CN116283987A - 一种苦参碱类生物碱及其制备方法和用途 - Google Patents
一种苦参碱类生物碱及其制备方法和用途 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
-
- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
- C07D471/16—Peri-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
- C07D471/18—Bridged systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
-
- 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
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Plant Pathology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Pest Control & Pesticides (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Insects & Arthropods (AREA)
- General Health & Medical Sciences (AREA)
- Dentistry (AREA)
- Health & Medical Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
本发明公开了一种苦参碱类生物碱及其制备方法和用途,苦参碱类生物碱选自新的化合物1~化合物16中的任意一种,从越南槐根茎中提取分离得或通过人工合成获得,还提供了苦参碱类生物碱的制备方法,验证了该苦参碱类生物碱在预防和治疗TSWV和毒杀TSWV传播媒介西花蓟马上具有显著的作用。
Description
技术领域
本发明涉及一种农作物的农药技术领域,特别是一种苦参碱类生物碱及其制备方法和用途。
背景技术
植物病毒病是仅次于植物真菌病害的第二大类植物病害,其专化性强、危害大,较难防治。每年在全世界造成的经济损失高达600亿美元,其中仅粮食作物就损失高达200亿美元。番茄斑萎病毒(Tomato spotted wilt virus,TSWV)是全球公认的排名前10种植物的病毒之一,也是番茄斑萎病毒属中唯一能感染植物的病毒。TSWV可侵染84个科1090种植物,尤其在农作物大豆、花生、芹菜、南瓜、辣椒、马铃薯和番茄等侵染严重,在温带和亚热带地区危害最为严重。我国最早于2003年在辣椒上检测出TSWV,随后危害范围逐年扩大,在四川、云南、广州、山东、云南、河北、宁夏、陕西、青海等地多种园艺植物上均有报道。目前,TSWV对农作物生产的危害日趋严重,大棚、温室和大田栽培番茄均有受损,严重时产量损失可超过80%甚至绝收。TSWV可以通过多种蓟马和机械传播,其中西花蓟马(Frankliniellaoccidentalis)为主要传播媒介。西花蓟马具有寄主范围广、取食性杂繁殖能力强等特点,且一旦携带病毒则终生带毒。TSWV可以在西花蓟马体内自行复制增殖,大大增强了病毒的传播效率。当前,随着西花蓟马和TSWV的频繁交叉侵染,以及TSWV病毒的易变性,使得当前防治措施仅能在一定程度上抑制或减少病毒在植物体内的增殖,而无法彻底清除TSWV。针对目前植物病毒病TSWV危害严重、抗植物病毒TSWV的药剂依然匮乏、病毒耐药性增强和农药残留污染环境等问题,开发高效、环保、安全的抗TSWV病毒剂已成为当前抗植物病毒TSWV新农药研发的重点和热点。
发明内容
本发明的目的在于,提供一种苦参碱类生物碱及其制备方法和用途。本发明从越南槐根茎中提取到一种新的苦参碱类生物碱,经药效试验证明具有有效防治TSWV和杀虫的特点。
本发明的技术方案:苦参碱类生物碱,所述苦参碱类生物碱选自以下化学结构的化合物1~化合物16中的任意一种:
所述苦参碱类生物碱是从越南槐根茎中提取分离获得或通过人工合成获得。
上述的苦参碱类生物碱的制备方法,包括以下步骤:
(1)取干燥的越南槐根茎以甲醇回流提取,合并提取液浓缩得浸膏,浸膏采用酸碱处理得总生物碱;
(2)取总生物碱经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为100:0~0:100为流动相进行梯度洗脱,得馏分A、馏分B、馏分C、馏分D和馏分E;
(3)将馏分D经MCI柱色谱分离,用甲醇:水以体积比为0:100~100:0为流动相进行梯度洗脱,得D-1、D-2、D-3和D-4;
(4)将D-1经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为100:1:0.125~1:1:0.005为流动相进行梯度洗脱得化合物14和化合物12;
(5)将D-2经甲醇凝胶纯化,再经半制备HPLC纯化,用甲醇:水以体积比为21:79为流动相进行洗脱,得化合物6、化合物7和化合物1;
(6)将D-3经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为50:1为流动相进行梯度洗脱,再经甲醇凝胶纯化,得化合物8、化合物9、化合物4和化合物5;
(7)将D-4经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为50:1:0.125~1:1:0.005为流动相进行梯度洗脱,得化合物10和化合物15;
(8)将馏分E经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为20:1~0:100为流动相进行梯度洗脱,得E-1、E-2、E-3、E-4和E-5;
(9)将E-1经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为25:1:0.005~1:1:0.005为流动相进行梯度洗脱,再经甲醇凝胶纯化,再经半制备HPLC纯化,最后用乙腈:水以体积比为17:83为流动相进行洗脱,得化合物11;
(10)将E-3经甲醇凝胶纯化,再通过300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为10:1:0.0.05~1:1:0.005为流动相进行梯度洗脱,得化合物16;
(11)将E-4经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为25:1为流动相进行梯度洗脱,再经半制备HPLC纯化,用甲醇:水以体积比为34:66为流动相进行洗脱,得化合物2和化合物3;
(12)将E-5经甲醇凝胶纯化,再经半制备HPLC纯化,用甲醇:水以体积比为36:64为流动相进行洗脱,得化合物13。
前述的制备方法中,步骤(1)中,所述越南槐为豆科槐属植物越南槐。
前述的制备方法中,步骤(1)中,所述回流提取为回流提取3次,每次3小时。
前述的制备方法中,步骤(1)中,所述酸碱处理具体为,先用0.1%的盐酸调pH值为1-3,然后用石油醚萃取除去非生物碱成分,酸水层再用25%~28%的浓氨水调pH为9-11,然后碱水层依次使用二氯甲烷和正丁醇萃取。
前述的制备方法中,步骤(2)中,所述馏分A是二氯甲烷:甲醇以体积比为50:1洗脱得到,馏分B是二氯甲烷:甲醇以体积比为20:1洗脱得到,馏分C是二氯甲烷:甲醇以体积比为10:1洗脱得到,馏分D是二氯甲烷:甲醇以体积比为5:1洗脱得到,E是二氯甲烷:甲醇以体积比为2:1洗脱得到。
前述的制备方法中,步骤(3)中,所述D-1是甲醇:水以体积比为20:80洗脱得到,D-2是甲醇:水以体积比为40:60洗脱得到,D-3是甲醇:水以体积比为60:40洗脱得到,D-4是甲醇:水以体积比为80:20洗脱得到。
前述的制备方法中,步骤(4)中,所述化合物14是石油醚:丙酮:二乙胺以体积比为20:1:0.005洗脱得到,化合物12是石油醚:丙酮:二乙胺以体积比为10:1:0.005洗脱得到。
前述的制备方法中,步骤(7)中,所述化合物10是石油醚:丙酮:二乙胺以体积比为10:1:0.005洗脱得到,化合物15是石油醚:丙酮:二乙胺以体积比为2:1:0.005洗脱得到。
前述的制备方法中,步骤(8)中,所述E-1是二氯甲烷:甲醇以体积比为15:1洗脱得到,E-2是二氯甲烷:甲醇以体积比为10:1洗脱得到,E-3是二氯甲烷:甲醇以体积比为5:1洗脱得到,E-4是二氯甲烷:甲醇以体积比为2:1洗脱得到,E-5是二氯甲烷:甲醇以体积比为1:1洗脱得到。
前述的制备方法中,步骤(10)中,所述化合物16是石油醚:丙酮:二乙胺以体积比为5:1:0.005洗脱得到。
上述方法制备得到的苦参碱类生物碱为白色粉末,化合物1-化合物16的核磁氢谱(1H-NMR)和碳谱(13C-NMR)数据如表1-表6,其中表1为化合物1的氢谱(1H-NMR)和碳谱(13C-NMR)数据,表2为化合物2-化合物6的氢谱(1H-NMR)数据,表3为化合物7-化合物11的氢谱(1H-NMR)数据;表4为化合物12-化合物16的氢谱(1H-NMR)数据;表5为化合物2-化合物9的碳谱(13C-NMR)数据;表6为化合物10-化合物16的碳谱(13C-NMR)数据。
表1.化合物1的1H和13C NMR数据(δinppm,JinHz)
a600MHzinCDCl3
表2.化合物2-6的1HNMR数据(δinppm,Jin Hz)
a150MHz in CDCl3;b150MHz in CD3OD.
表3.化合物7-11的1HNMR数据(δinppm,Jin Hz)
a150MHzinCDCl3;b150MHzinCD3OD.
表4.化合物12-16的1HNMR数据(δinppm,Jin Hz)
a150MHz in CDCl3;b150MHz in CD3OD.
表5.化合物2-9的13C NMR数据(δin ppm)
a150MHz in CDCl3;b150MHz in CD3OD.
表6.化合物9-16的13C NMR数据(δinppm)
Position | 10a | 11a | 12a | 13a | 14a | 15a | 16a |
2 | 46.2 | 44.5 | 57.1 | 50.2 | 50.2 | 55.8 | 56.2 |
3 | 23.1 | 22.3 | 20.8 | 19.7 | 19.8 | 20.8 | 22.4 |
4 | 27.3 | 35.0 | 27.6 | 24.1 | 24.1 | 26.5 | 37.5 |
5 | 113.9 | 208.4 | 35.6 | 115.0 | 114.6 | 34.1 | 71.6 |
6 | 171.7 | 169.4 | 63.7 | 152.2 | 152.0 | 63.4 | 70.7 |
7 | 205.3 | 45.6 | 42.6 | 113.8 | 113.4 | 60.1 | 71.5 |
8 | 36.3 | 26.9 | 26.8 | 23.9 | 22.0 | 208.9 | 34.0 |
9 | 29.0 | 19.5 | 20.6 | 19.6 | 19.7 | 40.0 | 21.5 |
10 | 46.3 | 47.4 | 57.2 | 49.6 | 49.6 | 56.5 | 56.4 |
11 | 64.3 | 64.2 | 59.7 | 148.6 | 148.9 | 52.5 | 56.7 |
12 | 25.3 | 30.0 | 27.0 | 30.2 | 29.7 | 26.6 | 19.0 |
13 | 17.5 | 17.1 | 20.8 | 23.0 | 23.6 | 18.4 | 21.6 |
14 | 33.1 | 31.0 | 33.9 | 34.7 | 32.8 | 32.7 | 32.6 |
15 | 169.2 | 172.2 | 173.9 | 175.2 | 173.7 | 169.2 | 173.3 |
17 | 122.2 | 60.9 | 40.8 | 134.8 | 135.1 | 41.4 | 47.0 |
OCH3 | - | - | 51.5 | - | 51.6 | - | - |
a150MHz in CDCl3;b150MHz in CD3OD.
经过上述结构鉴定,得化合物1-化合物16为:
化合物1:白色粉末;[α]25D–102.5(c 0.12,MeOH);UV(MeOH)λmax(logε):201(4.36)nm;IR(KBr)vmax 2935,1710,1640,1470,1334,1196,1101cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表1和表1;HRESIMS m/z 571.3134[M+H]+,(计算值C30H43N4O7,571.3126)。
化合物2:无色透明晶体;mp 182-83℃;[α]25D+65.9(c 0.04,MeOH);UV(MeOH)λmax(logε):200(4.17)nm;IR(KBr)vmax 2922,1624,1454,1415,1385,1129cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表2和表5;HRESIMS m/z 315.1674[M+Na]+(计算值C16H24N2O3Na,315.1679)。
化合物3:白色粉末;[α]25D+67.4(c 0.13,MeOH);UV(MeOH)λmax(logε):201(3.99)nm;IR(KBr)vmax 3409,2926,1622,1455,1385,1178,1127cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表2和表5;HRESIMS m/z 279.1695[M+H]+(计算值C15H23N2O3,279.1703)。
化合物4:白色粉末;[α]25D+61.8(c 0.09,MeOH);UV(MeOH)λmax(logε):200(4.05)nm;IR(KBr)vmax 3394,2943,1627,1541,1447,1372,1125cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表2和表5;HRESIMS m/z 342.1794[M+Na]+(计算值C17H25N3O3Na,342.1788)。
化合物5:白色粉末;[α]25D+87.7(c 0.12,MeOH);UV(MeOH)λmax(logε):200(3.97)nm;IR(KBr)vmax 2959,1664,1414,1351,1041,1026cm-1;1H and 13C NMR数据(CDCl3,600and150MHz)见表2和表5;HRESIMS m/z 313.1526[M+H]+(计算值C16H22N2O3Na,313.1523)。
化合物6:无色油状物;[α]25D+59.6(c 0.04,MeOH);UV(MeOH)λmax(logε):201(4.14)nm;IR(KBr)vmax 2940,1683,1635,1412,1385,1332,1206,1180,1132cm-1;1H and 13CNMR数据(CD3OD,600and150MHz)见表2和表5;HRESIMS m/z 299.1358[M+Na]+(计算值C15H20N2O3Na,299.1366)。
化合物7:白色粉末;[α]25D+51.5(c 0.03,MeOH);UV(MeOH)λmax(logε):200(4.10),238(3.58)nm;IR(KBr)vmax 2947,1682,1647,1384,1205,1180,1133,1085,801,722cm-1;1H and 13C NMR数据(CD3OD,600and 150MHz)见表3和表5;HRESIMS m/z 299.1357[M+H]+(计算值C15H20N2O3Na,299.1366)。
化合物8:白色粉末;[α]25D+73.8(c 0.12,MeOH);UV(MeOH)λmax(logε):201(3.95),231(3.55)nm;IR(KBr)vmax 2937,1748,1672,1634,1449,1310,1222,1013cm-1;1Hand 13C NMR数据(CDCl3,600and 150MHz)见表3和表5;HRESIMS m/z 359.1581[M+H]+(计算值C17H24N2O5Na,359.1577)。
化合物9:白色粉末;[α]25D–29.0(c 0.10,MeOH);UV(MeOH)λmax(logε):200(3.78)nm;IR(KBr)vmax 2938,1736,1669,1670,1645,1452,1213,1015cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表3和表5;HRESIMS m/z 359.1581[M+Na]+(计算值C17H24N2O5Na,359.1577)。
化合物10:无色透明晶体;mp 225-226℃;[α]25D+45.5(c 0.10,MeOH);UV(MeOH)λmax(logε):208(3.65),250(3.95)nm;IR(KBr)vmax 2945,1710,1639,1480,1404,1253cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表3和表6;HRESIMS m/z 299.1370[M+H]+(计算值C15H20N2O3Na,299.1366)。
化合物11:白色粉末;[α]25D+36.9(c 0.07,MeOH);UV(MeOH)λmax(logε):201(4.25),240(2.52)nm;IR(KBr)vmax 2932,1671,1644,1459,1396,1367,1273,1180cm-1;1Hand 13C NMR数据(CDCl3,600and 150MHz)见表3和表6;HRESIMS m/z 301.1515[M+Na]+(计算值C15H22N2O3Na,301.1523)。
化合物12:无色针状结晶;mp 195-196℃;[α]25D+65.5(c 0.03,MeOH);UV(MeOH)λmax(logε):200(3.91)nm;IR(KBr)vmax 2936,1737,1634,1444,1385,1202,1166,1096cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表4和表6;HRESIMS m/z332.1952[M+Na]+(计算值C16H27N3O3Na,332.1945)。
化合物13:白色粉末;UV(MeOH)λmax(logε):220(4.12),251(3.07),296(4.13)nm;IR(KBr)vmax 3375,2919,1670,1567,1475,1382cm-1;1H and 13C NMR数据(CDCl3,600and150MHz)见表4和表6;HRESIMS m/z 260.1760[M+H]+(计算值C15H22N3O,260.1757)。
化合物14:白色粉末;UV(MeOH)λmax(logε):221(3.87),251(3.13),297(3.87)nm;IR(KBr)vmax 2927,2358,1733,1635,1594,1557,1557,1201cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表4和表6;HRESIMS m/z 275.1755[M+Na]+(计算值C16H23N2O2,275.1754)。
化合物15:白色粉末;[α]25D+55.3(c 0.04,MeOH);UV(MeOH)λmax(logε):200(4.25),259(3.51)nm;IR(KBr)vmax 2932,1671,1664,1396,1273,1180,1140,1084cm-1;1Hand 13C NMR数据(CDCl3,600and 150MHz)见表4和表6;HRESIMS m/z 285.1580[M+Na]+(计算值C15H22N2O2Na,285.1574)。
化合物16:白色粉末;[α]25D+40.8(c 0.06,MeOH);UV(MeOH)λmax(logε):201(4.12)nm;IR(KBr)vmax 3426,2927,1626,1415,1384,1131,1040cm-1;1H and 13C NMR数据(CDCl3,600and 150MHz)见表4和表6;HRESIMS m/z 303.1675[M+Na]+(计算值C15H24N2O3Na,303.1679)。
本发明还提供一种药物组合物,包括上述的苦参碱类生物碱或其盐及其农药上可接受的载体。
所述的药物组合物为药物制剂。药物制剂包括:粉剂、可湿性粉剂、乳油、颗粒剂、悬浮剂、可溶性液剂、乳粉、水剂、烟剂、种衣剂、微囊剂等。
本发明还提供上述的苦参碱类生物碱或上述的药物组合物在制备抗植物病毒TSWV的农药或杀虫剂中的应用。
与现有技术相比:
本发明以越南槐根茎为原料,采用了一种新的提取分离方式,制备方法简便易操作,从中提取出了新化合物,结构新颖,鉴定为苦参碱类生物碱。经试验验证,该苦参碱类生物碱对番茄斑萎病毒病毒,在治疗和预防方面均具有较强的抑制作用,对于传毒媒介西花蓟马具有较强的毒杀作用。
具体实施方式
下面结合实施例对本发明作进一步的说明,但并不作为对本发明限制的依据。
实施例1:
苦参碱类生物碱,选自以下化学结构的化合物1~化合物16中的任意一种:
所述苦参碱类生物碱是从越南槐根茎中提取分离获得或通过人工合成获得。
苦参碱类生物碱的制备方法,包括以下步骤:
(1)取干燥的越南槐根茎45kg以甲醇回流提取3次,每次3小时,合并提取液浓缩得浸膏,浸膏采用酸碱处理,即先用0.1%的盐酸调pH值为2,然后用石油醚萃取除去非生物碱成分,酸水层再用26%的浓氨水调pH为10,然后碱水层再依次使用二氯甲烷和正丁醇萃取,得总生物碱为1.7kg;
(2)取总生物碱经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为100:0~0:100为流动相进行梯度洗脱,以体积比为50:1洗脱得馏分A、以体积比为20:1洗脱得馏分B、以体积比为10:1洗脱得馏分C、以体积比5:1洗脱得馏分D、以体积比2:1洗脱得馏分E;
(3)将馏分D经MCI柱色谱分离,用甲醇:水以体积比为0:100~100:0为流动相进行梯度洗脱,以体积比为20:80洗脱得D-1、以体积比为40:60洗脱得D-2、以体积比为60:40洗脱得D-3、以体积比为80:20洗脱得D-4;
(4)将D-1经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为100:1:0.125~1:1:0.005为流动相进行梯度洗脱,以体积比为20:1:0.125洗脱得化合物14为23.2mg,以体积比为10:1:0.005洗脱得化合物12为52.7mg;
(5)将D-2经甲醇凝胶纯化,再经半制备HPLC纯化,用甲醇:水以体积比为21:79为流动相进行洗脱,得化合物6为27.7mg、化合物7为21.6mg和化合物1为9.3mg;
(6)将D-3经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为50:1为流动相进行梯度洗脱,再经甲醇凝胶纯化,得化合物8为9.5mg、化合物9为13.2mg、化合物4为24.3mg和化合物5为8.5mg;
(7)将D-4经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为50:1:0.125~1:1:0.005为流动相进行梯度洗脱,以体积比为10:1:0.125洗脱得化合物10为23.5mg和以体积比为2:1:0.005洗脱得化合物15为8.4mg;
(8)将馏分E经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为20:1~0:100为流动相进行梯度洗脱,以体积比为15:1洗脱得E-1、以体积比为10:1洗脱得E-2、以体积比为5:1洗脱得E-3、以体积比为2:1洗脱得E-4和以体积比为1:1洗脱得E-5;
(9)将E-1经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为25:1:0.005~1:1:0.005为流动相进行梯度洗脱,再经过甲醇凝胶分离,再经半制备HPLC纯化,最后用乙腈:水以体积比为17:83为流动相进行洗脱,得化合物11为34.1mg;
(10)将E-3经甲醇凝胶纯化,再通过300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为10:1:0.005~1:1:0.005为流动相进行梯度洗脱,以体积比为5:1:0.005洗脱得化合物16为46.9mg;
(11)将E-4经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为25:1为流动相进行洗脱,再经半制备HPLC纯化,用甲醇:水以体积比为34:66为流动相进行洗脱,得化合物2为36.2mg和化合物11为43.9mg。
(12)将E-5经甲醇凝胶纯化,再经半制备HPLC纯化,用甲醇:水以体积比为36:64为流动相进行洗脱,得化合物13为26.1mg。
一种药物组合物,包括上述的苦参碱类生物碱或其盐及其农药上可接受的载体。
本发明还提供上述的苦参碱类生物碱或者药物组合物在制备抗植物病毒TSWV的农药或杀虫剂中的应用。
实验例1:
将实施例1得到的苦参碱型生物碱进行抗TSWV活性研究验证,具体方法如下:
a、寄主为普通烟栽培品种K326(Nicotianatabacum cv.K326),种子由本实验保存提供并栽培于玻璃温室中。番茄斑萎病毒(TSWV)毒源由本实验室保存提供。宁南霉素购于黑龙江德强生物股份有限公司。
b、接种病毒和施药:
先接病毒后施药:选择生长一致的5~6片真叶期的烟苗摩擦接种TSWV,TSWV接种于第4片和第5片真叶上,10min后用清水冲洗,24h后再分别涂抹1mL浓度为100μg/mL的化合物1~化合物16,涂抹均匀,10min后用清水冲洗;阳性对照2个,一个阳性对照为先摩擦接种TSWV,24h后涂抹DMSO(用无菌水稀释成100μg/mL),用于计算抑制率;另一个阳性对照为先摩擦接种TSWV,24h后涂抹宁南霉素,24h后涂抹DMSO作为空白对照。植物生长于无虫温室中。试验重复3次,涂抹3d后统计枯斑数,计算抑制率。
抑制率(%)=(阳性对照枯斑数-处理枯斑数)/阳性对照枯斑数×100%先施药后接病毒:选择生长一致的5~6片真叶期的烟苗,于第4片和第5片真叶上分别均匀涂抹1mL浓度为100μg/mL的化合物1~化合物16,10min后用清水冲洗,6h后摩擦接种TSWV,10min后用清水冲洗。阳性对照2个,一个阳性对照为先涂抹DMSO(100μg/mL),6h后接种TSWV,用于计算抑制率;另一个阳性对照为先施宁南霉素,6h后接种TSWV,只涂抹DMSO作为空白对照。烟苗生长于无虫温室中,重复3次,接种病毒3d后统计枯斑数,并计算抑制率。检测结果见表7。
表7化合物1-化合物16抗TSWV活性结果
a三次平均值;b宁南霉素作为阳性对照.
体内抗TSWV试验表明;化合物12(100μg/mL)在先施药后接种病毒的处理中表现出较强的预防作用,其抑制率为78.0%,高于阳性对照宁南霉素65.0%。
实验例2:
对上述的苦参碱类生物碱的杀虫活性进行研究验证,具体如下:
供试靶标:西花蓟马(Frankliniella occidentalis)系室内饲养多年的敏感品系,实验前收集西花蓟马的伪蛹,待其羽化后,选取同一天羽化的成虫供本试验用。
培养条件:供试靶标及试验后靶标的培养条件为温度27±1℃,相对湿度70±5%,光照周期16/8h(L/D)。
仪器设备:电子天平(感量万分之一)、喷雾塔、100ml烧杯、玻璃试管、量筒、培养皿、帕拉膜、海绵、滤纸、移液管、镊子、毛笔、显微镜等。
试验药剂:新化合物含量均以100%计。
试验浓度:200mg/L。
药剂配制:原药:用万分之一电子天平称取所需量;溶剂:N,N-二甲基甲酰胺(DMF),0.2%;乳化剂:Tween 80,0.2%;加入清水稀释到所需浓度。
试验方法:
将测试化合物溶于N,N-二甲基甲酰胺中,用含TW-80(2mL/L)的蒸馏水稀释至所需浓度,均设置4个重复。在直径200mm培养皿底部放入直径为180mm滤纸,将新鲜葱段切成长度120mm,置于不同浓度的药液中浸泡30s后取出后在自然条件下晾干,放入先准备好的培养皿中,每皿5根,并挑选50头生长健康大小一致的供试蓟马成虫分别于培养皿中,用保鲜膜进行封口并扎眼20孔保持培养皿内通风,将培养皿放置于上述人工气候箱中饲养,阳性对照为乙基多杀菌素,阴性对照为等量N,N-二甲基甲酰胺和TW-80浓度的溶液(CK)。
调查方法:
48h后观察记录各个浓度杀虫剂处理下的蓟马成虫的存活情况,观察时用细毛笔轻触碰虫体,两次不动则记录为蓟马死亡,记录结果见表8。
表8部分化合物的对西花蓟马的杀虫效果
以上表8的结果显示,化合物4、化合物10和化合物12在供试浓度下对西花蓟马表现出较好活性。
实验例3:
对化合物4、化合物10和化合物12对西花蓟马的杀虫活性进行进一步评价,具体方法如下:
分别设定200mg/L、100mg/L、50mg/L、25mg/L、12.5mg/L和6.25mg/L六个浓度的化合物4、化合物10和化合物12,采用实验例2的方法进行抗蚜虫活性研究。
数据统计分析:所有试验数据均采用DPS V 7.0统计软件进行分析。根据试验数据计算死亡率,求出毒力回归方程、相关系数(r)、LC50、LC90及其95%置信限,见表9。
表9 LC50化合物4、化合物10和化合物12对西花蓟马的活性测试结果
由表9可得,化合物4、化合物10和化合物12具有较高的杀虫活性。
Claims (10)
2.根据权利要求1所述的苦参碱类生物碱的制备方法,其特征在于:包括以下步骤:
(1)取越南槐根茎以甲醇回流提取,合并提取液浓缩得浸膏,浸膏采用酸碱处理得总生物碱;
(2)取总生物碱经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为100:0~0:100为流动相进行梯度洗脱,得馏分A、馏分B、馏分C、馏分D和馏分E;
(3)将馏分D经MCI柱色谱分离,用甲醇:水以体积比为0:100~100:0为流动相进行梯度洗脱,得D-1、D-2、D-3和D-4;
(4)将D-1经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为100:1:0.125~1:1:0.005为流动相进行梯度洗脱,得化合物14和化合物12;
(5)将D-2经甲醇凝胶纯化,再经半制备HPLC纯化,用甲醇:水以体积比为21:79为流动相进行洗脱,得化合物6、化合物7和化合物1;
(6)将D-3经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为50:1为流动相进行洗脱,再经甲醇凝胶纯化,得化合物8、化合物9、化合物4和化合物5;
(7)将D-4经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为50:1:0.125~1:1:0.005为流动相进行梯度洗脱,得化合物10和化合物15;
(8)将馏分E经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为20:1~0:100为流动相进行梯度洗脱,得E-1、E-2、E-3、E-4和E-5;
(9)将E-1经300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为25:1:0.005~1:1:0.005为流动相进行梯度洗脱,再经甲醇凝胶分离,再经半制备HPLC纯化,最后用乙腈:水以体积比为17:83为流动相进行洗脱,得化合物11;
(10)将E-3经甲醇凝胶纯化,再通过300~400目硅胶柱层析,用石油醚:丙酮:二乙胺以体积比为10:1:0.0.05~1:1:0.005为流动相进行梯度洗脱,得化合物16;
(11)将E-4经300~400目硅胶柱层析,用二氯甲烷:甲醇以体积比为25:1为流动相进行梯度洗脱,再经半制备HPLC纯化,用甲醇:水以体积比为34:66为流动相进行洗脱,得化合物2和化合物3;
(12)将E-5经甲醇凝胶纯化,再经半制备HPLC纯化,用甲醇:水以体积比为36:64为流动相进行洗脱,得化合物13。
3.根据权利要求2所述的制备方法,其特征在于:步骤(2)中,所述馏分A是二氯甲烷:甲醇以体积比为50:1洗脱得到,馏分B是二氯甲烷:甲醇以体积比为20:1洗脱得到,馏分C是二氯甲烷:甲醇以体积比为10:1洗脱得到,馏分D是二氯甲烷:甲醇以体积比为5:1洗脱得到,馏分E是二氯甲烷:甲醇以体积比为2:1洗脱得到。
4.根据权利要求2所述的制备方法,其特征在于:步骤(3)中,所述D-1是甲醇:水以体积比为20:80洗脱得到,D-2是甲醇:水以体积比为40:60洗脱得到,D-3是甲醇:水以体积比为60:40洗脱得到,D-4是甲醇:水以体积比为80:20洗脱得到。
5.根据权利要求2所述的制备方法,其特征在于:步骤(4)中,所述化合物14是石油醚:丙酮:二乙胺以体积比为20:1:0.005洗脱得到,化合物12是石油醚:丙酮:二乙胺以体积比为10:1:0.005洗脱得到。
6.根据权利要求2所述的制备方法,其特征在于:步骤(7)中,所述化合物10是石油醚:丙酮:二乙胺以体积比为10:1:0.005洗脱得到,化合物15是石油醚:丙酮:二乙胺以体积比为2:1:0.005洗脱得到。
7.根据权利要求2所述的制备方法,其特征在于:步骤(8)中,所述E-1是二氯甲烷:甲醇以体积比为15:1洗脱得到,E-2是二氯甲烷:甲醇以体积比为10:1洗脱得到,E-3是二氯甲烷:甲醇以体积比为5:1洗脱得到,E-4是二氯甲烷:甲醇以体积比为2:1洗脱得到,E-5是二氯甲烷:甲醇以体积比为1:1洗脱得到。
8.根据权利要求2所述的制备方法,其特征在于:步骤(10)中,所述化合物16是石油醚:丙酮:二乙胺以体积比为5:1:0.005洗脱得到。
9.一种药物组合物,包括权利要求1所述的苦参碱类生物碱或其盐及其农药上可接受的载体。
10.权利要求1所述的苦参碱类生物碱或权利要求9所示的药物组合物在制备抗植物病毒TSWV的农药或杀虫剂中的应用。
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