CN105669797B - A kind of method that burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H are separated from great burdock achene - Google Patents
A kind of method that burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H are separated from great burdock achene Download PDFInfo
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Abstract
The present invention provides a kind of method that burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H are separated from great burdock achene, and the method first passes through CO2Supercritical fluid extraction carries out degreasing, isolated burdock seed oil and degreasing great burdock achene powder to great burdock achene, carries out water alcohol extracting to degreasing great burdock achene powder and is concentrated to give extraction medicinal extract, and CH is carried out to extraction medicinal extract2Cl2Extraction and upper silica gel column chromatography, arctigenin, arctigenin-4'-gentiobioside E, arctiin and arctigenin-4'-gentiobioside H are obtained using dichloromethane methanol gradient elution, and by thin-layer chromatographic analysis, purifying.The present invention can complete the separation of multi-products using a route of continous way, take full advantage of raw material, resource is saved, skimming processes do not pollute the environment, obtained burdock seed oil nutritive value is high, arctigenin, arctiin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H purity are high, can be applied to medicine food field individually or with other material mixings as raw material.
Description
Technical field
The invention belongs to separation and extraction technology field, is related to one kind and burdock seed oil and wooden lipid chemical combination are separated from great burdock achene
The method of thing, and in particular to one kind separates burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H from great burdock achene
Method.
Background technology
Great burdock achene is Compositae Arctium lappa category（Arctium lappaL.）The dry mature fruit of biennial herb plant burdock,
It is distributed widely in East Asian countries, belongs to wind-heat-dispersing medicinal in diaphoretic medicine, has dispelling wind and heat from the body, facilitaing lung promoting eruption, the work(of subdhing swelling and detoxicating
Effect, to go through version《Chinese Pharmacopoeia》Included.Modern pharmacological research proves that great burdock achene has anti-inflammatory, antitumor, blood pressure lowering and adjusting
Immune isoreactivity, its chemical composition mainly contain volatile oil and lignanoids, wherein lignan component mainly including arctiin,
Arctigenin, arctigenin-4'-gentiobioside E, arctigenin-4'-gentiobioside H.These chemical compositions of bardane oil, arctigenin, arctiin are adapted in treatment in great burdock achene
There is certain difference in terms of disease, it is necessary to select corresponding target component according to therapeutic effect, bardane oil is mainly terpene and sesquialter
Terpene volatile oil and fatty acid compound, have preferable antibacterial effect；Arctiin and arctigenin are respectively provided with anti-inflammatory town
Bitterly, the effect of diabetic complication etc. is treated, but there is also otherness slightly, ox in arctiin and arctigenin curative effect
Burdock glycosides has well antitumor, anti-inflammatory, analgesic effect, and arctigenin has the work of good anti-diabetic vascular lesions
With.It can be seen from the above that burdock extract has good pharmacological effect, but how great burdock achene herb resource can be made full use of,
Component therein is obtained one by one be current great burdock achene extraction and separation process difficult point.
Patent CN101278940A is provided with arctiin and arctigenin composition different components ratio treatment diabetes
Property cardiovascular pathological changes effect, it is determined that optimal ratio, but the preparation method is complicated, need to be first with great burdock achene medicinal material through macropore tree
Fat obtains arctiin, then obtains arctigenin through silica gel column chromatography with great burdock achene medicinal material again, then by arctiin and burdock
The mixture of aglycon composition different proportion is screened, too cumbersome.Patent CN1323674C provides a kind of for treating
The preparation method of diabetic nephropathy or Fructus Arctii total lignans' glycoside extracts of diabetes, obtains Fructus Arctii total lignans' glycosides
Class material 50-90%（Determined by ultraviolet spectrophotometry）Extraction raw material, for treating diabetic nephropathy and diabetes, solve
The special-purpose medicaments of diabetic nephropathy or diabetes, but in this method raw material is total lignan, and do not have further purification, do not obtain
Main active arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H, the impurity such as polysaccharide is not also divided in extract
From there are certain deficiency.Patent CN1000443493C provides a kind of preparation method for preparing arctiin and arctigenin,
The patent obtains burdock seed oil using organic solvent degreasing, the method for polyamide column chromatography, at the same also obtain arctiin and
The mixture of its aglycon, but the technique, using organic solvent extraction great burdock achene medicinal material, consumption of organic solvent is big, the influence to environment
Also it is big, and the arctiin, the arctigenin that are obtained are mixture, it is impossible to select corresponding chemical composition group according to Therapy characteristics
Resulting mixture.
Therefore, further to make the modernization of Chinese medicine, lignan component application range and application effect are improved, it is necessary to right
Lignan constituents are separately separated, and obtain its main active, according to the needs in food and medicine field, individually
Using or apply in varing proportions on demand respectively, but at present make full use of great burdock achene medicinal material obtain at the same time bardane oil,
The method of arctiin, arctigenin and other lignanoids chemical compositions.
The content of the invention
For deficiencies of the prior art, the technical problems to be solved by the invention are：How to provide it is a kind of from
The method that burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H are separated in great burdock achene, can pass through one
Route isolates burdock seed oil, arctigenin, arctiin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H from great burdock achene at the same time, and has and fill
Divide and utilize the characteristics of raw material, separation method are simple efficiently, practicality is good, environmental pollution is small.
In order to solve the above-mentioned technical problem, the present invention adopts the following technical scheme that：One kind separates great burdock achene from great burdock achene
Oil, arctiin, arctigenin, the method for arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H, include the following steps：
1）CO is carried out after great burdock achene is crushed2When supercritical extract degreasing 5 ~ 7 is small, burdock seed oil and degreasing great burdock achene are obtained
Powder；
2）To step 1）Obtained degreasing great burdock achene powder refluxing extraction 3 ~ 4 times, filters the extracting solution extracted every time, merges filter
Liquid is simultaneously concentrated, and obtains extraction medicinal extract；Wherein, extracting solution is the water-alcohol solution that mass concentration is 50 ~ 95%, every time during extraction
The use quality of the extracting solution is 5 ~ 10 times of the degreasing great burdock achene silty amount, when each extraction time is 0.5 ~ 2 small；
3）Using CH2Cl2Extraction step 2）Obtained extraction medicinal extract 2 ~ 3 times, combining extraction liquid, obtains sample to be separated；Its
In, the CH2Cl2Mass volume ratio with the extraction medicinal extract is 2 ~ 3 mL:1 g；
4）By step 3）Sample silica gel mixed sample is mixed in obtained sample concentration to be separated, addition, is carried out silica gel column chromatography, is adopted
With gradient elution mode, the eluent that the gradient elution uses is followed successively by volume ratio 100:0、100: 2、100: 5、100:
8、100:10 and 0:100 dichloro methane-methanol, each 5 ~ 8 retention volumes of gradient elution, every 1/3 retention volume are received
Collect for a cut；Wherein, the mass ratio that sample silica gel and sample to be separated are mixed in the silica gel column chromatography is 1 ~ 2:1, fill out
Material silica gel and the mass ratio for mixing sample silica gel are 5 ~ 15:1；
5）To step 4）Collect each obtained cut and carry out thin-layer chromatographic analysis, be 100 with volume ratio:5 dichloro
Methane-methanol solvent detection arctigenin, volume ratio 100:8 methylene chloride-methanol solvent detects arctigenin-4'-gentiobioside E, body
Product is than being 100:10 methylene chloride-methanol solvent detects arctiin, volume ratio 100:15 methylene chloride-methanol expansion
Agent detects arctigenin-4'-gentiobioside H, merges the similar cut of tlc analysis result, respectively obtains arctigenin cut, arctigenin-4'-gentiobioside E cuts, ox
Burdock glycosides cut and arctigenin-4'-gentiobioside H cuts；
6）Respectively to step 5）Obtained arctigenin cut and arctiin cut is concentrated and dried, and obtains arctigenin
Sterling and arctiin sterling；
7）With alcohol dissolving step 5）Obtained arctigenin-4'-gentiobioside E cuts, and add and mix sample silica gel mixed sample, using mesolow positive silicon
Glue chromatography carries out gradient elution, and the eluent that the gradient elution uses is followed successively by volume ratio 100:8、100:12 and 0:100
Petroleum ether-ethyl acetate solution, each 5 ~ 8 retention volumes of gradient elution, the eluent to each gradient are with volume ratio
100:8 dichloro methane-methanol carries out thin-layer chromatographic analysis as solvent, and the similar cut of combined analysis result is simultaneously dense
Contracting, obtains arctigenin-4'-gentiobioside E sterlings；Wherein, the sample silica gel and the mass ratio of arctigenin-4'-gentiobioside E cuts mixed is 1 ~ 2:1, the mesolow is just
Filler silica gel and the mass ratio for mixing sample silica gel are 6 ~ 10 in phase silica gel chromatograph:1；
8）With alcohol dissolving step 5）Obtained arctigenin-4'-gentiobioside H cuts, and add and mix sample silica gel mixed sample, using mesolow positive silicon
Glue chromatography carries out gradient elution, and the eluent that the gradient elution uses is followed successively by volume ratio 100:3、100:5 and 0:100 second
Acetoacetic ester-methanol solution, each 5 ~ 8 retention volumes of gradient elution, the eluent volume ratio to each gradient are 100:15
Dichloro methane-methanol carry out thin-layer chromatographic analysis as solvent, the similar cut of combined analysis result and concentration obtain
To arctigenin-4'-gentiobioside H sterlings；Wherein, the sample silica gel and the mass ratio of arctigenin-4'-gentiobioside E cuts mixed is 1 ~ 2:1, the mesolow positive silicon
Filler silica gel and the mass ratio for mixing sample silica gel are 10 ~ 20 in glue chromatography:1.
Compared with prior art, the present invention has the advantages that：
1st, the method for the present invention uses CO2Supercritical extraction carries out degreasing to great burdock achene, obtains burdock seed oil, compared to existing
Have and the problem of environmental pollution that organic reagent degreasing is brought is avoided for technology, with more environment friendly, and use CO2It is super to face
Extraction yield higher of boundary's extracting process to burdock seed oil（It can obtain the burdock seed oil of great burdock achene raw material weight 12 ~ 18%）, avoid
The problem of there is interference because of preparation of the seed oil containing burdock on follow-up arctiin, arctigenin isoreactivity component and influence, makes at the same time
The burdock seed oil nutritive value higher obtained.
2nd, present invention firstly provides the separation that multi-products in great burdock achene are completed using a kind of continous way disjunctive path, knot
The property of arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H are closed, the lightning strip of each product has been obtained by innovative research
Part, has isolated the high sterling of arctiin best in quality, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H, purity 95% with
On, raw material is taken full advantage of, has saved resource, it is highly practical.
3rd, separation method of the present invention separative efficiency higher compared with the prior art, obtained each product purity is high, without into
The extra processing of row can be used as the bulk pharmaceutical chemicals for the treatment of correlative diseases medicine, can be individually or with other material mixings as former
Material is applied to drug and food field, the especially application in diabetes and diabetic nephropathy field, has more preferable market prospects,
Value of the product is high.
Embodiment
The method of the present invention is described further with reference to specific embodiment, but these embodiments must not be interpreted as any
Limiting to the claimed invention in meaning.
1 great burdock achene degreasing of embodiment prepares burdock seed oil
Great burdock achene, which is ground into, can cross the coarse powder of 10 mesh sieves, use CO2When supercritical extracting equipment progress degreasing 6 is small, obtain
Burdock seed oil and degreasing great burdock achene powder.CO2The process conditions of supercritical extract are, 30 MPa of extracting pressure, 40 DEG C of extraction temperature,
10 MPa of separating pressure, 40 DEG C of separation temperature, 500 L/h of carbon dioxide flow.
The chemical composition analysis of 2 burdock seed oil of embodiment
（1）The extraction of volatile oil component in burdock seed oil
Using volatile component in extraction by steam distillation burdock seed oil, 35.0 g of burdock seed oil is accurately weighed, with material
Liquor ratio 1:5 add 5% sodium chloride solution, 165 mL in round-bottomed flask, and heating, drip the first dropping liquid at the mouth of pipe of condenser pipe lower end
Start timing during body, control temperature, keep slight boiling condition, distill 5 h.After sufficiently cool, upper strata grease, 40 DEG C of water are drawn
Bath drying, weighs, in case GC-MS is analyzed.
（2）The esterification of fatty acid composition in burdock seed oil
Since fatty acid composition boiling point is higher, active carbon is now carried out, to reduce its boiling point, adapts to GC-MS inspections
Survey and require.Step（1）After the completion of burdock volatile oil extracting, 25 mL n-hexanes are added into flask, fat is separated using separatory funnel
Fat acid component, the accurate separated fatty acid compositions of 0.5 mL of drawing add 2.5 mL ether/n-hexane in 25 mL volumetric flasks
Mixed solvent（Volume ratio 2:1）, 2.5 mL methanol solutions and 2.5 mL potassium hydroxide solutions（0.8 mol/L）, 5 are stood after shaking up
Min, makes fully to react.After 5 min, distilled water is added to terminate reaction to volumetric flask scale.Upper-layer fat acid methyl ester product is drawn,
45 DEG C of water-bath dryings, weigh, in case GC-MS is analyzed.
（3）Great burdock achene oil component is analyzed
Fatty acid compound after deriving respectively to bardane oil volatile ingredient and esterification carries out GC-MS analyses.
It is using instrument:Agilent 7890A/5975C type chromatograph-mas spectrometers.GC conditions: HP-Chiral 20β
（25mm×0.25mm）Quartz capillary column, initial column temperature are set as 100 DEG C, and 3 min internal programs are warming up to 260 DEG C, pressure
It is set as that 50 kPa, He make carrier gas, 280 DEG C of temperature of vaporization chamber, 1.0 ml/min of flow, split ratio 10:1.Mass Spectrometry Conditions：Add
Hot hyperboloid level Four bar analyzer, ion gun use EI, and electron bombardment energy is 70 eV, accelerating potential 3kV, resolution ratio 500,
Sensitivity S/N >=400,240 DEG C of ion source temperature, 0.2 μ l of sample size, mass spectral database use NIST11.
GC-MS experiments identify the information such as 16 kinds of volatile oil components, its molecular formula, relative abundance and see annex table 1 altogether.By
Table 1 understands that great burdock achene volatile oil component mainly includes four constituents such as ester, acid, aldehyde and alkene.Wherein, methyl linoleate and oleic acid
Methyl esters is main volatile oil, accounts for 36.02 %, 21.10 % respectively, secondly hexahydrotoluene, relative amount is 17.57 %.Separately
Outside also containing a small amount of alcohol, ketone, sequiterpene and linear paraffin etc..
Fatty acid part identifies 44 kinds of compounds altogether, and wherein fatty acid composition has 19 kinds, and is carried out with area normalization method
Quantitative determination, the result is shown in annex table 2.As known from Table 2, a variety of aliphatic acid are contained in bardane oil, wherein linoleic content is most
Height, relative abundance are up to 57.25 %, are secondly 23.96 % of oleic acid, 5.24 % of palmitic acid, 2.39 % of stearic acid, other 2.49
%.Unrighted acid accounts for the overwhelming majority, is the 86.45% of total fatty acid content.
3 arctiin of embodiment, arctigenin, the preparation method of arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H
（1）The degreasing great burdock achene powder obtained to embodiment 1 carries out refluxing extraction 3 ~ 4 times, every using 150 ~ 300 mesh screens
The extracting solution of secondary extraction, merging filtrate are simultaneously concentrated, and obtain extraction medicinal extract；Wherein, extracting solution is the water that mass concentration is 95%
Alcoholic solution, the use quality of the water-alcohol solution is 10 times of the degreasing great burdock achene powder during extraction every time, each extraction time
For 2 it is small when；
（2）To 1 kg steps（1）CH is added in obtained arctinol concentrate extraction medicinal extract2Cl2（2.0 L）Extraction 3
It is secondary, extract is collected, is concentrated under reduced pressure, obtains CH2Cl2Crude extract medicinal extract（450.0 g）.
（3）To CH2Cl2Added in crude extract medicinal extract and mix sample silica gel mixed sample, carried out silica gel column chromatography, utilize dichloromethane
Alkane-methanol system is to great burdock achene CH2Cl2Crude extract carries out silica gel column chromatography（The blade diameter length ratio of silicagel column is 1:8）Separation, uses dichloro
Methane:Methanol（100:0, 100:2, 100:5, 100:8, 100:10, 0:100）Gradient elution, each gradient elution 5 ~ 8
A retention volume, every 1/3 retention volume are collected as a cut；Wherein, sample silica gel is mixed in the silica gel column chromatography with treating
The mass ratio for separating sample is 1:1, filler silica gel and the mass ratio for mixing sample silica gel are 5:1；
Thin-layer chromatographic analysis are carried out to each cut that collection obtains, are 100 with volume ratio:5 methylene chloride-methanol
Solvent detection arctigenin, volume ratio 100:8 methylene chloride-methanol solvent detects arctigenin-4'-gentiobioside E, volume ratio 100:
10 methylene chloride-methanol solvent detects arctiin, volume ratio 100:15 methylene chloride-methanol solvent detection burdock
Phenol H, merges the similar cut of tlc analysis result, and the results show arctigenin concentrates on dichloromethane:Methanol=100:2 gradients,
Arctigenin-4'-gentiobioside E concentrates on dichloromethane:Methanol=100:5 gradients, arctiin concentrate on dichloromethane:Methanol=100:8 gradients, burdock
Phenol H concentrates on dichloromethane:Methanol=100:10 gradients, respectively obtain arctigenin cut, arctigenin-4'-gentiobioside E cuts, arctiin cut
With arctigenin-4'-gentiobioside H cuts；
（4）100:2 gradients obtain arctigenin cut, after arctigenin cut is concentrated into 40g placements 10h,
There is white amorphous powder precipitation, obtain arctigenin sterling, purity 68.5%, yield 63%（Actual preparation amount and theoretical calculation
The ratio between amount, similarly hereinafter）；Again with methanol recrystallization arctigenin sterling 3 times, obtains white powder crystal, is arctigenin high sterling,
Yield 38.5%, purity 97.5%；
（5）100:5 gradients obtain 76.5 g of arctigenin-4'-gentiobioside E crude products, are dissolved to form true solution with methanol, 1 times of silicon
Glue mixes sample, and preparing chromatography using mesolow purification on normal-phase silica gel is separated, 600 g of filler silica gel.Use petroleum ether:Ethyl acetate
（100:8, 100:12, 0:100）Gradient elution, each 5~8 retention volumes of gradient elution, to the eluent of each gradient
It is 100 with volume ratio:8 dichloro methane-methanol carries out thin-layer chromatographic analysis as solvent, and combined analysis result is similar
Cut and concentration, obtain arctigenin-4'-gentiobioside E sterlings, through analysis, arctigenin-4'-gentiobioside E is concentrated mainly on petroleum ether:Ethyl acetate=100:12 ladders
Degree, purity 55.1%, yield 60.3%, obtains arctigenin-4'-gentiobioside E sterlings；Efficient liquid phase preparation further is carried out to above-mentioned arctigenin-4'-gentiobioside E sterlings,
With methanol:Water（61:39）For mobile phase, 3.0 ml/min of flow velocity, 280 nm of Detection wavelength, collect the eluent of appearance, and right
Eluent carries out recrystallizing methanol, finally gets arctigenin-4'-gentiobioside E high sterlings, purity 98.5%, yield 45.5%；
（6）100:8 gradients obtain arctiin cut, after placing 4 h, there is white needles precipitation, stand 12-24h,
Obtain arctiin sterling, purity 82.5%, yield 74.5%；Recrystallizing methanol further is carried out 3 times to arctiin sterling, is obtained white
The powdered crystal of color, arctiin high sterling, purity 98.3%, yield 58.5%；
（7）100:10 gradients obtain 5.3 g of arctigenin-4'-gentiobioside H crude products, are dissolved to methanol after forming true solution, 1 times
Silica gel mixed sample, prepares chromatography using mesolow purification on normal-phase silica gel and is separated, 110.0 g of filler silica gel, ethyl acetate:Methanol
（100:3, 100:5, 0:100）Gradient elution, each 5~8 retention volumes of gradient elution, use the eluent of each gradient
Volume ratio is 100:15 dichloro methane-methanol carries out thin-layer chromatographic analysis as solvent, and combined analysis result is similar
Cut and concentration, obtain arctigenin-4'-gentiobioside H sterlings, arctigenin-4'-gentiobioside H is mainly enriched in ethyl acetate:Methanol=100:5 gradients, purity
51.2%, yield 55.3%.High-efficient liquid phase color preparation is further carried out again to arctigenin-4'-gentiobioside H sterlings, with methanol:Water（40:60）For stream
Dynamic phase, flow velocity is 3.0 ml/min, 280 nm of Detection wavelength, and carries out recrystallizing methanol, be finally separating arctigenin-4'-gentiobioside H is high-purity
Product, purity 98.6%, yield 43.5%.
4 arctiin of embodiment, arctigenin, the Structural Identification of arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H
Structure is carried out to the lignan component being prepared in embodiment 3 with analysis method by use of spectrum such as UV, IR, NMR, MS
Identification.
（1）The Structural Identification of arctiin
White amorphous powder, C27H34O11, ESI-MS m/z: 557 [ M + Na]+.It is soluble in dichloromethane, first
The organic solvents such as alcohol, ethyl acetate, are insoluble in water.UV spectrum, which are shown at 210 nm and 280 nm, strong absorption, shows this change
Compound contains aromatic rings.There are 3350 cm in IR collection of illustrative plates-1（ν OH peaks）、3000 cm-1（Phenyl ring ν=CH peaks）、1750 cm-1（Ester
Carbonyl ν C=O peaks）、1650-1450 cm-1（Phenyl ring skeletal vibration ν C=C peaks）Deng characteristic peak, show exist in the compound structure
Aromatic ring and carbonyl.Analysis1H-NMR（400MHz, CD3OD）With13C-NMR（100MHz, CD3OD）Data, reference document report, together
Shi Yubiao product are total to thin layer comparison, and it is arctiin to identify the compound（arctiin）.
（2）The Structural Identification of arctigenin
White amorphous powder, C21H24O6, ESI-MS m/z: 395 [ M + Na]+.It is soluble in dichloromethane, acetic acid
The organic solvents such as ethyl ester, are insoluble in water.UV spectrum, which are shown at 210 nm and 280 nm, strong absorption, shows that this compound contains
Aromatic rings.There are 3350 cm in IR collection of illustrative plates-1（ν OH peaks）、3000 cm-1（Phenyl ring ν=CH peaks）、1750 cm-1（Ester carbonyl group ν C=O
Peak）、1650-1450 cm-1（Phenyl ring skeletal vibration ν C=C peaks）Deng characteristic peak, show that there are aromatic ring and carbonyl in the compound structure
Base.Analysis1H-NMR（400MHz, CD3OD）With13C-NMR（100MHz, CD3OD）Data, reference document report, while with marking product
Thin layer compares altogether, and it is arctigenin to identify the compound（Arctigenin）.
（3）The Structural Identification of arctigenin-4'-gentiobioside E
White amorphous powder, C30H34O10, ESI-MS m/z: 577[ M+Na]+.It is soluble in methanol, dichloromethane, second
The organic solvents such as acetoacetic ester, are insoluble in water.UV spectrum, which are shown at 210 nm and 280 nm, strong absorption, shows that it contains fragrance
Ring.And there are 3350cm-1 in IR collection of illustrative plates（ν OH peaks）、~3000cm-1（Phenyl ring ν=CH peaks）1750cm-1（Ester carbonyl group ν C=O peaks）、
1650-1450cm-1（Phenyl ring skeletal vibration ν C=C peaks）Deng characteristic peak, show that there are aromatic ring and carbonyl in the compound structure.Should
Compound1H-NMR（400MHz, CDCl3）、13C-NMR（100MHz, CDCl3）As a result it is arctigenin-4'-gentiobioside E also to identify this compound
（lappaol E）.
（4）The Structural Identification of arctigenin-4'-gentiobioside H
White amorphous powder, C40H46O14, ESI-MS: m/z: 773 [ M+Na]+.Be soluble in methanol, dichloromethane,
The organic solvents such as ethyl acetate, are insoluble in water.UV spectrum, which are shown at 210 nm and 280 nm, strong absorption, shows that it contains virtue
Fragrant ring.And there are 3350 cm in IR collection of illustrative plates-1（ν OH peaks）、~3000 cm-1（Phenyl ring ν=CH peaks）1750 cm-1（Ester carbonyl group ν C=O
Peak）、1650-1450 cm-1（Phenyl ring skeletal vibration ν C=C peaks）Deng characteristic peak, show in this compound structure there are aromatic ring and
Carbonyl.The compound1H-NMR（400MHz, CD3OD）、13C-NMR（100MHz, CD3OD）As a result it is ox also to identify this compound
Burdock phenol H（lappaol H）.
The above embodiment of the present invention is only example to illustrate the invention, and is not the implementation to the present invention
The restriction of mode.For those of ordinary skill in the field, other can also be made not on the basis of the above description
With the change and variation of form.Every technical scheme changes and variations that derived from that belong to still are located
In the row of protection scope of the present invention.
Claims (6)
1. a kind of method that burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside H are separated from great burdock achene, it is special
Sign is, includes the following steps：
1）CO is carried out after great burdock achene is crushed2When supercritical extract degreasing 5 ~ 7 is small, burdock seed oil and degreasing great burdock achene powder are obtained；
CO2The process conditions of supercritical extract are 30 MPa of extracting pressure, 40 DEG C of extraction temperature, 10 MPa of separating pressure, separation temperature
40 DEG C, 500 L/h of carbon dioxide flow；
2）To step 1）Obtained degreasing great burdock achene powder refluxing extraction 3 ~ 4 times, is carried using what 150 ~ 300 mesh screens extracted every time
Liquid is taken, merging filtrate is simultaneously concentrated, and obtains extraction medicinal extract；Wherein, extracting solution is the water-alcohol solution that mass concentration is 50 ~ 95%,
The use quality of the extracting solution is 5 ~ 10 times of the degreasing great burdock achene silty amount during extraction every time, and each extraction time is
0.5 ~ 2 it is small when；
3）Using CH2Cl2Extraction step 2）Obtained extraction medicinal extract 2 ~ 3 times, combining extraction liquid, obtains sample to be separated；Wherein,
The CH2Cl2Volume mass ratio with the extraction medicinal extract is 2 ~ 3 mL:1 g；
4）By step 3）Sample silica gel mixed sample is mixed in obtained sample concentration to be separated, addition, silica gel column chromatography is carried out, using ladder
Elution mode is spent, the eluent that the gradient elution uses is followed successively by volume ratio 100:0、100: 2、100: 5、100: 8、
100:10 and 0:100 dichloro methane-methanol, each 5 ~ 8 retention volumes of gradient elution, every 1/3 retention volume are collected
For a cut；Wherein, the mass ratio that sample silica gel and sample to be separated are mixed in the silica gel column chromatography is 1 ~ 2:1, filler
Silica gel and the mass ratio for mixing sample silica gel are 5 ~ 15:1；
5）To step 4）Collect each obtained cut and carry out thin-layer chromatographic analysis, be 100 with volume ratio:5 dichloromethane-
Methanol solvent detection arctigenin, volume ratio 100:8 methylene chloride-methanol solvent detects arctigenin-4'-gentiobioside E, volume ratio is
100:10 methylene chloride-methanol solvent detects arctiin, volume ratio 100:15 methylene chloride-methanol solvent detection
Arctigenin-4'-gentiobioside H, merges the similar cut of tlc analysis result, respectively obtains arctigenin cut, arctigenin-4'-gentiobioside E cuts, arctiin and evaporate
Divide and arctigenin-4'-gentiobioside H cuts；
6）Respectively to step 5）Obtained arctigenin cut and arctiin cut is concentrated and dried, and obtains arctigenin sterling
With arctiin sterling；
7）With alcohol dissolving step 5）Obtained arctigenin-4'-gentiobioside E cuts, and add and mix sample silica gel mixed sample, using mesolow purification on normal-phase silica gel
Chromatography carries out gradient elution, and the eluent that the gradient elution uses is followed successively by volume ratio 100:8、100:12 and 0:100 stone
Oily ether-ethyl acetate solution, each 5 ~ 8 retention volumes of gradient elution, the eluent volume ratio to each gradient are 100:8
Dichloro methane-methanol carry out thin-layer chromatographic analysis as solvent, the similar cut of combined analysis result and concentration obtain
To arctigenin-4'-gentiobioside E sterlings；Wherein, the sample silica gel and the mass ratio of arctigenin-4'-gentiobioside E cuts mixed is 1 ~ 2:1, the mesolow positive silicon
Filler silica gel and the mass ratio for mixing sample silica gel are 6 ~ 10 in glue chromatography:1；
8）With alcohol dissolving step 5）Obtained arctigenin-4'-gentiobioside H cuts, and add and mix sample silica gel mixed sample, using mesolow purification on normal-phase silica gel color
Spectrum carries out gradient elution, and the eluent that the gradient elution uses is followed successively by volume ratio 100:3、100:5 and 0:100 acetic acid second
Ester-methanol solution, each 5 ~ 8 retention volumes of gradient elution, the eluent volume ratio to each gradient are 100:The two of 15
Chloromethanes-methanol solution carries out thin-layer chromatographic analysis, the similar cut of combined analysis result and concentration as solvent, obtains ox
Burdock phenol H sterlings；Wherein, the sample silica gel and the mass ratio of arctigenin-4'-gentiobioside E cuts mixed is 1 ~ 2:1, the mesolow purification on normal-phase silica gel color
Filler silica gel and the mass ratio for mixing sample silica gel are 10 ~ 20 in spectrum:1.
2. burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside are separated from great burdock achene according to claim 1
The method of H, it is characterised in that step 1）It is middle that great burdock achene is crushed to 10 mesh.
3. burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside are separated from great burdock achene according to claim 1
The method of H, it is characterised in that step 4）Described in silicagel column blade diameter length ratio be 1:8.
4. burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside are separated from great burdock achene according to claim 1
The method of H, it is characterised in that respectively to step 6）Obtained arctigenin sterling and arctiin sterling carries out recrystallizing methanol 3
It is secondary, obtain arctigenin high sterling and arctiin high sterling.
5. burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside are separated from great burdock achene according to claim 1
The method of H, it is characterised in that by step 7）The arctigenin-4'-gentiobioside E sterlings loading high performance liquid chromatography, uses volume ratio as 61:39
Methanol-water solution as mobile phase, 3.0 ml/min of flow velocity, 280 nm of Detection wavelength, collect the eluent of appearance, and to washing
De- liquid carries out alcohol recrystallization, obtains arctigenin-4'-gentiobioside E high sterlings.
6. burdock seed oil, arctiin, arctigenin, arctigenin-4'-gentiobioside E and arctigenin-4'-gentiobioside are separated from great burdock achene according to claim 1
The method of H, it is characterised in that by step 8）The arctigenin-4'-gentiobioside H sterlings loading high performance liquid chromatography, with methanol:Water is than 40:60 are
Mobile phase, flow velocity are 3.0 ml/min, 280 nm of Detection wavelength, collect the eluent of appearance, and carry out alcohol to eluent and tie again
Crystalline substance, obtains arctigenin-4'-gentiobioside H high sterlings.
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| CN109061021B (en) * | 2018-07-09 | 2021-02-26 | 西南民族大学 | Method for separating or analyzing phenolic acid compounds from burdock |
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