CN102246829B - Clove extract for killing nematode and preparation method thereof - Google Patents
Clove extract for killing nematode and preparation method thereof Download PDFInfo
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- CN102246829B CN102246829B CN 201110214040 CN201110214040A CN102246829B CN 102246829 B CN102246829 B CN 102246829B CN 201110214040 CN201110214040 CN 201110214040 CN 201110214040 A CN201110214040 A CN 201110214040A CN 102246829 B CN102246829 B CN 102246829B
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Abstract
The invention discloses a clove extract for killing nematode and a preparation method thereof. The invention provides a method for preparing a clove absolute ethyl alcohol extract, and the method comprises the following steps: 1) evenly mixing clove with absolute ethyl alcohol to obtain a mixture; 2) carrying out ultrasonic treatment on the mixture obtained in step 1) and then carrying out pumping filtration and condensation to obtain the clove absolute ethyl alcohol extract. According to experiments in the invention, absolute ethyl alcohol is used to extract clove, tests of toxic effects of the extract on Meloidogyne spp. are carried out, and the test results show that the extract has strong toxic activity on Meloidogyne spp.; therefore, it is indicated that the extract provided in the invention provides a development and application basis for preparations for controlling Meloidogyne spp. in the future. In the invention, active components capable of killing nematode are extracted from the plant clove which is natural and harmless, which allows reduced usage of chemical pesticides, thereby reducing pollution to agricultural products and environment.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of clove extract for killing nematode 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 new direction that the nematode floristics will become future development of killing.
Cloves is the dry flower of plant clove of myrtaceae (Eugenia caryophyllata).When bud turns red by green, pluck, dry and be used as medicine.It is caryophyllus oil that bud contains volatile oil, mainly comprise Eugenol (Eugenol), B-caryophyllene (B-Caryophyllene), acetyleugenol, and methyl-n-amyl ketone, gaultherolin, benzaldehyde, benzylalcohol, NSC 43794, benzyl acetate and chavicol (Chavicol) etc.The application of its active substance on physianthropy is more, has antimycotic and bacterium, expelling parasite, the effects such as pain relieving.Relevant report seldom only has a small amount of report that is used for killing mite in agricultural production, has no the relevant report that cloves absolute ethyl alcohol 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 cloves absolute ethyl alcohol extract.
Method provided by the invention comprises the steps:
1) with cloves and absolute ethyl alcohol mixing, obtains mixture;
2) with step 1) mixture that obtains carries out ultrasonic processing, namely obtains cloves absolute ethyl alcohol extract.
Described cloves is the cloves bud.
Step 1) in, the proportioning of described cloves and absolute ethyl alcohol is 10g: (45-55) ml;
Step 2) in, described ultrasonic power is 300W-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 described cloves and absolute ethyl alcohol 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 described Ground Cloves is broken to 35 orders-45 order size; Described Ground Cloves is broken 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 cloves absolute ethyl alcohol extract.
Cloves absolute ethyl alcohol extract by described method preparation also is the scope of protection of the invention.
The application of described cloves absolute ethyl alcohol extract in killing nematode and/or anti-nematode also is the scope of protection of the invention.
The application of described cloves absolute ethyl alcohol extract in the biological prevention and control agent of preparation nematode also is the scope of protection of the invention.
Described nematode is root-knot nematode (Meloidogyne spp.).
Another object of the present invention provides 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 cloves absolute ethyl alcohol extract.
Described nematode is root-knot nematode (Meloidogyne spp.).
The present invention of experiment showed, of the present invention adopts absolute ethyl alcohol that cloves is extracted, and with the toxic effect test of extract to root-knot nematode, the result has strong cytotoxicity for this extract to root-knot nematode; Illustrate that extract of the present invention lays the foundation for the from now on development and application of root-knot nematode biological prevention and control agent.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The acquisition of embodiment 1, cloves absolute ethyl alcohol extract
High speed disintegrator, 6202 types, Beijing is encircled inferior Tianyuan Mechanical Technology Inc. and is made; 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 disposable Tissue Culture Plate in 96 holes, JET BIOFIL
Type, Canada; Electro-heating standing-temperature cultivator, SKP-02.420 type, Hengfeng Medical Instruments Co., Ltd., Huangsih City; Absolute ethyl alcohol (analyzing pure), the Beijing Chemical Plant;
Available from the large pharmacy of Beijing Tongrentang, belong to Myrtaceae for the cloves Eugenia caryophyllata that tries, use bud.
Gather the old complaint of tomato root-knot eelworm disease from morbidity plot, field for examination root-knot nematode (Meloidogyne spp.), (two kinds of agricultural chemicals are to the Toxicity Determination of Meloidogyne incognita according to people such as horse Zhe, 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, in 25 ℃ of insulating boxs, hatch 3d, collect 2 instar larvaes (be called for short J2), and add a certain amount of sterile water and it is mixed with finite concentration (root-knot nematode second instar larvae suspension 500/mL) is for subsequent use.
2, the preparation of plant extracts
Method one:
1) pulverizes
The cloves of drying is put into high speed disintegrator pulverize rear 40 mesh sieves of crossing, put into sealed bag for subsequent use.
2) extract
Adopt ultrasonic extraction, accurately take by weighing the plant drymeal 10g that crushes, add absolute ethyl alcohol 50mL in iodine flask, put into supersonic generator, ultrasonic power is 400W, operating frequency is 45KHz, and ultrasonic temperature is 25 ℃, behind the ultrasonic processing 30min, obtain the absolute ethyl alcohol extract of cloves, through suction filtration (use bottle,suction and Buchner funnel, fill up 2 metafiltration paper), then filtrate is evaporated to thick paste shape (cloves absolute ethyl alcohol 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 places 4 ℃ of refrigerators to save backup, and is plant extraction liquid (cloves absolute ethyl alcohol extract).
Method two:
1) pulverize: basic identical with method one, different is to cross 35 mesh sieves after pulverizing, and puts into sealed bag for subsequent use.
2) extract: basic identical with method one, that different is plant drymeal 10g, adds absolute ethyl alcohol 45mL, and ultrasonic power is 300W, and operating frequency is 28KHz, and ultrasonic temperature is 23 ℃, ultrasonic processing 25min; Obtain plant extraction liquid (cloves absolute ethyl alcohol extract).
Method three:
1) pulverize: basic identical with method one, different is to cross 45 mesh sieves after pulverizing, and puts into sealed bag for subsequent use.
2) extract: basic identical with method one, that different is plant drymeal 10g, adds absolute ethyl alcohol 55mL, and ultrasonic power is 500W, and operating frequency is 100KHz, and ultrasonic temperature is 28 ℃, ultrasonic processing 35min; Obtain plant extraction liquid (cloves absolute ethyl alcohol 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, mixing, 25 ℃ leave standstill, respectively under stereomicroscope, observing behind 12h, 24h and the 48h, check nematode anyway (needle contact method), 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 the result carries 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 and press (the 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) behind the column data, capitalization represents difference extremely significantly (p＜0.01, DMRT method).
When processing nematode take extract concentrations as 10mg/mL, cloves absolute ethyl alcohol extract is as follows to the cytotoxicity result of root-knot nematode:
After processing behind the 12h, cloves absolute ethyl alcohol extract to the cytotoxicity intensity of root-knot nematode is +++, corrected mortality is 61.38 ± 8.75bC,
After processing behind the 24h, cloves absolute ethyl alcohol extract to the cytotoxicity intensity of root-knot nematode is ++ ++, corrected mortality is 94.04 ± 3.91aA,
After processing behind the 48h, cloves absolute ethyl alcohol extract to the cytotoxicity intensity of root-knot nematode is ++ ++, corrected mortality is 98.48 ± 1.52aA.
From the above results as seen, the absolute ethyl alcohol extract of cloves shows strong cytotoxicity to root-knot nematode, and behind the processing 24h, the nematode corrected mortality is respectively 94.04%; After processing 48h, the nematode corrected mortality is respectively 98.48%.
Show according to the DPS the result of multiple comparisons, the absolute ethyl alcohol extract of cloves difference on 0.05 and 0.01 level is not remarkable, all shows strong cytotoxicity yet.
The employing same method detects, and 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 (9)
1. one kind prepares the method that cloves kills nematode absolute ethyl alcohol extract, comprises the steps:
1) with cloves and absolute ethyl alcohol mixing, obtains mixture; The proportioning of described cloves and described absolute ethyl alcohol is 10g: (45-55) ml;
2) with step 1) mixture that obtains carries out ultrasonic processing, and namely obtain cloves and kill nematode absolute ethyl alcohol extract; Described ultrasonic power is 300W-500W, and described ultrasonic operating frequency is 28KHz-100KHz, and the described ultrasonic time is 25min-35min; Described ultrasonic temperature is 23 ℃-28 ℃.
2. method according to claim 1 is characterized in that:
Step 1) in, the proportioning of described cloves and absolute ethyl alcohol 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 ℃.
3. method according to claim 2 is characterized in that:
In step 1) front, also comprise the step that described Ground Cloves is broken to 35 orders-45 order size; Described Ground Cloves is broken 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 cloves and kill nematode absolute ethyl alcohol extract.
4. the cloves by arbitrary described method preparation among the claim 1-4 kills nematode absolute ethyl alcohol extract.
5. cloves claimed in claim 4 kills the application of nematode absolute ethyl alcohol extract in killing nematode and/or anti-nematode.
6. cloves claimed in claim 4 kills the application of nematode absolute ethyl alcohol extract in the biological prevention and control agent of preparation nematode.
7. according to claim 5 or 6 described application, it is characterized in that: described nematode is root-knot nematode (Meloidogyne spp.).
8. the biological prevention and control agent of a nematode, its active component is that cloves claimed in claim 4 kills nematode absolute ethyl alcohol extract.
9. biological prevention and control agent according to claim 8, it is characterized in that: described nematode is root-knot nematode (Meloidogyne spp.).
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| CN 201110214040 CN102246829B (en) | 2011-07-28 | 2011-07-28 | Clove extract for killing nematode and preparation method thereof |
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| CN 201110214040 CN102246829B (en) | 2011-07-28 | 2011-07-28 | Clove extract for killing nematode and preparation method thereof |
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| CN102246829A CN102246829A (en) | 2011-11-23 |
| CN102246829B true CN102246829B (en) | 2013-05-01 |
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| CN102726455A (en) * | 2012-07-02 | 2012-10-17 | 黑龙江省科学院大庆分院 | Botanical nematocide and preparation method thereof |
| CN106359467A (en) * | 2016-08-31 | 2017-02-01 | 江苏星源生物科技有限公司 | Preparation method of plant-derived pesticide |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1144121A (en) * | 1996-04-18 | 1997-03-05 | 中国医药研究开发中心 | Chinese medicine composite for preventing dental caries |
| CN1190892A (en) * | 1995-05-26 | 1998-08-19 | 罗帕法姆公司 | Pharmaceutical compositions, based on etheric oils obtained from plants for use in the human and veterinary medical field |
| CN101966212A (en) * | 2010-09-28 | 2011-02-09 | 山东大学威海分校 | Preparation method and use of Syringa pubescens bark extract |
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2011
- 2011-07-28 CN CN 201110214040 patent/CN102246829B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1190892A (en) * | 1995-05-26 | 1998-08-19 | 罗帕法姆公司 | Pharmaceutical compositions, based on etheric oils obtained from plants for use in the human and veterinary medical field |
| CN1144121A (en) * | 1996-04-18 | 1997-03-05 | 中国医药研究开发中心 | Chinese medicine composite for preventing dental caries |
| CN101966212A (en) * | 2010-09-28 | 2011-02-09 | 山东大学威海分校 | Preparation method and use of Syringa pubescens bark extract |
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