A kind of method extracting moulting hormone from spinach
Technical field
The invention belongs to the extraction and separation technology field of botanical insect moulting hormone analogue, relate to a kind of method extracting moulting hormone from spinach.
Background technology
Phytoecdysone 1 is the very important Secondary metabolites of a class, is also the natural steroid compound with insect molting activity.Phytoecdysone can promote casting off a skin of insect, and the moulting hormone of high density can the normal physiological metabolism of interference insect, makes insect cast off a skin ahead of time and become small and weak or the individuality of deformity.Insect is quite responsive to moulting hormone, and the lighter can cause sterile insect technique, and severe one can cause death.Therefore, Phytoecdysone plays important role in plant resistant non-habitual plant-feed insect, and low-down concentration level just can stop the infringement of insect.The focus that moulting hormone becomes current production, research and development is already extracted from plant.From Radix Cyanotis vagae, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), extract the patent of moulting hormone and report of common occurrence, but at present not extract moulting hormone (beta-Ecdysone) with spinach (SpinaciaoleraceaL.) for raw material and measure the technical system of its content.Spinach few in number self can be synthesized and accumulate the farm crop of a large amount of Phytoecdysone.Set up the method for efficient, easy spinach moulting hormone abstraction and quantification, new Testing index can be provided for the spinach pest-resistant cultivar seed selection of routine, and then provide theoretical foundation for developing agricultural pest control new technology and breeding for pest resistance.
Summary of the invention
The object of the invention is to overcome the defect that above-mentioned technology exists, a kind of easy, method of extracting moulting hormone quickly from spinach is provided.The method extracting method is simple, and the rate of recovery is high, and can Trace bio-element.Its concrete technical scheme is:
From spinach, extract a method for moulting hormone, comprise the following steps:
(1) get Spinach Leaf, add liquid nitrogen, be ground to Powdered;
(2) powder is placed in the centrifuge tube of 50ml, adds the MeOH-CHCl that volume ratio is 9: 1
3mixed solution or 100% MeOH solution to 50ml, put into constant-temperature shaking incubator concussion 2h, shaking table temperature is set to 37 DEG C, and rotating speed is set to 180rpm;
(3) joined by diatomite 545 in ground Bu Shi sand core funnel, thickness is 2cm, and compacting pours said extracted liquid into funnel, uses circulating water type multiplex vacuum pump SHK-III suction filtration sample;
(4) pour in separating funnel by the sample that suction filtration is good, add hexane (Hexane) 50ml and a little NaCl, concuss, make it fully extract, then leave standstill 30min, take off a layer extraction liquid, upper layer of extraction liquid is outwelled, and this step repeats twice;
(5) get the solution extracted, use Rotary Evaporators dry sample, water temperature is 45-50 DEG C, and dried crystals is moulting hormone extract.
Further preferably, the Spinach Leaf described in step (1) is fresh Spinach Leaf.
Further preferably, need to record its quality after the Spinach Leaf grinding described in step (1).
Further preferably, the NaCl described in step (4) quality≤solution quality 5%.
Further preferably, the temperature of the dry sample described in step (5) is 45-50 DEG C.
The moulting hormone sample that a kind of method extracting moulting hormone from spinach of the present invention prepares.
Compared with prior art, beneficial effect of the present invention is: the present invention utilizes methyl alcohol, chloroform and hexane precipitation to combine and repeatedly repeats the technique of extraction, moulting hormone is extracted from spinach, not only easy and simple to handle, consuming time short but also extraction efficiency is high, can Trace bio-element.Moulting hormone extraction yield can reach 100%.Employing methyl alcohol is Extraction solvent, not only reduces extraction cost, it also avoid environmental pollution simultaneously.Adopt high effective liquid chromatography for measuring moulting hormone content, level of sensitivity can reach 5mg/L.Moulting hormone of the present invention passes through mixed extraction and the extraction of three kinds of organic solvents, make the method extraction rate reached to 100%, for the detection of this material in plant materials provides believable extraction detection method, can utilize in Molecular Detection conventional breeding, also can utilize in the research of gene function.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in more detail.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
(1) get Spinach Leaf, add liquid nitrogen, be ground to Powdered.
(2) powder is placed in the centrifuge tube of 50ml, adds MeOH-CHCl
3(9: 1, V/V), to 50ml, put into constant-temperature shaking incubator concussion 2h, shaking table temperature is set to 37 DEG C, and rotating speed is set to 180rpm.
(3) diatomite 545 is joined in Bu Shi sand core funnel, use circulating water type multiplex vacuum pump SHK-III suction filtration sample.
(4) pour in separating funnel by the sample that suction filtration is good, add hexane (Hexane) 50ml and a little NaCl, concuss, make it fully extract, after then leaving standstill 30min, 30min, take off a layer extraction liquid, upper layer of extraction liquid is outwelled.This step repeats twice.
(5) get the solution extracted, use Rotary Evaporators dry sample, water temperature is 45-50 DEG C, until by sample distilled-to-dryness.
(6) in sample, 1ml10% Virahol (Isopropanol) is added, KQ-500DE type numerical control ultrasonic cleaner is used to carry out shaking the dissolving accelerating sample, the concussion time is at about 5min, until the solid in flask walls all dissolves.
(7) the sample solution liquid-transfering gun sucking-off of will dissolve, utilizes disposable syringe to be filled in the brown chromatography column feed materials bottle of 2ml by the polyvinylidene fluoride microporous filtering film (F type) of solution by 0.45 μm.Be kept in 4 DEG C of refrigerators.
(8) high effective liquid chromatography for measuring moulting hormone content
Movement is 10%Isopropanol (0.1%TFA) mutually, and all reagent needs through filtering, and uses numerical control ultrasonic cleaner clean and use after bleeding.The instrument used is Agilent1100Series high performance liquid chromatograph, and chromatographic column (Column) is
c185 μm of 4.6 × 250mm, moving phase is 10% Virahol (Isopropanolalcohol), determined wavelength (Wavelength) is 248nm, column temperature (ColumnTemp) is 40 DEG C, flow velocity (Flowrate) is 1.2ml/min, sample size is 20 μ l, working time 45.0min.
The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.