CN102318651B - Nematicidal extract of radix-polygoni multiflori and preparation method thereof - Google Patents
Nematicidal extract of radix-polygoni multiflori and preparation method thereof Download PDFInfo
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- CN102318651B CN102318651B CN 201110214267 CN201110214267A CN102318651B CN 102318651 B CN102318651 B CN 102318651B CN 201110214267 CN201110214267 CN 201110214267 CN 201110214267 A CN201110214267 A CN 201110214267A CN 102318651 B CN102318651 B CN 102318651B
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Abstract
The invention discloses a nematicidal extract of radix-polygoni multiflori and a preparation method thereof, and provides a method for preparing ethyl acetate extract of radix-polygoni multiflori. The method comprises the following steps: 1) uniformly mixing radix-polygoni multiflori with ethyl acetate to obtain a mixture; and 2) ultrasonically treating the mixture obtained in the step 1), carrying out suction filtration and concentrating to obtain the ethyl acetate extract of radix-polygoni multiflori. The experiments prove that radix-polygoni multiflori is extracted by using ethyl acetate, the poisoning and killing effect of the extract on root-knot nematode is tested, and the test result shows that the extract has strong poisoning and killing activity on root-knot nematode; and the extract disclosed by the invention lays a foundation for the subsequent development and application of root-knot nematode biological prevention and control agents. The method has the advantages that nematicidal active substance is extracted by utilizing natural harmless plant materials, pollution caused by chemical pesticides is reduced, and the extraction method is simple and can be easily popularized and applied to the prevention and control of plant root-knot nematode disease.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of Nematicidal extract of radix-polygoni multiflori and preparation method thereof.
Background technology
Root-knot nematode (Meloidogyne spp.) is the root system of plant obligate parasite, is a current class important pathogen thing that affects agricultural production.
Since the eighties in 20th century, utilize the research of plant extraction liquid control nematode to cause gradually people's attention, so far the nematode plant that kills of having reported approximately has 102 sections 226 to belong to 316 kinds, wherein study more have composite family, Meliaceae, pulse family, Cruciferae, Solanaceae, grass family, Euphorbiaceae, Papaveraceae, Apocynaceae, Mimosaceae, Liliaceae etc., but the natural plant resource is very abundant, many plants with nematocidal effect are also undiscovered, " Chinese poisonous plant " book is listed poisonous plant kind more than 1300 in, wherein, numerous species all has insecticidal activity.A lot of plants can be gathered materials on the spot, it is convenient to use, successful, therefore, at present domestic have more research to aspects such as the screening of killing the nematode plant resources and cytotoxicity mensuration, fully investigates and excavate the how more effective direction that the nematode floristics will become future development of newly killing.
The fleece-flower root (Polygonum multiflorum) is the perennial winding of polygonaceae liane.Root is elongated, and end becomes loose piece root, and the appearance bronzing is to crineous, but dry piece root hyoscine.The piece root contains lecithin (lecithin) approximately 3.7%; Anthraquinone derivative approximately 1.1% is mainly Chrysophanol, archen, is secondly Rhein, Physcion, chrysophanic acid anthrone, rhaponticin; Also contain the materials such as resvertrol glucoside (polydatin) and tannin.The application of its active component on physianthropy is a lot, has reducing blood lipid, protects the liver kidney tonifying and the effect such as antibiotic.Have no relevant report in plant application medically, also have no the relevant report that fleece-flower root ethyl acetate extract is used for the plant root-knot nematodes control.
Summary of the invention
An object of the present invention is to provide a kind of method for preparing fleece-flower root ethyl acetate extract.
Method provided by the invention comprises the steps:
1) with the fleece-flower root and ethyl acetate mixing, obtain mixture;
2) with step 1) mixture that obtains carries out ultrasonic processing, namely obtains fleece-flower root ethyl acetate extract.
The described fleece-flower root is polygonum multiforum root tuber.
Step 1) in, the proportioning of the described fleece-flower root and ethyl acetate is 10g: (45-55) ml;
Step 2) in, described ultrasonic power is 300-500W, and described ultrasonic operating frequency is 28KHz-100KHz, and the described ultrasonic time is 25min-35min; Described ultrasonic temperature is 23 ℃-28 ℃.
Step 1) in, the proportioning of the described fleece-flower root and ethyl acetate is 10g: (45,50 or 55) ml;
Step 2) in, described ultrasonic power is 300W, 400W or 500W, and described ultrasonic operating frequency is 28KHz, 45KHz or 100KHz, and the described ultrasonic time is 25min, 30min or 35min; Described ultrasonic temperature is 23 ℃, 25 ℃ or 28 ℃.
In step 1) front, also comprise the step that the described fleece-flower root is crushed to 35 orders-45 order size; The described fleece-flower root is crushed to and is specially 35 orders, 40 orders or 45 orders size;
In step 2) after, also comprise the steps: the ultrasonic product that described ultrasonic processing obtains is concentrated into paste through suction filtration, filtrate successively, obtain fleece-flower root ethyl acetate extract.
Fleece-flower root ethyl acetate extract by described method preparation is also the scope of protection of the invention.
The application of described fleece-flower root ethyl acetate extract in killing nematode and/or anti-nematode is also the scope of protection of the invention.
The application of described fleece-flower root ethyl acetate extract in the biological prevention and control agent of preparation nematode is also the scope of protection of the invention.
Described nematode is root-knot nematode (Meloidogyne spp.).
Another object of the present invention is to provide the biological prevention and control agent of a kind of nematode.
Biological prevention and control agent provided by the invention, its active component are described fleece-flower root ethyl acetate extract.
Described nematode is root-knot nematode (Meloidogyne spp.).
The present invention of experiment showed, of the present invention adopts ethyl acetate that the fleece-flower root is extracted, and with the toxic effect test of extract to root-knot nematode, result has strong cytotoxicity for this extract to root-knot nematode; Illustrate that extract of the present invention lays the foundation for the development and application of root-knot nematode biological prevention and control agent from now on.
Embodiment
The experimental technique that uses in following embodiment is conventional method if no special instructions.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment 1, fleece-flower root ethyl acetate extract
High speed disintegrator, 6202 types, Beijing inferior Tianyuan of ring Mechanical Technology Inc. makes; Desk-top three frequency controlled ultrasonic wave washers, KQ-500VDV type, Kunshan Ultrasonic Instruments Co., Ltd.; Rotary Evaporators, RE-2000 type, Shanghai Yarong Biochemical Instrument Plant; Stereomicroscope, Motic SMZ-140 type, Maike Aodi Industry Group Co Ltd; The trace adjustable pipette, GILSON, France; The 96 disposable Tissue Culture Plates in hole, JET BIOFIL
Type, Canada; Electro-heating standing-temperature cultivator, SKP-02.420 type, Hengfeng Medical Instruments Co., Ltd., Huangsih City; Ethyl acetate (analyzing pure), the Beijing Chemical Plant;
Available from the large pharmacy of Beijing Tongrentang, belong to polygonaceae for the fleece-flower root Polygontm multiflorum that tries, use the piece root.
Gather the old complaint of tomato root-knot eelworm disease from morbidity plot, field for examination root-knot nematode (Meloidogyne spp.), according to the people such as horse Zhe (two kinds of agricultural chemicals Toxicity Determination to Meloidogyne incognita, Beijing Agricultural College's journal, 2009,24 (3): the method for 17-19) introducing, by choose egg capsule and hatching acquisition from old complaint.
1, the preparation of root-knot nematode second instar larvae suspension
Gather the old complaint of tomato root-knot eelworm disease from the field, get the Tomato Root System that produces a large amount of root knots, running water is rinsed well gently, with the dissecting needle milky egg capsule on the picking old complaint gently, put into the little culture dish of diameter 6cm, add a small amount of sterile water, hatch 3d in 25 ℃ of insulating boxs, collect 2 instar larvaes (being called for short J2), and (root-knot nematode second instar larvae suspension 500/mL) is standby to add a certain amount of sterile water that it is mixed with finite concentration.
2, the preparation of plant extracts
Method one:
1) pulverize
The fleece-flower root of drying is put into crossed 40 mesh sieves after high speed disintegrator is pulverized, put into sealed bag standby.
2) extract
Adopt ultrasonic extraction, accurately take the plant drymeal 10g that crushes, add ethyl acetate 50mL in iodine flask, put into supersonic generator, ultrasonic power is 400W, operating frequency is 45KHz, and ultrasonic temperature is 25 ℃, after ultrasonic processing 30min, obtain the acetic acid ethyl acetate extract of the fleece-flower root, through suction filtration (using bottle,suction and Buchner funnel, pad 2 metafiltration paper), then filtrate is evaporated to thick paste shape (fleece-flower root ethyl acetate extract) in rotary evaporator.Before carrying out insecticidal activity assay, paste is added water be mixed with the aqueous solution that concentration is 20mg/mL, the brown wide-mouth bottle sealing of the ground of then packing into is placed in 4 ℃ of refrigerators and saves backup, and is plant extraction liquid (fleece-flower root ethyl acetate extract).
Method two:
1) pulverize: basic identical with method one, different is to cross 35 mesh sieves after pulverizing, and puts into sealed bag standby.
2) extract: basic identical with method one, that different is plant drymeal 10g, adds ethyl acetate 45mL, and ultrasonic power is 300W, and operating frequency is 28KHz, and ultrasonic temperature is 23 ℃, ultrasonic processing 25min; Obtain plant extraction liquid (fleece-flower root ethyl acetate extract).
Method three:
1) pulverize: basic identical with method one, different is to cross 45 mesh sieves after pulverizing, and puts into sealed bag standby.
2) extract: basic identical with method one, that different is plant drymeal 10g, adds ethyl acetate 55mL, and ultrasonic power is 500W, and operating frequency is 100KHz, and ultrasonic temperature is 28 ℃, ultrasonic processing 35min; Obtain plant extraction liquid (fleece-flower root ethyl acetate extract).
Embodiment 2, cytotoxicity assay method
The employing method of tagging is measured, concrete steps are: get 96 porocyte culture plates, it is the plant extraction liquid 150 μ L that the method one by embodiment 1 step 2 of 20mg/mL obtains that every hole adds respectively the concentration for preparing, add again isopyknic root-knot nematode second instar larvae suspension that is obtained by step 1,25 ℃ standing, respectively at observing under stereomicroscope after 12h, 24h and 48h, check nematode (needle contact method) anyway, stiff or curling motionless polypide is considered as death, is calculated as follows the nematode corrected mortality.Take sterile water as contrast, every processing repeats 3 times, results averaged ± standard error.
Data are processed and analyzed: experimental data is by the DPS2000 analysis and arrangement, and result is carried out significance of difference analysis by the poor multiple comparison method of the new multipole of Deng Kenshi (Duncan ' s multiple range test, DMRT).Kill the strong and weak grade scale of nematode by (Chandravadana M V such as Chandravadana, Eugene S, Nidiry J, et al.Nematicidal activity of some plant extracts[J] .Indian J Nematol, 1996,26 (2): method 148-151.) is carried out, and concrete grade scale is: "-" expression non-activity, corrected mortality≤10%; "+" expression is weak active, and corrected mortality is 10%～30%; " ++ " expression medium activity, corrected mortality is 30%～50%; " +++" the strong activity of expression, corrected mortality is 50%～80%; " ++ ++ " the strong activity of expression, corrected mortality＞80%.With indicating different lowercase alphabet differentials different significantly (p＜0.05, DMRT method) after column data, capitalization represents difference extremely significantly (p＜0.01, DMRT method).
When processing nematode take extract concentrations as 10mg/mL, fleece-flower root ethyl acetate extract is as follows to the cytotoxicity result of root-knot nematode:
After processing after 12h, fleece-flower root ethyl acetate extract to the cytotoxicity intensity of root-knot nematode is ++ ++, corrected mortality is 86.74 ± 2.47aA,
After processing after 24h, fleece-flower root ethyl acetate extract to the cytotoxicity intensity of root-knot nematode is ++ ++, corrected mortality is 91.51 ± 0.42aA,
After processing after 48h, fleece-flower root ethyl acetate extract to the cytotoxicity intensity of root-knot nematode is ++ ++, corrected mortality is 93.39 ± 1.18abA.
From the above results as seen, the ethyl acetate extract of the fleece-flower root shows strong cytotoxicity to root-knot nematode, and after processing 24h, the nematode corrected mortality is respectively 91.51%; After processing 48h, the nematode corrected mortality is respectively 93.39%.
Show according to the DPS the result of multiple comparisons, the ethyl acetate extract of fleece-flower root difference on 0.05 and 0.01 level is not remarkable, all shows strong cytotoxicity yet.
Adopt same method to detect by the method two of embodiment 1 step 2 and the plant extraction liquid of method three acquisitions, result and method one are without significant difference.
Claims (4)
1. the application of fleece-flower root ethyl acetate extract in killing nematode and/or anti-nematode, the extracting method of described fleece-flower root ethyl acetate extract comprises the steps:
1) with the fleece-flower root and ethyl acetate mixing, obtain mixture; The proportioning of the described fleece-flower root and described ethyl acetate is 10g:(45-55) ml;
2) mixture that step 1) is obtained carries out ultrasonic processing, namely obtains the fleece-flower root and kills the nematode ethyl acetate extract; Described ultrasonic power is 300-500W, and described ultrasonic operating frequency is 28 KHz-100KHz, and the described ultrasonic time is 25 min-35min; Described ultrasonic temperature is 23 ℃-28 ℃;
Described nematode be root-knot nematode (
MeloidogyneSpp.).
2. application according to claim 1 is characterized in that: in the extracting method of described fleece-flower root ethyl acetate extract, in step 1), the proportioning of the described fleece-flower root and ethyl acetate is 10g:(45,50 or 55) ml; Before step 1), also comprise the step that the described fleece-flower root is crushed to 35 orders-45 order size;
Step 2) in, described ultrasonic power is 300 W, 400 W or 500W, and described ultrasonic operating frequency is 28 KHz, 45KHz or 100KHz, and the described ultrasonic time is 25 min, 30 min or 35min; Described ultrasonic temperature is 23 ℃, 25 ℃ or 28 ℃.
3. application according to claim 2 is characterized in that: in the extracting method of described fleece-flower root ethyl acetate extract, the described fleece-flower root is crushed to and is specially 35 orders, 40 orders or 45 orders size;
In step 2) after, also comprise the steps: the ultrasonic product that described ultrasonic processing obtains is concentrated into paste through suction filtration, filtrate successively, obtain fleece-flower root ethyl acetate extract.
4. the fleece-flower root kills the application of nematode ethyl acetate extract in the biological prevention and control agent of preparation nematode, and described fleece-flower root ethyl acetate extract is according to the extracting method preparation of the fleece-flower root ethyl acetate extract described in any one in claim 1-3.
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| CN102014940A (en) * | 2008-05-02 | 2011-04-13 | 株式会社太平洋 | Medicinal plants extract using processing of herbal medicine and composition of skin external application comprising the same |
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| CN102014940A (en) * | 2008-05-02 | 2011-04-13 | 株式会社太平洋 | Medicinal plants extract using processing of herbal medicine and composition of skin external application comprising the same |
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Inventor after: Wei Yanmin Inventor after: Ma Zhe Inventor after: Zhao Xiaoyan Inventor after: Shang Qiaoxia Inventor after: Liu Zhengping Inventor after: Song Qingfeng Inventor before: Wei Yanmin Inventor before: Ma Zhe Inventor before: Liu Zhengping Inventor before: Shang Qiaoxia Inventor before: Zhao Xiaoyan Inventor before: Song Qingfeng |
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Application publication date: 20120118 Assignee: The middle peasants lvkang Biotechnology Co. Ltd. (Beijing) Assignor: Beijing University Of Agriculture Contract record no.: 2013990000869 Denomination of invention: Nematicidal extract of radix-polygoni multiflori and preparation method thereof Granted publication date: 20130605 License type: Exclusive License Record date: 20131224 |
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