CN115414403A - 余甘子提取物在制备抗海洋弧菌药物中的用途 - Google Patents
余甘子提取物在制备抗海洋弧菌药物中的用途 Download PDFInfo
- Publication number
- CN115414403A CN115414403A CN202211227946.2A CN202211227946A CN115414403A CN 115414403 A CN115414403 A CN 115414403A CN 202211227946 A CN202211227946 A CN 202211227946A CN 115414403 A CN115414403 A CN 115414403A
- Authority
- CN
- China
- Prior art keywords
- extract
- leafflower fruit
- emblic leafflower
- vibrio
- emblic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000284 extract Substances 0.000 title claims abstract description 56
- 235000015489 Emblica officinalis Nutrition 0.000 title claims abstract description 45
- 244000119298 Emblica officinalis Species 0.000 title claims abstract description 43
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 27
- 239000003814 drug Substances 0.000 title claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 32
- 241000607618 Vibrio harveyi Species 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 241000607598 Vibrio Species 0.000 claims abstract description 14
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 3
- 240000009120 Phyllanthus emblica Species 0.000 claims abstract 2
- 235000019441 ethanol Nutrition 0.000 claims description 24
- 238000000605 extraction Methods 0.000 claims description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 14
- 229940079593 drug Drugs 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 11
- 230000003385 bacteriostatic effect Effects 0.000 claims description 10
- 239000009609 fructus phyllanthi Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 239000004480 active ingredient Substances 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 238000002137 ultrasound extraction Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 238000010025 steaming Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 238000000967 suction filtration Methods 0.000 claims description 2
- 239000000047 product Substances 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 19
- 244000052616 bacterial pathogen Species 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 15
- 241000411851 herbal medicine Species 0.000 abstract description 15
- 239000006286 aqueous extract Substances 0.000 abstract description 14
- -1 phenolic acid compounds Chemical class 0.000 abstract description 8
- 239000012675 alcoholic extract Substances 0.000 description 16
- 230000005764 inhibitory process Effects 0.000 description 12
- 239000000126 substance Substances 0.000 description 8
- 206010047400 Vibrio infections Diseases 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 238000009360 aquaculture Methods 0.000 description 6
- 244000144974 aquaculture Species 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 244000308495 Potentilla anserina Species 0.000 description 4
- 206010070834 Sensitisation Diseases 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000008313 sensitization Effects 0.000 description 4
- 241000205585 Aquilegia canadensis Species 0.000 description 3
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 3
- 241000522190 Desmodium Species 0.000 description 3
- 235000009728 Potentilla anserina subsp anserina Nutrition 0.000 description 3
- 240000007164 Salvia officinalis Species 0.000 description 3
- 235000002912 Salvia officinalis Nutrition 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 235000002020 sage Nutrition 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000009910 sargent gloryvine Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241000506964 Dichondra repens Species 0.000 description 2
- 241001289529 Fallopia multiflora Species 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- 235000003805 Musa ABB Group Nutrition 0.000 description 2
- 240000008790 Musa x paradisiaca Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 235000015266 Plantago major Nutrition 0.000 description 2
- 244000274050 Platycodon grandiflorum Species 0.000 description 2
- 235000006753 Platycodon grandiflorum Nutrition 0.000 description 2
- 241000219000 Populus Species 0.000 description 2
- 241000534017 Saururus chinensis Species 0.000 description 2
- 235000007212 Verbena X moechina Moldenke Nutrition 0.000 description 2
- 240000001519 Verbena officinalis Species 0.000 description 2
- 235000001594 Verbena polystachya Kunth Nutrition 0.000 description 2
- 235000007200 Verbena x perriana Moldenke Nutrition 0.000 description 2
- 235000002270 Verbena x stuprosa Moldenke Nutrition 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 230000008023 solidification Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241001253169 Asystasia gangetica Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 244000292697 Polygonum aviculare Species 0.000 description 1
- 235000006386 Polygonum aviculare Nutrition 0.000 description 1
- 235000016594 Potentilla anserina Nutrition 0.000 description 1
- 241000218206 Ranunculus Species 0.000 description 1
- 240000001341 Reynoutria japonica Species 0.000 description 1
- 235000018167 Reynoutria japonica Nutrition 0.000 description 1
- 241000195974 Selaginella Species 0.000 description 1
- 241000967218 Selaginella tamariscina Species 0.000 description 1
- 240000006688 Telosma cordata Species 0.000 description 1
- 235000017352 Telosma cordata Nutrition 0.000 description 1
- 241000607594 Vibrio alginolyticus Species 0.000 description 1
- 241000544286 Vibrio anguillarum Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 208000010824 fish disease Diseases 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 1
- 229960001180 norfloxacin Drugs 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229960005404 sulfamethoxazole Drugs 0.000 description 1
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010267 two-fold dilution method Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明是一种余甘子提取物在制备抗海洋弧菌药物中的用途，涉及中草药提取物的应用技术领域。所述余甘子提取物为以余甘子为原料，采用水或者醇提取所得的余甘子水提物或余甘子醇提取；所抑制的海洋弧菌为哈维氏弧菌或者副溶血弧菌。本发明筛选出前述的中草药中余甘子具有较好抑制海洋致病菌的作用，MIC值均为15.63mg/mL。本发明还确定抑制海洋致病菌的余甘子提取物的有效成分为酚酸类化合物。
Description
技术领域
本发明涉及中草药提取物的用途，特别涉及余甘子提取物的抗海洋弧菌的用途。
背景技术
中国作为世界第一的水产养殖国家，每年的养殖产量为世界养殖总产量的70％左右。但随着水产养殖环境的污染日益加剧，导致水产动物疾病爆发、病原微生物种类增多以及传播速度加快。目前，细菌性疾病在水产养殖，尤其是在密集型的养殖体系中发病率极高，严重阻碍了海水鱼类的养殖和发展，由致病性弧菌所引起的疾病，流行面积广，发病率高，更是给对水产养殖业造成了巨大危害。海洋致病菌主要包括副溶血弧菌、鳗弧菌、溶藻弧菌、哈维氏弧菌等。
目前，对于海洋致病菌多采用化学药物防治的方法，主要包括青霉素，诺氟沙星，新诺明等，但长期单一使用一种药物会产生抗性，出现耐药性菌株。中草药具有成分丰富、不易产生耐药性、绿色环保及功效多样等优点，在病毒性、细菌性等各类鱼病防治中均拥有独到的作用及良好的效果。
现有对中草药进行筛选来抑制海洋致病菌的研究，但是关于本发明的中草药在抑制海洋致病菌中的应用目前尚缺乏报道。因此，筛选中草药，寻找具有较好抑制海洋致病菌有效的中草药具有重要的意义。
发明内容
本发明的目的是针对现有技术的不足，提供一种效果显著的余甘子提取物在制备抑制海洋致病菌药物中的用途。
本发明所要解决的技术问题是通过以下的技术方案来实现的。本发明是余甘子提取物在制备抗海洋弧菌药物中的用途，其特点是，所述余甘子提取物为以余甘子为原料，采用水或者醇提取所得的余甘子水提物或余甘子醇提取；所抑制的海洋弧菌为哈维氏弧菌或者副溶血弧菌。
以上所述的用途，其进一步优选的技术方案是：所述的余甘子提取物的制备方法为：称取余甘子粉末，加入溶剂水或无水乙醇，在室温下超声提取10～90min，再在30～90℃下回流提取30～120min，抽滤，同法提取1～4次，收集滤液，浓缩使生药的最终浓度为1g/mL。
本发明优选的技术方案是：所述的余甘子提取物的提取方法如下：
(1)提取：称取余甘子粉末，加入无水乙醇，在室温下超声提取30min，再在50℃下回流提取30min，抽滤，同法提取2次，收集滤液，浓缩使生药的最终浓度为1g/mL；
(2)抑菌活性成分分离：对余甘子醇提物进行分离，余甘子醇提物旋蒸成浸膏后加入蒸馏水配成混浊液，分别用等体积石油醚、乙酸乙酯、正丁醇萃取，萃取至溶液澄清无色，每次萃取时间30min。萃取完毕后，将溶剂旋干得到干燥粉末，将不同萃取物干燥粉末，配置成10mg/mL的样品溶液，用牛津杯法测定样品溶液的抑菌活性；
(3)抑菌活性成分化学定性检测：使用化学定性的方法对4个组分进行检测，用盐酸+镁粉、溴甲酚绿溶液及三氯化铁铁氰化钾溶液进行化学定性检测；确定乙酸乙酯组为酚酸类化合物；确定酚酸类化合物为抑制海洋致病菌。
与现有技术相比，本发明具有以下有益效果：本发明将余甘子提取物用于制备抑制海洋致病菌的药物，余甘子醇提物对哈维氏弧菌和副溶血弧菌均有较好的抑制作用，MIC值均为15.63mg/mL。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1，筛选具有抑制海洋致病菌作用的中草药，确定余甘子为抑制海洋致病菌活性最好的中草药
1、抑制海洋致病菌中草药的筛选
初步选出桔梗、金钱草、余甘子、三白草、酸枣仁、紫花地丁、荔枝草、马鞭草、卷柏、忍冬藤、杨树花、马蹄金、忍冬叶、首乌藤、大血藤、猫须草、蕨麻、车前草水提物或醇提取物中的一种或多种18种中草药。称取中药粉末，加入溶剂(水、无水乙醇或石油醚)，在室温下超声提取30min，再在50℃下回流提取30min，抽滤，同法提取2次，收集滤液，浓缩使生药的最终浓度为1g/mL。
选取哈维氏弧菌、副溶血弧菌为目标菌株，采用牛津杯法，在无菌环境下，向平板中倒入约20mL已灭菌的牛肉膏蛋白胨琼脂培养基，凝固后，加入200μL的菌悬液，涂布棒涂布均匀，静置10min。将牛津杯垂直均匀放置于培养基上，在杯中加入200μL的提取物样品，每个样品进行3次平行实验，移至37℃恒温培养箱培养18h后，用电子数显卡尺测定抑菌圈直径，结果取三次测量结果的平均值。抑菌圈直径大于20mm为极敏，15～19mm为高敏，10～15mm为中敏，小于10mm为低敏(无活性)。
通过上述体外抑菌实验筛选出对哈维氏弧菌、副溶血弧菌抑制作用较强的中草药作为进一步实验对象。18种中草药提取物对两种弧菌的抑菌效果见表1：
表1 18种中草药提取物对两种弧菌的抑菌效果
注：“+++”表示极敏、“++”表示高敏、“+”表示中敏、“-”表示低敏(无活性)。
实验结果如表1所示，桔梗醇提物、余甘子水提物及醇提物、金钱草水提物及醇提物、酸枣仁醇提物、首乌藤水提物、大血藤醇提物、猫须草醇提物、蕨麻水提物均对副溶血弧菌有抑制作用；余甘子水提物及醇提物、金钱草醇提物、三白草醇提物、酸枣仁醇提物、紫花地丁水提物及醇提物、荔枝草水提物及醇提物、马鞭草水提物及醇提物、卷柏醇提物、忍冬藤水提物、杨树花水提物、马蹄金醇提物、首乌藤水提物及醇提物、大血藤醇提物、猫须草水提物及醇提物、蕨麻水提物及醇提物、车前草醇提物均对哈维氏弧菌有抑制作用。其中，余甘子水提物及醇提物对两种弧菌的抑菌圈直径均达到15mm以上；荔枝草醇提物、首乌藤水提物、蕨麻水提物对哈维氏弧菌的抑菌圈也达到15mm以上。余甘子醇提物抑制两种弧菌活性最强，抑菌圈直径最大，分别为(19.11±0.20)mm、(23.9±0.30)mm。
根据上述实验结果，选择抑制哈维氏弧菌、副溶血弧菌活性最好的余甘子醇提物作为进一步实验对象，测定其最小抑菌浓度(MIC)。
2、余甘子醇提物对两种弧菌的MIC测定
采用二倍稀释法测定余甘子醇提物对两种弧菌的MIC，即将1g/mL的余甘子醇提液分别稀释至生药浓度为500mg/mL、250mg/mL、125mg/mL、62.5mg/mL、31.25mg/mL、15.63mg/mL、7.82mg/mL。于30℃200r/min摇床振荡恒温培养24h后观察结果，把无菌生长稀释度最大的药液浓度作为MIC值。
表2余甘子醇提物对两种弧菌的最低抑菌浓度实验结果
注：“-”表示无活性。
实验结果如表2所示，余甘子醇提物对哈维氏弧菌和副溶血弧菌均有较好的抑制作用，MIC值分别均为15.63mg/mL。
实施例2余甘子抑制海洋致病菌有效成分的提取分离、检测分析。
1、抑菌活性成分提取
称取余甘子粉末，加入无水乙醇，在室温下超声提取30min，再在50℃下回流提取30min，抽滤，同法提取2次，收集滤液，浓缩使生药的最终浓度为1g/mL。
2、抑菌活性成分分离
对余甘子醇提物进行分离，余甘子醇提物旋蒸成浸膏后加入蒸馏水配成混浊液，分别用等体积石油醚、乙酸乙酯、正丁醇萃取，萃取至溶液澄清无色，每次萃取时间30min。萃取完毕后，将溶剂旋干后得到干燥粉末，将不同萃取物干燥粉末，配置成10mg/mL的样品溶液。选取哈维氏弧菌为目标菌株，采用牛津杯法，在无菌环境下，向平板中倒入约20mL已灭菌的牛肉膏蛋白胨琼脂培养基，凝固后，加入200μL的菌悬液，涂布棒涂布均匀，静置10min。将牛津杯垂直均匀放置于培养基上，分别在杯中加入200μL样品溶液溶液，每个样品进行3次平行实验，移至37℃恒温培养箱培养18h后，用电子数显卡尺测定抑菌圈直径，结果取三次测量结果的平均值。抑菌圈直径大于20mm为极敏，15～19mm为高敏，10～15mm为中敏，小于10mm为低敏(无活性)。
表3余甘子各组分抑制哈维氏弧菌结果
注：“+++”表示极敏、“++”表示高敏、“+”表示中敏、“-”表示低敏(无活性)。
通过对不同极性段的分离，得到四个组分。将四个组分进行抑菌实验，实验结果如表3所示，石油醚组对哈维氏弧菌没有抑制活性，正丁醇组与蒸馏水组对哈维氏弧菌均有抑制作用。乙酸乙酯组对哈维氏弧菌有较好的抑制作用。
3抑菌活性成分化学定性检测
使用化学定性的方法对乙酸乙酯组进行检测，用盐酸+镁粉、溴甲酚绿溶液及三氯化铁-铁氰化钾溶液进行化学定性检测。
表4余甘子抑制哈维氏弧菌活性成分的检测
实验结果如表4所示，乙酸乙酯组与溴甲酚绿溶液呈现阳性反应，说明其主要成分为羧酸类化合物；乙酸乙酯组又与三氯化铁-铁氰化钾溶液呈现阳性反应，说明其主要成分为酚酸类化合物。
通过上述分离、化学定性检测，初步确定余甘子抑制海洋致病菌的有效成分为酚酸类化合物。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (3)
1.余甘子提取物在制备抗海洋弧菌药物中的用途，其特征在于，所述余甘子提取物为以余甘子为原料，采用水或者醇提取所得的余甘子水提物或余甘子醇提取；所抑制的海洋弧菌为哈维氏弧菌或者副溶血弧菌。
2.根据权利要求1所述的用途，其特征在于，所述的余甘子提取物的提取方法为：称取余甘子制成的粉末，加入溶剂水或无水乙醇，在室温下超声提取10～90min，再在30℃～90℃下回流提取30～120min，抽滤，同法提取1～4次，收集滤液，浓缩使生药的最终浓度为1g/mL。
3.根据权利要求1所述的用途，其特征在于，所述的余甘子提取物的提取方法如下：
(1)称取余甘子粉末，加入无水乙醇，在室温下超声提取30min，再在50℃下回流提取30min，抽滤，同法提取2次，收集滤液，浓缩使生药的最终浓度为1g/mL；
(2)抑菌活性成分分离：对余甘子醇提物进行分离，余甘子醇提物旋蒸成浸膏后加入蒸馏水配成混浊液，用等体积乙酸乙酯萃取至溶液澄清无色，萃取时间30min；萃取完毕后，将溶剂旋干得到干燥粉末，即得。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211227946.2A CN115414403B (zh) | 2022-10-09 | 2022-10-09 | 余甘子提取物在制备抗海洋弧菌药物中的用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211227946.2A CN115414403B (zh) | 2022-10-09 | 2022-10-09 | 余甘子提取物在制备抗海洋弧菌药物中的用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115414403A true CN115414403A (zh) | 2022-12-02 |
| CN115414403B CN115414403B (zh) | 2023-06-20 |
Family
ID=84206786
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211227946.2A Active CN115414403B (zh) | 2022-10-09 | 2022-10-09 | 余甘子提取物在制备抗海洋弧菌药物中的用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115414403B (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1947525A (zh) * | 2006-11-28 | 2007-04-18 | 黄华信 | 余甘子中药复方防腐制剂及其制备和用途 |
| US20120107432A1 (en) * | 2009-06-29 | 2012-05-03 | Benny Antony | Composition of extract of emblica officinalis and method of preparing the same |
| CN103347529A (zh) * | 2010-12-09 | 2013-10-09 | Y&B妈妈之选有限公司 | 包含皂苷的制剂及其用途 |
| CN109276602A (zh) * | 2018-12-03 | 2019-01-29 | 四川省畜牧科学研究院 | 余甘子的用途及其组合物和组合物用途 |
| US20190365839A1 (en) * | 2016-11-20 | 2019-12-05 | Laila Nutraceuticals | Synergistic dietary supplement compositions of sphaeranthus indicus and terminalia chebula for liver health |
| US20200085904A1 (en) * | 2017-03-17 | 2020-03-19 | Kerala Ayurveda Limited (India) | Herbal composition |
-
2022
- 2022-10-09 CN CN202211227946.2A patent/CN115414403B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1947525A (zh) * | 2006-11-28 | 2007-04-18 | 黄华信 | 余甘子中药复方防腐制剂及其制备和用途 |
| US20120107432A1 (en) * | 2009-06-29 | 2012-05-03 | Benny Antony | Composition of extract of emblica officinalis and method of preparing the same |
| CN103347529A (zh) * | 2010-12-09 | 2013-10-09 | Y&B妈妈之选有限公司 | 包含皂苷的制剂及其用途 |
| US20190365839A1 (en) * | 2016-11-20 | 2019-12-05 | Laila Nutraceuticals | Synergistic dietary supplement compositions of sphaeranthus indicus and terminalia chebula for liver health |
| US20200085904A1 (en) * | 2017-03-17 | 2020-03-19 | Kerala Ayurveda Limited (India) | Herbal composition |
| CN109276602A (zh) * | 2018-12-03 | 2019-01-29 | 四川省畜牧科学研究院 | 余甘子的用途及其组合物和组合物用途 |
Non-Patent Citations (2)
| Title |
|---|
| HOANG-VIET-TRINH NGO ,ETC: "Effects of Phyllanthus amarus extract on nonspecific immune responses ,growth,and resistance to Vibro alginolyticus in white shrimp Litopenaeus vannamei", 《FISH & SHELLFISH IMMUNOLOGY》, pages 1 - 8 * |
| 周艳丽等: "红甜菜提取物抗菌潜力研究与应用", 《中国糖料》, pages 57 - 61 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115414403B (zh) | 2023-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102145066B (zh) | 一种从密茱萸中提取总黄酮的制备方法 | |
| CN101352180A (zh) | 用于抑制植物病原菌的博落回生物碱及其制备方法 | |
| CN101492706A (zh) | 一种提高蛹虫草液体发酵虫草菌素产量的方法 | |
| CN112807253A (zh) | 一种裂褶菌发酵产物及其制备方法、护肤品 | |
| CN101914586B (zh) | Don毒素的制备纯化方法 | |
| WO2021093425A1 (zh) | 黄芪-蝉拟青霉发酵菌质及其用途 | |
| CN113262245B (zh) | 山茱萸提取物在制备抑制海洋致病菌药物中的用途 | |
| CN115414403A (zh) | 余甘子提取物在制备抗海洋弧菌药物中的用途 | |
| CN114983997B (zh) | 一种治疗石斑鱼虹彩病毒病的药物 | |
| CN105481817B (zh) | 一种异香豆素类化合物及其制备方法和应用 | |
| CN105766377B (zh) | 一种提高黑木耳黄酮含量和种类的培养方法 | |
| CN112691125A (zh) | 一种药物组合物及其制备方法、护肤品 | |
| CN105348247B (zh) | 一种异香豆素类化合物及其制备方法和应用 | |
| CN115381873A (zh) | 牡丹皮提取物在制备抗海洋弧菌药物中的用途 | |
| CN105695544B (zh) | 一种从凝结芽孢杆菌中提取环(脯-甘)二肽的方法 | |
| CN105352902B (zh) | 一种快速测定蝉花样品中多球壳菌素含量的方法 | |
| CN114315812A (zh) | 海洋真菌来源的两种生物碱化合物及其制备方法和在制备抗炎药物中的应用 | |
| CN105837545A (zh) | 含卤素或苯并咪唑类功能基团的香豆素类衍生物及其制备方法和应用 | |
| CN114350529B (zh) | 一株产查尔酮类成分的甘草内生真菌及其应用 | |
| Mar'ie et al. | Antibacterial activity of Cassia alata stems ethanol extract against Staphylococcus aureus | |
| AU2020102410A4 (en) | Method for screening chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using tetrahymena thermophila | |
| CN109350648B (zh) | 一种连翘发酵原液及其制备方法 | |
| CN113173904B (zh) | 新的抑菌化合物以及用于制备该化合物的曲霉 | |
| CN102584806B (zh) | 天然植物中一个黄酮的制备方法 | |
| CN114276471B (zh) | 长茎葡萄蕨藻多糖的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |