本发明属于天然杀虫剂领域，具体地，涉及从天然植物中提取杀虫剂的工艺。 The present invention belongs to the field of natural pesticides, in particular, to extract pesticides from natural plant process.

随着人们对于合成农药固有的一些特性，如污染环境、杀伤自然、残留物对人畜毒性大的认识，人们对自我生存环境的保护意识的增强，天然杀虫剂已越来越为人们所看好，世界各国都在积极从植物中寻找天然杀虫活性物质，每年世界上农业化学费用约有一半用于杀虫剂的研究，其重要性是显而易见的。 With some of the features inherent to synthetic pesticides, such as environmental pollution, destruction of natural, human and animal toxicity residues understanding people's living environment of self-protection awareness, natural pesticides have been more and more people are optimistic Research, the world are actively looking for a natural insecticidal active substances from plants in the world each year, about half the cost of agricultural chemicals used in pesticides, and its importance is obvious. 印楝(Azadirachta indica)植物是楝科植物中一种具有很强杀虫活性的长青植物，而从中提取制备的印楝素是高活性的昆虫拒食剂，其杀虫活性大约在半个世纪之前已为人们所认识。 Neem (Azadirachta indica) plants are evergreen plants Meliaceae plants a strong insecticidal activity, while preparing to extract azadirachtin is highly active insect antifeedant, its insecticidal activity in about half a century previously known by the people. 系统科学地研究印楝起始于60年代末期，迄今一直是天然杀虫剂研究的一个热点。 Neem scientific research system starting in the late 1960s, so far has been a hot natural insecticide research. 国外天然产物化学研究人员已以印楝各部分分离鉴定了一百余个化合物，并且作了许多生物活性测定，其中以印楝素活性最强，常用来作为天然杀虫活性的评价标准。 Foreign natural product chemistry researchers have isolated and identified in the various parts of a hundred neem compounds and made many bioassay, which azadirachtin strongest activity, commonly used as evaluation criteria natural insecticidal activity. 从印楝植物中分离提取印楝素工艺已有报道，但存在流程复杂、成本昂贵、得率较低、所得产品不稳定等缺点，如EC Uebel等在Journal of Liquid Chromatography 2(6)875-882(1979)，R.BRYAN YAMASAKI在Journal of Chromatography 356(1986)220-226，W.Kraus在TetrahedronVOL 43 No.12 2828(1987)中报道的工艺，均采用比较昂贵的分离试剂和材料，如乙腈等试剂、制备性的HPLC等，得率均少于0.02％。 Isolated from neem plant extract azadirachtin process have been reported, but there is a complex process, expensive, low yield, the resulting product is unstable and other shortcomings, such as EC Uebel et al. Journal of Liquid Chromatography 2 (6) 875- 882 (1979), R.BRYAN YAMASAKI in Journal of Chromatography 356 (1986) 220-226, W.Kraus in TetrahedronVOL 43 No.12 2828 (1987) reported that the processes are using more expensive separation reagents and materials, such as acetonitrile and the like reagents, preparative HPLC and the like, have ratios are less than 0.02%.

本发明的目的在于提供一种与现有技术不同的，流程简单、成本低廉、得率较高、产品稳定的从印楝中提取印楝素的工艺。 The purpose of the present invention is to provide a different prior art, the process is simple, low cost, high yield, product stability extraction azadirachtin from neem in the process.

本发明的目的是这样来实现的：以印楝植物的种仁为原料，依次用脱脂、提取分配、上柱分离步骤制得印楝素，所说脱脂用石油醚冷浸1-3次；所说提取分配先用甲醇∶水4∶1-9∶1浸泡或回流提取1-3次，滤液回收，在含水甲醇层中用乙酸乙酯萃取；所说上柱分离用乙酸乙酯层上硅胶柱1-2次，然后上中压柱MPLC，以氯仿∶丙酮9∶1洗脱，最后经过Rp18反相柱，以甲醇∶水1∶1洗脱即可得印楝素。 Object of the present invention is achieved: the neem seeds of plants as raw materials, followed by degreasing, extractable, the column separation steps gave azadirachtin, said defatted with petroleum ether to cold soak 1-3 times; allocation of said extracted first with methanol: water 4:1-9:1 soak or reflux extraction 1-3 times, the filtrate was recovered, the aqueous methanol layer was extracted with ethyl acetate in; on said upper ethyl acetate layer was separated by column silica gel 1-2 times, then on medium pressure column MPLC, chloroform: acetone elution 9:1, and finally through Rp18 reversed phase column with methanol: water 1:1 eluting you can get azadirachtin.

上述工艺中，提取分配可优选用甲醇∶水6∶1-9∶1浸泡3次。 The above-described process, the extraction may preferably be assigned with methanol: water 6:1-9:1 immersion 3 times.

上述工艺中，提取分配可优选用甲醇∶水6∶1-9∶1回流提取3次。 The above-described process, the extraction may preferably be assigned with methanol: water and extracted 3 times 6:1-9:1 reflux.

与现有技术相比，本发明存在的有益效果如下：1、由于本发明在工艺的提取分配步骤采用乙酸乙酯萃取，取代了已有技术用氯仿萃取、直接上HPLC的步骤，因而使用的溶剂成本低，为常用溶剂，同时克服了用氯仿使产物不稳定的缺点；2、本发明在上柱分离步骤增加了上中压柱MPLC步骤，使产物得率达到0.18％，比现有技术得率均高，如比ECVebel在Journal of LiguidChromatagraphy 2(6)875(1979)报道的得率高十倍，比R BRYAN YAMASAKI在Jouranal of Chromatography 356(1986)220-226报道的得率高数十倍，比W.Kraus在Tetrahedron VOL 43 No.12 2817(1987)报道的得率也高数十倍；而且产物不发生转变，稳定性大大增高，克服了现有技术产物得率低，同时产物不稳定，易发生转变(如由印楝素(Azadirachtin)构型向印楝素异构体(Azadirachtinin)转变)的缺点。 Compared with the prior art, the present invention is the presence of the beneficial effects as follows: 1, due to the present invention in the extraction step of the process allocation extracted with EtOAc, replace the prior art was extracted with chloroform, HPLC step directly on, thus the use of low cost of solvent, as a common solvent, while overcoming the instability of the product with chloroform disadvantages; 2, the present invention is applied to the column in the separation step is added with a medium pressure column MPLC step, the product yield was 0.18%, compared with the prior art rate too high, as compared ECVebel in Journal of LiguidChromatagraphy 2 (6) 875 (1979) reported high yield ten times more than R BRYAN YAMASAKI in Jouranal of Chromatography 356 (1986) 220-226 reported dozens of high yield times, than W.Kraus in Tetrahedron VOL 43 No.12 2817 (1987) also reported the number of times a high yield; no transition occurs and the product, stability is greatly increased, to overcome the prior art product was low, while the product unstable, easily change (such as by azadirachtin (Azadirachtin) configuration to azadirachtin isomer (Azadirachtinin) transition) shortcomings occur.

为了更好地理解本发明的实质，下面用实施例来详细说明本发明的技术内容，但本发明的内容并不局限于此。 To better understand the essence of the invention, the following examples to illustrate the technical content of the present invention in detail, but the present invention is not limited thereto.

实施例一：取印楝(Azadirachta indica)种仁10kg粉碎，用石油醚冷浸3次，滤渣用甲醇∶水4∶1浸泡3次，滤液回收，在含水甲醇层中用乙酸乙酯萃取，乙酸乙酯层上硅胶柱，以石油醚-乙酸乙酯(1∶1)洗脱，以250ml/瓶收集，用21-29瓶上硅胶柱，以石油醚-丙酮(2∶1)洗脱，以50ml/瓶收集，用23-42瓶上中压柱MPLC，以氯仿∶丙酮9∶1洗脱，以50ml/瓶收集，用7-13瓶经过Rp18反相柱，以甲醇∶水1∶1洗脱即可得印楝素18克。 Example A: Take Neem (Azadirachta indica) Seeds 10kg pulverized, cold soak 3 times with petroleum ether, filtration residue with methanol: water 4:1 immersion 3 times, the filtrate was recovered, the aqueous methanol layer was extracted with ethyl acetate, The ethyl acetate layer on a silica gel column with petroleum ether - ethyl acetate (1/1) as eluent to 250ml / bottle collection, with 21-29 bottles on a silica gel column with petroleum ether - acetone (2:1) eluting to 50ml / bottle collection, with a 23-42 bottles of medium pressure column MPLC, chloroform: acetone elution 9:1 to 50ml / bottle collection bottle with 7-13 after Rp18 reversed phase column with methanol: water 1 :1 azadirachtin elution you can get 18 grams.

实施例二：取印楝(Azadirachta indica)种仁20kg粉碎，用石油醚冷浸2次，滤渣用甲醇∶水9∶1浸泡2次，滤液回收，在含水甲醇层中用乙酸乙酯萃取，乙酸乙酯层上硅胶柱，以石油醚-乙酸乙酯(1∶1)洗脱，以250ml/瓶收集，用21-29瓶上硅胶柱，以石油醚-丙酮(2∶1)洗脱，以50ml/瓶收集，用23-42瓶上中压柱MPLC，以氯仿∶丙酮9∶1洗脱，以50ml/瓶收集，用7-13瓶经过Rp18反相柱，以甲醇∶水1∶1洗脱即可得印楝素36克。 Example II: Take Neem (Azadirachta indica) Seeds 20kg pulverized, cold soak twice with petroleum ether, filtration residue with methanol: water 9:1 soaked for 2 times, the filtrate was recovered, the aqueous methanol layer was extracted with ethyl acetate, The ethyl acetate layer on a silica gel column with petroleum ether - ethyl acetate (1/1) as eluent to 250ml / bottle collection, with 21-29 bottles on a silica gel column with petroleum ether - acetone (2:1) eluting to 50ml / bottle collection, with a 23-42 bottles of medium pressure column MPLC, chloroform: acetone elution 9:1 to 50ml / bottle collection bottle with 7-13 after Rp18 reversed phase column with methanol: water 1 :1 azadirachtin elution you can get 36 grams.

实施例三：取印楝(Azadirachta indica)种仁10kg粉碎，用石油醚冷浸3次，滤渣用甲醇∶水4∶1回流提取3次，滤液回收，在含水甲醇层中用乙酸乙酯萃取，乙酸乙酯层上硅胶柱，以石油醚-乙酸乙酯(1∶1)洗脱，以250ml/瓶收集，用21-29瓶上硅胶柱，以石油醚-丙酮(2∶1)洗脱，以50ml/瓶收集，用23-42瓶上中压柱MPLC，以氯仿∶丙酮9∶1洗脱，以50ml/瓶收集，用7-13瓶经过Rp18反相柱，以甲醇∶水1∶1洗脱即可得印楝素18克。 Example Three: Take Neem (Azadirachta indica) Seeds 10kg pulverized, cold soak 3 times with petroleum ether, filtration residue with methanol: water 4:1 extracted three times under reflux, filtrate recovery, the aqueous methanol layer was extracted with ethyl acetate , the ethyl acetate layer silica gel column with petroleum ether - ethyl acetate (1/1) as eluent to 250ml / bottle collection, with 21-29 bottles on a silica gel column with petroleum ether - acetone (2:1) wash off to 50ml / bottle collection, with a 23-42 bottles of medium pressure column MPLC, chloroform: acetone elution 9:1 to 50ml / bottle collection bottle with 7-13 after Rp18 reversed phase column with methanol: water 1:1 azadirachtin elution you can get 18 grams.

实施例四：取印楝(Azadirachta indica)种仁30kg粉碎，用石油醚冷浸1次，滤渣用甲醇∶水8∶1浸泡2次，滤液回收，在含水甲醇层中用乙酸乙酯萃取，乙酸乙酯层上硅胶柱，以石油醚-乙酸乙酯(1∶1)洗脱，以250ml/瓶收集，用21-29瓶上硅胶柱，以石油醚-丙酮(2∶1)洗脱，以50ml/瓶收集，用23-42瓶上中压柱MPLC，以氯仿∶丙酮9∶1洗脱，以50ml/瓶收集，用7-13瓶经过Rp18反相柱，以甲醇∶水1∶1洗脱即可得印楝素52克。 Example Four: Take Neem (Azadirachta indica) Seeds 30kg pulverized, cold soak 1 times with petroleum ether, filtration residue with methanol: water 8:1 soaked for 2 times, the filtrate was recovered, the aqueous methanol layer was extracted with ethyl acetate, The ethyl acetate layer on a silica gel column with petroleum ether - ethyl acetate (1/1) as eluent to 250ml / bottle collection, with 21-29 bottles on a silica gel column with petroleum ether - acetone (2:1) eluting to 50ml / bottle collection, with a 23-42 bottles of medium pressure column MPLC, chloroform: acetone elution 9:1 to 50ml / bottle collection bottle with 7-13 after Rp18 reversed phase column with methanol: water 1 :1 azadirachtin elution you can get 52 grams.

实施例五：取印楝(Azadirachta indica)种仁10kg粉碎，用石油醚冷浸3次，滤渣用甲醇∶水6∶1浸泡3次，滤液回收，在含水甲醇层中用乙酸乙酯萃取，乙酸乙酯层上硅胶柱，以石油醚-乙酸乙酯(1∶1).洗脱，以250ml/瓶收集，用21-29瓶上硅胶柱，以石油醚-丙酮(2∶1)洗脱，以50ml/瓶收集，用23-42瓶上中压柱MPLC，以氯仿∶丙酮9∶1洗脱，以50ml/瓶收集，用7-13瓶经过Rp18反相柱，以甲醇∶水1∶1洗脱即可得印楝素19克。 Example Five: Take Neem (Azadirachta indica) Seeds 10kg pulverized, cold soak 3 times with petroleum ether, filtration residue with methanol: water 6:1 immersion 3 times, the filtrate was recovered, the aqueous methanol layer was extracted with ethyl acetate, The ethyl acetate layer on a silica gel column with petroleum ether - ethyl acetate (1/1) as eluent to 250ml / bottle collection, with 21-29 bottles on a silica gel column with petroleum ether - acetone (2:1) wash off to 50ml / bottle collection, with a 23-42 bottles of medium pressure column MPLC, chloroform: acetone elution 9:1 to 50ml / bottle collection bottle with 7-13 after Rp18 reversed phase column with methanol: water 1:1 azadirachtin elution you can get 19 grams.