白色念珠菌、酿酒酵母、红色毛癣菌、申克孢子丝菌)的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。
抗菌抑菌药敏试验及最低抑菌浓度和最低杀菌浓度表明，这种精油具有对细菌(大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、表皮葡萄球菌等)和真菌(白色念珠菌、申克孢子丝菌、红色毛癣菌、酿酒酵母等)的抑菌作用，且具有对细菌和真菌的广谱性抑制作用。
本发明通过分析化学、细胞生物学和微生物学等实验技术，分析了香雪兰精油的抑菌作用，为基于香雪兰精油的相关产品的开发和应用提供有价值的资料。
具体实施方式
下面通过具体实施例对本发明进行说明，但本发明并不局限于此，凡在本发明的精神和原则之内所做的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中采用菌种如下：大肠杆菌(Escherichia coli)[1]、枯草芽孢杆菌(Bacillus subtilis)[2]、金黄色葡萄球菌(Staphylococcus aureus)[3]、酿酒酵母(Saccharomyces cerevisiae)[4]和白色念珠菌(Candida albicans)[5]，表皮葡萄球菌(Staphylococcus epidermidis)购置于郑州精思威化工有限公司、红色毛癣菌[6]、申克孢子丝菌[7]。
参考文献：
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[3]杨英铎，陆大洪等.香樟精油对金黄色葡萄球菌的抑菌活性及抑菌机理研究.中国现代中药2017。
[4]王媛媛，任璇等.竹叶精油组分构成及其抑菌活性的研究.食品安全质量检测学报2016。
[5]王杨，陈菲，谢明杰.紫草素对白色念珠菌的抑制作用机制.微生物学报2018。
[6]赵明月，李治建.红色毛癣菌中羊毛甾醇含量的高效液相色谱法分析.时珍国医国药2012。
[7]万江帆，沈志斌等.香鳞毛蕨有效部位对申克孢子丝菌酵母相的体外抗真菌作用研究.中药新药与临床药理2014。
下述实施例中所使用的香雪兰，其品种为香雪兰园艺种Red
实施例1、水蒸气蒸馏法提取香雪兰精油
(1)称取香雪兰花朵100g，用漏斗装入1L圆底烧瓶中，再加入蒸馏水至淹没香雪兰花朵材料(约200-300ml蒸馏水)(花朵和蒸馏水的总体积不能超过圆底烧瓶容积的2/3，以防在蒸馏时材料因沸腾而冲出瓶口)。
(2)在油水分离器中加入适量的有机溶剂正己烷约4mL左右，加热约4小时结束蒸馏。
(4)静置20分钟左右，待精油溶于有机溶剂(正己烷)后，取油水分离器中上层有机溶剂，放入经过超声清洗的收集瓶中，然后再经过自然挥发去除有机溶剂最终得到香雪兰花朵精油，精油得率约为1％。
实施例2、香雪兰精油的抗菌活性研究
1培养基的制备
细菌培养采用LB培养基：配制每升培养基，应该在950mL去离子水中加入：胰化蛋白胨10g，酵母提取物5g，氯化钠10g，定容到1000mL摇动容器直至溶质溶解。用1mol/L NaOH调pH至7.0，分装后121℃蒸汽高压蒸汽灭菌20min。固体培养基：在液体培养基的基础上加入15g琼脂粉，分装后121℃高压蒸汽灭菌20min。
真菌培养采用YPD培养基：溶解酵母提取物10g，蛋白胨20g于900mL水中,如制平板需加入20g琼脂粉。分装后高压蒸汽121℃下灭菌20min，用时在加入100mL配制好的20％葡萄糖溶液。
底层琼脂：将配好的相应菌的固体培养基融化，倒入无菌的10cm平板中，每个平板约20～25mL，待其凝固备用。
含菌平板的制备：将配好的含0.7％琼脂粉的固体培养基融化，待温度降低至不烫手时取3mL放入5mL EP管内，再将1mL菌悬液放入EP管内混匀倒入底层琼脂平板内，待其温度降低备用。
含药琼脂平板制备：根据实验设计，将已倍比稀释的不同浓度的抗菌药物分别加入已加热溶解的琼脂内，并在45～50℃水浴中平衡琼脂，充分混匀倾倒灭菌平皿内，琼脂厚度3～4mm，根据需要来选择药物浓度范围。
2菌悬液制备
将储存在4℃冰箱中的菌体，用固体斜面培养基先进行活化培养，细菌(大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、表皮葡萄球菌)置于37℃培养箱培养24h，真菌(白色念珠菌和酿酒酵母)接种后置于30℃培养箱培养48h，真菌红色毛癣菌置于30℃培养箱培养6-7d，真菌申克孢子丝菌置于37℃培养箱培养6-7d，将菌种活化2-3次后，菌种的活性最佳。将活化后的菌种转移到营养液体培养基中在适宜的条件下进行进一步的培养，用营养液体培养基将菌液稀释并校正至0.5麦氏标准浓度(0.5MCF)，此时菌液的浓度约为1.5×108CFU/mL备用。
3试剂配制
用DMSO作为乳化剂将香雪兰精油配制成如下终浓度：0.4％、0.25％、0.125％、0.1％、0.05％、0.025％、0.005％v/v。
氨苄青霉素(100mg/mL)：将1g氨苄青霉素溶于ddH2O中，然后加ddH2O定容至10mL，在超净台内用0.22μm滤膜过滤除菌，之后放入-20℃冰箱保存。
两性霉素B(100mg/mL)：用电子天平秤取一定量的两性霉素B粉末，用二甲基亚砜溶解配制成(100mg/mL)的两性霉素B溶液。
4抑菌效果的测定
4.1香雪兰花朵精油的抑菌圈效果分析
采用滤纸片琼脂扩散法：取直径为6mm的圆形小滤纸片，分别向小滤纸片中央滴加6μL的精油和对照溶液(DMSO)为阴性对照、抗生素(细菌采用氨苄青霉素，真菌采用两性霉素B)为阳性对照，静止几分钟待滤纸片完全润湿后，等间距放入制备好的含菌平板上。最后，将贴好纸片的平板放入恒温培养箱中，经过适宜条件和时间培养。采用十字交叉法以游标卡尺测量抑菌圈直径，每个处理三次重复，具体数值如表1所示。表1为香雪兰花朵精油对八种供试菌的抑菌圈直径大小(mm)。其中，氨苄青霉素起始浓度为100mg/mL；两性霉素B起始浓度为100mg/mL；(1:60)代表稀释60倍；(1:5)代表稀释5倍；(0)代表未经稀释；“-”代表没有以其做对照；滤纸片直径为6mm；抑菌圈直径7-9mm为低敏感；10-15mm为中敏感；>15mm为高度敏感；<6mm为不敏感。每组实验重复3次，实验结果表示为抑菌圈直径平均值+标准偏差，滤纸片直径包括在内。
表1香雪兰花朵精油对供试菌的抑菌圈直径大小(mm)
结果表明：从细菌的抑菌圈直径大小，可以发现表皮葡萄球菌对精油高度敏感，金黄色葡萄球菌和枯草芽孢杆菌对精油中度敏感，大肠杆菌对精油低度敏感。从真菌的抑菌圈直径大小可以发现，白色念珠菌、酿酒酵母和红色毛癣菌对精油高度敏感，申克孢子丝菌对精油中度敏感。抑菌圈直径结果表明，真菌的抑菌效果要好于细菌的抑菌效果。
4.2香雪兰精油的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)
为了进一步确定香雪兰精油的最低抑菌浓度和最低杀菌浓度，在600nm波长下通过全波长酶标仪分析经过不同浓度(0.4％、0.25％、0.125％、0.1％、0.05％、0.025％、0.005％v/v)精油处理后菌的光密度。以此来作为菌生长的标准，进一步确定最低抑菌浓度。测定最低杀菌浓度方法是，利用测定最低抑菌浓度以上各试验管培养物，分别吸取0.1mL，移种在不含药物的琼脂培养基上，经适宜温度和时间孵育，分别菌落计数，其菌落数少于5个的药物浓度即为MBC。结果如表2所示。表2为香雪兰花朵精油最低抑菌(MIC)和最低杀菌(MBC)浓度。“/”代表少量菌生长。
表2香雪兰花朵精油最低抑菌(MIC)和最低杀菌(MBC)浓度
“/”代表少量菌生长。
结果表明：香雪兰精油对不同供试菌的MIC值和MBC值是不同的，但是均有抑制效果。在本发明的试验中，精油对红色毛癣菌的抑制效果最好，MIC值和MBC值分别为0.025％和0.05％。其次是枯草芽孢杆菌、白色念珠菌、酿酒酵母和申克孢子丝菌，它们的MIC值均为0.10％，除申克孢子丝菌的MBC值为0.125％，其余均为0.25％。金黄色葡萄球菌和表皮葡萄球菌的MIC值均为0.125％，大肠杆菌的MIC值为0.25％，以上三种菌在精油浓度为0.4％时，仍有少量菌生长，未能测定MBC值。经过测定香雪兰精油的抑菌圈直径大小、最低抑菌浓度和最低杀菌浓度，均表明精油具有对细菌和真菌的广谱性抑制作用，尤其对真菌的抑菌效果更优，是一种良好的天然抑菌杀菌剂。
Claims (10)
1.一种提取香兰雪精油的方法，包括下述步骤：采用水蒸气蒸馏法对香兰雪花朵进行蒸馏提取。
2.根据权利要求1所述的方法，其特征在于：所述香兰雪花朵为盛开期的香雪兰花朵。
3.根据权利要求1或2所述的方法，其特征在于：所述水蒸气蒸馏法中的蒸馏温度为100℃，蒸馏时间为3-4小时。
4.根据权利要求1-3中任一项所述的方法，其特征在于：所述蒸馏提取前将所述香兰雪花朵浸没在水中。
5.根据权利要求1-4中任一项所述的方法，其特征在于：所述蒸馏提取时，油水分离器中加入有机溶剂，蒸馏结束后经静置，待精油溶于所述有机溶剂后，取油水分离器中上层有机溶剂，放入经过超声清洗的收集瓶中，然后再经过挥发去除有机溶剂得到香雪兰花朵精油。
6.根据权利要求5所述的方法，其特征在于：所述有机溶剂为正己烷、丙酮、或石油醚。
7.权利要求1-6中任一项所述方法制备得到的香雪兰精油。
8.权利要求7所述的香兰雪精油在制备抑制和/或杀灭细菌和/或真菌产品中的应用。
9.根据权利要求8所述的应用，其特征在于：所述细菌选自下述至少一种：表皮葡萄球菌、枯草芽孢杆菌、金黄色葡萄球菌和大肠杆菌，优选为表皮葡萄球菌和枯草芽孢杆菌。
10.根据权利要求8所述的应用，其特征在于：所述真菌选自下述至少一种：白色念珠菌、酿酒酵母、红色毛癣菌和申克孢子丝菌，优选为白色念珠菌、酿酒酵母和红色毛癣菌。
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| CN115746957A (zh) * | 2022-11-21 | 2023-03-07 | 中国热带农业科学院热带作物品种资源研究所 | 一种诺丽精油在抑制奇异变形杆菌中的应用 |
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