CN101440054A - Method for preparing phenethyl isosulfocyanate from horseradish - Google Patents
Method for preparing phenethyl isosulfocyanate from horseradish Download PDFInfo
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Abstract
The invention discloses a method for preparing phenylethyl isothiocyanate from horseradish. The method comprises the following steps: the horseradish is cleaned, sliced and pulverized and is subjected to hydrolysis and filtering under the optimized hydrolysis condition; the filtrate is extracted for several times through a low-polarity solvent; the filter residue is distilled through water vapour, is adsorbed and enriched through macroporous resin and is extracted through an organic solvent; the organic solvent is reclaimed to obtain a coarse product; and the coarse product is subjected to extraction separation and column chromatography separation through the solvent so as to prepare the phenylethyl isothiocyanate with high purity of over 90 percent. The method has a simple process; and the obtained product has high content and is suitable for industrialized production.
Description
Technical field
The present invention relates to the plant milk extract technical field, particularly a kind of method of from Cruciferae Armoracia horseradish, extracting phenethyl isothiocyanate.
Background technology
Horseradish (Armoracia lapathifolia Gilib.) has another name called Western horseradish, and horseradish is Cruciferae Armoracia per nnial herb.Its fleshy root and subterraneous stem crust are thicker, yellow-white or meat white, and cylinder shape, all burying is about 30～60cm, and the thick about 5～6cm of stem originates in south of europe, based on the land cultivation.Horseradish is the very high vegetables of a kind of economic worth medicinal plants of holding concurrently.Pungency that it is unique and Xin Xiang pungent have the aquatic foods of proposing and germicidal action, are Japanese cuisine, cold vegetable dish in sauce, eat seasoning matter commonly used such as seafood raw.Simultaneously also be to make the requisite raw material of horseradish sauce, widely popular in Japan.
Glucosinolate is the sulfur-bearing secondary metabolite that extensively is present in the cresss such as horseradish, mustard and Semen Brassicae campestris.Common glucosinolate has more than 23 kinds, as methyl glucosinolate, alkene butyl glucosinolate, allyl group glucosinolate etc.The basic structure of glucosinolate is:
According to the literature, different thioglucose glycosides compound hydrolysates mainly contains allyl group isosulfocyanate (AITC), 3-butenyl lsothiocyanates, 4-pentenyl lsothiocyanates, 5-hexenyl lsothiocyanates, 5,7-first sulfurous base hexyl lsothiocyanates, beta-phenyl ethyl, phenyl and benzyl lsothiocyanates etc.The maximum cress of research has mustard, horseradish, horseradish (Japanese horseradish), Semen Brassicae campestris, wild cabbage, brussels sprouts, Cauliflower, radish or the like at present, and its pungent local flavor mainly comes from the allyl group isosulfocyanate (AITC) in the plant.Contain a large amount of strong pungent volatile flavor component AITC and crotononitrile in the wide report of her the rattan leaf mustard leaf.Domestic research is than later, and main object is the mustard wet goods.Jiang Zitao etc. point out that piquancy component is mainly AITC in the seasoning mustard oil, and its content is big more, and pungent is heavy more.Also mostly be allyl group isosulfocyanate at the activity research aspect the antibacterial desinsection, Hu Shangqin studies show that horseradish effective constituent allyl group isosulfocyanate is obvious to the effect of pathogenic bacteria, and to candida albicans, the highest kill ratio such as Streptococcus hemolyticus is 99%.Shik studies show that the allyl group isosulfocyanate in each position of Japanese horseradish (horseradish) (root, bar and leaf) can both effectively suppress Hp, and wherein the activity of leaf is better than root.Wu Hua etc. discover that 95% athomin crude oil (allyl group isosulfocyanate) all has stifling preferably biological activity to sitophilus zea-mais, lesser grain borer, red flour beetle and booklice.Allyl group isosulfocyanate (AITC) structure is:
Bibliographical information the hydrolysising condition and the detection method of lsothiocyanates.The yellow bright dark isothiocyanate content of studying each tissue site of horseradish, the result shows that the content in the rhizome is the highest, leaf is minimum.Zhang Qingfeng etc. have studied the stability to hydrolysis of lsothiocyanates in the horseradish, the result show when temperature high more, hydrolysis rate is fast more, under the ultrasonic wave condition, helps accelerating hydrolysis, and lsothiocyanates stable in properties in acetone soln.On detection method, Jiang Zitao proposes the method for many mensuration ITCs, as piperidines volumetry, chloramines 2T indirect oxidation method, morpholine volumetry, piperazine nonaqueous titrations, dithio carbon propylhomoserin forming method.Chu Xingdi etc. directly make standard with isothiocyanic acid propyl diester and the reaction of 1,2 diphenyl disulfide phenol, have set up the rp-hplc method of ITCs in the urine, and HPLC condition: HP1090M joins diode-array detector; Chromatographic column is Supelco C
18Post (150mm * 416mm), moving phase is methyl alcohol: water=70:30, flow velocity 1.75mL/min, and sample size 20 μ L detect wavelength 365nm.
At present in the majority for chemistry, extraction process, content distribution, the bioactivity research of allyl group isosulfocyanate, foreign patent (WO00/61163), the quantity research that contains of phenethyl isothiocyanate in the yellor rocket platymiscium seed different plant-sourceds such as (Caulis et Folium Brassicae capitatae, turnip, radish and Herba Nasturth officinalis) is provided, but horseradish phenethyl isothiocyanate (PEITC) is not studied as yet.And phenethyl isothiocyanate has anti-tumor activity, can prevent the cytopigment p450 enzyme in the I phase metabolism, promotes the DPNH I (QR, UDP-glucuronyl transferase and GST) in the II phase metabolism.
Summary of the invention
The object of the present invention is to provide a kind of from the natural phant horseradish method of extraction separation phenethyl isothiocyanate, this method technology is simple, the product content height that obtains is fit to suitability for industrialized production.
Technical scheme of the present invention is: the method for extraction separation phenethyl isothiocyanate from horseradish may further comprise the steps:
The first step raw materials pretreatment
The horseradish that one week of harvesting is interior is clean to dry section, is ground into fine powder after the oven dry;
The raw material hydrolysis of second step
Fine powder after pulverizing is added the distilled water hydrolysis with the ratio that 1 gram raw material adds 10-30mL distilled water, and hydrolysis temperature 30-40 ℃, pH is 5-7, the vitamins C addition is 1-10:1000 with horseradish fine powder raw materials quality ratio, hydrolysis time 60-140min after hydrolysis finishes, filters;
The preparation of the 3rd step crude product
The filtrate part: the filtrate that obtains with second step of low polar solvent equal-volume extraction 1-5 time, merge the low polar solvent part, vacuum concentration must the phenethyl isothiocyanate crude product;
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin adsorption, with the washing of 2-6BV, use the dehydrated alcohol wash-out of 2-6BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims;
B, the low polar organic solvent extraction a of the usefulness filter residue behind the steam distillation in the step, the ratio of filter residue and low polar organic solvent adds the low polar organic solvent of 5-25mL for the 1g filter residue, heated and stirred is extracted 1-4 time, each extraction time 1-3h, filters by temperature 20-40 ℃, merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and low polar organic solvent reclaims;
The 4th one-step refining
The phenethyl isothiocyanate crude product that the 3rd step was obtained merges column chromatography for separation after solvent extraction and separation:
A, the phenethyl isothiocyanate crude product that the 3rd step was obtained are dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 20-50mL methyl alcohol for the 1g crude product, remove insolubles, with the dissolve with methanol part, rotary evaporation in vacuo concentrates, reclaim methanol solvate, enriched material is dissolved in the sherwood oil again, the ratio of enriched material and sherwood oil adds the 20-50mL sherwood oil for the 1g enriched material, remove insoluble part, petroleum ether dissolution partial vacuum rotary evaporation is concentrated, reclaim sherwood oil, obtain phenethyl isothiocyanate, analyze the quality percentage composition more than 80% through HPLC;
B, the phenethyl isothiocyanate that a is obtained further carry out column chromatography for separation, the phenethyl isothiocyanate that a is obtained is dissolved in sherwood oil, after the absorption of polarity filled column, the elutriant of employing petroleum ether-ethyl acetate volume ratio 100:1-50 is gradient elution step by step, collect elutriant, obtain phenethyl isothiocyanate, analyze the quality percentage composition more than 90% through HPLC.
Beneficial effect:
(1) the present invention is by screening horseradish hydrolysising condition, the contrast different process, utilize the phenethyl isothiocyanate in steam distillation and the heated and stirred extraction method extraction horseradish, first by extracting and separating and column chromatography for separation preparation quality percentage composition at the phenethyl isothiocyanate more than 90%, and set up the HPLC detection method of this material on this basis.
(2) to produce horseradish with Linyi, China Shandong be raw material in the present invention, extraction separation phenethyl isothiocyanate from horseradish first, and identify its chemical structure.
(3) processing method of the present invention is simple, and the product content height that obtains is fit to suitability for industrialized production.
Description of drawings
Fig. 1 is the HPLC canonical plotting of phenethyl isothiocyanate.
Fig. 2 is the HPLC collection of illustrative plates of phenethyl isothiocyanate.
Fig. 3 is the mass spectrum of phenethyl isothiocyanate.
Fig. 4 is the infrared spectrum of phenethyl isothiocyanate.
Fig. 5 is the hydrogen nuclear magnetic resonance spectrogram of phenethyl isothiocyanate.
Fig. 6 is the carbon-13 nmr spectra figure of phenethyl isothiocyanate.
Fig. 7 is the total ion current figure of method 1 among the embodiment 2.
Fig. 8 is the total ion current figure of method 2 among the embodiment 2.
Fig. 9 is the total ion current figure of method 3 among the embodiment 2.
Embodiment
A kind of method for preparing phenethyl isothiocyanate from horseradish may further comprise the steps:
The first step raw materials pretreatment
With plucking all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100 μ m-1000 μ m fine powders, under 30-60 ℃ condition, to dry, moisture is lower than 10%wt.
The raw material hydrolysis of second step
Fine powder after pulverizing is added the distilled water hydrolysis with the ratio that 1 gram raw material adds 10-30mL distilled water, and hydrolysis temperature 30-40 ℃, pH is 5-7, the vitamins C addition is 1-10:1000 with horseradish fine powder raw materials quality ratio, hydrolysis time 60-140min after hydrolysis finishes, filters.
The preparation of the 3rd step crude product
The filtrate part: the filtrate that obtains with second step of low polar solvent equal-volume extraction 1-5 time, merge the low polar solvent part, vacuum concentration must the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin adsorption, with the washing of 2-6BV, use the dehydrated alcohol wash-out of 2-6BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims; Macroporous resin is AB-8, X-5, S-8, DM1400, DM130, DM301, DA201, D101.
B, the low polar organic solvent extraction a of the usefulness filter residue behind the steam distillation in the step, the ratio of filter residue and low polar organic solvent adds the low polar organic solvent of 5-25mL for the 1g filter residue, heated and stirred is extracted 1-4 time, each extraction time 1-3h, filters by temperature 20-40 ℃, merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and low polar organic solvent reclaims; Low polar solvent in the 3rd step is sherwood oil, normal hexane, ether, benzene, chloroform or methylene dichloride.
The 4th one-step refining
The phenethyl isothiocyanate crude product that the 3rd step was obtained merges column chromatography for separation after solvent extraction and separation:
A, the phenethyl isothiocyanate crude product that the 3rd step was obtained are dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 20-50mL methyl alcohol for the 1g crude product, remove insolubles, with the dissolve with methanol part, rotary evaporation in vacuo concentrates, reclaim methanol solvate, enriched material is dissolved in the sherwood oil again, the ratio of enriched material and sherwood oil adds the 20-50mL sherwood oil for the 1g enriched material, remove insoluble part, petroleum ether dissolution partial vacuum rotary evaporation is concentrated, reclaim sherwood oil, obtain phenethyl isothiocyanate, analyze the quality percentage composition more than 80% through HPLC.
B, the phenethyl isothiocyanate that a is obtained further carry out column chromatography for separation, the phenethyl isothiocyanate that a is obtained is dissolved in sherwood oil, after the absorption of polarity filled column, the elutriant of employing petroleum ether-ethyl acetate volume ratio 100:1-50 is gradient elution step by step, collect elutriant, obtain phenethyl isothiocyanate, analyze the quality percentage composition more than 90% through HPLC.
Adopt steam distillation to extract the horseradish phenethyl isothiocyanate in present method, through solvent extraction, resin absorption and desorb, polarizable medium separates and prepares percentage composition and reach 95% phenethyl isothiocyanate, through GC-MS, IR,
1H-NMR,
13C-NMR and MS are accredited as phenethyl isothiocyanate, this compound isolation identification first in horseradish, and its structure is:
Shown in Fig. 3～6, in this compound infrared spectrum, 3028,1347,749 and 699cm
-1Being respectively the characteristic absorbance of benzene, is a substituted benzene, 2921,2870 and 1454cm
-1Be the characteristic absorbance of methylene radical, 2198 and 2094 is the characteristic absorbance position of different thiocyanide.
1H-NMR (500MHz, CDCl
3) in the characteristic spectrum, δ: 2.95 (2H, H-3), 3.68 (2H, H-2), 7.19 (2H, H-5, H-9), 7.32 (2H, H-6, H-8), 7.25 (1H, H-7), and 7.19,7.32,7.25 these three characteristic displacements that displacement is a phenyl ring hydrogen, 2.95 and 3.68 is the characteristic displacement of methylene radical hydrogen.
13C-NMR (125MHz, CDCl
3) δ: 36.39 (C-3), 46.25 (C-2), 127.04 (C-7), 128.65 (and C-5, C-9), 136.89 (C-4), 128.59 (C-6, C-8).36.39 and 46.25 be the characteristic displacement of mesomethylene carbon, 127.04,128.65,128.59 and 136.89 is the characteristic displacement of carbon on the phenyl ring.In the mass spectrum of this material, contain the M+2 peak, infer that it contains sulfonium ion, and molecular weight 163 is an odd number, contain odd number nitrogen, 105 these fragment ion peaks be remove-N=C=S obtains, and contains the peculiar fragment ion peak of 91,77,65 these phenyl ring simultaneously, is phenethyl isothiocyanate so identify this compound.
Described raw material horseradish comes from Linyi, Shandong, and in preparation phenethyl isothiocyanate crude product and elaboration process, low polar solvent adopts sherwood oil, normal hexane, ether, benzene, chloroform or methylene dichloride, preferred sherwood oil, methylene dichloride.Described vacuum concentration is that rotary evaporation concentrates, and temperature is 30-60 ℃, and vacuum tightness is 0.02-0.09MPa.
The hydrolysising condition extracting mode of described horseradish sulphur glycosides adopts microwave extraction, thermal backflow extraction or ultrasonic extraction, preferred ultrasonic-assisted extraction.Power, time, pH, vitamins C addition (being that 1g horseradish fine powder raw material need add ascorbic mg number), solid-liquid ratio (every 1g horseradish fine powder raw material need add the mL number of distilled water) are carried out experiment of single factor, power (W) chooses 20,40,60,80,100,120 respectively, time, (min) chose 10,15,20,25,30,35,40, pH chooses 2,3,4,5,6,7,8 respectively, vitamins C addition (mg/g) chooses 0,0.2,2,20,200, and solid-liquid ratio (g/mL) is chosen 1:5,1:10,1:15,1:20,1:25.By glucosinolate hydrolysis L
16(4
4) orthogonal test, the result is as table 1-2.
Table 1 glucosinolate orthogonal experiments and analysis
Table 2 variance analysis
By the comprehensive evaluation to each factor, by the extreme difference size as seen, the primary and secondary of A, B, C, D4 factor affecting isothiocyanate content is in proper order: A〉C〉D〉B, promptly each factor is followed successively by the influence of isothiocyanate content: power〉pH〉solid-liquid ratio〉time.Optimised process is A as seen from Table 1
1B
3C
4D
1And A
1B
3C
4D2, promptly preferred power 20W, time 35min, pH are 7, and the vitamins C addition is 2mg/g, and solid-liquid ratio (g/mL) is 1:10.
Conciliate attached middle school in above-mentioned resin absorption, macroporous resin is chosen as any one among AB-8, X-5, S-8, DM1400, DM130, DM301, DA201, the D101, the Static Adsorption test of macroporous resin shows: that adsorption effect is best is DM1400, is X-5, AB-8 and DM130 secondly.Desorb effect best be D101, secondly be DA201, AB-8, S-8 and X-5.Carry out desorption with ethanol, desorption effect improves with the increase of alcohol concn, and the dehydrated alcohol elute effect is best.From taking all factors into consideration absorption and desorbing, preferred X-5 or AB-8 are as the resin of the enriching and purifying of horseradish lsothiocyanates.As show 3-4.
Table 3 different resins is to the Static Adsorption rate and the desorption rate of lsothiocyanates
Table 4 X-5 resin purification effect relatively
| Method Method | Sample volume (mL) (sample volume) | Isothiocyanate content (%) (isothiocyanates content) | Yield (%) (yield) |
| The X-5 resin absorption, dehydrated alcohol desorption X-5resin adsorption and |
500 | 46.9％ | 0.187％ |
| Dichloromethane |
500 | 40.5％ | 0.206％ |
The present invention adopts the polarizable medium separation and purification, and the polarity filler is silica gel, aluminum oxide, diatomite etc., preferred silica gel; The elutriant of column chromatography for separation 2～5 gradients employing petroleum ether-ethyl acetate volume ratio 100:1-50 is gradient elution step by step, and the 1st gradient is 100% sherwood oil wash-out, and the 2-5 gradient is respectively sherwood oil: ethyl acetate=15:1,10:1,10:3,10:5, the 6th gradient eluent ethyl acetate.
The present invention adopts gas/matter chromatographic condition:
GC conditions: pillar: Hp-20M, 25 * 0.32 * 0.3 μ m, injector temperature: 250 ℃, 50 ℃ of column temperatures again through 5 ℃/min temperature programming to 200 ℃, and keep 20min at 200 ℃.Carrier gas: He, flow velocity 1mL/min, sample size 1 μ L does not shunt.
Mass spectrum: ionization mode EI, ionization voltage 70ev, 250 ℃ of mass detectors, mass range: 50-550.
Infrared spectra: this sample is an oily matter, so the Potassium Bromide sheet glass is cleaned with dehydrated alcohol, again sample evenly is applied to glass sheet surface, useful range: 4000-400cm
-1, resolution is 2cm
-1
Nucleus magnetic resonance: adopt deuterochloroform to make solvent, the carbon spectrum scans under the condition of 125MHz, and the hydrogen spectrum scans under the condition of 500MHz.
The HPLC measuring method is standard substance with the phenethyl isothiocyanate, with Hypersil ODS2 5 μ m4.6mm*250mm chromatographic columns, is moving phase with the ratio liquid mixture prepared of volume ratio methyl alcohol: water=70:30, is to detect wavelength with 243nm.
Horseradish phenethyl isothiocyanate HPLC analytical procedure
(1) chromatographic condition: chromatographic column Hypersil ODS2 5 μ m 4.6mm*250mm are moving phase with the ratio liquid mixture prepared of volume ratio methyl alcohol: water=70:30, are to detect wavelength, flow velocity 1mL/min with 243nm.
(2) typical curve is drawn: accurately pipette allyl mustard oil 0.1mL, move to behind the dissolve with ethanol with 50%wt in the volumetric flask of 100mL, and with being settled to scale, getting its concentration is the reference liquid of 1 μ L/mL.Draw above-mentioned allyl mustard oil reference liquid 1,2,3,4,5,6,7,8,9,10mL respectively in the 100mL volumetric flask, be settled to scale with 50% ethanol again, be mixed with the reference liquid of 0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.10 μ L/mL.With 50% ethanolic soln is blank, surveys light absorption value at the 243nm place, and the drawing standard curve is as Fig. 1.Typical curve linear equation y=8.6273x-0.0036, r=0.9998, linearity range: 0.01～0.1 μ L.
(3) sample extraction and mensuration
The horseradish root of plucking in the week is standby after cleaning, cut into slices, dry, pulverizing, precision takes by weighing horseradish sample 2.5g and places Erlenmeyer flask, be hydrolyzed by factor and the level selected, it is transferred in the 500mL round-bottomed flask, connect the water vapour generator, slowly adding thermogenesis water vapour distills, phlegma receives with the 100mL volumetric flask that fills the 50mL dehydrated alcohol, and distillation stops distillation when scale, use the distilled water constant volume, shake up the back and survey absorption value in UV-light 243nm place.Obtain the phenethyl isothiocyanate content of sample according to typical curve.
(4) the phenethyl isothiocyanate raw product and the highly finished product of the present invention's preparation are analyzed content respectively at 40%-80% and 80%-100% through HPLC.Fig. 2 is the HPLC collection of illustrative plates of phenethyl isothiocyanate.
Embodiment 2
The preparation of horseradish glucosinolate hydrolysate
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100 μ m-1000 μ m fine powders, dry under 40-60 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Method 1: horseradish powder is under 30 ℃-40 ℃, the condition of pH5-7, solid-liquid ratio 1:10-25 (g/mL), adding the vitamins C amount is the 0.2%wt of horseradish powder, hydrolysis 1-3h in water, then through steam distillation, distillate is through cooled and filtered, dichloromethane extraction 4 times, and dichloromethane layer is dense to be done, preparation horseradish glucosinolate sample of hydrolysate is analyzed for GC-MS.
Method 2: horseradish powder is under 40 ℃, the condition of pH7, solid-liquid ratio 1:20 (g/mL), adding the vitamins C amount is the 0.1%wt of horseradish powder, hydrolyzed solution is volume ratio methylene dichloride: water=1:2-5, hydrolysis 2h, and material is after filtration after the hydrolysis, the filter residue part with eluent methylene chloride for several times, filtrate is partly used dichloromethane extraction 4 times, combined dichloromethane layer, dense doing, preparation horseradish glucosinolate sample of hydrolysate is analyzed for GC-MS.
Method 3: extract the myrosin in the horseradish powder earlier, extract the glucosinolate in the horseradish powder again.Both are put into 40 ℃, the water of pH6-7, solid-liquid ratio 1:10-20 (g/mL), and adding the vitamins C amount is the 0.1-0.2%wt of horseradish powder, hydrolysis 2h in water.Enzymolysis finishes after-filtration, filter residue part with eluent methylene chloride for several times, filtrate is through dichloromethane extraction 4 times, the combined dichloromethane layer, dense doing, preparation horseradish glucosinolate sample of hydrolysate is analyzed for GC-MS.
(3) purification process of horseradish phenethyl isothiocyanate
Sample with method 1 gained, be dissolved in sherwood oil, remove insoluble part, through silica gel column chromatography, use the petroleum ether-ethyl acetate gradient elution, collected volume is than being the eluting fraction of sherwood oil: ethyl acetate=10:1, reclaim solvent, sample is analyzed through GC-MS, and the simplification compound of preparation content 95% is through being accredited as phenethyl isothiocyanate.
(4) chromatographic condition
GC conditions: pillar: Hp-20M, 25 * 0.32 * 0.3 μ m, injector temperature: 250 ℃, 50 ℃ of column temperatures again through 5 ℃/min temperature programming to 200 ℃, and keep 20min at 200 ℃.Carrier gas: He, flow velocity 1mL/min, sample size 1 μ L does not shunt.
Mass spectrum: ionization mode: EI, ionization voltage 70ev, 250 ℃ of mass detectors, mass range: 50-550.
(5) chemical composition analysis of horseradish glucosinolate hydrolysate
3 kinds of method for hydrolysis gained volatile oil are analyzed through GC-MS coupling method, and its total ion current figure such as Fig. 7-9.The mass-spectrometric data of gained enters HP-586/66XM data processing hangar (American National Standard storehouse), retrieve and interpretation of mass spectra with the INCOS data system, fitting index and mass-spectrometric data are further confirmed, calculated the relative percentage composition of each composition with the peak area normalization method, its Analysis and Identification the results are shown in Table 5.
The composition of three kinds of method gained of table 5 horse radish oil is identified relatively
Table 5 analysis revealed: method 1,2,3 obtains 5,6,5 peaks respectively by the makings collection of illustrative plates, wherein the retention time unanimity at 5 peaks.With the comparison of standard spectrum picture library, contain phenethyl isothiocyanate, hexadecanoic acid, 9,12 octadecadienoic acid, 11,14 simultaneously, 17-eicosatrienoic acid methyl esters.Except allyl group isosulfocyanate and phenethyl isothiocyanate are horseradish isosulfocyanate materials, other is the fatty acid material.The relative content of the phenethyl isothiocyanate of method 1 gained is the highest, is 88.707%, because hydrolyzed solution passes through steam distillation, volatile oil component in the horseradish is taken out of, and the sample of gained, impurity is less.And method 2 is hydrolyzed by direct the contact with horseradish powder of methylene dichloride, the composition of the lsothiocyanates of gained is Duoed a kind of allyl group isosulfocyanate than other two kinds of methods, but the relative content of lsothiocyanates is only 60%, this be since methylene dichloride and water in the hydrolysis horseradish powder, methylene dichloride has also extracted some fat-soluble components in the horseradish powder, so the fatty acid substances content just increases.Method 3 is that glucosinolate and myrosin are extracted respectively, both are hydrolyzed, but the content of the phenethyl isothiocyanate that obtains has only 10.67%, mainly contains the material of fatty acid again.This is because in the process of extracting myrosin, the active part of myrosin is destroyed, and the extraction of glucosinolate is incomplete, so cause the content of phenethyl isothiocyanate minimum.
Embodiment 3
The preparation of phenethyl isothiocyanate
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 30 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 10mL distilled water with 1 gram raw material adds distilled water, 30 ℃ of temperature, and pH5, vitamins C addition and raw material ratio be hydrolysis 60min under the condition of 1mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with sherwood oil equal-volume extraction (2) 1 time, merge the sherwood oil part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin X-5 absorption, with the washing of 2BV, use the dehydrated alcohol wash-out of 2BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims;
B, the usefulness petroleum ether extraction a filter residue behind the steam distillation in the step, the ratio of filter residue and sherwood oil is that the 1g filter residue adds the 5mL sherwood oil, heated and stirred is extracted 1 time, extraction time 1h, 20 ℃ of temperature are filtered merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and sherwood oil reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 20mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 20mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 82%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 93%.
(5) the phenethyl isothiocyanate structure is identified
The HPLC chromatographic condition: chromatographic column Hypersil ODS2 5 μ m 4.6mm*250mm are moving phase with the ratio liquid mixture prepared of volume ratio methyl alcohol: water=70:30, are to detect wavelength, flow velocity 1mL/min with 243nm.
Infrared spectra: this sample is an oily matter, so the Potassium Bromide sheet glass is cleaned with dehydrated alcohol, again sample evenly is applied to glass sheet surface, useful range: 4000-400cm
-1, resolution is 2cm
-1
Nucleus magnetic resonance: adopt deuterochloroform to make solvent, the carbon spectrum scans under the condition of 125MHz, and the hydrogen spectrum scans under the condition of 500MHz.
Phenethyl isothiocyanate feature structure color atlas is Fig. 3-Fig. 6.Phenethyl isothiocyanate has following chemical structure.
Embodiment 4
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 60 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 30mL distilled water with 1 gram raw material adds distilled water, 40 ℃ of temperature, and pH7, vitamins C addition and raw material ratio be hydrolysis 140min under the condition of 10mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with methylene dichloride equal-volume extraction (2) 5 times, the combined dichloromethane part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin AB-8 absorption, with the washing of 6BV, use the dehydrated alcohol wash-out of 6BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims.
B, the usefulness dichloromethane extraction a filter residue behind the steam distillation in the step, the ratio of filter residue and methylene dichloride adds the 25mL methylene dichloride for the 1g filter residue, heated and stirred is extracted 4 times, each extraction time 3h, 40 ℃ of temperature are filtered, merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and methylene dichloride reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 50mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 50mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 87%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 96%.
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 45 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 20mL distilled water with 1 gram raw material adds distilled water, 35 ℃ of temperature, and pH6, vitamins C addition and raw material ratio be hydrolysis 100min under the condition of 5mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with sherwood oil equal-volume extraction (2) 3 times, merge the sherwood oil part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin S-8 absorption, with the washing of 4BV, use the dehydrated alcohol wash-out of 4BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims.
B, the usefulness petroleum ether extraction a filter residue behind the steam distillation in the step, the ratio of filter residue and sherwood oil adds the 15mL sherwood oil for the 1g filter residue, heated and stirred is extracted 3 times, each extraction time 2h, 30 ℃ of temperature are filtered, merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and sherwood oil reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 50mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 50mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 85%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 94%.
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 35 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 15mL distilled water with 1 gram raw material adds distilled water, 32 ℃ of temperature, and pH5.4, vitamins C addition and raw material ratio be hydrolysis 70min under the condition of 2mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with normal hexane equal-volume extraction (2) 3 times, merge the normal hexane part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin DM1400 absorption, with the washing of 5BV, use the dehydrated alcohol wash-out of 3BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims.
B, the usefulness n-hexane extraction a filter residue behind the steam distillation in the step, the ratio of filter residue and normal hexane adds the 10mL normal hexane for the 1g filter residue, heated and stirred is extracted 3 times, each extraction time 1.5h, 25 ℃ of temperature are filtered, merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and normal hexane reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 25mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 25mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 83%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 94%.
Embodiment 7
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 40 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 20mL distilled water with 1 gram raw material adds distilled water, 34 ℃ of temperature, and pH5.7, vitamins C addition and raw material ratio be hydrolysis 80min under the condition of 4mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with ether equal-volume extraction (2) 4 times, merge the ether part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin DM130 absorption, with the washing of 3BV, use the dehydrated alcohol wash-out of 5BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims.
B, with the extracted with diethyl ether a filter residue behind the steam distillation in the step, the ratio of filter residue and ether is that the 1g filter residue adds the 5mL ether, heated and stirred is extracted 3 times, at every turn extraction time 2h, 25 ℃ of temperature are filtered merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and ether reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 30mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 30mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 84%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 95%.
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 50 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 25mL distilled water with 1 gram raw material adds distilled water, 35 ℃ of temperature, and pH6.3, vitamins C addition and raw material ratio be hydrolysis 90min under the condition of 5mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with benzene equal-volume extraction (2) 2 times, the combined benzene part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin DM301 absorption, with the washing of 4BV, use the dehydrated alcohol wash-out of 6BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims.
B, with benzene extraction a filter residue behind the steam distillation in the step, the ratio of filter residue and benzene is that the 1g filter residue adds 10mL benzene, heated and stirred is extracted 3 times, at every turn extraction time 2.5h, 25 ℃ of temperature are filtered merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and benzene reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 35mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 30mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 84%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 94%.
Embodiment 9
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 50 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 25mL distilled water with 1 gram raw material adds distilled water, 35 ℃ of temperature, and pH6.5, vitamins C addition and raw material ratio be hydrolysis 110min under the condition of 7mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with chloroform equal-volume extraction (2) 5 times, the combined chloroform part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin DA-201 absorption, with the washing of 5BV, use the dehydrated alcohol wash-out of 2BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims.
B, with the chloroform extraction a filter residue behind the steam distillation in the step, the ratio of filter residue and chloroform is that the 1g filter residue adds the 25mL chloroform, heated and stirred is extracted 2 times, at every turn extraction time 3h, 30 ℃ of temperature are filtered merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and chloroform reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 40mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 50mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 83%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 95%.
(1) raw materials pretreatment
Pluck all interior horseradish roots through cleaning, cut into slices, dry, be ground into 100-1000 μ m fine powder, dry under 55 ℃ condition, moisture is lower than 10%wt.
(2) raw material hydrolysis
Raw material after pulverizing is put in the hydrolytic decomposition pot, and the ratio that adds 30mL distilled water with 1 gram raw material adds distilled water, 30 ℃ of temperature, and pH6.7, vitamins C addition and raw material ratio be hydrolysis 130min under the condition of 8mg/g.After hydrolysis finishes, filter.
(3) preparation of crude product
The filtrate part: the filtrate that obtains with methylene dichloride equal-volume extraction (2) 4 times, the combined dichloromethane part, vacuum concentration, the phenethyl isothiocyanate crude product.
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin D-101 absorption, with the washing of 3BV, use the dehydrated alcohol wash-out of 6BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims.
B, the usefulness dichloromethane extraction a filter residue behind the steam distillation in the step, the ratio of filter residue and methylene dichloride adds the 20mL methylene dichloride for the 1g filter residue, heated and stirred is extracted 4 times, each extraction time 2.5h, 35 ℃ of temperature are filtered, merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and methylene dichloride reclaims.
(4) refining
The above-mentioned phenethyl isothiocyanate crude product that obtains is carried out extracting and separating and column chromatography for separation.
A, the above-mentioned phenethyl isothiocyanate crude product that obtains is dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 45mL methyl alcohol for the 1g crude product, removes indissolvable component.The part that methyl alcohol is molten, rotary evaporation is dense to be done, and reclaims methyl alcohol.The molten component of methyl alcohol is dissolved in the sherwood oil, the ratio of enriched material and sherwood oil adds the 45mL sherwood oil for the 1g enriched material, removes insoluble part again, and the molten component rotary evaporation of sherwood oil is dense dried, reclaims sherwood oil.Obtain phenethyl isothiocyanate, HPLC content is 85%.
B, the phenethyl isothiocyanate that obtains is further carried out chromatographic separation.Adopt silica filler, sample adopts the molten sample of going up of sherwood oil, adds elutriant then, wash-out step by step, and elutriant is followed successively by sherwood oil and the ethyl acetate of 2 times of column volumes and carries out gradient elution, flow velocity 1.5mL/min, collection elutriant.Its elutriant is that the 1st gradient is pure sherwood oil wash-out, and gradient is volume ratio sherwood oil: ethyl acetate=15:1 thereafter, 10:1, and 10:3,10:5 uses eluent ethyl acetate at last.The content of analyzing phenethyl isothiocyanate through HPLC is 95%.
Claims (7)
1, a kind of method for preparing phenethyl isothiocyanate from horseradish is characterized in that, may further comprise the steps:
The first step raw materials pretreatment
Horseradish cleaned dry section, be ground into fine powder after the oven dry;
The raw material hydrolysis of second step
Fine powder after pulverizing is added the distilled water hydrolysis with the ratio that 1 gram raw material adds 10-30mL distilled water, and hydrolysis temperature 30-40 ℃, pH is 5-7, the vitamins C addition is 1-10:1000 with horseradish fine powder raw materials quality ratio, hydrolysis time 60-140min after hydrolysis finishes, filters;
The preparation of the 3rd step crude product
The filtrate part: the filtrate that obtains with second step of low polar solvent equal-volume extraction 1-5 time, merge the low polar solvent part, vacuum concentration must the phenethyl isothiocyanate crude product;
The filter residue part:
A, usefulness water vapor straight run distillation filter residue part, steam distillation liquid is cooled to room temperature, again after macroporous resin adsorption, with the washing of 2-6BV, use the dehydrated alcohol wash-out of 2-6BV again, dehydrated alcohol wash-out partial rotation evaporation concentration earlier, get the phenethyl isothiocyanate crude product, ethanol reclaims;
B, the low polar organic solvent extraction a of the usefulness filter residue behind the steam distillation in the step, the ratio of filter residue and low polar organic solvent adds the low polar organic solvent of 5-25mL for the 1g filter residue, heated and stirred is extracted 1-4 time, each extraction time 1-3h, filters by temperature 20-40 ℃, merging filtrate, vacuum concentration obtains the phenethyl isothiocyanate crude product, and low polar organic solvent reclaims;
The 4th one-step refining
The phenethyl isothiocyanate crude product that the 3rd step was obtained merges column chromatography for separation after solvent extraction and separation:
A, the phenethyl isothiocyanate crude product that the 3rd step was obtained are dissolved in the methyl alcohol, the ratio of crude product and methyl alcohol adds 20-50mL methyl alcohol for the 1g crude product, remove insolubles, with the dissolve with methanol part, rotary evaporation in vacuo concentrates, reclaim methanol solvate, enriched material is dissolved in the sherwood oil again, the ratio of enriched material and sherwood oil adds the 20-50mL sherwood oil for the 1g enriched material, remove insoluble part, petroleum ether dissolution partial vacuum rotary evaporation is concentrated, reclaim sherwood oil, obtain phenethyl isothiocyanate, analyze the quality percentage composition more than 80% through HPLC;
B, the phenethyl isothiocyanate that a is obtained further carry out column chromatography for separation, the phenethyl isothiocyanate that a is obtained is dissolved in sherwood oil, after the absorption of polarity filled column, adopt petroleum ether-ethyl acetate volume ratio 100: the elutriant of 1-50 is gradient elution step by step, collect elutriant, obtain phenethyl isothiocyanate, analyze the quality percentage composition more than 90% through HPLC.
2, from horseradish, prepare the method for phenethyl isothiocyanate according to claim 1, it is characterized in that,
Described horseradish raw material comes from Linyi, Shandong.
3, prepare the method for phenethyl isothiocyanate according to claim 1 from horseradish, it is characterized in that, described vacuum concentration is that rotary evaporation concentrates, and temperature is 30-60 ℃, and vacuum tightness is 0.02-0.09MPa.
4, prepare the method for phenethyl isothiocyanate according to claim 1 from horseradish, it is characterized in that, the low polar solvent in the 3rd step is sherwood oil, normal hexane, ether, benzene, chloroform or methylene dichloride.
5, prepare the method for phenethyl isothiocyanate according to claim 1 from horseradish, it is characterized in that, the 4th step Semi-polarity filler is silica gel, aluminum oxide or diatomite.
6, prepare the method for phenethyl isothiocyanate according to claim 1 from horseradish, it is characterized in that, macroporous resin is AB-8, X-5, S-8, DM1400, DM130, DM301, DA201, D101 in the 3rd step.
7, the method that from horseradish, prepares phenethyl isothiocyanate according to claim 1, it is characterized in that, HPLC measuring method in the 4th step is standard substance with the phenethyl isothiocyanate, with Hypersil ODS2 5 μ m 4.6mm*250mm chromatographic columns, ratio liquid mixture prepared with volume ratio methyl alcohol: water=70:30 is a moving phase, with 243nm is to detect wavelength.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603406A (en) * | 2012-03-28 | 2012-07-25 | 常熟市南洋化肥有限公司 | Horseradish soil fertilizer composition for preventing and curing soil-borne disease |
| CN104961667A (en) * | 2015-06-16 | 2015-10-07 | 赣州华汉生物科技有限公司 | Purifying method of sulforaphane |
| CN105384671A (en) * | 2015-12-16 | 2016-03-09 | 济宁医学院 | Method for extracting allyl isothiocyanate from horseradish |
| CN111850062A (en) * | 2020-07-06 | 2020-10-30 | 暨南大学 | Method for hydrolyzing glucosinolates in mustard into isothiocyanates under assistance of ultrahigh static pressure combined with conversion liquid |
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2008
- 2008-12-02 CN CN2008102354236A patent/CN101440054B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603406A (en) * | 2012-03-28 | 2012-07-25 | 常熟市南洋化肥有限公司 | Horseradish soil fertilizer composition for preventing and curing soil-borne disease |
| CN104961667A (en) * | 2015-06-16 | 2015-10-07 | 赣州华汉生物科技有限公司 | Purifying method of sulforaphane |
| CN105384671A (en) * | 2015-12-16 | 2016-03-09 | 济宁医学院 | Method for extracting allyl isothiocyanate from horseradish |
| CN111850062A (en) * | 2020-07-06 | 2020-10-30 | 暨南大学 | Method for hydrolyzing glucosinolates in mustard into isothiocyanates under assistance of ultrahigh static pressure combined with conversion liquid |
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| CN101440054B (en) | 2011-05-11 |
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