CN109557222A - A kind of Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE - Google Patents

A kind of Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE Download PDF

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CN109557222A
CN109557222A CN201910078215.8A CN201910078215A CN109557222A CN 109557222 A CN109557222 A CN 109557222A CN 201910078215 A CN201910078215 A CN 201910078215A CN 109557222 A CN109557222 A CN 109557222A
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radix curcumae
extraction
enrichment method
ionic liquid
bromination
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CN109557222B (en
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楚楚
王珊
颜继忠
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Zhejiang University of Technology ZJUT
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Zhejiang University of Technology ZJUT
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/062—Preparation extracting sample from raw material

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Abstract

The invention discloses a kind of Extraction and enrichment methods of Sesquiterpene Polyesterss in RADIX CURCUMAE, the sesquiterpenoids ingredient is the mixture of germacrone, furanodiene and beta-elemene, the following steps are included: 1) extract: RADIX CURCUMAE medicinal powder is mixed with ionic liquid aqueous solution, microwave abstracting is carried out again, obtained suspension centrifuging and taking supernatant, filtrate is filtered to take, RADIX CURCUMAE extracting solution is obtained；2) it is enriched with: lauryl sodium sulfate, carbon fluoride nano-tube and water is mixed, it is ultrasonic to obtain dispersion liquid, then RADIX CURCUMAE extracting solution is added in dispersion liquid, it is vortexed, filters after mixing, obtained solid is eluted, and collects eluent to get pregnant solution.The extracting method is simple, and solvent usage is few, and efficiently avoids the use of poisonous and harmful reagent, environmentally protective, while avoiding high temperature extraction and causing damages to low-boiling sesquiterpenoids ingredient, is very suitable for large-scale promotion application.

Description

A kind of Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE
Technical field
The invention belongs to natural drug extraction and analysis technical fields, and in particular to sesquiterpenoids ingredient in a kind of RADIX CURCUMAE Extraction and enrichment method.
Background technique
RADIX CURCUMAE (Curcuma wenyujin Y.H.Chen et C.Ling) is Zingiber curcuma, is famous One of genunie medicinal materials " eight Zhe's " have effects that relieving stagnant Qi promoting circulation of blood, clearing stagnation and killing pain and anticancer, antiviral, anti-inflammatory, anticoagulation, resist The physiological actions such as oxidation and liver protection.The main active of RADIX CURCUMAE includes sesquiterpenoids ingredient and curcumin two major classes, Middle sesquiterpenoids ingredient includes germacrone, furanodiene, beta-elemene etc..Existing pharmacology and clinical research discovery, germacrone, furan Mutter diene, beta-elemene etc. all has antiviral and anti-tumor activity, has for liver cancer, gastric cancer, lung cancer, breast cancer etc. and inhibits Effect；In addition, above-mentioned sesquiterpenoids ingredient also has good therapeutic effect for inflammation and thrombus disease.
With the development of science and technology, ionic liquid is widely paid close attention to by researcher, ionic liquid by organic cation and Inorganic or organic anion composition, is one of emerging research field of Green Chemistry.Conventional solvent can only be completed by concentration difference The extraction of effective component in Chinese medicine, and some ionic liquids are the fine solvents of cellulose, can by dissolution cellulose come Dissolution Activities of Some Plants cell wall achievees the purpose that extraction, to increase recovery rate and shorten extraction time, therefore, in traditional Chinese medicine extraction It has broad application prospects with use aspects.
In addition, Novel extraction method of many foundation on the basis of Solid Phase Extraction is come into being to improve extraction efficiency, point Dissipating micro- Solid Phase Extraction (DMSPE) is a kind of a kind of novel sample treatment technology established on the basis of Solid Phase Extraction, will extract, Concentration and extract introduce analysis system and settle at one go, can be used for separating and being enriched with target analytes, such as phenolic compound, three Piperazine, phytochemicals, selenium and pesticide etc..Compared with traditional solid phase extraction method, DMSPE is few using solid absorbent, section The about cost and organic solvent amount used is few, environmental pollution is few, and extraction time is short, and extraction efficiency is high, is increasingly becoming in recent years The popular domain studied both at home and abroad, has been widely used in Analysis of Natural Products detection field.Disperse micro- Solid Phase Extraction A committed step be adsorbent selection, currently, for disperse the sorbent material of micro- Solid Phase Extraction to include hybrid mesoporous Material, chitosan, graphene oxide and multi-walled carbon nanotube etc..
The fluorination of carbon material is and a kind of process for forming C-F key to be combined with carbon atom by breaking carbon-coating pi bond, is to repair Change and one of the most effective approach for controlling carbon material physicochemical properties.Currently, carbon fluoride nano-tube (FCNTs) can be by carbon nanometer Pipe is prepared by the methods of direct fluorination, plasma method.Carbon fluoride nano-tube is in addition to the absorption with carbon nanotube Outside performance, also enhance its surface polarity, electric conductivity and capacitance characteristic, be widely used in lithium battery, solid lubricant and In nanocomposite.Nearest research report shows that carbon fluoride nano-tube is not only shown in terms of removing chromium ion well Performance, and good suction-operated is also shown for organic compound, as sulfamethoxazole, Ofloxacin, Norfloxacin, Bis-phenol a etc..
Currently, there are many reports for the detection of pre-treatment and effective component for RADIX CURCUMAE, but at present before RADIX CURCUMAE Processing method is concentrated mainly in ultrasonic extraction, also has part using microwave extraction technology.Although the operation letter of these extractive techniques It is single, but a large amount of poisonous and hazardous organic reagents can be used during the extraction process, environmental pollution is serious, does not meet " Green Chemistry " Theory, and extraction time is long, low to the recovery rate of target component.Therefore, one kind is established for the active constituent in RADIX CURCUMAE The extraction and analytical method environmentally protective, operation is quick and easy has very important significance to the quality of control RADIX CURCUMAE source medicinal material.
Summary of the invention
It is an object of that present invention to provide a kind of extracted with ionic liquid assisted microwave synthesis to combine the micro- solid phase extraction techniques of dispersion To the Extraction and enrichment method of sesquiterpenoids ingredient in RADIX CURCUMAE, simplifies extraction step, the use for avoiding harmful reagent, improves The Extraction and enrichment effect of sesquiterpenoids ingredient, gained pregnant solution can be directly used in the content detection of sesquiterpenoids ingredient, fit Close large-scale promotion application.
In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:
A kind of Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE, the sesquiterpenoids ingredient are germacrone, furans two The mixture of alkene and beta-elemene, comprising the following steps:
(1) it extracts: RADIX CURCUMAE medicinal powder being mixed with ionic liquid aqueous solution, then carries out microwave abstracting, what is obtained is mixed Suspension centrifuging and taking supernatant, filters to take filtrate, obtains RADIX CURCUMAE extracting solution；
(2) be enriched with: lauryl sodium sulfate, carbon fluoride nano-tube and water mixed, ultrasonic dispersion liquid, by dispersion liquid with RADIX CURCUMAE extracting solution is vortexed after adding water to mix, and filtering obtained solid is eluted, and collects eluent to get the sesquialter is contained The pregnant solution of terpene component.
The excellent properties of the gap structure on carbon fluoride nano-tube surface and high surface area can be preferably enriched in RADIX CURCUMAE Three kinds of sesquiterpenoids ingredients, therefore its effect for dispersing micro- Solid Phase Extraction is fabulous.In addition, carbon fluoride nano-tube is as a kind of novel Material not only has good absorption property, but also safe and non-toxic, is very suitable for as the adsorbent for dispersing micro- Solid Phase Extraction.
The method of the present invention will be for the first time using ionic liquid as Microwave Extraction reagent, for extracting the sesquialter in RADIX CURCUMAE medicinal material Ter penoids, then using the carbon fluoride nano-tube dispersion liquid of lauryl sodium sulfate dispersion as adsorbent, dexterously combine dispersion Micro- solid phase extraction techniques are enriched with sesquiterpenoids ingredient, obtained pregnant solution can measurement for further analysis use, be The analysis measurement of three kinds of sesquiterpenoids ingredients provides a kind of more green, easy, fast and efficiently new method.
In step (1), the ionic liquid is selected from bromination 1- hexyl -3- vinyl imidazole, bromination 1- octyl -3- vinyl Imidazoles, bromination 1- decyl -3- vinyl imidazole, any one in bromination 1- dodecyl -3- vinyl imidazole, preferably bromine Change 1- decyl -3- vinyl imidazole.
Ionic liquid is different, and the effect of the microwave-assisted micro- Solid Phase Extraction of dispersion is also different, although four kinds of ionic liquids With anion of the same race, but because of long alkyl chains difference, extraction efficiency is different.Since the ionic liquid with longer carbon chain has Lower critical micelle concentration, therefore be easier to form micella in aqueous solution, so as long alkyl chains increase, extraction effect The significant raising of rate.But since target component is hydrophobic compound, under the ionic liquid effect of longer carbon chain, have Biggish distribution coefficient can be such that extraction efficiency reduces instead, this is because when alkyl chain increases to certain length, steric hindrance and The comprehensive function of hydrogen bond can be more obvious, so that extraction efficiency weakens, comprehensively considers, and selects bromination 1- decyl -3- vinyl miaow Azoles.
The amount of the substance of the ionic liquid is calculated as 0.0025~0.02mol/g with the dosage of RADIX CURCUMAE medicinal powder, excellent It is selected as 0.0025~0.005mol/g.This is because ionic liquid dosage effect of extracting in preferred scope is best, ionic liquid Dosage is excessively high, effect of extracting can be made to weaken, this is because ionic liquid dosage is excessive, ionic liquid viscosity increases, diffusivity Weaken, makes its medicinal material content more difficult to get access, extraction efficiency is caused to reduce.
The microwave abstracting time be 3~15min, preferably 5~10min, experiment show with the microwave abstracting time increase Add, extraction efficiency increases therewith, but when extraction time it is too long, will affect the extraction yield of furanodiene instead, therefore, comprehensively consider The slective extraction time is 5~10min.
The microwave abstracting temperature is 40~70 DEG C, preferably 50~60 DEG C.This is because microwave abstracting temperature is preferred The microwave-assisted micro- Solid Phase Extraction effect of dispersion is best in range, and with the raising of microwave abstracting temperature, extraction efficiency is also gradually mentioned Height, this is because the viscosity that higher extraction temperature helps to reduce ionic liquid enhance simultaneously the diffusivity of ionic liquid with Dissolubility promotes dissolution of the target compound from medicinal material to solvent.But when extraction temperature is excessively high, extraction efficiency weakens instead, This is because target compound structural instability at high temperature.
In step (2), the vortex time is 1~10min, preferably 1~2min.This is because in a certain range, As vortex time increases, effect of extracting is continuously increased, this is because the mass transfer of target compound on the sorbent with The increase of vortex time and increase.But when vortex time is too long, effect of extracting can gradually weaken instead, this is because too long Vortex time leads to target analytes too strong absorption on the sorbent, so that subsequent elution is difficult.
Any one of the eluant, eluent in methanol, acetonitrile, ethyl alcohol or acetone, preferably acetonitrile, different eluant, eluents Elution property it is different, due to the similar principle that mixes, acetonitrile is more close with the polarity of three kinds of sesquiterpenoids ingredients, therefore, washes Elution effect is best when de- agent is acetonitrile.
In step (2), in the dispersion liquid, the mass ratio of carbon fluoride nano-tube and lauryl sodium sulfate be 1:40~ 60。
Water is added to be mixed to get 10mL mixed liquor dispersion liquid and 1mL RADIX CURCUMAE extracting solution, the fluorocarbons nanometer in mixed liquor The concentration of pipe is 0.5~4.0 μ g/mL, preferably 1.5~2.5 μ g/mL.
The concentration of adsorbent carbon fluoride nano-tube is different, and the effect for dispersing micro- Solid Phase Extraction is also different, as adsorbent is dense Degree increases, and effect of extracting enhancing promotes target point this is because the adsorption site between adsorbent and target compound increases Analysis object is transferred in adsorbent from sample solution, to realize absorption completely.In addition, adsorption concentration is excessively high, effect of extracting is instead Weaken, this is because adsorbent excessively makes between analyte and adsorbent, adsorption capacity is too strong, causes elution process difficult.
The eluant, eluent volume is 80~200 μ L, preferably 90~100 μ L, this is because as eluting agent increases, Completely, but continuing growing with eluting agent, due to diluting effect, effect of extracting is instead gradually for target compound elution Weaken.
Compared with prior art, the invention has the following advantages:
(1) the method for the present invention is extracted to have obtained RADIX CURCUMAE extracting solution, sample extraction method using ionic liquid assisted microwave synthesis Simply, solvent usage is few, and efficiently avoids the use of poisonous and harmful reagent, environmentally protective, while avoiding high temperature extraction It causes damages to low-boiling sesquiterpenoids ingredient.
(2) the method for the present invention is enriched with sesquiterpenoids ingredient in RADIX CURCUMAE extracting solution herein in connection with micro- solid phase extraction techniques are dispersed, Its is easy to operate, saves the time, organic solvent and amount of samples are few；In addition, the present invention preferably uses carbon fluoride nano-tube as suction It is attached dose, good with absorption property, it is safe and non-toxic, the characteristics of chemical stability and good thermal stability, reproducibility, improve The selectivity of analysis method；Therefore, Extraction and enrichment method of the invention is significantly better than traditional extracting method.
Detailed description of the invention
Fig. 1 is the process flow chart of Extraction and enrichment of embodiment of the present invention method.
Fig. 2 be investigate the histogram of the micro- Solid Phase Extraction effect of dispersion indicated under different enrichment conditions with chromatographic peak area with Line chart, wherein 1,2,3 respectively represent germacrone, furanodiene, beta-elemene；(A) adsorbent species are investigated；(B) fluorine is investigated Carbon nano tube concentration；(C) vortex time is investigated；(D) eluant, eluent type is investigated；(E) eluant, eluent volume is investigated.
Fig. 3 is the extraction effect histogram investigated in Microwave Extraction technique；Wherein, 1,2 germacrone, furans two are respectively represented Alkene；(A) type of ionic liquid is investigated；(B) concentration of ionic liquid is investigated；(C) time of Microwave Extraction is investigated；(D) it investigates The temperature of Microwave Extraction.
Specific embodiment
Extraction detection method provided by the present invention is described in more detail by following instance.Since it is answered It is wide with range, therefore specific embodiment is also more, the contents of the present invention are made further in the discussion below in conjunction with several examples It illustrates.
Embodiment used silica gel, purchased from Town in Shanghai spectrum experiment Science and Technology Co., Ltd..
Al used in embodiment2O3, purchased from Town in Shanghai spectrum experiment Science and Technology Co., Ltd..
C used in embodiment18, it is purchased from Shanghai Zheng Ya Chemical Co., Ltd..
Carbon fluoride nano-tube used in embodiment, be purchased from Nanjing Xian Feng Nono-material Science & Technology Ltd., caliber 20-30nm, 2-10 μm of pipe range.
Embodiment uses the model Agilent 1290UHPLC of Ultra Performance Liquid Chromatography instrument (UHPLC), uses The chromatographic condition of UHPLC1290 are as follows: chromatographic column: InertSustain C18 chromatographic column (5 μm, 4.6 × 250mm), sample volume: 20 μ L, column temperature: 25 DEG C, flow velocity 1mL/min, mobile phase: A:0.1v% formic acid water, B:0.1v% formic acid acetonitrile.Gradient elution: 0~ 10min, 70%~90v% Mobile phase B；10~18min, 90%~97v% Mobile phase B；18~26min, 97%~98v% stream Dynamic phase B.
The preparation method specific steps of the RADIX CURCUMAE reference substance mixed liquor are as follows: precision weighs appropriate germacrone, furan respectively It mutters diene, beta-elemene reference substance, is settled in 1mL volumetric flask with methanol mixed dissolution, it is dense eventually that above-mentioned various reference substances are made Degree is respectively the reference substance mixed liquor of 1mg/mL, 1mg/mL, 1mg/mL.
When being enriched with using the micro- Solid Phase Extraction of dispersion, influence of the following parameter to micro- Solid Phase Extraction effect is dispersed is investigated:
(1) selection of adsorbent species
It takes 4 parts of 50.47mg lauryl sodium sulfate to be added separately in 4 groups of 100mL stuffed conical flasks in parallel, and weighs not With type adsorbent (silica gel, Al2O3, C18, carbon fluoride nano-tube) and each 1mg, it is added separately in 4 groups of stuffed conical flasks, respectively 10mL is added water to, ultrasonic 15min is uniformly mixed, and obtains four kinds of absorption agent dispersing liquids.
Take 4 parts of 10 μ L of 1mg/mL reference substance mixed liquor to be added separately in the centrifuge tube of 4 groups of 50mL specifications in parallel, respectively plus Enter to prepare four kinds of obtained 200 μ L of absorption agent dispersing liquid, adds water to 10mL respectively.By 4 groups of centrifuge tubes as on turbula shaker, Vortex 2min draws the solution in centrifuge tube with 10mL disposable syringe.Syringe needle is removed, organic mistake in 0.22 μm of aperture is loaded onto Syringe is placed on solid-phase extracting instrument and filters by filter, and until no water droplet, the filtering head containing adsorbent is taken Under.Filtering head is eluted with 100 μ L acetonitriles, eluent is collected in 1.5mL specification centrifuge tube, and 13000rpm is centrifuged 5min, is taken Clear liquid is packed into liquid phase bottle, analyzes result with UHPLC.
Fig. 2A shows the effect of extracting histogram under different adsorbent species, and 1,2,3 respectively represent different sesquialters in figure Ter penoids, respectively germacrone, furanodiene, beta-elemene；Silica gel is silica gel in figure, and FCNTs receives for fluorocarbons Mitron.
The results show that comparing and other three kinds of adsorbents, chromatographic peak of the carbon fluoride nano-tube to three kinds of sesquiterpenoids ingredients Area is maximum, therefore it is best to the micro- Solid Phase Extraction effect of the dispersion of three kinds of sesquiterpenoids ingredients.
(2) selection of carbon fluoride nano-tube concentration
It takes 50.47mg lauryl sodium sulfate and 1mg carbon fluoride nano-tube to be added in 100mL stuffed conical flask, adds water To 10mL, ultrasonic 15min is uniformly mixed, and obtains carbon fluoride nano-tube dispersion liquid.
Take 4 parts of 10 μ L of 1mg/mL reference substance mixed liquor to be added separately in the centrifuge tube of 4 groups of 50mL specifications in parallel, respectively plus The carbon fluoride nano-tube dispersion liquid (50 μ L, 100 μ L, 200 μ L, 400 μ L) for entering different volumes, adds water to 10mL, so that fluorine respectively The concentration of carbon nano tube is respectively 0.5 μ g/mL, 1.0 μ g/mL, 2.0 μ g/mL, 4.0 μ g/mL.By 4 groups of centrifuge tubes as vortex On oscillator, vortex 2min draws the solution in centrifuge tube with 10mL disposable syringe.Syringe needle is removed, 0.22 μm of hole is loaded onto The organic filter head of diameter, syringe is placed on solid-phase extracting instrument and is filtered, and until no water droplet, will contain adsorbent Filtering head is removed.Filtering head is eluted with 100 μ L acetonitriles, eluent is collected in 1.5mL specification centrifuge tube, 13000rpm centrifugation 5min takes supernatant to be packed into liquid phase bottle, analyzes result with UHPLC.
Fig. 2 B shows the effect of extracting histogram under carbon fluoride nano-tube various concentration, and 1,2,3 respectively represent not in figure With sesquiterpenoids ingredient, respectively germacrone, furanodiene, beta-elemene.
The results show that when concentration increases to 2.0 μ g/mL, effect of extracting enhancing from 0.5 μ g/mL；But work as concentration from 2.0 μ g/ When mL increases to 4.0 μ g/mL, effect of extracting weakens.
(3) selection of vortex time
It takes 50.47mg lauryl sodium sulfate and 1mg carbon fluoride nano-tube to be added in 100mL stuffed conical flask, adds water To 10mL, ultrasonic 15min is uniformly mixed, and obtains carbon fluoride nano-tube dispersion liquid.
Take 6 parts of 10 μ L of 1mg/mL reference substance mixed liquor to be added separately in the centrifuge tube of 6 groups of 50mL specifications in parallel, respectively plus Enter 200 μ L of carbon fluoride nano-tube dispersion liquid, adds water to 10mL respectively.6 groups of centrifuge tubes are different as being vortexed on turbula shaker Time (1min, 2min, 4min, 6min, 8min, 10min), with 10mL disposable syringe draw centrifuge tube in solution. Syringe needle is removed, the organic filter head in 0.22 μm of aperture is loaded onto, syringe is placed on solid-phase extracting instrument and is filtered, until anhydrous Until drop, the filtering head containing adsorbent is removed.With 100 μ L acetonitriles elute filtering head, eluent be collected in 1.5mL specification from In heart pipe, 13000rpm is centrifuged 5min, and supernatant is taken to be packed into liquid phase bottle, analyzes result with UHPLC.
Fig. 2 C shows the effect of extracting histogram under different vortex times, and 1,2,3 respectively represent different sequiterpenes in figure Constituents, respectively germacrone, furanodiene, beta-elemene.
The results show that the chromatographic peak area of three ingredients increases, shows as vortex time from 1min increases to 2min Effect of extracting is continuously increased；But when vortex time increases to 10min from 2min, effect of extracting then slowly weakens.
(4) selection of eluant, eluent type
It takes 50.47mg lauryl sodium sulfate and 1mg carbon fluoride nano-tube to be added in 100mL stuffed conical flask, adds water To 10mL, ultrasonic 15min is uniformly mixed, and obtains carbon fluoride nano-tube dispersion liquid.
Take 4 parts of 10 μ L of 1mg/mL reference substance mixed liquor to be added separately in the centrifuge tube of 4 groups of 50mL specifications in parallel, respectively plus Enter 200 μ L of carbon fluoride nano-tube dispersion liquid, adds water to 10mL respectively.By 4 groups of centrifuge tubes as on turbula shaker, it is vortexed 2min draws the solution in centrifuge tube with 10mL disposable syringe.Syringe needle is removed, the organic filter in 0.22 μm of aperture is loaded onto Syringe is placed on solid-phase extracting instrument and filters by head, and until no water droplet, the filtering head containing adsorbent is removed. Filtering head is eluted with 100 μ L methanol, acetonitrile, ethyl alcohol, acetone respectively, eluent is collected in 1.5mL specification centrifuge tube, 13000rpm is centrifuged 5min, and supernatant is taken to be packed into liquid phase bottle, analyzes result with UHPLC.
Fig. 2 D shows the effect of extracting line chart under different eluant, eluent types, and 1,2,3 respectively represent different sesquialters in figure Ter penoids, respectively germacrone, furanodiene, beta-elemene；Abscissa is followed successively by methanol, acetonitrile, second from a left side from the right side in figure Alcohol, acetone.
The results show that the elution effect of acetonitrile is best.
(5) selection of eluant, eluent volume
It takes 50.47mg lauryl sodium sulfate and 1mg carbon fluoride nano-tube to be added in 100mL stuffed conical flask, adds water To 10mL, ultrasonic 15min is uniformly mixed, and obtains carbon fluoride nano-tube dispersion liquid.
Take 4 parts of 10 μ L of 1mg/mL reference substance mixed liquor to be added separately in the centrifuge tube of 4 groups of 50mL specifications in parallel, respectively plus Enter 200 μ L of carbon fluoride nano-tube dispersion liquid, adds water to 10mL respectively.By 4 groups of centrifuge tubes as on turbula shaker, it is vortexed 2min draws the solution in centrifuge tube with 10mL disposable syringe.Syringe needle is removed, the organic filter in 0.22 μm of aperture is loaded onto Syringe is placed on solid-phase extracting instrument and filters by head, and until no water droplet, the filtering head containing adsorbent is removed. Filtering head is eluted with the acetonitrile of different volumes (80 μ L, 100 μ L, 150 μ L, 200 μ L) respectively, eluent is collected in 1.5mL specification In centrifuge tube, 13000rpm is centrifuged 5min, and supernatant is taken to be packed into liquid phase bottle, analyzes result with UHPLC.
Fig. 2 E shows the effect of extracting line chart under different eluant, eluent volumes, and 1,2,3 respectively represent different sesquialters in figure Ter penoids, respectively germacrone, furanodiene, beta-elemene.
The results show that failing sufficiently to elute three kinds of target compounds from adsorbent when eluant, eluent volume is lower than 100 μ L Get off, and when volume increases to 100 μ L, target substance can be eluted complete.
Embodiment 1
(1) extract: it is 0.250M bromination 1- decyl -3- ethylene that 10mL concentration, which is added, in 0.5g RADIX CURCUMAE source medicinal powder In base imidazole ion liquid, mixed solution is placed in Microwave Extraction Apparatus, under the conditions of 60 DEG C, microwave abstracting 10min extracts function Rate is 200W, and the suspension after microwave abstracting is transferred in the centrifuge tube of 50mL, and 4000rpm centrifugation 20min takes supernatant, on Clear liquid crosses 0.22 μm of membrane filtration, and taking filtrate is RADIX CURCUMAE extracting solution；
(2) it takes 50.47mg lauryl sodium sulfate and 1mg carbon fluoride nano-tube to be added in 100mL stuffed conical flask, adds Water is uniformly mixed to 10mL, ultrasonic 15min, obtains carbon fluoride nano-tube dispersion liquid；
(3) it is enriched with: taking the centrifuge tube of 50mL specification, RADIX CURCUMAE extracting solution 1mL is added, add carbon fluoride nano-tube dispersion 200 μ L of liquid, adds water to 10mL；Centrifuge tube is placed on turbula shaker again, vortex 2min, is drawn with 10mL disposable syringe Solution in centrifuge tube removes syringe needle, loads onto the organic filter head in 0.22 μm of aperture, it is enterprising that syringe is placed in solid-phase extracting instrument Row filters, and until no water droplet, the filtering head containing adsorbent is removed, and elutes filtering head with the acetonitrile of 100 μ L, collection is washed De- liquid is pregnant solution.
Pregnant solution is placed in 1.5mL specification centrifuge tube, 13000rpm is centrifuged 5min, and take supernatant to be packed into liquid phase bottle, I.e. available ultra performance liquid chromatography (UHPLC) analysis measurement component content.
Embodiment 2
Specific reaction condition is consistent with 1 the method for embodiment, in addition to by ionic liquid bromination 1- decyl -3- vinyl miaow Azoles replaces with bromination 1- hexyl -3- vinyl imidazole, bromination 1- octyl -3- vinyl imidazole, bromination 1- dodecyl -3- respectively Vinyl imidazole.
Fig. 3 A shows the effect of extracting histogram under different ionic liquid type, and 1,2 respectively represent different sesquialters in figure Ter penoids, respectively germacrone, furanodiene；[HVIM] Br is bromination 1- hexyl -3- vinyl imidazole, [OVIM] Br in figure For bromination 1- octyl -3- vinyl imidazole, [C10VIM] Br be bromination 1- decyl -3- vinyl imidazole, [C12VIM] Br is bromine Change 1- dodecyl -3- vinyl imidazole.
The results show that as long alkyl chains are from C6Increase to C10, the significant raising of extraction efficiency, and when long alkyl chains from C10Increase to C12, extraction efficiency significant decrease.
Embodiment 3
Specific reaction condition is consistent with 1 the method for embodiment, in addition to by bromination 1- decyl -3- vinyl imidazole ionic liquid The concentration of body aqueous solution replaces with 0.125M, 0.500M, 1.000M respectively.
Fig. 3 B shows the effect of extracting histogram under different ionic liquid concentration, and 1,2 respectively represent different sesquialters in figure Ter penoids, respectively germacrone, furanodiene.
The results show that effect of extracting is optimal when bromination 1- octyl -3- vinyl imidazole concentration increases to 0.250M, and When continuing growing ionic liquid concentration, effect of extracting weakens.
Embodiment 4
Specific reaction condition is consistent with 1 the method for embodiment, in addition to by the time of microwave abstracting replace with respectively 3min, 5min、15min。
Fig. 3 C shows the effect of extracting histogram under the different microwave abstracting times, and 1,2 respectively represent different sesquialters in figure Ter penoids, respectively germacrone, furanodiene.
The results show that as the microwave abstracting time from 3min increases to 10min, the extraction efficiency of germacrone and furanodiene Increase, however when the microwave abstracting time increasing to 15min from 10min, the extraction yield of germacrone increased, but furanodiene Extraction yield be obviously reduced.
Embodiment 5
Specific reaction condition is consistent with 1 the method for embodiment, in addition to by the temperature of microwave abstracting replace with respectively 40 DEG C, 50℃、70℃。
Fig. 3 D shows the effect of extracting histogram under the different microwave abstracting times, and 1,2 respectively represent different sesquialters in figure Ter penoids, respectively germacrone, furanodiene.
The results show that extraction efficiency is gradually increased when microwave abstracting temperature rises to 60 DEG C, and when temperature continues to be increased to 70 DEG C, extraction efficiency weakens.
The quantitative determination of sesquiterpenoids ingredient in RADIX CURCUMAE:
1, using the Extraction and enrichment method of embodiment 1, extraction and quantitative analysis are carried out to the RADIX CURCUMAE of three different batches, The quantified results for obtaining sesquiterpenoids ingredient in three kinds of different batches RADIX CURCUMAEs are as shown in table 1 below.
The measurement result (mg/g) of sesquiterpenoids ingredient in 1 three kinds of different batches RADIX CURCUMAEs of table
aIt is collected in the pharmacy of Wenzhou District of Zhejiang Province
2, the formulation of standard curve:
Precision weighs germacrone reference substance 3.00mg, furanodiene reference substance 0.90mg and beta-elemene reference substance respectively 48mg dissolves and is settled to methanol 1mL and takes 20 μ L of mixed liquor in the volumetric flask of 10mL specification after ultrasound mixes, use methanol 10mL is diluted and be settled to, is configured to containing 6.00 μ g/mL of germacrone, 1.80 μ g/mL of furanodiene, 96.00 μ g/mL of beta-elemene Mixing reference substance stock solution, it is spare.The mixing reference substance for drawing 1 μ L, 2 μ L, 4 μ L, 7 μ L, 10 μ L, 15 μ L and 20 μ L respectively is molten Liquid injection liquid chromatograph is analyzed, and peak area is measured.Using the concentration of analyte as abscissa, peak area is ordinate, is drawn Standard curve processed.
Germacrone, the standard curve of furanodiene and beta-elemene, linearly dependent coefficient, the range of linearity and detection limit are as follows Table 2.
Each ingredient standard curvilinear equation of table 2, linearly dependent coefficient, the range of linearity and detection limit
3, repeatability is investigated
According to the parallel sample preparation of 1 the method for embodiment 6, its repeatability is investigated under the chromatographic condition, the results are shown in Table 3。
4, precision is investigated
Under the chromatographic condition, precision draws mixed reference substance solution interior repetition sample introduction 6 times on the same day, is in a few days accurate Degree；Under the chromatographic condition, precision draws mixed reference substance solution, and daily sample introduction 2 times for three days on end are day to day precision, knot Fruit is shown in Table 3.
3 repeatability of table, precision, stability experiment result
5, sample recovery rate is investigated
0.5g RADIX CURCUMAE powder is weighed, is placed in the container of 80mL Microwave Extraction device.Addition 10mL concentration is 0.250M Bromination 1- decyl -3- vinyl iminazole ionic liquid aqueous solution (contain 10.18 μ g/mL germacrones, 8.795 μ g/mL furans two Alkene, 16.28 μ g/mL beta-elemenes).Mixed liquor is placed in Microwave Extraction Apparatus, temperature is set as 60 DEG C, and power is set as 200W, micro- Wave extracts 10min.In the centrifuge tube that suspension after microwave abstracting is shifted to 50mL specification respectively, 4000rpm centrifugation 20min is taken Supernatant, supernatant cross 0.22 μm of membrane filtration, take filtrate up to mark-on RADIX CURCUMAE extracting solution.The RADIX CURCUMAE extracting solution that will be obtained After being extracted by enrichment method described in embodiment 1, result is analyzed with UHPLC.
0.5g RADIX CURCUMAE powder is weighed, is placed in the container of 80mL Microwave Extraction device.Addition 10mL concentration is 0.250M Bromination 1- decyl -3- vinyl iminazole ionic liquid aqueous solution (contain 40.72 μ g/mL germacrones, 35.18 μ g/mL furans two Alkene, 65.12 μ g/mL beta-elemenes).Mixed liquor is placed in Microwave Extraction Apparatus, temperature is set as 60 DEG C, and power is set as 200W, micro- Wave extracts 10min.In the centrifuge tube that suspension after microwave abstracting is shifted to 50mL specification respectively, 4000rpm centrifugation 20min is taken Supernatant, supernatant cross 0.22 μm of membrane filtration, take filtrate up to mark-on RADIX CURCUMAE extracting solution.The RADIX CURCUMAE extracting solution that will be obtained After being extracted by enrichment method described in embodiment 1, result is analyzed with UHPLC.
Under the chromatographic condition, to blank RADIX CURCUMAE extract liquor and mark-on RADIX CURCUMAE extract liquor, sample introduction is analyzed, according to it Germacrone, furanodiene, the recovery of standard addition of beta-elemene and its relative standard is calculated in peak area and calibration curve equation Deviation the results are shown in Table 4.
Sample recovery rate experimental result such as the following table 4:
The experiment of 4 sample recovery rate of table
Data are shown in upper table, and the method for the present invention rate of recovery is high.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims (9)

1. a kind of Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE, the sesquiterpenoids ingredient is germacrone, furanodiene With the mixture of beta-elemene, which comprises the following steps:
(1) it extracts: RADIX CURCUMAE medicinal powder being mixed with ionic liquid aqueous solution, then carries out microwave abstracting, obtained suspension Centrifuging and taking supernatant, filters to take filtrate, obtains RADIX CURCUMAE extracting solution；
(2) it is enriched with: lauryl sodium sulfate, carbon fluoride nano-tube and water is mixed, ultrasonic dispersion liquid, then by dispersion liquid and temperature Radix Curcumae extracting solution is vortexed after adding water to mix, and filtering obtained solid is eluted, and collects eluent to get the sequiterpene is contained The pregnant solution of ingredient.
2. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 1, which is characterized in that step (1) In, the ionic liquid is selected from bromination 1- hexyl -3- vinyl imidazole, bromination 1- octyl -3- vinyl imidazole, the bromination 1- last of the ten Heavenly stems Any one in base -3- vinyl imidazole, bromination 1- dodecyl -3- vinyl imidazole.
3. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 1, which is characterized in that step (1) In, the amount of the substance of the ionic liquid is calculated as 0.0025~0.02mol/g with the dosage of RADIX CURCUMAE medicinal powder.
4. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 1, which is characterized in that step (1) In, the ionic liquid is bromination 1- decyl -3- vinyl imidazole, and the amount of the substance of ionic liquid is with RADIX CURCUMAE medicinal powder Dosage is calculated as 0.0025~0.005mol/g.
5. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 1, which is characterized in that step (1) In, the microwave abstracting time is 3~15min, and microwave abstracting temperature is 40~70 DEG C.
6. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 1, which is characterized in that step (2) In, the vortex time is 1~10min.
7. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 1, which is characterized in that step (2) In, any one of eluant, eluent used in the elution in methanol, acetonitrile, ethyl alcohol or acetone.
8. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 1, which is characterized in that step (2) In, add water to be mixed to get 10mL mixed liquor dispersion liquid and 1mL RADIX CURCUMAE extracting solution, the carbon fluoride nano-tube in mixed liquor it is dense Degree is 0.5~4.0 μ g/mL.
9. the Extraction and enrichment method of Sesquiterpene Polyesterss in RADIX CURCUMAE according to claim 8, which is characterized in that the elution Agent volume is 80~200 μ L.
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