CN109438221A - A method of preparing syringic acid from spirulina - Google Patents
A method of preparing syringic acid from spirulina Download PDFInfo
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Abstract
The method that the invention discloses a kind of to prepare syringic acid from spirulina, comprising the following steps: Step 1: extracting spirulina crude extract with ether；Step 2: crude extract isolate and purify for the first time with sephadex column；It is separated Step 3: crude extract carries out second with reverse phase silica gel column；Step 4: crude extract carries out third time separation with sephadex column；Step 5: crude extract carries out the 4th separation with reverse phase silica gel column；Step 6: crude extract carries out the 5th separation with normal phase silicagel column, compound A is obtained；Step 7: Structural Identification compound A is syringic acid, syringic acid is obtained.The present invention has isolated syringic acid from spirulina for the first time, and active constituent of the syringic acid as spirulina is significant for the secondary development of spirulina.
Description
Technical field
The present invention relates to the technical field of active medicine more particularly to a kind of methods that syringic acid is prepared from spirulina.
Background technique
Syringic acid, also known as 3,5- dimethoxy-4 '-hydroxybenzoic acid.Syringic acid and its derivative are important among medicine
Body, agricultural chemicals and photosensitizer etc., demand is larger in the international market.Syringic acid is the chemistry list extracted in dendrobium nobile
Body because containing phenolic hydroxyl group, therefore is easier to the variations such as generation aoxidizes, polymerize, place is closed, with preferably anti-oxidant, removing free radical
Ability.Syringic acid also has medicineization effect.Medicineization effect mainly includes antibacterial action and central inhibitory action.Wherein antibacterial is made
With the main antibiotic effective ingredient that syringic acid is Artemisia stelleriana is embodied in, there are antibacterium and fungi；Central inhibitory action embodies
There is calm drawn game anaesthetic effect in syringic acid；Syringic acid widely applies to medicine, fragrance, chemistry of pesticide and organic synthesis work
Industry.But in the prior art, not yet have been reported that syringic acid is present in spirulina.
In view of this, the present inventor studies and devises a kind of method for preparing syringic acid from spirulina, this case is thus
It generates.
Summary of the invention
The method that the purpose of the present invention is to provide a kind of to prepare syringic acid from spirulina, by being added in spirulina
After extract gradually extracts, and by the multiple chromatographic isolation of reverse phase silica gel column and normal phase silicagel column, with finally from spirulina
Syringic acid is made.
To achieve the above object, the present invention the technical solution to solve the technical problem is that:
A method of preparing syringic acid from spirulina, comprising the following steps:
It is placed in the triangular flask of 1L Step 1: weighing 50g spirulina powder, 500mL anhydrous ether is added and shakes up, extracts 4h,
It filters, collects filtrate；500mL anhydrous ether is added into precipitating again and carries out second of extraction 4h, filters, collects filtrate；So
Extraction 3 times, merging filtrate, vacuum-concentrcted is evaporated, and obtains crude extract；
Step 2: after crude extract is dissolved with methanol, upper Sephadex LH-20 gel column, use methanol as eluant, eluent into
Row elution, coutroi velocity 8s/drop, every 0.5h collect a pipe, collect 216 pipes altogether, every pipe component is with thin-layer chromatography TLC points
Analysis merges the component containing same substance；It is 5mg/mL that component will be merged again with methanol dilution to concentration, is measured and is closed with DPPH method
And the antioxidant activity of component, it selects the highest merging component of antioxidant activity to be labeled as T1, is evaporated, 4 DEG C of storages are spare；
Step 3: after component T1 is dissolved with proper amount of methanol, upper C18 reverse phase silica gel column is eluted by the way of gradient elution
Sample, gradient are set as 95% water: 5% methanol, 75% water: 25% methanol, 50% water: 50% methanol, 25% water: 75% first
Alcohol, 100% methanol；Coutroi velocity is 8mL/min, and in first four gradient, each gradient collects 5 pipes, the last one gradient is collected
16 pipes, every pipe collect 7min, collect 36 pipes altogether；Every pipe component is analyzed with thin-layer chromatography TLC, merges the group containing same substance
Point；The biggish merging component of selected amount is labeled as T2, is 5mg/mL with methanol dilution to concentration, measures component T2 with DPPH method
Antioxidant activity, be evaporated, 4 DEG C of storages are spare；
Step 4: after component T2 is dissolved with proper amount of methanol, upper Sephadex LH-20 gel column uses methanol as eluant, eluent
It is eluted, coutroi velocity 13s/drop, every 0.5h collect a pipe, collect 40 pipes altogether, every pipe component is with thin-layer chromatography TLC points
Analysis merges the component containing same substance, is labeled as T3；It is again 5mg/mL with methanol dilution to concentration by component T3, uses DPPH
Method measures the antioxidant activity of component T3, is evaporated, 4 DEG C of storages are spare；
Step 5: upper C18 reverse phase silica gel column is eluted by the way of gradient elution after component T3 adds proper amount of methanol to dissolve
Sample, gradient are set as 25% water: 75% methanol, 15% water: 85% methanol, 5% water: 95% methanol, 100% methanol；Control
Flow velocity is 8mL/min, and in first three gradient, each gradient collects 5 pipes, the last one gradient collects 8 pipes, and every pipe collects 7min,
23 pipes are collected altogether；Every pipe component is analyzed with thin-layer chromatography TLC, merges the component containing same substance, is labeled as T4, and component T4 is used
Methanol dilution to concentration is 5mg/mL, with the antioxidant activity of DPPH method measurement component T4, is evaporated, 4 DEG C of storages are spare；
Step 6: crossing purification on normal-phase silica gel post separation after component T4 adds proper amount of methanol to dissolve；Sample, silica gel stone are mixed with silica white
Fill column after oily ether saturation, after sample upper prop, the elution samples by the way of gradient elution, gradient is set as petroleum ether and acetone
Volume ratio is 300:1,250:1,200:1,150%:1,80:1,50:1,20:1,10:1,5:1；First with petroleum ether: acetone is
The elution of 300:1 shares eluent 300mL, and every 9mL collects a pipe, while tracking target point with TLC, if not sending out
Existing target point, then change next gradient；Target point is in petroleum ether: acetone is that the eluent of 10:1 is eluted, and is continued
With the eluent of this gradient, judge to elute terminal with TLC colour developing, merges the 3rd to the 8th pipe of this gradient, carry out TLC
Analysis, by vacuum-concentrcted, obtains compound A；
Step 7: compound A is fitted into nuclear magnetic tube, solvent evaporated, deuterated reagent is added, using TMS as internal standard, uses
The nuclear-magnetism of 400MH nmr determination compound A is composed, and is parsed by the data composed to nuclear-magnetism, learns that compound A is cloves
Acid obtains syringic acid.
According to embodiments of the present invention, the present invention after adopting the above technical scheme, by spirulina be added extract by
Step extracting, and by the multiple chromatographic isolation of reverse phase silica gel column and normal phase silicagel column, syringic acid to be finally made from spirulina,
The present invention is isolated to syringic acid for the first time from spirulina.
As the preferred embodiment of embodiment, include: in the step 6
A, silica white activates:
It takes 200~300 mesh silica whites in reagent bottle, wraps bottleneck with the masking foil for pricking hole, be placed in 110 DEG C of baking ovens living
Change 2~3h, seals bottleneck after cooling and be placed in drier, it is spare；
B, sample is mixed:
According to sample: silica white is that 1:1.2 carries out sample to mix sample, with rubber head dropper pipette samples drop to round-bottomed flask
In silica white on, stir evenly rapidly and interval heated to round-bottomed flask so that solvent quickly volatilizes with hair dryer, make sample
It in uniform powder, so repeats, until sample is all adsorbed on silica white, continues to heat, keep the solvent in sample complete
Volatilization is placed on spare in drier with filter paper sealing；
C, column is filled:
Using wet method dress post, weighs after 1.8g silica white is saturated with petroleum ether and fill column；
D, loading:
It uses a dry method on a sample, the silica white for being adsorbed with sample is filled in chromatographic column, make its uniform settlement in layer of silica gel table
Valve completely to sample sedimentation is opened in face, collects eluent.
As the preferred embodiment of embodiment, the thin-layer chromatography TLC tracking are as follows: by GF254 silica gel plate in 110 DEG C of baking ovens
Activate 2~3h, taken out after its cooled to room temperature, be placed in it is spare in drier, with capillary glass tube draw appropriate amount of sample,
Point is marked in sampling point lower end with pencil on silica gel plate baseline, on silica gel plate baseline distance board bottom end distance for 0.8~
1cm, the distance between two sampling points are not less than 0.8cm, after drying up solvent with hair dryer, are placed in the chromatography cylinder equipped with saturation chromatographic solution
Middle expansion is taken out when solvent front is opened up to away from lamellae top about 1cm with tweezers, solvent is dried up, with uv analyzer and iodine
Cylinder dyeing, sulfuric acid ethyl alcohol color developing agent develop the color.
Detailed description of the invention
Fig. 1 is the thin-layer chromatography result of compound A；
Fig. 2 is the structure chart of the compounds of this invention A；
Fig. 3 is the compounds of this invention A1The nmr spectrum of H-NMR；
Fig. 4 is the compounds of this invention A13C-NMR nmr spectrum.
Specific embodiment
Instrument and equipment used in the present invention is as shown in table 1 below:
The experiment of table 1 key instrument and manufacturer
Main agents used in the present invention and drug are as shown in table 2 below:
Table 2 experiment main reagent and drug
The preparation of colour reagent used in the present invention:
(1) iodine colour reagent: iodine grain and silica gel mixed proportion about 1:3, i.e. 10g iodine grain match 30g silica white, with about
Half bottle of the glass jar of 500ml, adequately shaking up makes iodine steam full of bottle body.
(2) 5% ethanol solution of sulfuric acid: taking the 25ml concentrated sulfuric acid to be added in 500ml dehydrated alcohol, mixes.
Anti-oxidant test method used herein:
(1) DPPH free radical scavenging activity is tested
The preparation of DPPH test fluid
Take DPPH 1mg to be dissolved in about 20mL ethyl alcohol, ultrasonic 5min, shake well, make sure up and down each section it is uniform.Take 1mL
The DPPH solution surveys light absorption value at 519nm, makes best between A=1.2-1.3.The DPPH solution is preferably kept in dark place, and 3.5
It is finished in hour.
The preparation of sample liquid
Sample is dissolved with suitable solvent, for that can be made into 1mg/mL concentration convenient for calculating.
A0The measurement of value
It takes DPPH solution 2mL to be added in small test tube, adds dehydrated alcohol 1mL, be sufficiently mixed, survey A value (519nm), this A value
For A0。
The measurement of A value
Take DPPH solution 2mL to be added in small test tube, add sample liquid x μ L, then plus (1000-x) μ L dehydrated alcohol, mixing,
After standing 30 minutes, survey A value (519nm)
DPPH clearance rate calculates
(2) measurement of total antioxidant capacity
180uL FRAP working solution is added in each detection hole of 96 orifice plates.
The appropriate solution such as 5uL distilled water or PBS is added in blank control wells；It is various that 5uL is added in standard curve detection hole
The FeSO4 standard solution of concentration；The Trolox of the various samples of 5uL or 0.15-1.5mM are added in sample detection hole as positive right
According to.It mixes gently.
A593 is measured after 37 DEG C of incubation 3-5min.If measurement A593 have any problem, can also within the scope of 585-605nm into
Row measurement.
The total antioxidant capacity of sample is calculated according to standard curve.If sample determines the absorbance come in standard song
Other than line range, it is measured again after sample need to suitably being diluted.
The representation of total antioxidant capacity: for FRAP method, the concentration of total antioxidant capacity FeSO4 standard solution
To indicate.Such as the absorbance that the measurement of certain plasma sample obtains is identical with the absorbance of 1mM FeSO4 standard solution, then the blood plasma
The total antioxidant capacity of sample is 1mM；For another example the absorbance and 0.65mM FeSO4 standard that the measurement of certain blood serum sample obtains are molten
Liquid phase is same, then the total antioxidant capacity of the blood serum sample is 0.65mM；For another example certain cell homogenates liquid measurement obtain absorbance and
0.3mM FeSO4 standard solution is identical, and the protein concentration of the homogenate is 0.15mg/ml, then the cell sample is total anti-
Oxidability is 0.3mM/0.15mg/ml, i.e. 2mmol/g；For another example certain polyphenoils of 0.2mM its measurement obtain absorbance and
The absorbance that the ferrous salt measurement of 1mM obtains is identical, then it is 5 with respect to total antioxidant capacity.
(3) xanthine oxidase inhibitor
Phosphate buffer (0.1moL/L pH7.0)
Accurately weigh disodium hydrogen phosphate (Na2HPO4.2H2O) 2.066g, sodium dihydrogen phosphate (NaH2PO4.12H2O)
0.659g, sodium chloride (Nacl) 0.900g, are settled to 100ml with distilled water after dissolution, room temperature preservation is spare.
The xanthine (ready-to-use) of 1mM
Xanthine powder 15.211mg is accurately weighed, is dissolved in 2ml 0.1M NaOH, then be dissolved in the PBS buffer solution of 0.1M
In the volumetric flask of 100ml, saved under room temperature.
Xanthine oxidase
The xanthine oxidase stoste of existing 10U/mL, being configured to concentration with PBS is 0.1U/mL, 4 DEG C be kept in dark place it is standby
With.
Allopurinol (positive control, 10 μ g/ml are best)
Accurately weigh 10.000mg in allopurinol dry powder, be first dissolved in 1Ml 0.1M NaOH, be re-dissolved in the appearance of 10ml
Up to the stock solution of 1mg/mL in measuring bottle, the used time is diluted with PBS up to working solution.
Test and calculating
1 C2 C3 C4 of reagent C；
PBS/uL 175 125 100 175；
Sample/uL- -25 25；
Xanthine oxidase/uL 25 25 25-；
37 DEG C, it is incubated for 10min；
Xanthine/uL-50 50-；
(UV-96WELL) is detected immediately at vibration plate 30s, 295nm wavelength.
In formula: C1- zeroing group；
C2- reaction group completely；
C3- example reaction group (positive control)；
C4- sample controls group.
Embodiment
The method that present invention discloses a kind of to prepare syringic acid from spirulina, comprising the following steps:
Spirulina polyphenol crude extract is enriched with ether:
It accurately weighs 50g spirulina powder to be placed in the triangular flask of 1L, 500mL anhydrous ether is added and shakes up, extracts 4h, filters
Extract liquor collects filtrate；500mL anhydrous ether is added into precipitating again and carries out second of extraction 4h, filters extract liquor, collects filter
Liquid；So extraction 3 times, merging filtrate, vacuum-concentrcted is evaporated, and is 6.6828g according to the quality that difference assay obtains crude extract.
The first time of crude extract isolates and purifies:
Sephadex LH-20 gel column is selected in the first time separation of crude extract, and eluant, eluent selects methanol, first with elution
Pillar is rinsed in agent, and suitable methanol dissolution, loading to the Sephadex LH- rinsed are then added into 6.6828g crude extract
20 gel columns start to elute, and coutroi velocity is 8s/ drop, and every 0.5h collects a pipe, collect 216 pipes altogether；Every pipe component will use
TLC carries out substance tracking, determines that best solvent is chloroform: methanol=10:1 by groping, according to ultra-violet analysis, iodine cylinder
The colour developing situation of dyeing and sulfuric acid ethyl alcohol merges the component containing same substance, 4 kinds of components is mainly obtained, by four kinds of components
It is evaporated to obtain Y1 (28-41) 2.1733g, Y2 (42-53) 0.3909g, Y3 (54-70) 0.3018g, Y4 (71-83) respectively
0.2804g, it is 5mg/mL that four kinds of components, which are distinguished dissolved dilution to concentration, and then three kinds of methods used by above are surveyed respectively
The antioxidant activity of fixed four kinds of components, as shown in table 3- table 5.It is Y4 component labeled as T1 that it is highest, which to select antioxidant activity,；It surveys
Component T1 is evaporated after complete, 4 DEG C of storages are spare.
Clearance rate of 3 Y1-Y4 of table, the tetra- kinds of components to DPPH free radical
The total antioxidant capacity of 4 tetra- kinds of components of Y1-Y4 of table
Inhibiting rate of 5 Y1-Y4 of table, the tetra- kinds of components to XOD enzyme
Crude extract isolates and purifies for the second time:
C18 reverse phase silica gel column is selected in second of purifying, and suitable methanol dissolved constituent T1 is added, and loading extremely pre-processes
RP-C18 column, the elution samples by the way of gradient elution, gradient is set as 95% water: 5% methanol, 75% water: 25% first
Alcohol, 50% water: 50% methanol, 25% water: 75% methanol, 100% methanol；Set maximum pressure 1MPa, coutroi velocity 8mL/
Min, in first four gradient, each gradient collects 5 pipes, the last one gradient collects 16 pipes, and every pipe collects 7min, collects 36 altogether
Pipe；Pillar 40min is rinsed with+95% methanol of 5% water after collection, then rinses pillar 40min with 100% methanol, has been separated
Finish；By each pipe component of collection, 45 DEG C of vacuum-concentrcteds are transferred in small test tube respectively, and contact plate carries out TLC analysis respectively,
According to the colour developing situation of ultra-violet analysis, the dyeing of iodine cylinder and sulfuric acid ethyl alcohol, merge the component containing same substance, vacuum decompression is dense
Y5 (the 2-4 pipe of the 3rd gradient) 14.9mg and Y6 (the 2-3 pipe of the 5th gradient) 42.2mg is obtained after contracting, selected amount is larger
Y6 component, be labeled as T2, be added proper amount of methanol be dissolved to concentration be 5mg/mL, then measure its anti-oxidant work with DPPH method
Property, the results are shown in Table 6；Sample is evaporated after having surveyed, 4 DEG C of storages are spare.
Clearance rate of the 6 T2 component of table to DPPH free radical
The third time of crude extract isolates and purifies:
Suitable methanol dissolution, loading to the Sephadex LH-20 gel column rinsed in advance, choosing is added in T2 component
Methanol is eluted as eluant, eluent, and adjusting flow velocity is 13s/drop, and every 0.5h collects a pipe, collects 40 pipes altogether；Every pipe component
All distinguish contact plate and carry out TLC analysis, analyze three kinds of colour developing situations, component of the merging containing same substance, vacuum-concentrcted it
After obtain Y7 (15-17) 15.2mg, be labeled as component T3, be added proper amount of methanol be dissolved to concentration be 5mg/mL, then use DPPH
Method measures its antioxidant activity, and the results are shown in Table 7；Sample is evaporated after having surveyed, 4 DEG C of storages are spare.
Clearance rate of the 7 T3 component of table to DPPH free radical
The 4th time of crude extract isolates and purifies:
C18 reverse phase silica gel column is selected in 4th purifying, is rinsed the general 0.5h of pillar with+75% methanol of 25% water first, is added
After entering suitable methanol sample dissolution, it is splined on processed RP-C18 column, the elution samples by the way of gradient elution,
Gradient is set as 25% water: 75% methanol, 15% water: 85% methanol, 5% water: 95% methanol, 100% methanol；Coutroi velocity is
8mL/min；Maximum pressure 1MPa, coutroi velocity 8mL/min are set, in first three gradient, each gradient is collected 5 and managed, finally
One gradient collects 8 pipes, and every pipe acquisition time is 7min, collects 23 pipes altogether；It is rinsed after collection with+95% methanol of 5% water
Pillar 40min, separation finish；Each pipe component of collection is distinguished into 45 DEG C of vacuum-concentrcteds into small test tube, respectively contact plate
TLC analysis is carried out, according to the dyeing of iodine cylinder, the colour developing situation of sulfuric acid ethyl alcohol, it is dense to merge the component vacuum decompression containing same substance
Y8 (the 3rd gradient 2-3 pipe) 10.6mg is obtained after contracting, is labeled as component T4, component T4 is added proper amount of methanol and is dissolved to concentration
For 5mg/mL, its antioxidant activity then is measured with DPPH method, the results are shown in Table 8；Sample is evaporated after having surveyed, 4 DEG C of storages
It hides spare.
Clearance rate of the 8 T4 component of table to DPPH free radical
The 5th time of crude extract isolates and purifies:
According to the separating effect of front, this time normal phase silicagel column is selected in purifying；First silica white is activated: taking 200~300 mesh
Silica white wraps bottleneck in reagent bottle, with the masking foil for pricking hole, is placed in 2~3h of activation in 110 DEG C of baking ovens, seals bottle after cooling
Mouth is placed in drier, spare.Carry out mixing sample again: according to sample: silica white is that 1:1.2 carries out sample to mix sample, is dripped with rubber head
Pipe pipette samples drop stirs evenly rapidly on the silica white into round-bottomed flask and interval is heated with hair dryer to round-bottomed flask
So that solvent quickly volatilizes, makes sample in loose mixture, so repeat, until sample is all adsorbed on silica white, after
Continuous heating, makes the solvent in sample volatilize completely, is placed on filter paper sealing spare in drier.Then by 1.8g purification on normal-phase silica gel
Powder fills column after being saturated with petroleum ether.After sample upper prop, the elution samples by the way of gradient elution, gradient be set as petroleum ether and
The volume ratio of acetone is 300:1,250:1,200:1,150:1,80:1,50:1,20:1,10:1,5:1；First with petroleum ether: third
Ketone is the elution of 300:1, shares eluent 300mL, every 9mL collects a pipe, while tracking target point with TLC, if not having
It is found target point, then changes next gradient；Target point is in petroleum ether: acetone is that the eluent of 10:1 is eluted,
Then continue the eluent with this gradient, judge to elute terminal with TLC colour developing, merges the 3rd to the 8th pipe of this gradient,
It carries out TLC analysis and obtains compound A by vacuum-concentrcted.
In addition, TLC analysis of the present invention is that GF254 silica gel plate is activated to 2~3h in 110 DEG C of baking ovens, to it
Taken out after cooled to room temperature, be placed in it is spare in drier, with capillary glass tube draw appropriate amount of sample, put in silica gel plate baseline
On, and marked in sampling point lower end with pencil, baseline distance board bottom end distance is 0.8~1cm on silica gel plate, between two sampling points
Distance is not less than 0.8cm, after drying up solvent with hair dryer, is placed in the chromatography cylinder equipped with saturation chromatographic solution and is unfolded, before solvent
It when extending to away from lamellae top about 1cm, is taken out with tweezers, dries up solvent, shown with uv analyzer and other color developing agents
Color.
The Structural Identification of compound:
Nuclear magnetic tube is impregnated with potassium bichromate first, after impregnating 12h, the cleaning of potassium bichromate solution again with methanol is thrown away, if first
Alcohol does not wash clean to be washed with chloroform again, and methanol rinses several times for using chromatographic grade after wash clean again, nuclear magnetic tube dries up after washing
Claim its weight, sample deuterated methanol is dissolved into sucking nuclear magnetic tube, then evaporative flask vacuum decompression is placed on filter paper sealing and is evaporated
Solvent, then claim its weight, the weight by the compound A finally detected known to difference assay is 3.5mg, finally uses " 400MHz-
NMR " measures the nuclear-magnetism spectrum of compound.
Compound A is fitted into nuclear magnetic tube, solvent evaporated, deuterated reagent is added using TMS as internal standard and uses 400MH core
Magnetic resonance device measures the nuclear-magnetism spectrum of compound A, and compound A nuclear-magnetism modal data is as shown in table 9.
The nuclear-magnetism modal data of 9 compound A of table
The ESI-MS:m/z=221.39 of compound A known to after progress mass spectroscopy, the nuclear-magnetism modal data of comprehensive compound A
And mass spectrometric data and it is compared with document, thus it is speculated that compound A (molecular formula C9H10O5,Molecular weight be 198.23) for syringic acid,
Structure is as shown in Figure 2.Compound syringic acid1H-NMR、13C-NMR nuclear magnetic spectrogram is as shown in Figures 3 and 4.
The present invention mainly passes through gel filtration chromatography and positive reversed-phase silica gel column chromatography divides the active constituent in spirulina
From, and syringic acid has been isolated for the first time, active constituent of the syringic acid as spirulina, for the secondary development meaning weight of spirulina
Greatly.
All deformations that those skilled in the art directly can export or associate from the disclosure of invention, should all
It is considered protection scope of the present invention.
Claims (3)
1. a kind of method for preparing syringic acid from spirulina, it is characterised in that: the following steps are included:
It is placed in the triangular flask of 1L Step 1: weighing 50g spirulina powder, 500mL anhydrous ether is added and shakes up, extracts 4h, filters,
Collect filtrate；500mL anhydrous ether is added into precipitating again and carries out second of extraction 4h, filters, collects filtrate；So extraction 3
Secondary, merging filtrate, vacuum-concentrcted is evaporated, and obtains crude extract；
Step 2: after crude extract is dissolved with methanol, upper Sephadex LH-20 gel column uses methanol to be washed as eluant, eluent
De-, coutroi velocity is 8 s/drop, and every 0.5h collects a pipe, collects 216 pipes altogether, and every pipe component is analyzed with thin-layer chromatography TLC, is closed
And the component containing same substance；It is 5mg/mL that component will be merged again with methanol dilution to concentration, measures merging group with DPPH method
The antioxidant activity divided is selected the highest merging component of antioxidant activity to be labeled as T1, is evaporated, and 4oC storage is spare；
Step 3: after component T1 is dissolved with proper amount of methanol, upper C18 reverse phase silica gel column, the elution samples by the way of gradient elution,
Gradient is set as 95% water: 5% methanol, 75% water: 25% methanol, 50% water: 50% methanol, 25% water: 75% methanol, 100% methanol；Control
Flow velocity processed is 8mL/min, and in first four gradient, each gradient collects 5 pipes, the last one gradient collects 16 pipes, and every pipe is collected
7min collects 36 pipes altogether；Every pipe component is analyzed with thin-layer chromatography TLC, merges the component containing same substance；Selected amount is biggish
Merge component, be labeled as T2, be 5mg/mL with methanol dilution to concentration, with the antioxidant activity of DPPH method measurement component T2, steams
Dry, 4oC storage is spare；
Step 4: after component T2 is dissolved with proper amount of methanol, upper Sephadex LH-20 gel column uses methanol to carry out as eluant, eluent
Elution, coutroi velocity are 13 s/drop, and every 0.5h collects a pipe, collects 40 pipes altogether, and every pipe component is analyzed with thin-layer chromatography TLC,
Merge the component containing same substance, is labeled as T3；It is again 5mg/mL with methanol dilution to concentration by component T3, is surveyed with DPPH method
The antioxidant activity for determining component T3, is evaporated, and 4oC storage is spare；
Step 5: after component T3 adds proper amount of methanol to dissolve, upper C18 reverse phase silica gel column, the elution samples by the way of gradient elution,
Gradient is set as 25% water: 75% methanol, 15% water: 85% methanol, 5% water: 95% methanol, 100% methanol；Coutroi velocity is 8mL/
Min, in first three gradient, each gradient collects 5 pipes, the last one gradient collects 8 pipes, and every pipe collects 7min, collects 23 pipes altogether；
Every pipe component with thin-layer chromatography TLC analyze, merge the component containing same substance, be labeled as T4, component T4 with methanol dilution extremely
Concentration is 5mg/mL, with the antioxidant activity of DPPH method measurement component T4, is evaporated, 4oC storage is spare；
Step 6: crossing purification on normal-phase silica gel post separation after component T4 adds proper amount of methanol to dissolve；Sample, silica gel petroleum ether are mixed with silica white
Fill column after saturation, after sample upper prop, the elution samples by the way of gradient elution, gradient is set as the volume of petroleum ether and acetone
Than for 300:1,250:1,200:1,150%:1,80:1,50:1,20:1,10:1,5:1；First with petroleum ether: acetone 300:1
Elution, share eluent 300mL, every 9mL collects a pipe, while tracking target point with TLC, if not finding target
Point then changes next gradient；Target point is in petroleum ether: acetone is that the eluent of 10:1 is eluted, and continues to use this
The eluent of gradient judges elution terminal with TLC colour developing, merges the 3rd to the 8th pipe of this gradient, carries out TLC analysis,
By vacuum-concentrcted, compound A is obtained；
Step 7: compound A is fitted into nuclear magnetic tube, solvent evaporated, deuterated reagent is added, using TMS as internal standard, uses
The nuclear-magnetism of 400MH nmr determination compound A is composed, and is parsed by the data composed to nuclear-magnetism, learns that compound A is cloves
Acid obtains syringic acid.
2. a kind of method for preparing syringic acid from spirulina as described in claim 1, it is characterised in that: in the step 6
Include:
A, silica white activates:
It takes 200 ~ 300 mesh silica whites in reagent bottle, wraps bottleneck with the masking foil for pricking hole, be placed in 110 DEG C
2 ~ 3h is activated in baking oven, is sealed bottleneck after cooling and is placed in drier, it is spare；
B, sample is mixed:
According to sample: silica white is that 1:1.2 carries out sample to mix sample, with rubber head dropper pipette samples drop into round-bottomed flask
On silica white, stirs evenly rapidly and intermittently heated to round-bottomed flask so that solvent quickly volatilizes with hair dryer, it is in equal for making sample
It is even powdered, it so repeats, until sample is all adsorbed on silica white, continues to heat, the solvent in sample is made to volatilize completely,
It is placed on filter paper sealing spare in drier；
C, column is filled:
Using wet method dress post, weighs after 1.8g silica white is saturated with petroleum ether and fill column；
D, loading:
It uses a dry method on a sample, the silica white for being adsorbed with sample is filled in chromatographic column, make its uniform settlement in silica gel layer surface, to
Sample sedimentation completely, opens valve, collects eluent.
3. a kind of method for preparing syringic acid from spirulina as described in claim 1, it is characterised in that: the thin-layer chromatography
TLC tracking are as follows: GF254 silica gel plate is activated into 2 ~ 3h in 110 DEG C of baking ovens, takes out, is placed in dry after its cooled to room temperature
It is spare in dry device, appropriate amount of sample is drawn with capillary glass tube, point carries out mark with pencil in sampling point lower end on silica gel plate baseline
Remember, baseline distance board bottom end distance is 0.8 ~ 1cm on silica gel plate, and the distance between two sampling points are not less than 0.8cm, are dried up with hair dryer
It after solvent, is placed in the chromatography cylinder equipped with saturation chromatographic solution and is unfolded, when solvent front is opened up to away from lamellae top about 1cm, use
Tweezers take out, and dry up solvent, are developed the color with uv analyzer and the dyeing of iodine cylinder, sulfuric acid ethyl alcohol color developing agent.
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| CN1520837A (en) * | 2003-01-30 | 2004-08-18 | 普文英 | Plant extract and compound for treating endotoxin blood disease, and its extraction method and application |
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